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Focal but reversible diastolic sheet dysfunction reflects regional calcium mishandling in dystrophic mdx mouse hearts Ya-Jian Cheng ( ), 1,2 * Di Lang ( ), 2 * Shelton D. Caruthers, 1 Igor R. Efimov, 2 Junjie Chen, 1 and Samuel A. Wickline 1 1 Cardiovascular Division, Washington University School of Medicine, Saint Louis, Missouri: and 2 Department of Biomedical Engineering, Washington University in Saint Louis, Saint Louis, Missouri Submitted 17 April 2012; accepted in final form 26 June 2012 Cheng YJ, Lang D, Caruthers SD, Efimov IR, Chen J, Wickline SA. Focal but reversible diastolic sheet dysfunction reflects regional calcium mishandling in dystrophic mdx mouse hearts. Am J Physiol Heart Circ Physiol 303: H559 –H568, 2012. First published July 9, 2012; doi:10.1152/ajpheart.00321.2012.—Cardiac dysfunction is a primary cause of patient mortality in Duchenne muscular dystrophy, potentially related to elevated cytosolic calcium. However, the re- gional versus global functional consequences of cellular calcium mishandling have not been defined in the whole heart. Here we sought for the first time to elucidate potential regional dependencies between calcium mishandling and myocardial fiber/sheet function as a mani- festation of dystrophin-deficient (mdx) cardiomyopathy. Isolated-per- fused hearts from 16-mo-old mdx (N 10) and wild-type (WT; N 10) were arrested sequentially in diastole and systole for diffusion tensor MRI quantification of myocardial sheet architecture and func- tion. When compared with WT hearts, mdx hearts exhibited normal systolic sheet architecture but a lower diastolic sheet angle magnitude (||) in the basal region. The regional diastolic sheet dysfunction was normalized by reducing perfusate calcium concentrations. Optical mapping of calcium transients in isolated hearts (3 mdx and 4 WT) revealed a stretch-inducible regional defect of intracellular calcium reuptake, reflected by a 25% increase of decay times (T 50 ) and decay constants, at the base of mdx hearts. The basal region of mdx hearts also exhibited greater fibrosis than did the apex, which matched the regional sheet dysfunction. We conclude that myocardial diastolic sheet dysfunction is observed initially in basal segments along with calcium mishandling, ultimately culminating in increased fibrosis. The preservation of relatively normal calcium reuptake and diastolic/ systolic sheet mechanics throughout the rest of the heart, together with the rapid reversibility of functional defects by reducing cytosolic calcium, points to the significance of regional mechanical factors in the progression of the disease. diffusion tensor magnetic resonance imaging; myocardial sheet; Duchenne muscular dystrophy; calcium transients DUCHENNE MUSCULAR DYSTROPHY (DMD) is a fatal X-linked genetic disease that affects 1:3,500 of new-born boys who manifest unremitting deterioration of all muscle groups over time. Cardiac dysfunction now accounts for 1020% of mor- tality in DMD, making it the second leading cause of patient death following respiratory failure (32), because of advances in respiratory support and patient care that have allowed many patients to survive into their late 20s and 30s (1, 38, 73). Accordingly, cardiac complications now are commanding closer scrutiny in the management of advanced disease in patients with DMD (24, 34, 46, 56). The clinical DMD phenotype reflects the absence of normal dystrophin as a consequence of mutations of the dystrophin- encoding gene on the X chromosome. Dystrophin is a key component of the transmembrane dystrophin-associated glyco- protein complex that bridges intracellular actin and the extra- cellular matrix to facilitate mechanical coupling and signal transduction and to stabilize the sarcolemma membrane (25, 56, 66). Our previous work using magnetic resonance (MR) tagging uncovered occult abnormal wall strains occurring re- gionally at the base of heart in DMD patients (3). A subsequent report by Li et al. (44) also suggested that cardiac mechanical strain abnormalities are more prominent in the basal segments of hearts in muscular dytrophy (mdx) mice. These observed regional dependencies for DMD cardiac mechanical dysfunc- tion remain to be explained, as the mutation is present homo- geneously in all cardiomyocytes (5, 11, 70). The heart comprises a syncytium of cardiomyocytes that are organized in unique three-dimensional (3-D) fiber-sheet archi- tecture that is critical for optimizing a coordinated sequence of excitation, contraction, and relaxation (15, 62). Many reports have shown that sheet function produces a sliding motion that is accompanied by dynamic sheet reorientation in systole, which contributes 50% of focal wall thickening (14, 16, 41, 42). Because one role of dystrophin is to anchor cardiomyo- cytes to the extracellular matrix to coordinate contraction- relaxation coupling, we propose that dystrophin deficiency could adversely affect sheet function. Given that ventricular wall strain/stress vary regionally, and in light of the expected vulnerability of dystrophin deficient cardiomyocytes to stretch- induced membrane injury, we sought to examine the role of sheet function in the regional mechanical defects reported in the dystrophin-deficient cardiomyopathy (3, 44). Diffusion tensor MRI (DTI) is an established method for rapid 3-D reconstruction of myocardial microarchitecture that can depict structural changes across the heart cycle in the intact heart. (14, 61). The technique involves a pixel-by-pixel esti- mation of the eigenvectors of water diffusion tensor from seven or more MR images (6, 7). The primary eigenvector, or the eigenvector corresponding to the largest eigenvalue, is consid- ered to be aligned with the local orientation of cardiac myofi- bers, and thus defines the fiber angle () (27, 35, 36, 60). The secondary eigenvector, which corresponds to the second larg- est eigenvalue, is aligned parallel to sheet surfaces (68) and thus defines the sheet angle (). This definition system is based on the fact that eigenvalues of a diffusion tensor are also the apparent diffusion coefficients describing microscopic diffu- sion movements, which is largest in the fiber direction and second largest in the sheet direction (13, 60). In addition to the orientation information, the diffusion tensor also provides * Y.-J. Cheng and D. Lang, co-first authors, contributed equally to the work. Address for reprint requests and other correspondence: S. A. Wickline, Washington Univ. School of Medicine, Campus Box 8215, 660 S. Euclid Ave., St. Louis, MO, 63110 (e-mail: [email protected]). Am J Physiol Heart Circ Physiol 303: H559–H568, 2012. First published July 9, 2012; doi:10.1152/ajpheart.00321.2012. 0363-6135/12 Copyright © 2012 the American Physiological Society http://www.ajpheart.org H559 by 10.220.32.247 on October 6, 2016 http://ajpheart.physiology.org/ Downloaded from

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Page 1: Focal but reversible diastolic sheet dysfunction reflects ...€¦ · Focal but reversible diastolic sheet dysfunction reflects regional calcium mishandling in dystrophic mdx mouse

Focal but reversible diastolic sheet dysfunction reflects regional calciummishandling in dystrophic mdx mouse hearts

Ya-Jian Cheng ( ),1,2* Di Lang ( ),2* Shelton D. Caruthers,1 Igor R. Efimov,2 Junjie Chen,1

and Samuel A. Wickline1

1Cardiovascular Division, Washington University School of Medicine, Saint Louis, Missouri: and 2Department of BiomedicalEngineering, Washington University in Saint Louis, Saint Louis, Missouri

Submitted 17 April 2012; accepted in final form 26 June 2012

Cheng YJ, Lang D, Caruthers SD, Efimov IR, Chen J, WicklineSA. Focal but reversible diastolic sheet dysfunction reflects regionalcalcium mishandling in dystrophic mdx mouse hearts. Am J PhysiolHeart Circ Physiol 303: H559–H568, 2012. First published July 9,2012; doi:10.1152/ajpheart.00321.2012.—Cardiac dysfunction is aprimary cause of patient mortality in Duchenne muscular dystrophy,potentially related to elevated cytosolic calcium. However, the re-gional versus global functional consequences of cellular calciummishandling have not been defined in the whole heart. Here we soughtfor the first time to elucidate potential regional dependencies betweencalcium mishandling and myocardial fiber/sheet function as a mani-festation of dystrophin-deficient (mdx) cardiomyopathy. Isolated-per-fused hearts from 16-mo-old mdx (N � 10) and wild-type (WT; N �10) were arrested sequentially in diastole and systole for diffusiontensor MRI quantification of myocardial sheet architecture and func-tion. When compared with WT hearts, mdx hearts exhibited normalsystolic sheet architecture but a lower diastolic sheet angle magnitude(|�|) in the basal region. The regional diastolic sheet dysfunction wasnormalized by reducing perfusate calcium concentrations. Opticalmapping of calcium transients in isolated hearts (3 mdx and 4 WT)revealed a stretch-inducible regional defect of intracellular calciumreuptake, reflected by a 25% increase of decay times (T50) and decayconstants, at the base of mdx hearts. The basal region of mdx heartsalso exhibited greater fibrosis than did the apex, which matched theregional sheet dysfunction. We conclude that myocardial diastolicsheet dysfunction is observed initially in basal segments along withcalcium mishandling, ultimately culminating in increased fibrosis.The preservation of relatively normal calcium reuptake and diastolic/systolic sheet mechanics throughout the rest of the heart, together withthe rapid reversibility of functional defects by reducing cytosoliccalcium, points to the significance of regional mechanical factors inthe progression of the disease.

diffusion tensor magnetic resonance imaging; myocardial sheet;Duchenne muscular dystrophy; calcium transients

DUCHENNE MUSCULAR DYSTROPHY (DMD) is a fatal X-linkedgenetic disease that affects 1:3,500 of new-born boys whomanifest unremitting deterioration of all muscle groups overtime. Cardiac dysfunction now accounts for 10�20% of mor-tality in DMD, making it the second leading cause of patientdeath following respiratory failure (32), because of advances inrespiratory support and patient care that have allowed manypatients to survive into their late 20s and 30s (1, 38, 73).Accordingly, cardiac complications now are commandingcloser scrutiny in the management of advanced disease inpatients with DMD (24, 34, 46, 56).

The clinical DMD phenotype reflects the absence of normaldystrophin as a consequence of mutations of the dystrophin-encoding gene on the X chromosome. Dystrophin is a keycomponent of the transmembrane dystrophin-associated glyco-protein complex that bridges intracellular actin and the extra-cellular matrix to facilitate mechanical coupling and signaltransduction and to stabilize the sarcolemma membrane (25,56, 66). Our previous work using magnetic resonance (MR)tagging uncovered occult abnormal wall strains occurring re-gionally at the base of heart in DMD patients (3). A subsequentreport by Li et al. (44) also suggested that cardiac mechanicalstrain abnormalities are more prominent in the basal segmentsof hearts in muscular dytrophy (mdx) mice. These observedregional dependencies for DMD cardiac mechanical dysfunc-tion remain to be explained, as the mutation is present homo-geneously in all cardiomyocytes (5, 11, 70).

The heart comprises a syncytium of cardiomyocytes that areorganized in unique three-dimensional (3-D) fiber-sheet archi-tecture that is critical for optimizing a coordinated sequence ofexcitation, contraction, and relaxation (15, 62). Many reportshave shown that sheet function produces a sliding motion thatis accompanied by dynamic sheet reorientation in systole,which contributes �50% of focal wall thickening (14, 16, 41,42). Because one role of dystrophin is to anchor cardiomyo-cytes to the extracellular matrix to coordinate contraction-relaxation coupling, we propose that dystrophin deficiencycould adversely affect sheet function. Given that ventricularwall strain/stress vary regionally, and in light of the expectedvulnerability of dystrophin deficient cardiomyocytes to stretch-induced membrane injury, we sought to examine the role ofsheet function in the regional mechanical defects reported inthe dystrophin-deficient cardiomyopathy (3, 44).

Diffusion tensor MRI (DTI) is an established method forrapid 3-D reconstruction of myocardial microarchitecture thatcan depict structural changes across the heart cycle in the intactheart. (14, 61). The technique involves a pixel-by-pixel esti-mation of the eigenvectors of water diffusion tensor from sevenor more MR images (6, 7). The primary eigenvector, or theeigenvector corresponding to the largest eigenvalue, is consid-ered to be aligned with the local orientation of cardiac myofi-bers, and thus defines the fiber angle (�) (27, 35, 36, 60). Thesecondary eigenvector, which corresponds to the second larg-est eigenvalue, is aligned parallel to sheet surfaces (68) andthus defines the sheet angle (�). This definition system is basedon the fact that eigenvalues of a diffusion tensor are also theapparent diffusion coefficients describing microscopic diffu-sion movements, which is largest in the fiber direction andsecond largest in the sheet direction (13, 60). In addition to theorientation information, the diffusion tensor also provides

* Y.-J. Cheng and D. Lang, co-first authors, contributed equally to the work.Address for reprint requests and other correspondence: S. A. Wickline,

Washington Univ. School of Medicine, Campus Box 8215, 660 S. Euclid Ave.,St. Louis, MO, 63110 (e-mail: [email protected]).

Am J Physiol Heart Circ Physiol 303: H559–H568, 2012.First published July 9, 2012; doi:10.1152/ajpheart.00321.2012.

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shape information within the microdiffusion environmentbased on fractional anisotropy (FA) (22).

With respect to pathophysiological features of dystrophindeficiency potentially responsible for mechanical dysfunction,several recent studies in isolated cardiomyocytes of mdx mousehave suggested that intracellular Ca2� concentration ([Ca2�]i)mishandling is a key causative factor (2, 23, 75). However,how calcium mishandling influences cardiac mechanical dys-function in whole heart preparations has not been clarified,especially taking into account cellular and mechanical eventsthat are spatio-temporally distributed (e.g., excitation-contrac-tion coupling) and regionally heterogeneous (e.g., stress andstrain) (4, 17). Given the recognized abnormal and regionallyheterogeneous wall strains (3) and fibrosis (3, 76, 77) in DMDhearts and the similarly heterogeneous wall strains (44) andfibrosis (44, 63) in mdx mouse heart, the present work isfocused on the hypothesis that the functional consequences ofdystrophin deficiency play out regionally in DMD hearts overtime, despite the ubiquitous genetic abnormality in all cardio-myocytes.

Accordingly, we sought to elucidate such regional mechan-ical consequences of the disease on local myocardial fiber-sheet function with the use of DTI, in concert with whole heartoptical calcium mapping. The complete global 3-D informationset acquired in isolated perfused beating mdx hearts shouldallow delineation of both the locus (regional and intramural)and the cycle dependence (diastole vs. systole) of the principlecardiac defects in dystrophin-deficiency and potentially shedlight on whether these defects might be reversible (i.e., func-tional) consequences of calcium mishandling or be nonrevers-ible consequences of fibrosis (i.e., remodeling).

MATERIALS AND METHODS

Animals

Adult male mdx (C57BL/10ScSn-Dmdmdx/J, N � 14) and age-matched male wild-type (WT: C57BL/10SnJ, N � 10; and C57BL/6,N � 3) mice were purchased from the Jackson Laboratory (BarHarbor, ME) and the National Institute on Aging (18) and housedunder an American Association for Accreditation of Laboratory An-imal Care-approved facility to 16 mo of age. All mice were housed inthe same animal facility and cared for under approval by WashingtonUniversity’s Animal Study Committee. All mice were separated intotwo groups for DTI and optical mapping studies.

DTI

Isolated heart preparation for DTI. Ten mdx mice and 10 WT mice(C57BL/10SnJ) were used for DTI measurement. Mice were heparin-ized (1,000 U/kg) and anesthetized with 0.05 isoflurane. Hearts wereexcised and cannulated for retrograde perfusion using a proceduremodified from Chen et al. (14). Briefly, hearts were first perfused withKrebs buffer at 60 mmHg constant pressure for �5 min to wash outthe residue blood. Five mdx hearts and five WT hearts were perfusionarrested in diastole (perfusion pressure � 60 mmHg) with the regularSt. Thomas’ cardioplegic solution containing normal [Ca2�] of 1.2mmol/l (denoted as mdx-NC and WT-NC, respectively) under roomtemperature. DTI of diastolic arrested hearts were performed for 1.5 h.After that, hearts were reperfused with 37°C Krebs buffer to resumebeating. Upon stabilization of cardiac work (�6 min, up to 300beats/min), 2.5 mmol/l Ba2� were introduced to induce systoliccardiac contracture while Ca2� was lowered to 0.078 mmol/l (14, 49,67). The systolic arrest of mdx-NC, WT-NC, mdx-LC, and WT-LC(defined below) are all achieved with the same modified Tyrodesolution containing 2.5 mmol/l Ba2� and 0.078 mmol/l Ca2� (13). Allsolutions were equilibrated with 95% O2-5% CO2 (pH 7.4) andcontained adenosine (45 �mol/l) to maximally dilate the coronary

Fig. 1. Representative diffusion tensor MRI (DTI) andhistology images of sheet architecture in wild-type(WT) mouse hearts. A: a local cylindrical coordinatesystem was used to determine left ventricular (LV)myocardial structures. The primary eigenvector of thediffusion tensor is characterized by helix angle (�),which is the angle between the circumferential axis(CA) and the projection of the first eigenvector (EV1)onto the circumferential-longitudinal plane; the � shownin A is set as negative. The secondary eigenvector (EV2)is characterized by sheet angle (�), which is the anglebetween the secondary eigenvector and the radial axis(RA); the � shown in A, toward base and towardendocardium, is set as negative. B and C: short-axisviews of DTI determined sheet angle (�) in a basal sliceof WT heart that was sequentially arrested in diastole(B) and systole (C). The posterior-lateral wall of LVmanifested two populations of �, exhibiting positive(red) and negative (blue) values. D: histology of sheetorganization in a WT heart. Images were acquired fromthe basal-lateral wall, which is highlighted on the he-matoxylin and eosin-stained image (inset). Two popula-tions of � were observed. RV, right ventricle; PA, papillarymuscle; P, posterior; L, left; R, right; A, anterior.

H560 CALCIUM AND SHEET DYSFUNCTION IN MDX HEARTS

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vessels (39). Bovine serum albumin (61 �mol/l) was added to theperfusate to minimize interstitial edema (20, 74). DTI of systolichearts was imaged for �1.5 h. Upon the completion of MRI, all heartswere perfused with 10% formalin to fix in systole for histologicalstudy.

To evaluate the effect of [Ca2�] on the diastolic cardiomyocytearchitecture in mdx mice, another five mdx hearts and five WT heartswere perfusion arrested in diastole with a modified cardioplegicsolution containing low [Ca2�] of 0.078 mmol/l (denoted as mdx-LCand WT-LC, respectively). The 0.078 mmol/l [Ca2�] was chosen overthe calcium-free condition to minimize the risk of complete depletionof intracellular calcium, which can cause calcium overload uponreperfusion with solutions containing normal [Ca2�], thereby exertingunfavorable effects on certain ion channels (49, 55).

A MR-compatible, viable mouse heart perfusion system. A dedi-cated MRI-compatible, retrograde mouse heart perfusion system wasused to acquire high signal-to-noise diffusion tensor MR images. Thissystem provides a quantitative and nondestructive assessment ofdiastolic and systolic myocardial architecture from the same heart.

The system includes 1) a custom-built 15-mm-diameter solenoid coil,which provides high signal to noise (65 for nondiffusion-weightedimages, and 40 for diffusion-weighted images) and 2) a Langendorffconstant-pressure perfusion apparatus that allows arrest of a viableheart sequentially in diastole and then in systole.

MRI procedure. All MRI experiments were performed on a Varian11.7-T small animal MR scanner (Varian Associates; Palo Alto, CA),and a custom-built 10-mm-diameter solenoid coil was used to acquireimages. Long-axis scout images were acquired as previously de-scribed (14). A multislice spin-echo sequence with diffusion-sensitiz-ing bipolar gradient was used to acquire short-axis, diffusion-weighted images. Diffusion-sensitizing gradients were applied in sixnoncolinear directions. Imaging parameters are as follows: echo time,46 ms; time interval between diffusion-sensitizing gradients, 30 ms; bvalue, 0 and 800 s/mm2; in-plane resolution, 125 � 125 �m2;repetition time, 3 s; and image-acquisition time, 1 h. For diastolic-arrested hearts, eight contiguous slices (thickness � 0.7 mm) extend-ing from the base to apex were acquired. For systolic-arrested hearts,eight contiguous slices were acquired with slice thickness adjusted to

Fig. 2. DTI-determined diastolic and systolicmagnitude of sheet angle (|�|) in short-axisslices. A: muscular dystrophy (mdx) hearts ex-hibited abnormally lower diastolic |�| that wasrecovered to normal value after reducing [Ca2�]in the perfusate. For hearts perfusion arrested indiastole with normal calcium solution, mdxhearts (mdx-NC) exhibited lower |�| than didWT (WT-NC) hearts with the maximal �10°difference occurred at the basal region. In con-trast, mdx hearts perfused with the low calciumsolution (mdx-LC) exhibited normal diastolic |�|.Varying [Ca2�] had no effect on diastolic |�| inWT hearts (WT-LC). B: systolic |�| exhibited nodifference among 4 groups of hearts. C: diastolic|�| in the epicardium, midcardium, and endocar-dium at the base of WT and mdx hearts. In allregions, diastolic |�| of mdx-LC hearts werecomparable with that of WT-LC and WT-NChearts, but all were higher than that of mdx-NChearts. The largest difference in diastolic |�|between mdx-LC and mdx-NC was observed inthe endocardium. D and E: representative DTI-determined diastolic |�| maps on short-axis slicesat the base of mdx-NC and mdx-LC hearts. Thelower |�| in the mdx-NC heart, indicated byfewer regions with red color, was visually ap-preciable. F: in the posterior-lateral region (theareas enclosed by dashed line in D and E), thediastolic |�| of mdx-NC hearts was approximated20° lower than that of mdx-LC hearts and of allWT hearts. Data represent means � SE; N � 5for each group. *P 0.05 and †P 0.005 forthe indicated short-axis location.

H561CALCIUM AND SHEET DYSFUNCTION IN MDX HEARTS

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0.55–0.65 mm based on the fraction of longitudinal shortening afterbarium-induced contracture. Attention was paid to keep the sliceselection and the heart orientation the same between diastole andsystole imaging.

DTI data analysis. All MR images were processed using custom-developed software written in Matlab (MathWorks) as previouslydescribed (14). Fiber helix angle (�) and sheet angle (�) are calculatedfrom the tensor according to the literature (14) (see the DTI paragraphin Introduction for background). Briefly, the fiber helix angle (�) wascalculated as the angle between the circumferential cardiac axis andthe projection of the first eigenvector of the diffusion tensor onto thecircumferential-longitudinal plane; the right-handed helix was set aspositive (60). Sheet angle (�) was determined as the angle between thesecondary eigenvector and the radial axis; the sheet inclined towardthe base from endocardium to epicardium was set as positive (Fig. 1)(60, 68). Because regional wall thickening is accompanied by areduced magnitude of the sheet angle (|�|) despite the sign of sheetangle (14, 42), the |�| was used to analyze regional diastolic andsystolic sheet function. Left ventricular (LV) wall thickness wascalculated as the mean distance between epicardial and endocardialborders. The through-wall difference of �, defined as the difference inendocardial and epicardial helix angles, i.e., � � �endocardium ��epicardium, was used to quantify transmural changes of fiber orienta-tion.

Histology. After DTI acquisition, five mdx and five WT hearts wererapidly fixed in 10% formalin. Afterward, fixed hearts were sliced intofour blocks with 2 mm thickness from base to apex. Each slice wasembedded in paraffin and sectioned at 4 �m thickness. The tissuesections were stained with picrosirius red for the identification offibrosis (71). Quantitative light microscopic analysis of histological

samples was performed by video microscopy. The extent of myocar-dial fibrosis was quantified from digitized histological images using athresholding algorithm implemented in MatLab. Regions that demon-strate stronger red-to-green ratio in picrosirius red staining wereconsidered to represent interstitial fibrosis. The percentage of tissuefibrosis was calculated as the ratio of pixels representing fibrotic tissueversus. pixels of the entire short-axis slice, and the average value intwo adjacent slices were used in analysis.

Optical Mapping

Isolated heart preparation and calcium dye loading. Seven Lan-gendorff-perfused hearts from 16-mo-old mice were separated intotwo groups [mdx, N � 4; and WT (C57BL/6), N � 3]. The isolatedheart preparation was according to a Langendorff perfusion protocolmodified for murine hearts (40). In brief, after anesthesia and heartexcision, a short section of aorta was attached to a 21-gauge cannula.After cannulation, hearts were superfused and retrogradely perfusedwith oxygenated (95% O2-5% CO2) constant temperature (37 � 1°C),modified Tyrode solution consisting of (in mmol/l) 128.2 NaCl, 4.7KCl, 1.19 NaH2PO4, 1.05 MgCl2, 1.3 CaCl2, 20.0 NaHCO3, and 11.1glucose (pH 7.3) that was passed through a 5-�m filter (Millipore,Billerica, MA). Perfusion was performed using a peristaltic pump(Peri-Star, WPI, Sarasota, FL) under a constant aortic pressure of60–80 mmHg.

The isolated heart was pinned at the edge of ventricular apex to theSylgard bottom of the chamber to prevent stream-induced movement.A small silicon tube was inserted into the left ventricle through thepulmonary vein, left atria, and tricuspid valve to prevent solutioncongestion and subsequent ischemia. A small black tape was used to

Fig. 3. Averaged wall thicknesses in each short-axis slice are similar in all WT and mdx groups,both in diastole (A) and in systole (B). Averagedthrough-wall myofiber helix angle differences(�) in each short-axis slice are similar in allWT and mdx groups, both in diastole (C) and insystole (D). Overall, � is increased in systole,agreeing with a prior publication (14). No sig-nificance was detected in all comparisons in eachpanel.

H562 CALCIUM AND SHEET DYSFUNCTION IN MDX HEARTS

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cover the atria to avoid fluorescence scattering pollution from theatria. Excitation-contraction uncoupler blebbistatin (10 �M, TocrisBioscience) was used to prevent the effect of motion artifact on theaction potential duration estimation. The heart was then stained witha calcium indicator (30 �l of 1 mg/ml dimethyl sulfoxide, 1:1 mixedwith Fluronic F127, Invitrogen, Carlsbad, CA, in Tyrode solution;Rhod-2 AM, Invitrogen) for 5–7 min.

Optical imaging system. The excitation light was generated by ahalogen lamp (Newport Oriel Instruments, Stratford, CT; SciMedia,Costa Mesa, CA) and passed through a heat filter, shutter, andexcitation band-pass filter (520 � 45 nm). A flexible light guidedirects the band-pass filtered light onto the preparation. The opticalmapping apparatus comprised an MiCAM Ultima-L CMOS camera(SciMedia) with high-spatial (100 � 100 pixels, 230 � 20 �m perpixel) and temporal (1,000–3,000 frames/s) resolution. A band-passfilter (590 � 15 nm, Thorlabs, Newton, NJ) was fixed in front of thecalcium-imaging camera.

Optical mapping protocols. After isolation and cannulation, motionsuppression, and dye staining, prepared hearts were equilibrated for5–10 min before imaging. The optical mapping signals were recordedsequentially under unloaded and stretched conditions. The unloadedcondition was achieved by a pressure releasing silicon tube insertedinto the LV chamber of the Langendorff heart preparation. Thestretched condition was achieved by inflating the LV chamber withTyrode solution to 80 mmHg pressure through the silicon tube

inserted into the left ventricle. The perfusion pressure was increasedfrom 60 to 100 mmHg to maintain the direction of the retrogradeperfusion and the viability of the perfused heart. A steady-staterestitution (S1-S1) (40) pacing protocol was applied under bothconditions.

Optical mapping data processing. A custom-designed MatLabprogram was used to analyze the optical mapping signals. The signalswere filtered with a low-pass Butterworth filter at 256 Hz. Regionalcalcium dynamics were quantified in WT and mdx mouse ventricles(base vs. apex) under both unloaded and stretched conditions. Hand-drawn regions of interest were used to define basal (upper 1/3 area ofleft ventricle) versus apical regions (lower 1/3 area of left ventricle).Because the calcium relaxation process is not monoexponential, onlythe latter half of the calcium signal decay could be fitted exponentiallyto derive the decay constant. Typically, both 50% relaxation decaytime (T50) and the decay constant are used to characterize calciumrelaxation kinetics (12, 29, 47, 48, 57, 58, 64, 72, 75, 80). Specifically,the Ca2� relaxation time from the calcium transient peak value to 50%relaxation (T50) was calculated to define the calcium reuptake phase,and Ca2� decay constant was calculated using an exponential curvefitting program from the 50% calcium peak value to full relaxation.Taken together, the T50 (for the early relaxation phase) and the decayconstant (for the late relaxation phase) represent the whole process ofCa2� reuptake-dependent relaxation.

Fig. 4. A–D: representative LV fractional anisot-ropy (FA) maps at base of a WT and a mdx heartsequentially arrested in diastole and then in sys-tole. When compared with FA in diastole, lowerFA in systole is visually appreciable. E andF: when compared with WT-NC hearts, mdx-NChearts exhibit �15% lower FA in diastole (E)and �30% lower FA in systole (F). Reducing[Ca2�] in cardioplegic solution recovered dia-stolic FA in mdx-LC hearts to normal value.*P 0.05. In barium-arrested systole, bothmdx-LC and mdx-NC hearts exhibit lowered FAthan do WT-LC and WT-NC, indicating a moreisotropic tissue organization/cell shape in systoledespite similar sheet angles. Please see MATERI-ALS AND METHODS for the systolic arrest condi-tion of all 4 groups.

H563CALCIUM AND SHEET DYSFUNCTION IN MDX HEARTS

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Statistics. Statistical differences were assessed with the use ofanalysis of variance (ANOVA) or multivariate analysis of variance(MANOVA) as appropriate. Bonferroni correction was applied to posthoc comparisons. A value of P 0.05 was considered significant.Data were expressed as means � SE. All statistics were conductedwith the IBM SPSS statistics program.

RESULTS

mdx Hearts Manifest Abnormal but Recoverable RegionalDiastolic Sheet Function

DTI defined two populations of � with opposite signs in theLV wall of WT and mdx mice, as confirmed by severalprevious reports (14, 28, 33, 41, 52, 68). The |�| value isdecreased in systole (Figs. 1, B and C, and 2, A and B),reflecting the reorientation of myocardial sheets, which con-tributes �50% of systolic focal wall thickening (14, 16, 41,42). Under conditions of barium-induced systolic arrest, the

systolic |�| was comparable among all groups of hearts [P �not significant (NS)]. However, the diastolic |�| of mdx hearts(mdx-NC) was �10° lower than that of WT hearts (WT-NC) atthe base when perfused with the regular cardioplegic solution(P 0.05, Fig. 2). The |�| at the apex of mdx-NC heartsappeared normal (P � NS compared with WT-NC at eachcorresponding short-axis imaging slice), demonstrating thatsheet diastolic dysfunction in mdx hearts occurred focally at thebase.

By the reduction of the [Ca2�] of the cardioplegic solutionto 7% of its regular concentration, a complete and rapidrestoration of diastolic |�| in mdx hearts (mdx-LC) to normalvalues was observed. No effect of calcium manipulation onWT diastolic |�| was observed (WT-LC, P � NS comparedwith WT-NC at each corresponding short-axis imaging slice),indicating that the diastolic sheet dysfunction in mdx heartswas substantially calcium dependent. These data suggest that

Fig. 5. Calcium relaxation for mdx and WT. A: representative calcium transients at the base of a WT (blue line) and a mdx (red line) heart. Signals were averagedfrom adjacent 25 charge-coupled device elements. Definitions of T50 and decay constant � are illustrated. B and C: representative T50 and decay constant mapsin a mdx and a WT heart under unloaded vs. stretched condition. D and E: under unloaded conditions, both T50 and decay constant were comparable betweenmdx and WT hearts. Ventricular stretch caused regional increases of T50 and decay constant at the base of mdx hearts. However, WT hearts exhibit similar T50

and decay constant. *P 0.05; **P 0.01.

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even at this advanced age, a principal mechanical abnormalityin mdx mice likely is associated with incomplete sarcomererelaxation that is regionally dependent.

At the base, further regional analysis indicated that themaximal decrease of diastolic |�| in mdx-NC hearts occurred inthe endocardium and in the posterior-lateral wall. When com-pared with WT-NC, mdx-NC exhibited 13° lower |�| (75 � 2vs. 62 � 3°, P 0.005) in the endocardium (inner 1/3 walldepth in transmural direction), 10° lower |�| (74 � 1 vs. 64 �2°, P 0.05) in the midcardium (middle 1/3 wall depth intransmural direction), and 8° lower |�| (68 � 2 vs. 60 � 2°,P 0.05) in the epicardium (outer 1/3 wall depth in transmuraldirection) (Fig. 2C). In the posterior-lateral wall, the diastolic|�| was 51 � 6° in mdx-NC (Fig. 2F). This value was �20°lower than that of mdx-LC (70 � 4°), as well as that of WT-NC(72 � 4°) or WT-LC (72 � 6°) groups (P 0.05 for allcomparisons). Together, these findings indicate a clear regionaland transmural dependence of incomplete relaxation despitethe ubiquitous dystrophin mutation.

mdx and WT hearts exhibit similar transmural fiber organi-zation and function but lower FA. Wall thickness and through-wall differences of myofiber helix angle (�) were similarbetween mdx and WT mice (Fig. 3), suggesting that the majormechanical abnormality was confined to sheets rather thanfibers. The normal wall thickness agrees with data of formerpublications (44, 63). In all hearts, systolic FA was �40%lower than diastolic FA (P 0.05 for all comparisons, Fig. 4),indicating that a more isotropic diffusion environment waselicited by cardiac contraction (19, 26), as expected from fibersshortening in the long axis and expanding in the short axis (i.e.,becoming rounder or less oblong).

Optical imaging reveals longer calcium decay time (T50)with regional dependencies on stretch. Figures 5 illustrates thecalcium T50, as defined by optical mapping, which representsthe duration of [Ca2�]i decay from 100 to 50% of its peakvalue. Under unloaded conditions, T50 was comparable be-tween mdx and WT hearts. However, ventricular stretch elic-ited an �25% increase in T50 (from 40 � 2 to 51 � 3 ms, P 0.05) and �50% increase of decay constant (from 25 � 1 to37 � 1 ms, P 0.05) at the base of mdx hearts, confirmingabnormal calcium uptake in this region. Ventricular stretch,

however, did not affect the T50 or decay constant at the apex ofmdx hearts, nor in the entire left ventricle of WT hearts,indicating a heterogeneous regional dependency on the devel-opment of abnormal calcium handling in mdx.

Mdx hearts exhibit higher fibrosis content at base comparedwith apex. Figure 6 illustrates representative histopathology inpicrosirius red stained mdx hearts. Scattered interstitial fibrosisstaining was observed throughout the entire myocardium.Quantitative analysis demonstrated that WT mice exhibitedsimilar collagen content at the base versus the apex. However,mdx mice exhibited approximately three times more collagen atthe base compared with the apex, agreeing with prior patientstudies (76, 77) and mdx mice studies (44, 63).

DISCUSSION

This study shows for the first time that mdx hearts exhibit anabnormal myocardial sheet angle magnitude |�| in relaxationthat is calcium dependent and rapidly reversible. Furthermore,regional and intramural gradients of diastolic dysfunction areevident, primarily in the more basal segments where abnormalcalcium reuptake has been demonstrated. These colocalizedabnormalities in calcium reuptake and diastolic sheet functionstrongly suggest that features other than the simple absence ofdystrophin are critical to the evolution of heart failure in DMD.Indeed, it is well recognized that the mechanical defect in thedystrophin-encoding gene does not independently elicit DMDcardiac functional defects such as reduced circumferential wallstrain (3), which emerge in a regionally heterogeneous fashionover time and not at all loci where dystrophin is missing.

Given that cardiac contraction induces sheet sliding, exten-sion, and reorientation leading to the reduced |�|, the observedlower diastolic |�| may reflect the state that mdx cardiomyo-cytes are not fully relaxed in diastole. The low-diastolic FA ofmdx hearts, indicating more round shaped fiber/sheets organi-zation (19, 26), also implies this diastolic mechanical defect ofmdx hearts. Systolic FA of mdx is lower than that of WT,indicating while the mechanical sheet orientation is not af-fected by the absence of dystrophin in systole, the microstruc-tural shape of muscular architecture is affected, which requiresfurther investigation. We also showed that this regional diastolic

Fig. 6. Regional variations of interstitial fi-brosis in mdx hearts. A: representative picro-sirius red stained slices at the base and apexof WT and mdx hearts. B: the percentage offibrotic area at the base and the apex of mdx(N � 5) and WT (N � 5) hearts. Originaldata points are marked as dots. Thick bar,mean value; thin bar, means � SE. †P 0.005 for all comparisons to the base seg-ment of mdx hearts.

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sheet dysfunction in mdx hearts is a likely consequence of persis-tently elevated cytosolic calcium, because it is quickly reversibleupon perfusion with a lower level of calcium (Fig. 2).

The prior recognition of stretch-induced calcium mishan-dling in isolated mdx cardiomyocytes informs the present work(23, 75). Specifically, mechanical stretch increases the openingchance of stretch-activated channels (75) and develops tran-sient membrane microruptures in mdx cardiomyocytes (65, 66).The resultant leakage of extracellular calcium into the cytosolthen compromises calcium clearance during relaxation andrenders mdx cardiomyocytes vulnerable to stretch-induced in-jury (59). In this study of intact perfused hearts, we employeda stretch protocol to mimic cardiomyocyte activity under mod-erate strain regimes by inflating LV pressure to 80 mmHg,which is similar to that used for investigating fibrillation byseveral groups (10, 21, 37, 50, 78). Under the stretched con-dition, mdx hearts exhibited regional deficiency in calciumuptake, as evidenced by the prolonged T50 and decay timeconstant, at the base where the sheet functional defect wasobserved. Thus our results illustrate a regional correspondencebetween abnormal diastolic sheet mechanics and disturbedcalcium kinetics.

Calcium kinetic signals from the optical mapping dyes canbe recorded from up to 300-�m depth of the tissue preparation,among which about half of the total signals come from first 100�m (30). Although optical mapping data may not reliablyrepresent the tissue beyond 300-�m depth, the surface signalprovides a reliable and well-accepted surrogate measure ofregional tissue calcium kinetics. Regional calcium mishan-dling, together with the abnormal � and FA, are observedtogether in all mdx hearts but not in any WT heart, which offersa suitable negative control.

The prolonged T50 and decay constant observed in mdxmouse hearts may reflect calcium leakage through the desta-bilized sarcolemma as a direct effect of the lack of infrastruc-tural integrity that otherwise would be provided by the dystro-phin-associated glycoprotein complex. Accordingly, a thera-peutic strategy to inhibit calcium leakage during the restingstate (i.e., unrelated to the operation of voltage-gated L-typechannels for cell excitation), could be effective in restoringcardiac function in myopathic mdx heart. The promising resultswith several novel compounds such as P188 that is claimed torepair sarcolemma microruptures (54), and S107 that stabilizesryanodine receptor 2 (8, 9), together with the ineffectiveness ofL-type calcium channel blockers as demonstrated in earlierclinical trials (51), attests to the potential clinical utility of theapproach.

Several potential limitations merit discussion. The �10°difference of diastolic |�| may not contribute to MR detectablewall thickness change because of the limited image resolutionrelative the wall thickness of mouse hearts and the partialvolume effect on edge detection common to MRI exams.Because the measurement of |�| in ventricular wall is notsensitive to these limitations, our results suggest that |�| ismore sensitive than is MR-measured wall thickness to detectsubtle changes in myocardial organization. Quantitative histo-logical validation of DTI-measured sheet angle was not con-ducted because it has been reported and validated in numerousearlier studies (33, 36, 60, 68). Because mdx mice can only bepurchased from commercial vendors at 2 mo of age, theconsiderable time, high cost, and susceptibility to sudden death

during even light anesthesia militated against acquiring in vivocardiac functional readout before DTI experiments. Finally,although the current study did not investigate regional cardiacmechanics, the previous work of our group and others haveshown such abnormal regional wall strain is detectable in bothpatients with DMD (3) and in mdx mice before ventricularfunction is abnormal (44).

Although the perfused viable whole heart preparation maynot entirely replicate the in vivo condition, the present obser-vations have elucidated striking functional differences betweenmdx and WT hearts and provided useful insights into theregional macroscopic ventricular mechanical dysfunction andrelated cellular calcium kinetics that cannot be defined byisolated cell preparations. Because sheet function is a keydeterminant of ventricular wall strain (16, 69) and calciummishandling in mdx cardiomyocytes is associated with cellularinjury (45, 53, 75), the observed stretch-induced regionalcalcium mishandling in mdx hearts may be responsible in partfor the local progression of diastolic sheet dysfunction. Incombination with the expected heterogeneous distribution ofwall strain and stress, increased diastolic stiffness may facili-tate a vicious cycle of continuing damage to fragile cellmembranes that ultimately results in global heart failure.(17,31, 43, 44, 79).

ACKNOWLEDGMENTS

We thank Lei Zhang and Lingzhi Hu for suggestions regarding MRI;Noriko Yanaba and Huiying Zhang for assistance with histology; Dr. Lung-Chang Chien for assistance with statistical analysis; Bor-Yiing Su for helpfulsuggestions on the imaging segmentation algorithm; and Matthew Goette, KirkHou, and Jan Kubanek for helpful discussions and manuscript review.

GRANTS

This study was supported by American Heart Association Grant12PRE9310044 (to Y.-J. Cheng) and National Institutes of Health GrantsR01-HL-085369 (to I. R. Efimov), R21-HL-108617 (to I. R. Efimov), andR01-AR-056223 (to S. A. Wickline).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Y.-J.C., D.L., and J.C. conception and design of research; Y.-J.C. and D.L.performed experiments; Y.-J.C. and D.L. analyzed data; Y.-J.C., D.L., S.D.C.,I.R.E., J.C., and S.A.W. interpreted results of experiments; Y.-J.C. and D.L.prepared figures; Y.-J.C., D.L., and J.C. drafted manuscript; Y.-J.C., D.L.,S.D.C., I.R.E., J.C., and S.A.W. edited and revised manuscript; Y.-J.C., D.L.,I.R.E., J.C., and S.A.W. approved final version of manuscript.

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