TitleEchocardiographic Evaluation of Myocardial Function in Dogswith Dilated Cardiomyopathy Using Two-dimensional TissueTracking Method( 本文(Fulltext) )

Author(s) 濵部, 理奈

Report No.(DoctoralDegree) 博士(獣医学) 甲第416号

Issue Date 2014-03-13

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/49039

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

1

Echocardiographic Evaluation of Myocardial Function in

Dogs with Dilated Cardiomyopathy

Using Two-dimensional Tissue Tracking Method

2013

The United Graduate School of Veterinary Sciences, Gifu University

(Tokyo University of Agriculture and Technology)

HAMABE, Lina

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Table of Contents

List of Abbreviations ………………………………………………………………. 4

Prolusion …...………………………………………………………………………. 6

Chapter 1 Evaluation of left ventricular myocardial function observed in canine

myocardial dysfunction model using two-dimensional tissue tracking

Introduction …………………………………………………………………... 11

Materials and Methods ………………………………………………….......... 13

Results ………………………………………………………………………… 17

Discussion …………………………………………………………………….. 24

Conclusion ……………………………………………………………………. 29

Chapter 2 Comparative evaluation of inotropic agents, pimobendan and

SCH00013, on the myocardial function of canine myocardial

dysfunction model

Introduction …………………………………………………………………… 30

Materials and Methods ………………………………………………………... 32

Results …………………………………………………………………….…... 35

Discussion …………………………………………………………………….. 44

Conclusion ……………………………………………………………………. 48

Chapter 3 Assessment of myocardial function in retrievers with dilated

cardiomyopathy using two-dimensional tissue tracking

Introduction ………………………………………………………………….... 49

Materials and Methods ………………………………………………………... 50

Results ………………………………………………………………………… 53

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Discussion …………………………………………………………………….. 58

Conclusion ………………………………………………………………......... 61

Chapter 4 Evaluation of myocardial changes observed before and after closure

of patent ductus arteriosus in dogs using two-dimensional tissue

tracking

Introduction …………………………………………………………………… 62

Materials and Methods ………………………………………………………... 64

Results ………………………………………………………………………… 67

Discussion …………………………………………………………………….. 72

Conclusion ………………………………………………………………..........75

Summery ...…………………………………………………………………………. 76

General Conclusion …………………………………………………………………79

Acknowledgement …………………………………………………………………. 81

References ………………………………………………………………………….. 82

4

List of Abbreviations

− 2DTT: Two-dimensional tissue tracking

− A: Late ventricular filling velocity

− Aa: Late myocardial velocity

− BP: Blood pressure

− bpm: Beats per minute

− cAMP: Cyclic adenosine monophosphate

− CHF: Congestive heart failure

− DCM: Dilated cardiomyopathy

− E: Early ventricular filling velocity

− Ea: Early myocardial velocity

− ECG: Electrocardiography

− FS: Fractional shortening

− IVS: Interventricular septum

− IVSd: Interventricular septal thickness in diastole

− IVSs: Interventricular septal thickness in systole

− LA/Ao: Left atrium to aorta ratio

− LV: Left ventricle

− LV dP/dtmax: Maximum rate of LV systolic pressure

− LVIDd: Left ventricular end-diastolic diameter

− LVIDs: Left ventricular end-systolic diameter

− LVOT pV: Aortic velocity

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− LVFW: Left ventricular free wall

− LVFWd: Left ventricular free wall thickness in diastole

− LVFWs: Left ventricular free wall thickness in systole

− MRI: Magnetic resonance imaging

− PDA: Patent ductus arteriosus

− PDE: Phosphodiesterase

− PEP:ET: Systolic time intervals

− PIE: Positive inotropic effect

− RV: Right ventricle

− Sa: Systolic myocardial velocity

− SD: Standard deviation

− STI: Synchrony time index

− SV: Stroke volume

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Prolusion

Cardiomyopathy is a disease of the myocardium, and canine dilated

cardiomyopathy (DCM) is the most common form of cardiomyopathy in dogs, which is

often observed in adult large breed dogs (22, 51). DCM is characterized by progressive

dilation and myocardial hypokinesis of the left ventricle (LV) or both ventricles, in

absence of other cardiac abnormalities (22, 51, 65). These myocardial changes

eventually results in development of congestive heart failure (CHF) with accompanying

clinical signs including syncope and exercise intolerance, which inevitably leads to

death (22, 51, 65). Echocardiographic diagnosis of DCM requires the demonstration of

dilation, myocardial hypokinesis and increased sphericity of the LV (22, 51, 65).

Diagnosis of symptomatic DCM using standard echocardiography is relatively

straightforward, and a previous study of 70 dogs in CHF was able to diagnose DCM

with a sensitivity of 93%, with the final diagnosis made based on post mortem criteria

(65).

DCM has a prolonged duration of asymptomatic phase, which is not

associated with any evident clinical signs (22, 51). This phase is characterized by the

presence of myocardial or electrical abnormalities, with variable duration that may last

for months to years until the appearance of the first clinical signs (22, 51, 65). Standard

echocardiography is not always sensitive enough for the detection of early subtle

myocardial changes, and the diagnosis of asymptomatic phase remains a challenge (22,

51). However, development of CHF and sudden death as the first clinical sign are

common, especially sudden death is as common as 40% in Doberman Pinschers (51).

For such reasons, early diagnosis and prompt treatment of DCM is crucial to the

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outcome of the disease (51).

Tagged-magnetic resonance imaging (MRI) is considered the gold standard

for quantitative assessment of myocardial function, but factors including high costs,

poor availability, and the need of general anesthesia limit its routine clinical use in

veterinary medicine (20). On the other hand, standard echocardiography has been

widely used as a non-invasive method for the assessment of myocardial function, and

commonly used parameters of systolic function include fractional shortening (FS) (9,

17). FS is calculated from myocardial motion of two specific LV segments in short-axis,

therefore it is a rough estimate of the radial global function (9). However, ventricular

function involves a multitude of interrelated factors of systolic and diastolic function,

ventricular synchrony and regional myocardial function, and additionally the systolic

myocardial mechanics occurs in radial, circumferential, and longitudinal directions (46,

54). Therefore, there is an increasing need for comprehensive quantitative assessment of

the myocardial function.

Strain analysis has recently emerged as a more comprehensive and reliable

assessment method of myocardial function (20). Strain is a parameter of strain analysis,

and it is a measurement of deformation (4, 20, 32, 46). Strain allows measurement of

local deformation to assess both global and regional myocardial function, and it can be

measured from either tissue Doppler-derived or two-dimensional tissue tracking (2DTT)

strain analysis (4, 20, 32, 46). However, tissue Doppler-derived strain analysis faces a

number of limitations including angle of dependency, which the strain can only be

measured along the ultrasound beam (4, 20, 32, 46). On the other hand, 2DTT strain

analysis tracks the natural acoustic reflections and interference patterns, known as

“speckles”, within an ultrasonic window to analyze myocardial motion (4, 32). Since

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“speckles” can be tracked in any directions, it allows measurements of strain in radial,

circumferential, and longitudinal directions (4, 32). Strain derived from 2DTT has been

validated using sonomicrometry and tagged-MRI (32, 46).

In humans, 2DTT has been used for the detection of sub-clinical diseases and

the assessment of myocardial damage such as myocardial infarction, coronary artery

disease and cardiomyopathies, and the prediction of patient outcome with heart failure

and response to cardiac resynchronization therapy (20, 32, 46). In veterinary medicine,

the numbers of reports on 2DTT has been limited, and most are on the evaluation of

myocardial function in healthy animals (15, 17, 19, 33, 54, 64). These reports have

demonstrated the applicability of 2DTT for the evaluation of myocardial function in

veterinary medicine. In order to apply the strain analysis for the diagnosis of DCM in

dogs, first the ability of 2DTT to evaluate the LV myocardial dysfunction associated

with DCM needs to be determined in fundamental study. Therefore, in chapter 1, an

epicardial pacemaker was implanted at the right ventricle (RV) of a dog, and canine

myocardial dysfunction model was created by performing sustained high-electrical

pacing at the rate of 250 beats per minute (bpm) for three consecutive weeks. The

ability of 2DTT to evaluate the changes in myocardial function associated with

sustained high-electrical pacing was evaluated using both standard echocardiography

and 2DTT.

In veterinary medicine, inotropic agents, such as digoxin and pimobendan, are

used to improve contractility, and play an important role in the treatment of DCM (10,

51). Pimobendan is a benzimidazole-pyridazinone derivative, and with its actions of

calcium sensitization and phosphodiesterase (PDE) inhibition, it exerts both positive

inotropic effect (PIE) and vasodilation (11). There have been numerous studies on the

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effect of pimobendan in dogs with DCM that have demonstrated beneficial effects, but

the evaluations were based mainly on the clinical signs of CHF and the survival time

(29, 49, 60). SCH00013 is also an inotropic agent with similar actions to that of

pimobendan (25). In order to assess the myocardial response to these medications,

myocardial function needs to be evaluated. Therefore, in chapter 2, myocardial response

to pimobendan and SCH00013 was evaluated in canine myocardial dysfunction model

using standard echocardiography and 2DTT.

Once the applicability of 2DTT for the evaluation of myocardial function has

been established using the experimental model, the strain analysis was applied to

clinical cases of DCM in chapter 3. Body size, breed, and somatotype are known to

influence the parameters of both standard echocardiography and 2DTT (21, 37, 64). FS

and strain parameters decrease with increased body weights, unrelated to systolic

function, and this is especially important for diagnosis of myocardial hypokinesis in

DCM (64). Therefore, in chapter 3, the breed of dog was limited to retrievers, including

Golden and Labrador retrievers. Breed predisposition of DCM include Doberman

pinscher, Irish Wolfhound, Scottish Deerhound, and Great Dane (51). However, these

breeds are uncommon in Japan, whereas Golden and Labrador retrievers are the most

and secondly most commonly owned large breed dogs in Japan according to Japan

Kennel Club. For such reason, in Japan, DCM is often observed in retrievers. Therefore,

in chapter 3, difference in myocardial function was observed between the clinical

retrievers with DCM and healthy retrievers using the standard echocardiography and

2DTT.

Patent ductus arteriosus (PDA) is a congenital cardiovascular defect, which

blood flow from aorta to pulmonary artery through the persistent ductus arteriosus,

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resulting in over-circulation in the lungs and left side of the heart, causing left-sided

volume overload (8, 12, 13, 58). FS is known to be influenced by loading condition, and

closure of PDA results in reduction of FS, which is unrelated to reduced contractility (9).

The influence of loading condition has also been reported in 2DTT as well, but since

strain analysis provides a comprehensive evaluation, it allows better quantitative

assessment of the myocardial function (14, 19, 70). Therefore, in chapter 4, myocardial

changes observed before and after the closure PDA was evaluated using standard

echocardiography and 2DTT, in aims to evaluate the short-term echocardiographic

changes in dogs before and after the surgical ligation of PDA.

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Chapter 1 Evaluation of left ventricular myocardial function observed in canine

myocardial dysfunction model using two-dimensional tissue tracking

Introduction

Sustained high-electrical pacing has been shown to produce dilated,

congestive cardiomyopathy in dogs that closely resemble the hemodynamic and

neurohumoral changes observed with myocardial dysfunction of both human and dog,

and this canine myocardial dysfunction model has been widely used to investigate CHF

(55, 56, 72, 73, 76). Therefore, evaluation of the myocardial function of this model may

allow better understanding of myocardial function in DCM.

Standard echocardiography is a non-invasive method of assessing cardiac

function that has become one of the most important diagnostic tools for cardiovascular

disorders (9, 18, 28). Commonly used standard echocardiographic parameters for the

assessment of LV function are obtained from M-mode measurements, and these include

LV chamber dimensions and FS (9, 28). However, FS may not reflect the true global

function, since it is a measurement of two specific LV segments at the anteroseptal and

posterior walls, and it only assesses the radial deformation (4, 46, 52, 64). In addition,

LV function is influenced by a number of factors, including regional myocardial

function and ventricular synchrony, which cannot be completely assessed based on

standard echocardiographic parameters alone (54).

2DTT is a relatively new strain analysis that provides a novel approach to the

assessment of myocardial function (32, 45, 64). 2DTT is based on the formation of

‘speckles’ caused by reflection, scattering, and interference between tissue and

ultrasound beams in the standard grey scale, two-dimensional echocardiographic images

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(4, 17, 20, 24, 32, 41, 45, 52, 54). Tracking these ‘speckles’ from one frame to another

enables analysis of myocardial movement throughout the entire cardiac cycle and

calculation of strain, which is a measurement of deformation of the myocardium over

time, expressed as the percent change from the original (4, 17, 20, 24, 32, 45, 46, 52,

54). Advantages of 2DTT include simultaneous evaluation of global and regional

myocardial function independent of the angle of insonation and cardiac translational

movement, and analysis of myocardial deformation in three spatial directions (4, 20, 32,

45).

The ability of 2DTT to evaluate the LV myocardial function associated with

sustained high-electrical pacing has not yet been assessed. Therefore, the aim was to

investigate the ability of 2DTT to evaluate (1) global myocardial function, (2) regional

myocardial function, and (3) the degree of dyssynchrony in the canine myocardial

dysfunction model.

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Materials and Methods

Animals

This experiment was carried out in accordance with the standards established

by the Tokyo University of Agriculture and Technology and its Guide for the Care and

Use of Laboratory Animals. All experimental procedures were approved by the Animal

Experimental Committee of Tokyo University of Agriculture and Technology

(acceptance No. 21–30). Five female Beagles with approximately one year of age were

used. The dogs had body condition scores of 4 to 6 in a 9-point scale, and their body

weights ranged from 8.75 to 10.00 kg. All dogs received complete physical examination,

blood count and plasma biochemical analysis, blood pressure (BP) measurement,

electrocardiography (ECG), radiography, and echocardiography prior to initiating the

experiment to rule out the presence of any other abnormalities.

Study Design

Thoracotomy was performed on each dog for the implantation of an epicardial

pacemaker at the RV apex, after which sustained high-electrical pacing of 250 bpm was

applied for three consecutive weeks. For each dog, an oscillometric BP measurement

(BP-100D, FUKUDA M.E KOGYO, Japan), standard 6-lead ECG (α8000, FUKUDA

M.E KOGYO, Japan), and standard echocardiography and 2DTT were performed at the

baseline and weeks 1, 2, and 3 of pacing. The entire examination was performed during

a temporary cessation of pacing, which the pacemaker was turned off for at least an

hour prior to the examination. All pacemakers were checked for accurate pacing at the

end of the each examination.

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Instrumentation

Each dog was pre-medicated with subcutaneous administration of atropine

and intravenous buprenorphine and midazolam, after which anaesthesia was induced

with intravenous propofol and general anaesthesia was maintained by the inhalation of

isoflurane. Thoracotomy was performed under general anaesthesia with the dog

positioned on the right lateral recumbency via an incision made at the fourth intercostal

space. The heart was exposed through pericardectomy, after which the pacemaker

electrode (TF204-062, KMY Medical, Japan) was implanted at the RV apex. The lead

was then tunnelled to the back and exited immediately ventral to the vertebral process,

where it was connected to a pulse generator (EV4543, Pace Medical, USA). After

recovery from general anaesthesia, the dog was returned to a post-surgical care facility,

where it received a standard diet and free access to water. Following surgery, each dog

was given a week of recovery period before initiating the pacing.

Echocardiography

An ultrasonography unit (ALOKA prosound α 10, Hitachi Aloka Medical,

Ltd., Japan) equipped with a 5MHz phased array transducer probe (UST52108, Hitachi

Aloka Medical, Ltd., Japan) was used for all echocardiographic examinations, which

were performed with concurrent ECG monitoring by the same trained examiner for all

dogs. All examinations were performed on awake dogs, using only gentle restraint. A

mean of three measurements of each parameter was obtained from three consecutive

cardiac cycles.

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Standard Echocardiography

The examinations were performed in accordance with the methods described

by Boon (9). LV measurements were taken from the right parasternal short axis view at

the level of the papillary muscle using M-mode. Measurements included LV

end-diastolic (LVIDd) and end-systolic (LVIDs) diameters, LV free wall thickness in

diastole (LVFWd) and in systole (LVFWs), interventricular septal thickness in diastole

(IVSd) and in systole (IVSs), and FS. The percentage of wall thickening of the

interventricular septum (IVS) and left ventricular free wall (LVFW), which is a

parameter of regional contractility of the myocardium evaluated by the changes in wall

thickening, were calculated as: (systolic thickness – diastolic thickness) / (diastolic

thickness) × 100. Additionally, diastolic indices including early (E) and late (A)

ventricular filling velocity, E/A ratio, and deceleration time were obtained from the

trans-mitral flow profile at the left parasternal apical four chamber view.

Two-Dimensional Tissue Tracking

The right parasternal short axis view at the level of the papillary muscle with

a rate of 70–110 frames/s was acquired and saved onto a hard disk and then analysed

off-line (DAS-RS1 software 1.1v, Hitachi Aloka Medical, Ltd., Japan). The observer

was unaware of the measurements of standard echocardiographic parameters while

analysing the strain analysis. Strain analysis involved multiple steps. Initially, the

endocardium and the epicardium were manually traced at the end-systole by placing a

number of points at the borders of the myocardium. The software then automatically

tracked these points on a frame-by-frame basis, after which it divided the LV into six

segments (anterior, lateral, posterior, inferior, septal, and anterior septal). Regional peak

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systolic strains of the six segments and global peak systolic strains, which are the mean

of the six segments, were calculated for both the radial and circumferential directions.

LV synchrony was also assessed by calculating the synchrony time index (STI), which

is the difference in timing of peak strains from the earliest to the latest segments.

Statistical Analysis

Variables are expressed as mean ± standard deviation (SD). Parameters of

standard echocardiography and 2DTT were tested for normality using the

Kolmogorov-Smirnov test and the visual inspection of histograms. The baseline value

for each parameter was compared to the values of week 1, 2, and 3 for statistical

significance using one-way repeated-measures ANOVA for parametric variables and

Friedman test for non-parametric variables. When a significant difference was detected,

further testing was performed using Dunnett’s multiple comparison tests to compare the

baseline measurements with weeks 1, 2, and 3. Significant difference was defined as p<

0.05. Statistical analysis was performed using statistical software (Prism 5.0v,

GraphPad Software, USA and JMP 10.0.2, SAS Institute, USA).

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Results

Pacemaker implantation was successful and recovery was uneventful in all

five dogs. Additionally, all dogs were able to maintain adequate physical condition and

survived until the last day of pacing. The resting heart rate with cessation of pacing at

each examination revealed no significant changes from the baseline. BP measurements

showed declining trends, but significant differences were only observed with the mean

and diastolic BP, which the differences were observed from week 1 of pacing. No

abnormalities on the ECG were observed during any examinations.

Standard Echocardiography (Table 1-1)

M-mode parameters including diastolic and systolic LVID, LVFW, and IVS

revealed significant differences from week 1 of pacing when compared to the baseline

value, with the exception of LVFWd. Pacing also resulted in significant difference in

FS from week 1. Reduced percent wall thickening was observed at IVS and LVFW, but

significant difference was only observed with LVFW, which was observed from week 1.

Among parameters of diastolic function, only A differed significantly at week 3, while

deceleration time showed a significant difference from week 2.

Two-Dimensional Tissue Tracking (Table 1-2)

Figure 1-1 shows examples of radial and circumferential strain analysis at

baseline and week 3 of pacing. Compared to the baseline, pacing caused significant

reduction of both global radial and circumferential strains from week 1. Regionally, in

the radial direction, the inferior segment showed significant change of strain from week

1, the anterior segment showed significant change from week 2, and the lateral,

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Table1-1. Temporal comparison of heart rate, blood pressure measurements, and standard echocardiographic parameters (mean ± SD) of a canine myocardial dysfunction model.

Parameters Baseline Week 1 Week 2 Week 3

Heart rate (beats/min) 145.00 ± 38.00 139.00 ± 11.00 151.00 ± 29.00 162.00 ± 37.00

Blood pressure (mmHg): Systolic 142.14 ± 17.88 134.27 ± 7.99 134.45 ± 8.79 126.40 ± 12.74

Mean 102.64 ± 14.81 91.15 ± 8.44* 86.80 ± 9.09** 83.12 ± 13.53**

Diastolic 83.80 ± 16.07 69.20 ± 10.73* 64.20 ± 15.64** 61.60 ± 14.48**

Conventional parameters: LVIDd (mm) 29.40 ± 3.08 33.09 ± 3.45** 36.87 ± 2.84*** 39.18 ± 4.84***

LVIDs (mm) 19.22 ± 3.03 26.49 ± 3.03*** 30.00 ± 2.11*** 32.16 ± 3.87***

LVFWd (mm) 7.82 ± 1.35 5.96 ± 1.13 6.20 ± 1.44 6.67 ± 2.43

LVFWs (mm) 10.60 ± 1.64 7.16 ± 1.52*** 6.78 ± 1.72*** 7.28 ± 2.54***

IVSd (mm) 7.76 ± 0.94 6.78 ± 0.90* 6.69 ± 0.63* 5.91 ± 0.96***

IVSs (mm) 11.16 ± 1.52 9.04 ± 1.02* 8.83 ± 0.52** 8.43 ± 1.17**

FS (%) 34.70 ± 4.60 19.90 ± 4.00*** 18.60 ± 1.40*** 17.80 ± 0.80***

IVS thickening (%) 43.67 ± 6.96 34.03 ± 10.55 32.55 ± 11.00 43.28 ± 8.61

LVFW thickening (%) 36.22 ± 10.39 19.80 ± 4.97* 9.27 ± 14.28** 9.38 ± 8.01**

E (cm/s) 78.79 ± 34.48 63.26 ± 27.70 65.24 ± 32.42 71.30 ± 33.09

A (cm/s) 56.03 ± 27.76 56.97 ± 23.39 40.34 ± 19.19 32.15 ± 15.77*

E/A 1.46 ± 0.64 1.12 ± 0.48 2.00 ± 1.58 2.58 ± 1.65

Deceleration time (ms) 74.07 ± 30.70 52.33 ± 25.42 45.33 ± 22.06* 46.20 ± 31.68*

*: p < 0.05, **: p < 0.01, ***: p < 0.001 versus corresponding baseline values.

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Table1-2. Temporal comparison of two-dimensional tissue tracking parameters (mean ± SD) of canine myocardial dysfunction model.

Parameters Baseline Week 1 Week 2 Week 3

Radial strains: Global strain (%) 31.96 ± 7.12 22.62 ± 4.98** 17.88 ± 6.62*** 13.52 ± 3.49***

Regional anterior septal strain (%) 33.31 ± 10.57 17.56 ± 9.90 19.71 ± 5.18 22.33 ± 7.93

Regional anterior strain (%) 23.10 ± 13.85 18.43 ± 10.96 13.23 ± 10.62** 13.29 ± 7.46**

Regional lateral strain (%) 26.17 ± 11.58 27.32 ± 10.71 16.43 ± 8.06 10.41 ± 5.20**

Regional posterior strain (%) 28.29 ± 11.50 27.15 ± 10.92 17.07 ± 6.28 9.77 ± 6.97*

Regional inferior strain (%) 35.63 ± 11.21 20.61 ± 8.43* 13.76 ± 8.89** 6.89 ± 3.91***

Regional septal strain (%) 45.35 ± 14.39 24.79 ± 13.15 27.08 ± 9.95 18.36 ± 10.02**

STI (ms) 44.25 ± 17.60 60.50 ± 10.93 71.56 ± 42.34* 85.23 ± 39.28***

Circumferential strains: Global strain (%) −15.44 ± 1.50 −11.60 ± 2.95** −9.32 ± 1.61*** −7.62 ± 1.47***

Regional anterior septal strain (%) −25.31 ± 8.25 −17.50 ± 9.34 −15.67 ± 4.00 −14.18 ± 3.92*

Regional anterior strain (%) −25.55 ± 4.75 −17.03 ± 5.01** −12.52 ± 2.66*** −11.21 ± 1.24***

Regional lateral strain (%) −21.55 ± 4.61 −17.43 ± 3.00 −15.40 ± 5.50 −10.83 ± 4.90**

Regional posterior strain (%) −27.25 ± 4.00 −17.89 ± 7.04* −15.79 ± 4.92** −9.35 ± 2.88***

Regional inferior strain (%) −20.28 ± 4.55 −15.97 ± 3.65 −11.43 ± 2.04** −7.77 ± 1.09***

Regional septal strain (%) −24.21 ± 3.73 −14.94 ± 4.11*** −11.62 ± 4.37*** −12.38 ± 3.02***

STI (ms) 41.63 ± 12.68 61.19 ± 22.93 54.00 ± 28.24 59.16 ± 27.11

*: p < 0.05, **: p < 0.01, ***: p < 0.001 versus corresponding baseline values.

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Figure 1-1. Examples of radial and circumferential strain profiles obtained from the

right parasternal short axis view at the level of the papillary muscle using

two-dimensional tissue tracking. Radial strain represents thickening motion of the

myocardium in the radial direction, and circumferential strain represents shortening

motion along the circular perimeter. In systole, the myocardial deformation increases in

the radial direction, thus the radial strain becomes positive, and the myocardial

deformation decreases in the circumferential direction, causing the circumferential

direction to becomes negative. Radial (A) and circumferential (B) strains at baseline,

and radial (C) and circumferential (D) strains at week 3 of sustained high-electrical

pacing.

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posterior, septal segments showed significant change at week 3, and anterior septal

segment did not show any significant change from the baseline (Figure 1-2). Conversely,

circumferential direction showed earlier changes, and all of the segments revealed

significant changes by the third week of pacing. The anterior, posterior and septal

segments showed significant changes of strain from week 1, while the inferior segment

showed significant changes from week 2, and the anterior septal and lateral segments

showed significant changes at week 3. The STI values of the radial strains revealed

significant increases from week 2, whereas no significant changes were observed in the

circumferential strains (Figure 1-3).

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Figure 1-2. Diagrams of the left ventricle with its six myocardial segments showing

temporal changes in regional radial (A) and circumferential (B) strain analysis. *: p <

0.05, **: p < 0.01, and ***: p < 0.001 versus corresponding baseline values.

Baseline Week 1 Week 2

A

Week 3

****

**

*

** ****

**

Baseline Week 1 Week 2

B

Week 3

*

***

*****

**

******

***

***

***

** ***

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Figure 1-3. Temporal changes in radial (A) and circumferential (B) synchrony time

index (mean ± SD), which is the indicator used to assess the degree of dyssynchrony.

**: p < 0.01, ***: p < 0.001 versus corresponding baseline values.

24

Discussion

2DTT was recently developed as an alternative technique to the tissue

Doppler-derived strain analysis, which was previously introduced as a method to

quantify myocardial function (4, 17, 18, 24, 41, 47, 54). 2DTT overcomes a number of

problems encountered with tissue Doppler-derived strain analysis, including angle

dependency, cardiac translation, noise interference, substantial intra-observer and

inter-observer variability, and inability to measure strain in the azimuth or perpendicular

planes (3, 4, 16, 17, 20, 24, 32, 41, 45, 54). In addition, 2DTT allows simultaneous

assessment of global and regional myocardial movements, as well as quantitative

evaluation of ventricular torsion and dyssynchrony (17, 24, 32, 41, 45, 52, 54). The

strain parameters derived from 2DTT have been validated in dogs, and shown to be well

correlated with tissue Doppler-derived strain, sonomicrometry, MRI, and measurement

of the maximum rate of LV systolic pressure (LV dP/dtmax) obtained from the

pressure-volume loop (2, 17, 19, 71). However, most studies of 2DTT in veterinary

medicine have been limited to clinically normal dogs, and there have not been many

reports on cardiac disorders. Therefore, the present experiment was conducted as a

pre-clinical evaluation to test the applicability of 2DTT on DCM. Specifically,

experimentally induced canine myocardial dysfunction model was used to assess the

ability of 2DTT to evaluate LV myocardial function in association with sustained

high-electrical pacing by analysing; (1) global myocardial function, (2) regional

myocardial function, and (3) the degree of dyssynchrony.

Human and animal studies have established that RV apical pacing results in

dilated, congestive cardiomyopathy characterized by LV systolic and diastolic

dysfunction and LV dyssynchrony (3, 55, 66, 67, 72-74, 76). The degree of myocardial

25

dysfunction is dependent on the time and the rate of pacing (55, 76). In the present

experiment, a three-week protocol with a pacing rate of 250 bpm was chosen to ensure

adequate LV structural changes and dysfunction during the three-week period that could

be detected by echocardiography.

Sustained high-electrical pacing resulted in a successful canine myocardial

dysfunction model, with LV dilation and thinning of the myocardium. Systolic

dysfunction, indicated by the reduction of FS, was observed immediately after the onset

of pacing. Diastolic dysfunction, which was observed after the onset of systolic

dysfunction, was characterized by reduction of A wave velocity and deceleration time,

which was consistent with impaired compliance. These changes were also accompanied

by reduced cardiac output, as indicated by the reduced BP.

Pacing caused significant reduction of both global radial and circumferential

strains. Mainly, the subendocardial function is governed by the longitudinal mechanics,

the subepicardial function by the circumferential mechanics, and transmural function by

the combination of longitudinal, circumferential and radial mechanics (32). Therefore,

reduction of radial strain indicates transmural dysfunction, whereas circumferential

strains suggest transmural and subepicardial dysfunction. Additionally, according to

Wang et al., systolic failure is characterized by the reduction of longitudinal, radial, and

circumferential strains, whereas diastolic failure is characterized by the reduction of

longitudinal and radial strains with preserved circumferential strains (69). In the present

experiment, the significant myocardial changes observed in both radial and

circumferential directions were most likely the result of both systolic and diastolic

dysfunction, and indicative of transmural and subepicardial dysfunction. Conventionally,

FS has been used to evaluate the global function of the LV (9, 28, 64). However, it may

26

not reflect the true global function since it is only measured at the anteroseptal and

posterior segments, and it only assesses radial deformation (4, 46, 52, 64). Conversely,

global radial and circumferential strains can be assessed simultaneously. These

parameters are the averages of measurements taken at multiple myocardial segments,

therefore, they provide a better representation of the global myocardial function (4, 32,

45). For example, dogs with uneven myocardial movements are sometimes encountered

in clinical settings, such as when a dog has sustained myocardial movement only at the

IVS, resulting in the values of FS remaining adequate. In such cases, 2DTT may be

better suited for evaluation of LV myocardial function. However, further investigations

using clinical cases are required to explore this possibility.

Results of the regional strain analysis have shown pacing to have a

detrimental effect on the regional myocardium in both the radial and circumferential

directions. It appears that more segments showed earlier myocardial changes in the

circumferential directions in comparison to the radial direction. As mentioned earlier,

circumferential mechanics is known to involve mainly the subepicardial and transmural

function (32). The earlier changes in the circumferential direction suggest the greatest

impact to be at the epicardium, which is most likely due to the epicardial insult

generated by the epicardial pacemaker lead. Regionally, the segments closest the lead

was hypothesized to receive the most influence from the pacing. However, in both

directions, the effect of the pacing was greatest at the LVWP, which were the segments

furthest from the lead. Percent wall thickening is the difference in wall thickness at

diastole and systole, a parameter similar to radial strain (72). This parameter allows

quantitative analysis of the regional myocardial movements at the IVS and LVFW, and

can be used to evaluate the regional contractility of the myocardium (72). In this

27

experiment, consistent results of maintained myocardial movements at the IVS and

reduced movements at the LVFW were observed in both percent wall thickening and

radial strains, confirming the results of the regional strain analysis. Wilson et al.

similarly looked at the changes in wall thickening in canine myocardial dysfunction

models with LV pacing and found impaired wall thickening at the LVFW, which were

the segments closest to the lead (72). Such findings suggest that the greatest myocardial

dysfunction occur at the LVWP, regardless to the location of the pacemaker lead. The

mechanism underlying the higher susceptibility of LVFW cannot be elucidated from the

results of this experiment. However, it could simply be due to the structural differences

of IVS, which involves both right and left ventricular myocardial component that better

able to sustain the myocardial function. Additionally, such finding is important in the

evaluation of myocardial function, since regional myocardial dysfunction may results in

uncoordinated myocardial contraction with reduced cardiac output, as observed in this

experiment, which is an important contributing factor in the clinical signs of CHF.

The use of 2DTT in the field of human medicine is widely increasing as a

method for assessment of cardiac resynchronization therapy and evaluation of

ventricular dyssynchrony caused by permanent pacemaker devices (40, 47, 66, 67, 74).

A number of human studies have demonstrated an association between increased

dyssynchrony and decreased LV function (27, 40, 67, 74). In the present experiment,

LV dyssynchrony was measured in terms of STI, which is the difference in timing of

peak strain from the earliest to the latest segments. Significant differences from the

baseline were observed in the radial direction, whereas no significant differences were

observed in the circumferential direction. Similar results have been observed in humans,

where a greater degree of myocardial deformation was observed in the radial direction

28

(27, 74). These findings suggest that dyssynchrony is easier to detect in the radial

direction. Overall, this experiment demonstrated the ability of the 2DTT to detect and

measure dyssynchrony in canine myocardial dysfunction models. The ability to measure

dyssynchrony would aid in the evaluation and monitoring of myocardial disorders.

There are several limitations to this experiment. First is the small number of

animals involved in the experiment. Secondly is employment of the high pacing rate.

Past reports of 2DTT have shown its use in regional analysis, and strain is thought to be

a more sensitive parameter of LV function that allow earlier detection of the myocardial

dysfunction (64). However, due to such a high pacing rate causing acute and severe

myocardial changes, FS and 2DTT parameters both demonstrated immediate changes

from the first week of pacing. 2DTT also has its own technical limitations. Specifically,

the accuracy of the strain values depends on the quality of the image and the frame rates

(32). In addition, the inadequate small search area of the tracking points may cause the

speckles to move in and out of the search area, decreasing the reliability and the ability

to track these speckles. Therefore, in some studies, there have been reports of an

inability to measure 2DTT parameters in as many as 5% of the myocardial segments

(6).

29

Conclusion

This experiment has shown the ability of 2DTT to measure strain in radial and

circumferential direction from the right parasternal short axis view, and to evaluate

changes in global and regional myocardial movement and the degree of dyssynchrony

in canine myocardial dysfunction model. The additional assessments of the regional

myocardial function and the degree of dyssynchrony are great advantages of 2DTT,

which facilitates better understanding of myocardial function.

30

Chapter 2 Comparative evaluation of inotropic agents, pimobendan and

SCH00013, on the myocardial function of canine myocardial

dysfunction model

Introduction

Inotropic agents have been indicated for the treatment of CHF with reduced

cardiac output and peripheral hypo-perfusion (44, 53). Classic inotropic agents,

including cardiac glycosides, catecholamines, and PDE III inhibitors, improve impaired

cardiac pump function by elevating intracellular calcium concentration in myocardial

cells to directly improve the myocardial contractility (25, 26, 48, 53). While they

effectively enhance the cardiac pump function, these agents have also been associated

with major side effects such as cardiac arrhythmia, induction of myocardial cell injury,

and unfavourable increase of myocardial energy consumption (26, 48, 53).

Calcium sensitizer is a relatively new class of inotropic agents, which enhance

myocardial contractility by increasing sensitivity of contractile protein to calcium (26,

48, 53). Unlike the classic inotropic agents, these agents augment the affinity of calcium

to contractile proteins, resulting in more force to be generated for a given intracellular

calcium concentration (26, 48, 53). Advantages include lack of intracellular calcium

overload and preserved energy consumption, and additionally the ability of the

myofilaments to respond to calcium sensitization is preserved in heart failure (26, 48,

53). Most of these agents are not pure calcium-sensitizer, and possess additional

mechanisms of action (48).

Pimobendan, a benzimidazole-pyridazinone derivative, elicits its PIE by (1)

calcium sensitization by increasing the affinity of the regulatory site on troponin C for

31

calcium, and (2) PDE III inhibition (7, 26, 30, 43, 50). The PIE of pimobendan is

mostly due to cyclic adenosine monophosphate (cAMP) dependent mechanism, and the

calcium sensitization plays a minor role (43). SCH00013, a pyridazinone derivative,

also possesses both actions of calcium sensitization by increasing myofilament calcium

sensitivity, and PDE III inhibition (25, 36, 75). However in contrary to pimobendan,

calcium sensitization is the main contributor of PIE (36, 59). The effects of these two

calcium sensitizers have not been comparatively evaluated in clinical model of CHF.

Although these two agents possess both actions of calcium sensitization and PDE II

inhibition, they differ in action mechanism, including the proportion of the contributing

actions of PIE. Therefore, we hypothesized that pimobendan and SCH0013 would have

a different effect on the myocardial function. This experiment was conducted to

comparatively evaluate the cardioprotective effect of pimobendan and SCH00013 on

the myocardial function of canine myocardial dysfunction model, therefore, the oral

administration of pimobendan and SCH00013 was initiated during the high-electrical

pacing and myocardial function was assed using echocardiography.

32

Materials and Methods

Study Design

Twenty female Beagles were used to create canine myocardial dysfunction

model, and of these dogs, fifteen received medication and the remaining five served as

control. Treatment included pimobendan (Vetmedin; Nippon Boehringer Ingelheim Co.,

Ltd., Japan), and two different doses of SCH00013 (Zenyaku Kogyo Co., Ltd., Japan),

with five dogs in each treatment groups. Implantation of external pacemaker lead to the

RV apex was performed via thoracotomy as described in chapter 1, and myocardial

dysfunction was created by sustained high-electrical pacing of 250 bpm for three

consecutive weeks (35). Administration of medications was initiated from the first day

of pacing in the following treatment groups; pimobendan (0.25 mg/kg PO q12hrs),

Low-SCH (SCH00013, 1 mg/kg PO q12hrs) and High-SCH (SCH00013, 3 mg/kg PO

q12hrs). Cardiac evaluation including indirect oscillometric BP measurements, standard

6-lead ECG, standard echocardiography and 2DTT were performed at the baseline and

weeks 1, 2, and 3 of pacing, for a total of four occasions. The entirety of the evaluation

was performed during a temporary cessation of pacing, at least an hour after the

pacemaker was turned off to minimize the influence of pacing. All of the pacemakers

were checked for accurate pacing at the end of each evaluation. The experiment was

carried out in accordance with the standards established by the Tokyo University of

Agriculture and Technology, and as described in its Guide for the Care and Use of

Laboratory Animals. All experimental procedures were approved by the Animal

Experimental Committee of Tokyo University of Agriculture and Technology

(acceptance no. 21-30).

33

Echocardiography

An ultrasonography unit equipped with a 5MHz phased array transducer

probe was used as described in chapter 1. All echocardiographic examinations were

performed with concurrent ECG monitoring, and by the same trained examiner for all

dogs. All examinations were performed on conscious dogs, using only gentle restraint.

A mean of five measurements of each parameter was obtained from five consecutive

cardiac cycles.

Standard Echocardiography

The examinations were performed in accordance with the methods described

by Boon (9). LV measurements were taken from the right parasternal short axis view at

the level of the papillary muscle using M-mode. Measurements included LVIDd, LVIDs,

LVFWd, LVFWs, IVSd, IVSs, and FS. Additionally, diastolic parameters including E,

A, E/A ratio, and deceleration time were obtained from the trans-mitral flow profile at

left parasternal apical four chamber view.

Two-Dimensional Tissue Tracking

Right parasternal short axis view at the level of the papillary muscle with the

frame rate of 70-110 frames/s were acquired and saved onto a hard disk, which was then

analysed off-line. Strain analysis was performed as described in chapter 1. Global and

regional peak systolic strains for both radial and circumferential directions were

calculated, and LV synchrony was also assessed by calculating the STI in the radial

direction.

34

Statistical Analysis

Variables are expressed as mean ± SD. Normality of the variables was tested

using the Kolmogorov-Smirnov test and the visual inspection of histograms. Clinical

and echocardiographic variables of the baseline values were compared that of week 1, 2,

and 3 using one-way repeated-measures ANOVA for parametric variables, and

Friedman test for non-parametric variables. Once significant difference was detected,

Dunn’s multiple comparison test was used to further compare each time point with the

baseline. Additionally, two-way ANOVA in conjunction with Dunn’s multiple

comparison tests was used to detect significant difference among groups using the

percent change from the baseline. Significant difference was defined as P < 0.05.

Statistical analyses were performed using statistical software, Prism 5.0v.

35

Results

The pacemaker implantation was successful and the recovery was uneventful

in all twenty dogs. They were able to maintain adequate physical condition and survived

until the last day of pacing.

The resting heart rate with cessation of pacing at each examination revealed

no significant changes from the baseline in all four groups. BP measurements showed

declining trends in all four groups. However, significant reductions were only seen with

control and High-SCH group (Figure 2-1). In control, significant reductions of mean

and diastolic pressure were observed from week 2 and week 3 respectively (mean =

0.001, diastolic = 0.007), and in High-SCH group, significant reductions of systolic

pressure and mean were observed from week 1and week 2 of pacing respectively

(systolic p = 0.002, MAP = 0.02). Additionally, no abnormalities on the ECG were

observed at each examination.

Standard Echocardiography (Table 2-1)

Sustained high-electrical pacing in control had resulted in immediate

alteration of LV morphology, including significant thinning of LV walls, with the

exception of LVFWd, and significant dilation of LV from week 1 of pacing.

Additionally, significant reduction of FS was also observed from week 1. Furthermore,

diastolic variables revealed significant reduction of A (p = 0.02) at week 3 and

deceleration time (p = 0.02) from week 2 of pacing.

Similar morphological changes of LV, including significant thinning of LV

wall and dilation of LV diameter, and reduction of FS were observed in both Low- and

High-SCH groups from week 1 of pacing, with the exception of IVSd in High-SCH

36

Figure 2-1. Temporal changes of blood pressure measurements observed in canine

myocardial dysfunction model (A) without treatment (control), and (B) with concurrent

administration of high dose of SCH00013 (1mg/kg and 3mg/kg PO q12hrs). *: p < 0.05,

**: p < 0.01 versus corresponding baseline values.

37

Table 2-1. Temporal comparison of the M-mode parameters (mean ± SD) in control, and with the administration of pimobendan (0.25 mg/kg PO q12hrs) and two doses of SCH00013 (1 mg/kg and 3 mg/kg PO q12hrs) of canine myocardial dysfunction model.

Parameters Baseline Week 1 Week 2 Week 3

LVIDd (mm): Control 29.40 ± 3.08 33.09 ± 3.45** 36.87 ± 2.84***††† 39.18 ± 4.84***†††

Pimobendan 33.82 ± 14.81 33.68 ± 14.34 31.51 ± 13.93 35.03 ± 14.52

Low-SCH 31.70 ± 13.18 35.90 ± 14.89** 36.38 ± 15.02*** 37.34 ± 15.44***¶

High-SCH 31.02 ± 12.80 34.00 ± 14.18* 35.86 ± 14.90**†† 37.62 ± 15.61***††

LVIDs (mm): Control 19.22 ± 3.03 26.49 ± 3.03*** 30.00 ± 2.11***††† 32.16 ± 3.87***†††

Pimobendan 21.26 ± 9.72 26.01 ± 11.33 24.04 ± 11.48 25.74 ± 10.80

Low-SCH 20.14 ± 8.61 26.14 ± 10.93** 27.80 ± 11.68*** 28.88 ± 12.08***

High-SCH 19.78 ± 8.18 26.10 ± 10.90** 26.98 ± 11.24*** 28.66 ± 12.28***

LVFWd (mm): Control 7.82 ± 1.35 5.96 ± 1.13 6.20 ± 1.44 6.67 ± 2.43

Pimobendan 6.31 ± 2.67 6.61 ± 2.79 6.43 ± 2.98 6.34 ± 2.91

Low-SCH 7.15 ± 3.16 6.35 ± 2.67* 6.33 ± 2.68* 5.96 ± 2.49**

High-SCH 7.56 ± 3.25 5.79 ± 2.58* 5.06 ± 2.13** 5.13 ± 2.19**

LVFWs (mm): Control 10.60 ± 1.64 7.16 ± 1.52*** 6.78 ± 1.72*** 7.28 ± 2.54***

Pimobendan 10.46 ± 4.60 8.42 ± 3.65 8.85 ± 4.18 8.60 ± 3.68

Low-SCH 10.58 ± 4.64 8.40 ± 3.66** 7.89 ± 3.30** 7.96 ± 3.30**

High-SCH 10.07 ± 4.15 7.61 ± 3.26*** 7.21 ± 3.10*** 6.79 ± 3.32***

IVSd (mm): Control 7.76 ± 0.94 6.78 ± 0.90* 6.69 ± 0.63* 5.91 ± 0.96***

Pimobendan 6.30 ± 2.76 6.73 ± 2.87 6.78 ± 2.98 6.02 ± 2.88

Low-SCH 6.87 ± 3.12 5.30 ± 2.22* 5.05 ± 2.11** 5.66 ± 2.41

High-SCH 7.32 ± 3.35 6.70 ± 2.95 6.70 ± 2.97 5.66 ± 2.34

IVSs (mm): Control 11.16 ± 1.52 9.04 ± 1.02* 8.83 ± 0.52** 8.43 ± 1.17**

Pimobendan 9.89 ± 4.43 9.14 ± 3.91 9.15 ± 4.25 8.74 ± 3.73

Low-SCH 9.75 ± 4.25 7.85 ± 3.30* 7.37 ± 3.10** 7.40 ± 3.10**

High-SCH 11.47 ± 5.01 9.72 ± 4.12 9.44 ± 4.06* 8.99 ± 3.73*

38

FS (%):

Control 34.70 ± 4.60 19.90 ± 4.00*** 18.60 ± 1.40*** 17.80 ± 0.80***

Pimobendan 37.25 ± 16.84 23.03 ± 10.20* 24.46 ± 14.87* 23.68 ± 11.09

Low-SCH 36.58 ± 15.69 27.16 ± 11.72** 23.72 ± 10.27*** 22.72 ± 9.56***

High-SCH 35.48 ± 14.60 23.16 ± 10.24*** 24.86 ± 10.74*** 23.92 ± 11.92***

*: p < 0.05, **: p < 0.01, ***: p < 0.001 versus corresponding baseline values. ††: p < 0.01, †††: p < 0.001 versus corresponding values of pimobendan. ¶: p < 0.05 versus corresponding values of control.

39

group. In regards to diastolic function, significant reduction of A (p = 0.01) and

deceleration time (p = 0.04) were briefly observed at week 2 in Low-SCH group, but no

other diastolic changes were observed in either treatment groups with SCH00013.

In pimobendan group, significant thinning of LV wall and dilation of LV was

not evident, but showed significant reduction of FS from week 1 of pacing. Moreover,

no significant change to the diastolic variables was observed.

Significant difference in the degree of LV dilation was observed between

groups (Figure 2-2). Significant differences in LVIDd (p = 0.001) were observed

between pimobendan and control and High-SCH group from week 2, and significant

difference between control and Low-SCH group was seen at week 3. For LVIDs (p =

0.03), significant difference was observed between control and pimobendan group from

week 2.

Two-Dimensional Tissue Tracking (Table 2-2)

In control, pacing had resulted in significant reduction of global strains in

both radial and circumferential directions from week 1. Regionally, reduction of strains

was observed in all six myocardial segments in both directions by the week 3 of pacing

(p < 0.02 for radial and p < 0.01 for circumferential directions), with the exception of

anterior septal segment in the radial direction (Figure 2-3). Moreover, significant

increase in STI was seen from week 1.

Similarly in Low- and High- SCH groups, global strains in both directions

resulted in significant reduction from week 1 of pacing, except for circumferential strain

in Low-SCH group, which was only observed from week 2. Regionally in Low-SCH

group, significant reductions of strains were observed in radial anterior septal (p =

40

Figure 2-2. Comparison of left ventricular (A) end-diastolic and (B)

end-systolic diameters between treatment groups at week 3 of pacing, values illustrated

as % change from baseline. *: p < 0.05, **: p < 0.01, and ***: p < 0.001 versus

corresponding baseline values.

41

Table 2-2. Temporal comparison of the global strain and synchronicity parameters (mean ± SD) in control, and with the administration of pimobendan (0.25mg/kg PO q12hrs) and two doses of SCH00013 (1mg/kg and 3mg/kg PO q12hrs) of canine myocardial dysfunction model.

Parameters Baseline Week 1 Week 2 Week 3

Radial strain (%):

Control 31.96 ± 7.12 22.62 ± 4.98** 17.88 ± 6.62*** 13.52 ± 3.49***

Pimobendan 32.72 ± 17.63 19.56 ± 10.95* 20.63 ± 9.15 20.78 ± 12.46

Low-SCH 42.46 ± 19.29 27.72 ± 12.32** 21.95 ± 9.85*** 25.44 ± 11.99**

High-SCH 50.47 ± 22.79 27.40 ± 14.89* 27.20 ± 15.38* 26.38 ± 17.11*

Circumferential strain (%):

Control -15.44 ± 1.50 -11.60 ± 2.95 -9.32 ± 5.42 -7.62 ± 1.47**†

Pimobendan -15.53 ± 6.48 -11.99 ± 5.89 -11.31 ± 5.42 -12.24 ± 5.32

Low-SCH -15.71 ± 6.80 -12.61 ± 5.39 -12.25 ± 5.42* -11.50 ± 5.13*

High-SCH -19.43 ± 7.95 -8.55 ± 4.91* -11.03 ± 5.13 -11.36 ± 6.18*

Radial STI (ms):

Control 44.25 ± 17.60 60.50 ± 10.93 84.57 ± 34.04 107.40 ± 37.31**

Pimobendan 36.42 ± 10.83 61.29 ± 16.36* 64.29 ± 15.84* 71.44 ± 18.39**

Low-SCH 55.30 ± 8.27 56.61 ± 19.06 62.46 ± 13.09 65.07 ± 7.64

High-SCH 49.44 ± 5.34 58.26 ± 12.51 78.73 ± 25.85* 81.33 ± 19.66*

*: p < 0.05, **: p < 0.01, ***: p < 0.001 versus corresponding baseline values. †: p < 0.05 versus corresponding values of pimobendan.

42

Figure 2-3. Diagrams of left ventricle with its six myocardial segments showing

significant changes between baseline and week 3 in regional radial (A) and

circumferential (B) strain analysis for each group. *: p < 0.05, **: p < 0.01, and ***: p

< 0.001 versus corresponding baseline values.

A

Control

****

*

***

**

Pimobendan

*

Low-SCH

*

**

**

High-SCH

**

*

**

B

Low-SCHPimobendanControl

*

***

**

***

******

High-SCH

***

43

0.02), posterior (p = 0.01) and inferior segments (p = 0.01) by week 3 of pacing, and no

changes were observed in the circumferential directions. In High-SCH group,

significant reductions of strains were observed in radial posterior (p = 0.05), inferior (p

= 0.01) and septal segments (p = 0.02), and circumferential anterior (p = 0.02) and

septal segments (p = 0.004) by week 3. For STI, while no significant change was

observed in Low-SCH group, significant increase was observed in High-SCH group

from week 2.

On the other hand, in pimobendan group, significant reduction of global strain

was observed in radial direction only. Regionally, regional anterior septal segments (p =

0.009) showed significant reduction from week 2, and no other segments showed any

significant changes. Similar to control and High-SCH groups, pimobendan group

showed significant increase of STI, which was observed from week 1 of pacing.

Between groups, significant difference was observed in global circumferential

strain between control and pimobendan at week 3. Regionally, circumferential anterior

and lateral segments showed significant differences, where anterior segment (p = 0.007)

showed significant difference between control and Low-SCH group from week 2, and

between pimobendan and control and High-SCH group at week 3. For lateral segment

(p = 0.02), significant difference was seen between control and pimobendan group at

week 3.

44

Discussion

In this experiment, the effect of oral administration of pimobendan and

SCH00013 on the myocardial function of canine myocardial dysfunction model was

evaluated using echocardiography. From past reports, sustained high-electrical pacing

has shown to produce dilated, congestive cardiomyopathy in dogs, and this canine

myocardial dysfunction model has been widely used to study CHF (55, 72, 73, 76). In

the control of this experiment, three weeks of sustained high-electrical pacing at the rate

of 250 bpm had resulted in CHF, characterized by thinning of the LV wall and dilation

of LV, and impaired systolic function indicated by the reduction of FS and strain values.

Additionally, diastolic dysfunction consistent with impaired compliance, and LV

dyssynchrony were also observed.

From past reports, administrations of pimobendan (0.25m g/kg i.v.) and

SCH00013 (0.3 – 3 mg/kg i.v.) in canine myocardial dysfunction model are known to

elicit PIE by increasing LVdP/dtmax (50, 62). In this experiment, even though

impairment of the systolic function was observed in all treatment groups indicated by

the reduction of FS, global strain analysis revealed the administration of pimobendan

was able to sustain myocardial function in the circumferential direction, where

SCH00013 could not. Additionally, significant echocardiographic changes indicative of

LV dysfunction, including thinning of the LV wall and dilation of the LV, were

observed in all treatment groups, with the exception of group treated with pimobendan.

Such result may suggest pimobendan to have a stronger PIE in comparison to

SCH00013.

Interestingly, regional strain analysis revealed less segments to be impaired in

the circumferential direction in comparison to radial direction in all treatment groups.

45

Especially in groups treated by pimobendan and low-dose of SCH00013, no significant

changes from the baseline were observed in the circumferential direction.

Circumferential strain indicates changes in the transmural and epicardial function,

whereas radial strain indicates mostly the changes in transmural function (32). Lack of

significant changes in the circumferential direction may suggest the effect of the

treatment to be the greatest at the epicardium. It can only be speculated, but the reason

for increased effect of the treatment at the epicardium could be explained by the course

of the coronary artery, which runs across the surface of the epicardium. Since the

epicardium is closest to the coronary artery, perhaps received the greatest influence of

the treatments in comparison to the trans-myocardium and endocardium, which are

further away from the coronary artery.

Intravenous and oral administration of SCH00013 (0.3 – 10mg/kg) in normal

dogs have been demonstrated to elicit PIE in a dose-dependent manner, shown by the

increase of LVdP/dtmax (75). Similarly, dose-dependent increase in LVdP/dtmax is

observed with intravenous administration of SCH00013 (0.3 – 3mg/kg) in canine

myocardial dysfunction model (62). The results of this experiment showed progressive

deterioration of systolic function, demonstrated by significant reduction of FS and

global strains in both SCH00013 treated groups. However, regional strain analysis

revealed myocardial impairments in both directions in High-SCH group, whereas

Low-SCH group showed regional myocardial impairment only in radial direction. Such

results indicate the low dose of SCH00013 was better able to sustain PIE, a result

contrary to the previous reports. PIE of SCH00013 is produced mainly by the increased

calcium sensitivity, and partially by the PDE III inhibition, however, PDE III inhibition

of SCH00013 increases with increased concentration (36, 59). This is supported by the

46

evidence of decreased BP observed with high dose of SCH00013, which was also seen

in this experiment (75). Additionally, failing myocardium in CHF is known to have

impaired response to PDE III inhibitors, due to reduced basal cAMP formation (7).

Therefore, increased PDE III inhibition, together with impaired response to PDE III

inhibitors in CHF might have resulted in failure to sustain PIE at high dose of

SCH00013 in this experiment. The results of this experiment had also shown that

pimobendan was better able to sustain PIE in CHF. Similar results have been seen

where pimobendan was able to persist PIE in CHF, which is thought to be due to the

effect of calcium sensitization (50).

LV mechanical dyssynchrony is known to be a sensitive indicator of

myocardial dysfunction, and increased dyssynchrony has been shown to correlate with

severity of LV function in patients with DCM (20, 27, 40). For such reasons, it has been

widely used for patient selection and assessment of the response to cardiac

resynchronization therapy (20, 32, 46). In this experiment, significant LV dyssynchrony

was observed in both pimobendan and High-SCH groups. On the contrary, Low-SCH

group was able to maintain LV synchronicity. This may suggest that even though the

PIE of SCH00013 may not be as potent as pimobendan, low dose of SCH00013 also has

a beneficial effect on the LV myocardial function. Additionally, such results allow us to

further speculate that this ability to sustain LV synchronicity in low dose SCH00013

may be due to the effect of the calcium sensitization.

Calcium sensitizers with concomitant PDE III inhibitory action possess

hypotensive effect due to its vasodilation of the PDEIII inhibition, and reduction of BP

with the administration of pimobendan has been reported (7, 23). However, in this

experiment, administration of pimobendan did not result in reduced BP. Similar results

47

have been observed by Ohte et al., where administration of pimobendan (0.25 mg/kg

i.v.) to dogs with pacing-induced myocardial dysfunction did not reduce the end systolic

BP, because of the markedly increased stroke volume (SV) (50, 61). From the results of

this experiment, it can also be speculated that increased PIE of pimobendan allowed

enough cardiac output to sustain the BP. Additionally, species differences in

vasodilatory effect of pimobendan has been reported, where pimobendan had a more

potent cardiac effect than vascular effect (23).

48

Conclusion

Results of this experiment demonstrated varying effect of pimobendan and

SCH00013 on the myocardial function in canine myocardial dysfunction model.

Additionally, the effect of SCH00013 on the myocardium differed depending on the

dose, most likely caused by the different proportion of calcium sensitizing and PDE III

inhibiting actions. SCH00013 has a higher proportion of calcium sensitization, and in

this experiment, low dose of SCH00013 showed sustained LV synchronicity, although

PIE was not as potent. However, at a higher dose, the action of PDE III inhibition

predominated, which was suggested by the reduction of BP, and the resulted in

deterioration of both PIE and LV synchronicity. The results have suggested pimobendan

to have a potent PIE on the pacing-induced myocardial dysfunction, however, little

effect on the LV dyssynchrony. On the other hand, although the PIE is not as potent,

low dose of SCH00013 is thought to be effective in sustaining LV synchronicity.

49

Chapter 3 Assessment of myocardial function in retrievers with dilated

cardiomyopathy using two-dimensional tissue tracking

Introduction

DCM is the most common form of canine cardiomyopathy, which is

characterized by progressive ventricular dilation and loss of myocardial contractility,

and is most often seen in adult large breed dogs (22, 51, 65). High prevalence of CHF

and sudden death in affected dogs, and the need for aggressive and comprehensive

medical therapy warrant for early diagnosis (51). However, the presence of an

asymptomatic phase with prolonged duration makes the diagnosis of DCM difficult (22,

51).

Standard echocardiography has been widely used for the assessment of

myocardial changes observed with DCM, and the diagnosis is made based on

echocardiographic evidence of LV dilation, myocardial hypokinesis, and increased

sphericity (22, 51, 65). Unfortunately, since accurate quantitative assessment of regional

myocardial function cannot be adequately made, standard echocardiography is not

particularly sensitive in detecting subtle myocardial changes observed in early

asymptomatic phase, nor in monitoring of the disease progression (4, 51).

To our knowledge, the sensitivity and the clinical relevance of the 2DTT have

not been studied in the context of clinical DCM in dogs. Therefore, the aim of this

experiment was to evaluate the myocardial dysfunction observed in dogs with DCM

using 2DTT, and to illustrate the difference in the myocardial function in comparison to

the healthy dogs.

50

Materials and Methods

Study Population

This prospective experiment comprised of two cohorts of retrievers. The first

cohort consisted of eight client-owned retrievers with DCM (DCM group) presented at

the Tokyo University of Agriculture and Technology Animal Medical Centre for the

diagnosis and treatment of DCM. Diagnosis was made based on (1) radiographic

evidence of left-sided or biventricular cardiac enlargement in association with

pulmonary oedema or pleural effusion; (2) echocardiographic evidence of LV dilation,

increased sphericity, and FS < 25%; and with or without (3) electrocardiographic

evidence of arrhythmia (22, 51). Dogs were excluded if there was evidence of

congenital or acquired cardiac disorder in addition to DCM, or other serious concurrent

disorder that may affect the cardiac function.

The second cohort consisted of eight healthy client-owned retrievers (control

group) that were admitted for the cardiac screening test. Screening tests including BP

measurements, ECG, and echocardiography were performed, and dogs with (1) FS <

25%; (2) arrhythmia; and (3) evidence of any congenital or acquired cardiac disorder

were excluded. These dogs were selected based on breed, gender, age and bodyweight

to match the DCM group.

Study Protocol

The experiment was approved and conducted in accordance with the

standards established by the Tokyo University of Agriculture and Technology Animal

Medical Centre, and the informed consent was obtained from the owners.

Echocardiographic evaluations, including the standard echocardiography and 2DTT,

51

were performed using an ultrasonography unit equipped with a 5MHz phased array

transducer probe. A mean of at least five measurements was obtained from consecutive

cardiac cycles in sinus rhythm for each parameter.

Standard Echocardiography

The examinations were performed in accordance with the methods described

by Boon (9). LV chamber dimensions were measured from the right parasternal short

axis view using M-mode. Measurements including LVIDd, LVIDs, LVFWd, LVFWs,

IVSd, and IVSs were measured at the level of the papillary muscle. LV systolic function

was evaluated using FS and systolic time intervals, which is the ratio of pre-ejection

period and ejection time (PEP:ET) measured from spectral Doppler aortic velocity

(LVOT pV) from left caudal parasternal five-chamber view. SV was also measured

from the LVOT pV. LV diastolic function was assessed by E, A, E/A ratio, and

deceleration time obtained from the trans-mitral flow profile at left parasternal apical

four chamber view. Additionally, systolic (Sa) and early (Ea) and late (Aa) diastolic

myocardial velocities were measured at IVS and LVFW of mitral annulus in the left

apical views using pulsed tissue Doppler imaging, and E/Ea was also calculated.

Two-Dimensional Tissue Tracking

Right parasternal short axis view at the level of the papillary muscle with the

frame rate of 70-110 frames/s was acquired, and was then analysed off-line. Strain

analysis was performed as described in chapter 1. Obtained parameters include global

and regional peak systolic strains for both radial and circumferential directions, and STI

52

in the radial direction. Additionally, pattern of contraction, which is analyzed from

graphic illustration of the average time to peak for each myocardial segment, was

evaluated from radial regional strains.

Statistical Analysis

Variables are expressed as mean ± SD. The normality of distribution for each

parameter was tested using D'Agostino & Pearson omnibus normality test. Significant

difference between DCM and Control groups was evaluated using unpaired Student’s

t-test for normally distributed parameters and Mann-Whitney test for parameters not

normally distributed. Significant difference was defined as P < 0.05. Statistical analysis

was performed using statistical software, Prism 5.0v.

53

Results

Study Population

Both DCM and control groups consisted of two Golden and six Labrador

retrievers, and two males and six females. The average age and bodyweight were 11.29

years old (range 8 – 13 years old) and 27.44 kg (range 22.00 – 32.15 kg) respectively

for DCM group, and 10.38 years old (range 7 – 12 years old) and 25.28 kg (range 19.00

– 31.20 kg) respectively for control group. There was no statistical difference with age

and bodyweight between groups (p = 0.21 for age and p = 0.32 for bodyweight).

Five dogs from the DCM group were presented with one or more of the

following clinical signs of CHF; cough (n = 3), depression (n = 2), and exercise

intolerance (n = 3), and ascites was found on physical examination in one dog. Among

dogs with clinical signs, four dogs were already being treated with one or a combination

of the following medications: angiotensin converting enzyme inhibitor (n = 2),

furosemide (n = 2), levothyroxine sodium (n = 1), pimobendan (n = 3), and taurine (n =

2). Cardiac auscultation revealed a left apical systolic murmur suggestive of mitral

regurgitation in three dogs. ECG abnormality of isolated ventricular premature beats

was observed in two dogs.

Standard Echocardiography (Table 3-1)

Standard echocardiographic evaluation of the control group revealed all

parameters to be within the reference range, but in DCM group, LVIDd was above and

FS was below the reference range (9). Comparison between groups showed significant

dilation of LV and thinning of LV walls with the DCM group. Additionally,

significantly lower values of FS, PEP/ET and IVS S’ were observed with the DCM

54

Table 3-1: Comparison of the parameters of standard echocardiography (mean ± SD) of retrievers with DCM and clinically normal retrievers.

Parameters DCM Control

IVSd (mm) 8.83 ± 2.12 10.43 ± 1.07

IVSs (mm) 11.78 ± 2.38 14.75 ± 1.57**

LVIDd (mm) 45.54 ± 6.63 42.98 ± 6.22

LVIDs (mm) 36.08 ± 6.60 27.82 ± 3.83**

LVFWd (mm) 8.01 ± 1.34 9.34 ± 1.35

LVFWs (mm) 10.96 ± 1.68 13.40 ± 2.44*

FS (%) 20.71 ± 5.60 35.15 ± 3.33***

PEP/ET 0.43 ± 0.09 0.27 ± 0.07**

SV (ml) 10.14 ± 3.36 13.23 ± 4.69

E (cm/s) 56.18 ± 23.98 57.40 ± 9.56

A (cm/s) 40.45 ± 7.85 51.04 ± 9.02*

E/A 1.45 ± 0.62 1.18 ± 0.29

DecT (ms) 146.10 ± 46.55 120.40 ± 18.66

S’ IVS (cm/s) 6.74 ± 3.42 11.91 ± 2.8**

E’ IVS (cm/s) 6.83 ± 2.03 8.06 ± 1.93

A’ IVS (cm/s) 5.58 ± 1.51 8.38 ± 1.91**

E/E’ IVS (cm/s) 9.65 ± 2.64 7.63 ± 2.07

S’ LVFW (cm/s) 9.43 ± 3.75 13.09 ± 2.37

E’ LVFW (cm/s) 7.93 ± 3.29 11.39 ± 2.25*

A’ LVFW (cm/s) 7.78 ± 2.74 9.90 ± 1.40

E/E’ LVFW (cm/s) 8.04 ± 3.05 5.40 ± 1.25

*: p < 0.05, **: p < 0.01, ***: p < 0.001 between DCM and control groups.

55

group indicating LV systolic dysfunction (9). In regards to LV diastolic function,

significant reduction of A and A’ at IVS were observed with DCM group in comparison

to the control group, suggestive of a degree of LV diastolic dysfunction. Moreover,

mitral valve regurgitation secondary to mitral annulus dilation was observed in three

dogs with DCM.

Two-Dimensional Tissue Tracking (Table 3-2)

Comparison of 2DTT between DCM and control groups revealed global strain

values to be significantly lower in DCM group in both radial and circumferential

directions. Regionally, significantly lower strain values were observed with DCM group

at posterior segment in radial direction, and at posterior, inferior and septal segments in

circumferential directions in comparison to the control group. Evaluation of the pattern

of contraction at the radial direction showed earliest time to peak at posterior segment

for DCM group and at septal segment for control group (Figure 3-1). Lastly, there was

no significant difference of STI between the groups.

56

Table 3-2: Comparison of the parameters of two-dimensional tissue tracking (mean ± SD) of retrievers with DCM and clinically normal retrievers.

Parameters DCM Control

Global radial strain (%) 19.53 ± 8.08 29.50 ± 9.79*

Global circumferential strain (%) -8.75 ± 3.06 -12.94 ± 2.12**

Regional radial anterior septal strain (%) 18.05 ± 13.44 29.83 ± 11.49

Regional radial anterior strain (%) 18.59 ± 11.75 23.63 ± 9.39

Regional radial lateral strain (%) 20.85 ± 9.67 30.96 ± 15.95

Regional radial posterior strain (%) 17.82 ± 9.38 34.86 ± 10.20**

Regional radial inferior strain (%) 19.69 ± 8.15 27.50 ± 10.23

Regional radial septal strain (%) 22.18 ± 6.36 30.22 ± 15.55

Regional circ. anterior septal strain (%) -11.05 ± 4.86 -14.11 ± 4.43

Regional circ. anterior strain (%) -8.87 ± 4.66 -10.97 ± 3.54

Regional circ. lateral strain (%) -8.98 ± 4.09 -11.80 ± 2.41

Regional circ. posterior strain (%) -8.97 ± 5.03 -14.36 ± 1.07*

Regional circ. inferior strain (%) -6.88 ± 2.57 -11.97 ± 3.34**

Regional circ. septal strain (%) -7.80 ± 1.97 -14.43 ± 3.15***

STI (ms) 50.70 ± 26.75 42.89 ± 25.27

*: p < 0.05, **: p < 0.01, ***: p < 0.001 between DCM and control groups.

57

Figure 3-1: Time to peak values for the regional myocardial segments in radial

direction illustrating the pattern of contraction of A) clinically normal retrievers, and B)

retrievers with DCM.

58

Discussion

This experiment illustrated the difference in myocardial function between

eight retrievers with DCM and eight clinically normal retrievers with comparable age,

sex and body weight. Even though the DCM group included three dogs in the

asymptomatic phase and four dogs receiving medical treatment, significant differences

in echocardiographic evaluations between the DCM and the control groups were

observed. From the standard echocardiography, dogs with DCM showed significant

dilation and thinning of the LV and myocardial hypokinesis in comparison to the

clinically normal dogs.

Comparison of global analysis of 2DTT between groups showed significantly

lower strain values in dogs with DCM in both radial and circumferential directions,

indicating significant myocardial dysfunction. It is known that radial deformation

indicates transmural dysfunction, whereas circumferential deformation indicates

transmural and subepicardial dysfunction (32). Therefore, the reduction of both radial

and circumferential strains in dogs with DCM in this experiment demonstrates that at

least the transmural and subepicardial dysfunction is involved in the disease state of

DCM. The presence of subendocardial dysfunction should be confirmed by evaluation

of the longitudinal strain, since subendocardial function is illustrated by longitudinal

deformation.

Regional analysis of 2DTT revealed significantly lower strain values focusing

around the LVFW (including the posterior, inferior and septal segments) in both radial

and circumferential directions in dogs with DCM in comparison to the normal dogs.

This is most likely because while the LVFW reflects the changes in LV, IVS reflects the

changes in both LV and RV. In most cases, especially in the early stages of DCM, the

59

RV changes are less evident in comparison to the LV changes (51). Therefore, it may be

presumed that changes in myocardium indicative of DCM are more evident in LVFW.

Such result is also in agreement with the result of the myocardial dysfunction model.

Whether similar finding is also observed in other cardiac disorder is unknown, however,

it appears that the LVFW has increased susceptibility to myocardial dysfunction in both

experimental and clinical DCM. Interestingly, the results of S’ of the tissue Doppler

analysis revealed differing results, where S’ at IVS was significantly lower in dogs with

DCM, while S’ at LVFW did not show any significant differences. Such dissimilarity

could be due to the fact that S’ is a measurement of longitudinal myocardial movements,

and differs from the radial and circumferential deformations.

It has been reported in humans that the normal LV contraction occurs in

homogenous pattern with the septal segment contracting slightly earlier than the lateral

and posterior segments (27). Similar result was also observed in the normal retrievers,

where on average the septal segment was the first and lateral segment was the last to

reach peak strain. On the contrary, in dogs with DCM, on average the posterior segment

was the first and the anterior segment was the last to reach peak strain, showing

heterogeneous pattern of contraction. Amongst dogs with DCM, only one dog, which

was asymptomatic, showed the normal homogeneous pattern of contraction. It can only

be a speculation, since it is based on a single observation, however, such result may

suggest the possibility of association between the pattern of contraction and the

appearance of the clinical signs. Heterogeneous pattern of contraction ultimately results

in smaller SV (27). Interestingly, difference in SV between groups was not evident in

this experiment, which may indicate that the heterogeneous pattern of contraction may

be an indication of myocardial dysfunction, which occurs before the reduction of SV

60

and the onset of clinical signs, are observed. Further study is warrant to define such

association, under a condition of increased myocardial demands, for example using an

exercise intolerance test. DCM has a prolonged period of asymptomatic phase with

myocardial hypokinesis may be the only detectable evidence (22, 51, 65). Therefore, the

evaluation of the pattern of contraction may be used as additional criteria for the

evaluation of DCM.

In humans, LV mechanical dyssynchrony is known to be an important

prognostic factor in patients with DCM (27). The orientation of the cardiac motion is

largely radial and circumferential, and reduction in systolic function is associated with

increased radial mechanical dyssynchrony in patients with DCM (27, 40). However, the

result of this experiment did not show any significant difference in STI between groups,

regardless of the demonstration of heterogeneous pattern of contraction in dogs with

DCM. This could be explained by a number of points. First, half of the dogs with DCM

had been treated with medications, which may have a positive effect on the

synchronicity. Three dogs with DCM were asymptomatic, therefore the degree of

dyssynchrony was minimal. Thirdly, since the control group consisted of older dogs to

match the DCM group, some degree of dyssynchrony was observed which is an

age-related change. Therefore, the dogs with DCM may have had lower than expected

value of STI, and the normal dogs may have had a higher value of STI, and hence

resulted in lack of significant differences between groups. A study by Lopez-Alvarez

has also evaluated the mechanical synchrony in Doberman Pinschers with DCM using

TDI, which failed to show differences between the normal dogs and dogs with DCM, a

result similar to this experiment (42). Such result suggests another possibility that

mechanical dyssynchrony is less evident in dogs with DCM.

61

Conclusion This experiment has demonstrated the ability of 2DTT to evaluate the

myocardial function in retrievers and differentiate between dogs with DCM and

clinically normal dogs. The results of global strain analysis indicate that DCM in

retrievers is associated with significant reduction of strain in both radial and

circumferential directions. Regional strain analysis suggested the possibility of

increased susceptibility of the LVFW to the radial and circumferential myocardial

dysfunction. Moreover, dogs with DCM showed heterogeneous pattern of contraction,

which may be associated progression of myocardial dysfunction and with possible

development of clinical signs. Regardless to the heterogeneous pattern of contraction in

dogs with DCM, significant mechanical dyssynchrony was not observed, which may

indicate that mechanical dyssynchrony is less evident in dogs with DCM.

62

Chapter 4 Evaluation of myocardial changes observed before and after closure

of patent ductus arteriosus in dogs using two-dimensional tissue

tracking

Introduction

PDA is one of the most common congenital cardiovascular defects in dogs (8,

12, 13). It occurs as a result of failure of ductus arteriosus to closure, a normal fetal

structure that shunts blood from the pulmonary artery to the aorta, which normally

closes after birth (8, 12, 13, 58). After birth, rise in the systemic pressure and fall in the

pulmonary artery pressure allows the blood to flow from aorta to pulmonary artery

through the ductus, and this left-to-right shunt induces over-circulation in the lungs and

left side of the heart causing left-sided volume overload (8, 12, 13, 58). Treatment of

PDA involving ductal closure is the most effective and strongly recommended in

left-to-right shunting PDA (8, 12, 13, 58). However, after the closure of ductal

arteriosus, systolic dysfunction of the LV is a common occurrence in both humans and

dogs (9, 31, 34, 38, 39, 63). This systolic dysfunction is the result of sudden changes in

the loading conditions caused by the attenuation of PDA flow, and is not a reflection of

the actual myocardial contractility (9, 31, 34, 38, 39, 63).

FS is a parameter that is widely used as a measurement of LV function (9).

However, FS is affected by the loading conditions, thus it is strictly a measurement of

LV function rather than a measure of contractility (9). Although previous reports have

demonstrated the influence of loading condition on strain values, 2DTT provides

comprehensive evaluation, which allows better quantitative assessment of the

myocardial function (14, 19, 70).

63

In humans, transient changes in LV function in association with closure of

PDA have been extensively reviewed (31, 34, 38, 39, 63). However, there have been a

limited number of comprehensive reports of echocardiographic changes observed with

the PDA closure in dogs (1, 57, 68). The purpose of this experiment was to illustrate the

short-term echocardiographic changes in dogs before and after the surgical ligation of

PDA, and evaluate the myocardial changes observed using standard echocardiography

and 2DTT.

64

Materials and Methods

Study Population

Seventeen client-owned dogs with PDA, presented at the Tokyo University of

Agriculture and Technology Animal Medical Centre from August 2010 to August 2013

for the definitive diagnosis and treatment of PDA, were prospectively evaluated.

Diagnosis was made based on the demonstration of PDA by colour Doppler

echocardiography in all dogs, and was confirmed by thoracotomy during the surgical

ligation of PDA. Dogs were included in the experiment if they had an uncomplicated

left-to right shunting PDA, without concurrent congenital or acquired cardiac disorders

and arrhythmias. Dogs with echocardiographic evidence of pulmonary hypertension,

bidirectional or reversed (right-to-left) PDA, or had other concurrent cardiac disorders

in addition to PDA were excluded.

Study Protocol

Informed consent was obtained from the owners, and the experiment was

approved and conducted in accordance with the standards established by the Tokyo

University of Agriculture and Technology Animal Medical Centre. Signalment, body

weight and clinical signs at the time of diagnosis, type and duration of medical

treatments were recorded. Echocardiographic evaluations, including the standard

echocardiography and 2DTT, were performed before and within three days of the

surgical ligations of PDA. An ultrasonography unit equipped with a 5MHz phased array

transducer probe was used. A mean of at least five measurements was obtained from

consecutive cardiac cycles in sinus rhythm for each parameter.

65

Standard Echocardiography

The examinations were performed in accordance with the methods described

by Boon (9). Cardiac dimensions were measured from the right parasternal short axis

view using M-mode. Measurements including LVIDd and LVIDs were measured at the

level of the papillary muscle, and left atrium to aorta ratio (LA/Ao) was measured at the

heart base level.

LV systolic function was evaluated using FS and LVOT PEP:ET measured

from LVOT pV at left caudal parasternal five-chamber view. Additionally, diastolic

function was assessed by E and A, E/A ratio, and deceleration time obtained from the

trans-mitral flow profile at left parasternal apical four chamber view. Mitral

regurgitation was diagnosed if there was a turbulent flow at the mitral valve.

Two-dimensional Tissue Tracking

Right parasternal short axis view at the level of the papillary muscle with the

frame rate of 70-110 frames/s were acquired and saved onto a hard disk, which was then

analysed off-line. Strain analysis was performed as described in chapter 1. Global and

regional peak systolic strains for both radial and circumferential directions were

calculated, and LV synchrony was also assessed by calculating the STI in the radial

direction. As an addition to the regional analysis, the pattern of contraction was also

evaluated from the average time to peak strain in the radial direction.

Statistical Analysis

Variables are expressed as mean ± SD. The normality of distribution for each

parameter was tested using D'Agostino & Pearson omnibus normality test. Significant

66

changes before and after the surgical ligation of PDA were evaluated using paired

Student’s t-test for continuous variables and Wilcoxon matched pairs test for data not

normally distributed. Significant difference was defined as P < 0.05. Statistical analyses

were performed using statistical software, Prism 5.0v.

67

Results

Seventeen dogs underwent surgical ligation of PDA and echocardiographic

examinations were performed before and after the closure of PDA. There were 8 breeds

represented with Pomeranians (n = 6), Toy Poodles (n = 4), Chihuahua (n = 2), and each

of Jack Russell Terrier, Maltese, Papillon, Pembroke Welsh Corgi, and Shetland Sheep

Dog. There were 10 females (59%) and 7 males (41%). The average age at the time of

diagnosis was 7 months (range 2 – 25 months), where only 2 dogs were one year or

older (12%). Average body weight was 2.5 kg (range 1.00 – 6.15 kg). 3 dogs showed

clinical signs of cough, 1 with exercise intolerance, and 1 with breathing difficulties,

and the remaining 12 dogs did not show any clinical signs. 7 dogs were treated with

angiotensin-converting enzyme inhibitors with (n=5) or without diuretics (n = 2),

Pimobendan (n = 2) and a prostacyclin analog (n = 1) for over a period of one week

prior to surgery.

Standard Echocardiography (Table 4-1)

Pre-surgical examination revealed increased LVIDd, LVIDs and LA/Ao,

suggestive of LV and left atrial dilation, and other values were within the reference

range (9, 28). Closure of PDA had resulted in significant reduction of LVIDd and

LA/Ao. With the reduction of LVIDd and LA/Ao, LA/Ao returned to within the

reference range, however, LVIDd and LVIDs still remained above the reference range.

FS significantly decreased and LV PEP:ET significantly increased, with FS at below the

reference range indicating LV systolic dysfunction. Additionally, significant reduction

of LVOT pV, E, and A were also observed. 7 dogs (4 females (57%) and 3 males

(43%)) had mitral regurgitation prior to surgery, which persisted to after the surgery.

68

Table 4-1: Comparison of the parameters of standard echocardiographic changes (mean ± SD) observed before and after the surgical ligation of PDA in 17 dogs.

Before After Reference range

LVIDd (mm) 24.64 ± 5.90 20.74 ± 3.87*** 13.05 - 18.61

LVIDs (mm) 14.83 ± 4.36 14.38 ± 3.30 7.86 - 13.67

LA/Ao 1.71 ± 0.40 1.28 ± 0.19* 0.83 - 1.13

FS (%) 40.33 ± 5.81 30.85 ± 8.06*** 33.7 - 45.90

LVOT pV (cm/s) 109.56 ± 26.39 68.34 ± 13.32** < 200.00

LVOT PEP/ET 0.19 ± 0.05 0.34 ± 0.14** < 0.40

E (cm/s) 74.53 ± 16.86 59.89 ± 16.66** 50.00 - 100.00

A (cm/s) 56.93 ± 14.05 34.04 ± 8.7** 30.00 - 60.00

*: p < 0.05, **: p < 0.01, ***: p < 0.001 between before and after the PDA ligation.

69

Two-dimensional Tissue Tracking (Table 4-2)

Global radial and circumferential strains before the closure of PDA were

33.47 ± 15.68 (reference range 22.30 – 71.10) and -14.43 ± 2.79 (reference range not

available), respectively (17). Closure of PDA had resulted in reduction of strains in both

directions, with the radial strains below the reference range. Regional strain analysis

had revealed reduction of strains in all segments in both directions with the exception of

anterior (p = 0.054) and inferior (p = 0.052) segments in radial directions after the

closure of PDA. STI value was initially within the reference range (STI reference range

0 – 45ms), but significantly increased following the closure of PDA, which was then

just above the reference range (17). Evaluation of the pattern of contraction, illustrated

by the average time to peak for each myocardial segment in the radial direction showed

septal segment to be the first segment to peak before the closure of PDA, whereas

lateral segment was the first segment to peak after the PDA closure (Figure 4-1).

70

Table 4-2: Comparison of the parameters of two-dimensional tissue tracking (mean ± SD) observed before and after the surgical ligation of PDA in 17 dogs. Parameters Before After

Global radial strain (%) 33.47 ± 15.68 21.34 ± 6.8**

Global circumferential strain (%) -14.43 ± 2.79 -10.86 ± 2.97***

Regional radial anterior septal strain (%) 36.88 ± 17.19 21.72 ± 11.81**

Regional radial anterior strain (%) 30.07 ± 20.11 20.79 ± 8.47*

Regional radial lateral strain (%) 36.14 ± 17.05 25.76 ± 9.52*

Regional radial posterior strain (%) 35.84 ± 15.2 24.79 ± 9.09**

Regional radial inferior strain (%) 30.24 ± 19.83 17.98 ± 5.98*

Regional radial septal strain (%) 31.68 ± 20.08 17.05 ± 10.94**

Regional circ. anterior septal strain (%) -14.58 ± 4.53 -11.29 ± 3.98**

Regional circ. anterior strain (%) -15.88 ± 3.67 -12.76 ± 4.22**

Regional circ. lateral strain (%) -14.47 ± 3.39 -11.48 ± 2.67**

Regional circ. posterior strain (%) -17.05 ± 4.12 -11.91 ± 3.59***

Regional circ. inferior strain (%) -12.17 ± 2.75 -8.40 ± 2.93***

Regional circ. septal strain (%) -12.43 ± 4.13 -9.31 ± 3.74**

STI (ms) 29.42 ± 15.66 45.90 ± 21.02*

*: p < 0.05, **: p < 0.01, ***: p < 0.001 between before and after the PDA ligation.

71

Figure 4-1. Time to peak values for the regional myocardial segments in radial

direction illustrating the pattern of contraction A) before, and B) after the surgical

ligation of PDA.

72

Discussion

This experiment demonstrated the presence of LV volume overload in dogs

with PDA, indicated by LV and LA dilation. Attenuation of PDA flow had resulted in

immediate reduction of LVIDd without LVIDs, and LA/Ao, and reduction of mitral

inflow and aortic outflow velocities, all suggestive of reduced preload. Additionally,

deterioration of LV systolic function, indicated by the significant reduction in FS and

increase of LV PEP:ET, was observed. Such reduction in preload and accompanied

deterioration of LV systolic function has been well documented after the attenuation of

hemodynamically significant PDA flow (31, 34, 38, 39, 63).

Study by Barlow et al. compared the LV contractility of human infants with

significant PDA to control using load independent parameter of LV contractility, the

rate-corrected velocity of fiber shortening, and their results failed to show differences

between the control and infants with PDA (5). Additionally, Takahashi et al. evaluated

the changes of LV contractility by mean normalized systolic ejection rate, another load

independent parameter, and demonstrated that it did not change while EF decreased

with the closure of PDA in human infants (63). These studies strongly suggest that LV

contractility is not impaired in the presence of volume overload, and the post closure

systolic dysfunction is the result of reduced preload.

2DTT strain analysis was developed as an assessment method of myocardial

function, and it has been validated with tagged-MRI, which is considered to be the gold

standard for the assessment of LV contractility (4, 19, 20, 32, 46). However, recent

reports have indicated that strain may not only reflect the LV contractility (14, 20, 70).

Several studies have compared the strain values to invasive measurements during

various hemodynamic and inotropic conditions, and they have demonstrated that the

73

strain is closely related to SV, and thus influenced by loading conditions (14, 70). In

this experiment, global analysis revealed reduction of strains in both radial and

circumferential directions after the closures of PDA in similar fashion to FS, indicating

the influence of preload. Regional strain analysis revealed significant reduction of strain

in all myocardial segments in both directions, with the exception of anterior and inferior

segments in the radial directions. However, these two segments also showed a declining

trend, with the p-values just shy of significance. There is a possibility that a larger

number of animals would have resulted in significant different at all of the segments.

Such results suggest that the influence of loading condition to be uniform, without any

regional differences.

Normally, LV mechanical contraction is synchronous, with the septal segment

contracting slightly earlier than the lateral and posterior segments (27). The results of

this experiment showed on average similar homogeneous pattern of contraction prior to

the attenuation of PDA flow, with the exception in two dogs, which the earliest

segments to contract were lateral, and inferior segments. The two dogs that did not

show the typical homogenous pattern of contraction prior to the closure of PDA had

also shown clinical signs including cough and exercise intolerance. On the contrary, on

average heterogeneous pattern of contraction was observed after the closure of PDA, of

which lateral segment was the earliest to contract. Six dogs were able to sustain the

homogenous pattern of contraction after the closure of PDA, and of these dogs, four

dogs were receiving treatment prior to the surgery. The hemodynamics of the dogs in

this experiment is influenced by a number of factors, including the received medications

and the presence of MR, therefore, the results of this study only allows speculations.

However, the results of this experiment illustrated the tendency of heterogeneous

74

pattern of contraction observed prior to surgery to be associated more often with dogs

showing clinical signs. Additionally, the administration of treatments such as ACE-I

may be aiding in the sustainment of the homogenous pattern of contraction after the

closure of PDA. A larger study needs to be conducted to clarify the potential association

between the pattern of contraction with the clinical signs and treatments.

One of the limitations of this experiment was the lack of strain rate analysis.

Strain rate is the rate of myocardial deformation, and although like strain, it may be load

dependent, but some studies suggest strain rate to be less influenced by loading

conditions (14, 19, 70). Another limitation was the lack of follow-up to evaluate the

outcome of surgical ligation of PDA. The immediate LV systolic dysfunction observed

after occlusion of PDA is known to be transient, however, LV dysfunction may persist

with delayed closure for example in older patients (38). Since most of the cases were

less than a year old, it is less likely that the LV dysfunction persists. However,

follow-up should be performed to check the recovery of the LV systolic function.

75

Conclusion

This experiment demonstrated that the surgical ligation of PDA causes

reduction of preload due to the alleviation of LV volume overload, which in turn result

in deterioration of LV systolic function observed by significant echocardiographic

changes. Additionally, it also showed that the strain analysis is influenced by acute

changes in loading conditions; therefore, factors such as preload and afterload should be

taken into consideration when interpreting strain measurements as an indicator of LV

systolic function. Furthermore, the attenuation of PDA flow had caused the previously

homogenous pattern of contraction to become heterogeneous, which suggested the

possibility of association between the pattern of contraction with the clinical signs and

treatments.

76

Summery

DCM is the most common cardiomyopathy in dogs, which is characterized by

progressive dilation and myocardial hypokinesis of the LV. The diagnosis of DCM is

complicated by the presence of asymptomatic phase, which comprehensive myocardial

evaluation is essential. However, standard echocardiography is often inadequate since

most commonly used parameter of systolic function, such as FS, is not a true

representative of the global function, and furthermore regional function can not be

evaluated. On the other hand, 2DTT strain analysis, which is a novel echocardiographic

technique, enables comprehensive evaluation of myocardial function. 2DTT allows

quantitative analysis of global, and regional myocardial function from multiple

directions including radial and circumferential directions, and also evaluates the degree

of synchronicity. The main objective of this study was to evaluate the LV myocardial

function of canine DCM using 2DTT.

Firstly, the aim of chapter 1 was to investigate the ability of 2DTT to evaluate

the global and regional myocardial function and the degree of dyssynchrony using the

canine myocardial dysfunction model. 2DTT was able to measure radial and

circumferential strain parameters from the right parasternal short axis view in the canine

model. Moreover, 2DTT allowed the analysis of the global and regional myocardial

changes, and the degree of dyssynchrony in association with the progressive myocardial

dysfunction. The additional assessments of the regional myocardial function and the

degree of dyssynchrony are great advantages of 2DTT, and allowed a more

comprehensive evaluation of the myocardial function in comparison to the standard

echocardiography.

77

Secondly in chapter 2, two inotropic agents, pimobendan and SCH00013,

were comparatively evaluated in aims to assess the effect of medical treatments on the

myocardial function of canine myocardial dysfunction model using 2DTT. 2DTT was

able to evaluate the varying effect of pimobendan and SCH00013 on the myocardial

function of the canine model, which these varying effects were most likely due to the

different proportion of calcium sensitizing and PDE II inhibiting actions. Pimobendan

had a potent positive inotropic effect, with little effect on synchronicity, while low dose

of SCH00013 had a weaker positive inotropic effect but was able to sustain

synchronicity. This ability of the 2DTT to evaluate the varying effect of these inotropic

agents suggests the potential for the application of 2DTT for the monitoring of the

response to medical treatments.

The ability of 2DTT to comprehensively evaluate the myocardial function in

canine model was established with chapter 1 and 2. Therefore, the aim of chapter 3 was

to evaluate the myocardial function in clinical dogs with DCM using 2DTT. 2DTT was

able to evaluate the myocardial function in dogs with DCM, and distinguish the dogs

with DCM from the clinically normal dogs. Regional strain analysis showed the

increased susceptibility of the LVFW to the myocardial dysfunction of DCM. This

chapter also looked at the pattern of contraction from the regional strain analysis, which

demonstrated a difference in the pattern of contraction between dogs with DCM and

clinically normal dogs. These results of the regional strain analysis illustrate the

importance of the regional assessment.

PDA is a commonly encountered congenital cardiovascular defect in dogs,

caused by the persistent ductus arteriosus, and the treatment involves closure of the

ductus arteriosus. Deterioration of systolic function is commonly observed post surgery,

78

which occurs as a result of the alleviation of LV volume overload rather than

myocardial dysfunction. The aim of chapter 4 was to evaluate myocardial function of

PDA before and after closure using 2DTT. Surgical ligation of PDA resulted in

reduction of preload due to the alleviation of LV volume overload, which in turn caused

deterioration of LV systolic function. Such changes were also reflected on the strain

parameters, indicated 2DTT as load-dependent analysis.

79

General Conclusion

The results of this study demonstrated the ability of 2DTT strain analysis to

allow comprehensive evaluation of the myocardial function. The global strain analysis

showed good agreement with the standard echocardiographic parameters of systolic

function, where the results of global strains were similar to the results of FS in both

canine model and clinical dogs with DCM and in clinical dogs with PDA, which is a

non-myocardial cardiovascular disorder. The regional strain analysis and the evaluation

of the synchronicity are one of the great advantages of 2DTT strain analysis, and these

additional assessments provide further information, which enables a more detailed

evaluation of the myocardial function in comparison to the parameters of standard

echocardiography. The regional strain analysis of both canine model and clinical dogs

with DCM indicated the possibility of increased susceptibility of LVFW to myocardial

damage in DCM. Additionally, the evaluation of synchronicity, which is known to be a

sensitive indicator of myocardial dysfunction, has also shown signs of deterioration in

both canine model and clinical dogs with DCM. Regional myocardial function and

synchronicity are meaningful in the evaluation of DCM, since regional myocardial

dysfunction and uncoordinated myocardial contraction will result in reduced cardiac

output, which is an important contributing factor in the clinical signs of CHF. Moreover,

simultaneous evaluation of regional myocardial function and synchronicity allowed

differentiation of the effect of two differing inotropic agents, suggesting the potential

for the application of 2DTT for the monitoring of the response to medical treatments.

Furthermore, the assessment of the pattern of contraction revealed a new insight into a

possibly a new method of evaluating the myocardial function. This study also illustrated

80

the influence of loading conditions on strain parameters, therefore, as with FS, the

influence of preload and afterload needs to be considered when assessing myocardial

function. Regardless, the additional assessment of regional myocardial function and

synchronicity are great advantages of 2DTT. Such assessments allow detailed

evaluation of the myocardial function, which hold a great potential for its use as a

diagnostic and evaluation tool of many cardiac disorders, and it can also be used as a

monitoring tool for the response to treatments in the veterinary field.

81

Acknowledgements

First and foremost, I am extremely grateful to my research advisor, Dr. Ryo

Tanaka, Associate Professor of Veterinary Surgery at the Tokyo University of

Agriculture and Technology, for his valuable guidance and scholarly inputs I received

throughout the research work. I would also like to express my deepest appreciation and

gratitude to Dr. Toshiroh Iwasaki, Professor of Veterinary Internal Medicine, and Dr.

Ryuji Fukushima, Associate Professor of Veterinary Surgery, at the Tokyo University

of Agriculture and Technology.

I would also like to thank my chief examiner, Dr. Noboru Machida, Professor

of Veterinary Clinical Pathology at the Tokyo University of Agriculture and

Technology, and associate examiners, Dr. Hitoshi Kitagawa, Professor of Veterinary

Internal Medicine at the Gifu University, Dr. Jun Yasuda, Professor of Veterinary

Clinical Medicine at Iwate University, and Dr. Kazurou Miyahara, Professor of Animal

Medical Centre at Obihiro University, for their expert, sincere and valuable guidance.

Much of my experimental work would have not been completed without the

assistance of the current and graduated PhD students and the undergraduate students of

the department of Veterinary Surgery at the Tokyo University of Agriculture and

Technology, to whom I will forever be thankful.

And finally to my family, who has always been there through my ups and

downs, and provided me with moral and emotional support.

82

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