ATORVASTATIN PREVENTS SEPSIS-INDUCED DOWNREGULATION OFMYOCARDIAL "1-ADRENOCEPTORS AND DECREASED CAMP RESPONSE

IN MICE

Ramasamy Thangamalai,* Kannan Kandasamy,* Susanth V. Sukumarn,*Narasimha Reddy,* Vishakha Singh,* Soumen Choudhury,* Subhashree Parida,*

Thakur Uttam Singh,* Raja Boobalan,† and Santosh Kumar Mishra**Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Bareilly, Uttar

Pradesh; and †Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar,Chidambaram, Tamilnadu, India

Received 13 Nov 2013; first review completed 26 Nov 2013; accepted in final form 8 Jan 2014

ABSTRACT—Impaired cardiac "-adrenoceptor signaling is an important cause of sepsis-induced myocardial depression inman and experimental animals. We examined the effect of atorvastatin (ATR) pretreatment on myocardial "1-adrenoceptor("1-AR) expressions and postYreceptor signaling in a mouse model of sepsis (cecal ligation and puncture [CLP]). After 20 T

2 h of surgery, hearts were isolated for the measurement of left ventricular functions (left ventricular developed pressure, dp/dtmax and dp/dtmin) using Langendorff setup. Western blot was used to determine "1-AR and G proteinYcoupled receptorkinase 2 protein expressions. Real-time polymerase chain reaction was done to determine "1-AR mRNA expression.Atorvastatin prevented sepsis-induced decrease in left ventricular functions, such as left ventricular developed pressure(CLP 75.90 T 0.53 vs. ATR 100.24 T 1.64 mmHg), dp/dtmax (CLP 3,742 T 71 vs. ATR 4,291 T 88 mmHg/s), and dp/dtmin

(CLPj1,010 T 24 vs. ATRj1,346 T 84 mmHg/s). Associated with functional impairments, sepsis decreased both myocardial"1-AR protein and mRNA expressions by 52% T 9% and 62% T 7%, respectively. However, ATR treatment of CLPmice (ATR)preserved "1-AR protein (96% T 11%) and mRNA (88% T 14%) expressions comparable to sham-operated level. Fur-thermore, it not only attenuated sepsis-induced decrease in basal cardiac adenosine 3¶,5¶-cyclic monophosphate content(CLP 1.30 T 0.27 vs. ATR 6.30 T 0.67 pmol/mg protein), but also prevented its refractoriness to dobutamine stimulation(CLP 1.72 T 0.27 vs. ATR 10.83 T 1.37 pmol/mg protein). Atorvastatin also inhibited sepsis-induced increase in cardiac GproteinYcoupled receptor kinase 2 protein expression (CLP 1.73 T 0.18-fold vs. ATR 1.10 T 0.18-fold), protein kinase Aactivity (CLP 1.12 T 0.14 vs. ATR 0.66 T 0.08 U/mg protein) and plasma catecholamines (CLP 138 T 22 vs. ATR 59 T 2 pg/mL).In conclusion, ATR seems to improve left ventricular functions in vitro through the preservation of "1-AR signaling in sepsis.

KEYWORDS—"1-adrenoceptor, catecholamines, GRK2, heart, sepsis, statin

INTRODUCTION

Sepsis, the systemic inflammatory response syndrome to

infection, is the leading cause of death in critically ill patients.

Sepsis frequently affects cardiovascular system, and according

to some clinical studies, myocardial dysfunction is one of the

major predictors of morbidity and mortality in sepsis (1, 2).

Echocardiographic studies have demonstrated impaired left

ventricular systolic and diastolic functions, reduced ejection

fraction, but normal or increased cardiac output in septic pa-

tients (3). These human studies along with in vivo and in vitrostudies in experimental animals have clearly established that

decreased contractility and impaired myocardial compliance

are major factors that give rise to myocardial dysfunction in

sepsis (4).

Downregulation of the "1-adrenoceptor ("1-AR) and adren-

ergic response are considered hallmarks of human heart failure

due to elevated circulating catecholamines in plasma (5). The

two most important mechanisms include the following: (a) G

proteinYcoupled receptor kinase 2 (GRK2) phosphorylating

agonist-bound "-AR leading to "-arrestin recruitment and

uncoupling of the receptor from the Gs protein and (b) the

receptor density that can be decreased due to reduced receptor

mRNA and protein expressions. Consequently, the "-AR down-

stream signaling is inhibited. Evidence has emerged that there

is an increased level of plasma catecholamines, which in-

duces sustained activation of "-AR, resulting in abnormal-

ities in cardiac "-adrenoceptor signaling in sepsis. The possible

mechanisms include down-regulation of "1-AR, decreased ex-

pression of Gs! protein, and depressed postYreceptor signaling

pathways (6).

In recent years, there is growing evidence that statins, the in-

hibitors of hydroxymethylglutaryl coenzyme A reductase (HMG

CoA-reductase), possess some beneficial effects in the preven-

tion of sepsis. These drugs exhibit both immunomodulatory and

anti-inflammatory effects independent of their lipid-lowering ac-

tions (7, 8). Almog and colleagues (9) demonstrated that previous

treatment with statins significantly reduced the rate of severe

sepsis and intensive care unit admissions of patients with acute

bacterial infection. Using mouse model of polymicrobial sepsis,

it was shown that statins completely preserved myocardial

functions and reversed sepsis-induced refractoriness of hemo-

dynamic responses to "1-AR agonist dobutamine in mouse (10).

However, it is not clear how statins would alter cardiac "1-AR

expression/signaling in sepsis. Therefore, the aim of the present

study was to evaluate the effect of atorvastatin (ATR) treatment

Address reprint requests to Santosh Kumar Mishra, PhD, Division of Pharmacology

and Toxicology, Indian Veterinary Research Institute, Izatnagar-243122, Bareilly,

Uttar Pradesh, India. E-mail: smishra@ivri.res.in.

DOI: 10.1097/SHK.0000000000000138

Copyright � 2014 by the Shock Society

406

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on cardiac "1-AR expression and postYreceptor signaling in

cecal ligation and puncture (CLP) model of sepsis in mouse,

which closely resembles natural sepsis in humans.

All three subtypes of "-ARs, "1, "2, and "3, are expressed in

the heart. However, "1-AR subtype is the most prominent one

and is responsible for positive chronotropic and inotropic ef-

fects of endogenous catecholamines. The classic pathway of

"1-AR signaling includes activation of adenylyl cyclase via Gs

protein resulting in increased intracellular adenosine 3¶,5¶-cyclic

monophosphate (cAMP) accumulation; cAMP primarily tar-

gets protein kinase A (PKA), which upon activation phos-

phorylates several proteins such as L-type calcium channels,

phospholamban, and troponin I to increase myocardial con-

tractility. In the present study, we evaluated the effect of ATR

pretreatment on left ventricular functions, myocardial "1-AR

expression, cAMP response, GRK2 protein expression, PKA

activity, and plasma catecholamine level in the mouse model

of CLP-induced sepsis.

MATERIALS AND METHODS

AnimalsHealthy adult male Swiss Albino mice (30Y35 g) were procured from the

Laboratory Animal Resource Section, Indian Veterinary Research Institute,Izatnagar, Uttar Pradesh, India. Mice were housed in different polypropylenecages with free access to feed and water in the divisional animal house. Ani-mals were handled according to the approved protocols of Institutional AnimalEthics Committee of the Indian Veterinary Research Institute, Izatnagar.

ATR treatmentAfter 7-day acclimatization period, mice were divided into four groups: (a)

sham-operated (SO), (b) ATR + SO, (c) CLP, and (d ) ATR + CLP. Atorva-statin was dissolved in ethanol at a concentration of 10 mg/mL and diluted withNaCl 0.9% in a ratio of 1:1,000 to yield a final concentration of 10 Hg/mL ATRcarrier. NaCl 0.9%Ybased carrier solution for the placebo group was preparedaccordingly to include ethanol only (i.e., without statin) at a concentration of1:1,000 (10). Mice were treated with either vehicle or ATR (0.2 Hg/g bodyweight) intraperitoneally at 18 and 3 h before surgery. The dose of ATR wasselected based on the previously published reports (10, 11). The pretreatmentschedule was chosen based on our earlier report that ATR pretreatment, but notthe posttreatment, improved survival in the mouse model of sepsis (11).

Induction of SepsisCecal ligation and puncture was produced as described by Wichterman

et al. (12). Mice were fasted overnight before the induction of sepsis, butallowed water ad libitum. The animals were anesthetized by injection ofxylazine (10 Hg/g body weight intraperitoneal) and ketamine (80 Hg/g bodyweight intraperitoneal), and a 2-cm ventral midline incision was performed.Then the cecum was exposed and ligated with 3Y0 silk just distal to theileocecal valve to avoid intestinal obstruction, punctured once with a 21-gaugeneedle, and returned to the abdomen. The abdominal incision was closedin layers. Normal saline (1 mL) was given subcutaneously to all the mice toprevent dehydration. Sham-operated mice had undergone the same surgicalprocedure except CLP.

Determination of serum lactate levelAfter 20 T 2 h of surgery, blood samples were collected by cardiac puncture

from mice of different groups, and serum was separated by centrifuging at1,717g for 10 min. Serum samples were kept at j80-C until further use. Serumlactate was estimated by enzyme-linked immunosorbent assay kit (Sigma-Aldrich, St. Louis, Mo) as per the manufacturer instruction. Serum lactate anal-ysis was based on the formation of a product, due to oxidation of lactate by lactatedehydrogenase, which interacts with a probe to produce red dye. The intensity ofthe dye was determined at 570 nm, and results were expressed as mmol/L. Briefly,2 HL of serum sample was added to lactate assay buffer in a 96-well plate to which50 HL of master mix was added to a final volume of 100-HL reaction mixture. After30-min incubation at room temperature, the absorbance was measured at 570 nm.The serum lactate concentration (in mmol/L) was determined from the standardcurve (0, 2, 4, 6, 8, and 10 mmol/L) as per the manufacturer guidelines.

Langendorff experimental setupThe preparation of murine hearts and retrograde perfusion were performed

essentially as described by Flogel and coworkers (13). In brief, after 20 T 2 hsurgery, the mice (sham/CLP) were anesthetized with urethane (1.5 g/kg) in-traperitoneally and injected with 250 U of heparin intraperitoneally. Thora-cotomy was performed, and hearts were rapidly excised into ice-cold perfusionfluid. The aorta was cannulated on a shortened and blunted 21-gauge needleand perfusion initiated in a recirculating Langendorff mode at constant pres-sure (100 mmHg) with modified Krebs-Henseleit solution (MKHS) containing(in mmol) NaCl 118, KCl 4.7, MgSO4 1.2, NaHCO3 25, CaCl2 2.3, KH2PO4

1.2, and glucose 11, equilibrated with 95% O2 and 5% CO2 (pH 7.4, 37-C). Afluid-filled balloon catheter was introduced into the left ventricle through anincision in the atrial appendage. The ventricular balloon was connected viafluid-filled tubing to a pressure transducer (MLT844; AD Instruments, CastleHill, Mew South Wales, Australia) for continuous assessment of ventricularperformance and was inflated to yield a left ventricular end-diastolic pressureof 5 mmHg during the initial 30 min of stabilization, after which it was notadjusted further. Hearts were immersed in warmed perfusate in a jacketed bathmaintained at 37-C, and perfusate delivered to the coronary circulation wasmaintained at the same temperature. Organ bath and perfusate temperatureswere continuously maintained using a thermostat. The left ventricular pressureof different groups was measured continuously at the baseline (14). The leftventricular pressure signals were recorded using Power Lab data acquisitionsystem (ML866; AD Instruments). Left ventricular pressure was digitallyprocessed to yield left ventricular developed pressure (LVDP), and the peakpositive and negative differentials of pressure change with time dp/dtmax anddp/dtmin, respectively.

Protein expression studyCardiac membrane preparation—Cardiac membrane was prepared from

the whole heart as described previously with some modifications (15, 16).Tissues were collected after 20 T 2 h of surgery (sham/sepsis) and washed withice-cold phosphate-buffered saline (PBS; pH 7.4). Tissue was homogenizedusing a homogenizer in ice-cold Tris-HCl buffer (10 mM Tris-HCl, pH 8.0;100 mM phenylmethylsulfonyl fluoride [PMSF]; 10 HL of 100� protease in-hibitor cocktail). The homogenate was incubated on ice for 20 min in thepresence of 1 mM KCl to dissolve the myofilament proteins. The homogenatewas filtered through layers of cheese cloth, and filtrate was centrifuged at45,000g, 4-C for 20 min. The pellet was resuspended in Tris-HCl buffer, ho-mogenized, and resedimented by the same speed of centrifugation. The finalpellet was dissolved in ice-cold detergent buffer (100 mM Tris-HCl, pH 7.5;100 mM EDTA, pH 7.5; 150 mM NaCl; 100 mM PMSF; 1% Triton X-100;10 HL of 100� protease inhibitor cocktail). The cell membrane homogenateswere aliquoted to 100 HL and stored at j80-C until use. Protein concentrationwas determined by Bradford protein assay kit method.

Cardiac cytosolic fraction preparationMyocardial cytosolic fractions were prepared by homogenization of excised

heart in ice-cold lysis buffer (100 mM Tris-HCl, pH 7.5; 100 mM EDTA,pH 7.5; 100 mM PMSF; 1% Triton X-100; 10 HL of 100� protease inhibitorcocktail) and centrifuged at 48,000g for 30 min (17). The supernatant, whichcontained soluble kinases, was collected. The cytosolic fraction was aliquotedto 100 HL and stored at j80-C until use. Protein concentration was determinedby Bradford protein assay kit method.

Western blot analysisAliquots of the samples (60 Hg of cell membrane protein for "1-AR and

80 Hg of cytosolic protein for GRK2) were diluted in loading buffer and de-natured for 3 min at 100-C in boiling water bath. Samples were separated bygradient sodium dodecyl sulfateYpolyacrylamide gel electrophoresis using aGeiNei, high power, DC power supply system. Proteins were electrophoreti-cally transferred onto polyvinylidene difluoride membrane using GeNei,Electro Transfer Midi Dual- 08-04 system (2 h at 100 V in 25 mM Tris, 192 mMglycine, and 20% methanol). After transfer, the membrane was washed four timesfor 20-min duration with PBSYTween-20 (PBS-T containing PBS, pH 7.4, and0.05% Tween-20 vol/vol) and blocked for 2 h at room temperature inblocking buffer containing 5% skimmed milk powder in PBS (wt/vol). Blots werethen incubated for overnight at 4-C with primary antibody in PBS against"1-AR (NB100-92439; Novus Biologicals, Littleton, Colo) at 1:500 dilution,GRK2 (SC-562; Santa Cruz Biotechnology, Santa Cruz, Calif) at 1:200 dilutionand GAPDH (SC-25778; Santa Cruz Biotechnology) at 1:200 dilutions in PBS-T.The primary antibody was removed, and blot was washed four times for 20-minduration with PBS-T. Blots were then incubated for 2 h at room temperature withthe goat antiYrabbit secondary antibody coupled to horseradish peroxidase (SC-2004; Santa Cruz Biotechnology) at 1:200 dilutions in PBS-T. Following removalof the secondary antibody, membrane was washed four times for 20-min duration

SHOCK MAY 2014 STATIN & "1-ADRENOCEPTOR SIGNALING IN SEPSIS 407

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with PBS-T and developed using DAB system (GeNei; Merck, Mumbai, India).Densitometric analysis was conducted for the protein bands from immunoblot-ting to compare the relative amounts of particular protein under different treat-ments by using Image J software (NIH, Bethesda, Md).

mRNA expression studyTotal RNA isolation—Tissues were collected after 20 T 2 h of surgery (sham/

sepsis). The heart was isolated in 1% diethyl pyrocarbonateYtreated autoclavedPBS. It was cleaned of surrounding adipose tissue and stored in RNA later atj20-C. Total RNA was isolated with Mini RNA isolation kit as per themanufacturer’s instructions. The samples were treated with RNase free-DNase,and the DNase was subsequently inactivated by heating at 56-C for 10 min andimmediately chilled to 4-C. The purity of the RNA was checked by A260/A280

ratio and quantified as follows 1 OD = 40 Hg/mL.

Quantitative real-time polymerase chain reactionReal-time polymerase chain reaction (PCR) was conducted using SYBR

Green I master mix (Maxima SYBR Green quantitative PCR master mix [2�];Thermo Scientific, Waltham, Mass). Each sample was run in duplicate in a20-HL reaction. The 20-HL reaction mixture consisted of 10 HL SYBR Greenmaster mix, 0.4 HL ROX Low, 1.0 HL from 10 pmol/HL stock solution of eachof the gene-specific forward and reverse primers, and 1 HL of cDNA, and volumewas made up to 20 HL with RNAse-free water. The real-time PCR reaction startedwith initial incubation at 95-C for 10 min followed by 40 cycles of amplificationwith denaturation at 95-C for 30 s, annealing ("1-AR at 62-C; GAPDH at 62-C)for 30 s, and extension at 72-C for 30 s each. The optimum annealing temper-atures determined by PCR for the respective gene using the specific primers wereas follows: for the "1-AR gene: forward 5¶-ACGCTCACCAACCTCTTCAT-3¶,reverse 5¶-AGGGGCACGTAGAAGGAGAC-3¶ (440 base pairs) at 62-C;for the GAPDH gene: forward 5¶-AACTTTGGCATTGTGGAAGG-3¶, reverse5¶-ACACATTGGGGGTAGGAACA-3¶ (223 base pairs) at 57-C.

To assess the specificity of the amplified product, a dissociation curve wasgenerated at temperatures of 55-C through 95-C. The results were expressed asthreshold cycle values (CT). This value is the cycle number when the fluo-rescence of the reporter dye is appreciably higher than the background fluo-rescence. The threshold, automatically adjusted by the instrument, was usedfor the generation of CT values.

cAMP measurementFor cAMP measurement, tissues were collected 20 T 2 h after surgery

(SO/sepsis). Heart pieces (n = 5 for each group), equilibrated in MKHS for30 min at 37-C under continuous bubbling with medical gas, were exposed to100 HM 3,7-dihydro-1-methyl-3-(2-methylpropyl)-1H-purine-2,6-dione (IBMX)for the next 30 min, and quickly snap frozen in liquid nitrogen. Both basaland dobutamine (1 HM; 2 min)Ystimulated tissue cAMP levels were determined(18, 19). Liquid nitrogen snap frozen heart tissues were homogenized in 1 mLchilled trichloroacetic acid (6%) and centrifuged at 1,500g for 10 min. Super-natant, thus obtained, was extracted for five times with water-saturated diethylether, and residual ether was removed from aqueous layer by heating the sampleat 70-C for 5 to 10 min. The neutralized supernatant was then used for cAMPassay using enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor,Mich) following the manufacturer’s instructions. Tissue pellets obtained aftercentrifugation were dissolved in 1 N NaOH for protein estimation by Lowrymethod (20). Concentrations of cAMP were expressed as pmol/mg protein.

PKA activity assayFor PKA activity measurement, tissues were collected 20 T 2 h after surgery

(SO/sepsis). Heart pieces (n = 5 for each groups), equilibrated in MKHS for30 min at 37-C under continuous bubbling with medical gas, were exposed to100 HM IBMX for the next 30 min and quickly snap frozen in liquid nitrogen.The basal PKA activity was determined. Liquid nitrogen snap-frozen hearttissues were weighed, and 10% homogenate prepared in activated cell lysisbuffer and centrifuged at 9,519g for 10 min. The supernatant was carefullyaspirated and stored at j80-C for analysis later. The supernatant was then usedfor PKA activity assay using EIA kit (Arbor Assays, Ann Arbor, Mich) fol-lowing the manufacturer’s instructions. The protein was estimated by Lowrymethod (20). Protein kinase A activity was expressed as U/mg protein.

Determination of plasma catecholamine levelsAfter 20 T 2-h surgery, the mice (sham/sepsis) were anaesthetized with

urethane (1.5 g/kg) intraperitoneally, and blood was collected in EDTA-containing tubes and centrifuged at 1,000g for 15 min at 4-C. The plasmasamples were separated and immediately aliquoted and stored at j80-C for cate-cholamine measurement using mouse catecholamine EIA kit (BlueGene Biotech,Shanghai, China). Concentration of catecholamines was expressed in pg/mL.

Drugs and chemicalsDobutamine and IBMX were purchased from Sigma Chemical (St Louis,

Mo). IBMX was dissolved in dimethylsulfoxide. All other drugs were dissolvedin distilled water. Atorvastatin was dissolved in ethanol at a concentration of10 mg/ mL and diluted with NaCl 0.9% in a ratio of 1:1,000 to yield a finalconcentration of 10 Hg ATR per mL carrier.

Statistical analysisResults are expressed as mean T SEM. Differences in repeated mea-

surements were analyzed by two-way analysis of variance (ANOVA) followedby multiple comparisons with Bonferroni post hoc test. Otherwise, one-wayANOVA followed by Newman-Keuls post hoc analysis was applied (GraphPadPrism 6 GraphPad Software, La Jolla, Calif). To study the relative changein gene expression, the 2j$$C

T method was used as described previouslyby Livak and Schmittgen (21). The formula used to calculate the foldchange in gene expression was Bfold change = 2j$$C

T,[ where $$CT =(CT,target gene j CT,GAPDH) treatment j (CT,target gene j CT,GAPDH) control.The gene-specific amplification was corrected for the difference in inputof RNA by taking housekeeping gene GAPDH to account. For CLP andATR + CLP groups, evaluation of 2j$$C

T indicates the fold change in geneexpression relative to SO control (i.e., fold change in sham control = 1). Theresults were analyzed in comparison with the CT (minimum threshold ofamplification) value of the target gene and the reference gene (GAPDH).

RESULTS

Determination of serum lactate level

Serum lactate was measured as a marker of sepsis taking blood

from different groups of mice at 20 h after surgery. In CLP mice,

the serum lactate level (CLP 4.51 T 0.53 mmol/L [n = 6] vs. SO

1.33 T 0.28 mmol/L [n = 5]) was significantly (P G 0.05) higher

than that of the SO animals. Atorvastatin pretreatment signifi-

cantly (P G 0.05) prevented rise in serum lactate level (ATR +

CLP 1.80 T 0.43 mmol/L [n = 6] vs. CLP 4.51 T 0.53 mmol/L

[n = 6]) in CLP mice. Thus, there was no significant difference

in the serum lactate levels of ATR-treated CLP mice in com-

parison to SO mice. We, however, observed that ATR treatment

significantly (P G 0.05) increased serum lactate (ATR + SO 3.17 T0.31 mmol/L [n = 5] vs. SO 1.33 T 0.28 mmol/L [n = 5]) level in

SO animals.

Left ventricular functions in mouse isolated heart

Figure 1 illustrates the effect of ATR pretreatment on CLP-

induced cardiac dysfunctions in isolated mouse heart obtained

at 20 T 2 h after surgery. As shown in Figure 1A, the basal

LVDP was 115.50 T 1.30 mmHg (n = 6) in SO mice. Atorva-

statin treatment had no significant effect on LVDP (109.86 T1.80 mmHg; n = 6) in SO mice. In comparison to SO con-

trol, CLP significantly (P G 0.001) decreased the LVDP (75.90 T0.53 mmHg; n = 6). However, ATR treatment of CLP mice

markedly prevented the decrease in the LVDP (100.24 T1.64 mmHg; n = 6). The results are summarized in Figure 1A.

Figure 1B depicts the effect of ATR treatment on the levels

of dp/dtmax in SO and CLP mice. The basal dp/dtmax in SO mice

and that in ATR plus SO mice were 4,529 T 42 mmHg and

4,490 T 118 mmHg (n = 6), respectively, which were not sta-

tistically different. In CLP mice, however, there was a signifi-

cant (P G 0.001) decrease in the dp/dtmax (3,742 T 71 mmHg).

Atorvastatin treatment of CLP mice preserved dp/dtmax (4,291 T89 mmHg) to SO control values.

We also evaluated the effect of ATR treatment on the diastolic

function of SO and CLP mice. The basal values of dp/dtmin in

SO (j1,518 T 121 mmHg) and ATR plus SO animals (j1,579 T68 mmHg; n = 6) were not statistically significant. However,

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CLP significantly (P G 0.001) decreased the dp/dtmin (j1,010 T24 mmHg) in comparison to SO controls. In CLP mice treated

with ATR, the dp/dtmin (j1,346 T 84 mmHg) was comparable

to SO control. The results are shown in Figure 1C.

"1-AR protein and mRNA expression in mouse heart

Figure 2A depicts the "1-AR protein expression in the cardiac

tissues taken from SO, ATR plus SO, CLP, and ATR-treated

CLP mice. The relative unit of protein expression in SO animals

was set to 1.0 (n = 4). The alteration in the protein expression in

other groups has been expressed as arbitrary units accordingly.

Cecal ligation and puncture markedly decreased the cardiac

"1-AR protein expression (0.48 T 0.09; n= 4). However, the

level of "1-AR protein expression in ATR-treated CLP mice

(0.96 T 0.11; n = 4) was comparable to that of SO animals

(1.04 T 0.11; n = 4). Furthermore, ATR treatment of SO animals

had no significant effect on "1-AR protein expression when

compared with untreated SO animals.

We then determined the influence of sepsis and treatment

with ATR on "1-AR mRNA expressions in the mouse heart.

Heart tissue from different groups, such as SO, ATR + SO,

CLP, and ATR + CLP, was obtained 20 T 2 h after surgery.

The cardiac "1-AR mRNA level in the SO animals was set as

1.00 (n = 4). Thus, the fold changes in the mRNA expression in

other groups were expressed, accordingly. Cecal ligation and

puncture caused a profound decrease in the level of "1-AR

mRNA expression (0.38 T 0.07; n = 4). Atorvastatin treatment

preserved the mRNA level (0.88 T 0.15; n = 4) nearly to the

level observed in SO mice. Atorvastatin treatment of SO mice

had no significant effect on "1-AR mRNA expression (0.84 T0.20 fold; n = 4). The results of mRNA expression are shown

in Figure 2B.

FIG. 1. Left ventricular functions measured in vitro. A, Left ventriculardeveloped pressure was decreased (*P G 0.05, n = 6) in CLP mice as com-pared with SO. Atorvastatin treatment of CLP mice improved the LVDP (#P G0.05, n = 6). B, dP/dtmax was decreased in CLP as compared with SO mice(*P G 0.001, n = 6). Atorvastatin prevented the decrease in dP/dtmax (#P G0.05 n = 6) in CLP mice. C, dP/dt min was decreased in CLP as compared withSO mice (*P G 0.001, n = 6). Atorvastatin prevented the decrease in dP/dt min

in CLP mice (#P G 0.05, n = 6). Results are expressed as mean T SEM. Datawere analyzed using one-way ANOVA followed by multiple comparisons withNewman-Keuls post hoc test.

FIG. 2. "1-Adrenoceptor protein and mRNA expressions. A, Immuno-blots depict "1-AR protein expression on the plasma membrane of SO, ATR +SO, CLP, and ATR + CLP groups. Upper and middle panels show the rep-resentative blots of "1-AR and GAPDH, respectively. Lower panel depictsrelative expression of "1-AR. "1-Adrenoceptor protein expression was signif-icantly (*P G 0.01) decreased in CLP animals as compared with SO. However,protein expression was significantly (#P G 0.01) increased in ATR-pretreatedCLP mice versus untreated CLP mice (n = 4 for each group). The ratio of"1-AR/GAPDH was set as 1 for the SO group, and the relative density ofother groups were calculated, accordingly B, Relative mRNA expressionshowing significant (*P G 0.05) decrease of "1-AR mRNA expression in CLPversus SO. mRNA expression was significantly (#P G 0.05) decreased in CLPversus ATR-treated CLPmice (n = 4 for each group). Results are expressed asmean T SEM. Data were analyzed using one-way ANOVA followed byNewman-Keuls post hoc test.

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Basal and dobutamine-stimulated cAMP determination inheart tissue

Figure 3 shows the basal and dobutamine (1 HM)Ystimulated

tissue cAMP contents in mouse heart obtained 20 T 2 h after

surgery. In SO mice, the basal cAMP content was 6.30 T1.14 pmol/mg protein (n = 5), which was significantly (P G 0.01)

decreased (1.30 T 0.27 pmol/mg protein; n = 5) in CLP mice.

Atorvastatin treatment of CLP mice preserved the basal cAMP

level (6.30 T 0.67 pmol/mg protein; n = 5) comparable to SO

control. Atorvastatin treatment of SO mice had no significant

effect on the basal tissue cAMP content (6.21 T 0.60 pmol/mg

protein; n = 5) in comparison to SO animals. We then evalu-

ated the effect of "1-AR agonist dobutamine (1 HM) on cAMP

responses in different groups. There was a marked increase in

tissue cAMP content to dobutamine stimulation in SO (11.90 T2.0 pmol/mg protein; n = 5), ATR plus SO (12.02 T 1.71 pmol/mg

protein), and ATR-treated CLP (10.83 T 1.37 pmol/mg protein;

n = 5) mice in comparison to their respective basal values.

However, the heart tissues, taken from CLP mice, were refrac-

tory to change in tissue cAMP level (1.72 T 0.27 pmol/mg pro-

tein; n = 5) in response to dobutamine stimulation.

GRK2 protein expression and PKA activity in heart

Figure 4A depicts the influence of ATR treatment on the

GRK2 protein expression in the mouse heart obtained from

SO, ATR-treated SO, CLP, and ATR-treated CLP mice. The

relative unit of protein expression in SO animals was set to 1.0

(n = 4). Atorvastatin significantly (P G 0.05) attenuated (1.10 T0.11; n = 4) CLP-induced marked increase (1.73 T 0.18; P G0.05; n = 4) in GRK2 expression in the heart tissue. The statin

treatment, however, had no significant effect on GRK2 ex-

pression (1.10 T 0.18; n = 4) in SO animals.

Figure 4B illustrates the basal PKA activity in the mouse

isolated heart. The basal PKA activity in the heart tissue from

SO animals was 0.41 T 0.1 U/mg protein (n = 5). It was sig-

nificantly elevated in CLP (1.12 T 0.14 U/mg protein; P G0.001; n = 5) mice. Atorvastatin treatment of CLP mice,

however, attenuated the PKA activity (0.66 T 0.08 U/mg pro-

tein; P G 0.05; n = 5). Atorvastatin treatment had no significant

effect on basal PKA activity in SO mice (0.40 T 0.10 U/mg

protein; n = 5).

Determination of plasma catecholamine levels

Figure 5 shows the effect of ATR on plasma catecholamine

levels (norepinephrine and epinephrine) in sepsis. In comparison

to the plasma catecholamine levels in SO animals (74 T 7 pg/mL;

n = 6), there was a significant (P G 0.01) increase in its level in

CLP mice (138 T 22 pg/mL; n = 6). Atorvastatin treatment sig-

nificantly (P G 0.01) prevented the rise in plasma catecholamine

levels in CLP animals (59 T 2 pg/mL; n = 5). Atorvastatin

treatment, however, had no significant effect on the plasma cat-

echolamine levels of SO animals (67 T 3 pg/mL; n = 5).

DISCUSSION

Both in patients with septic shock and animal models of

sepsis, impaired "-AR stimulation of cAMP is accompanied by

hyporesponsiveness to catecholamines and reduced cardiac

contractility. We found that CLP significantly decreased the

left ventricular contraction (dp/dtmax) and relaxation (dp/dtmin)

in the hypodynamic phase of sepsis in mice. Atorvastatin pre-

treatment completely preserved the left ventricular functions

in CLP mice. This observation is in agreement with a previous

report that showed that another lipophilic statin simvastatin

prevented sepsis-induced impairment in myocardial contractility

and cardiac output in the mouse model of sepsis (10).

We further studied the role of ATR-induced alterations in

the myocardial "1-AR signaling pathway to explain the mech-

anisms of the preservation of cardiac functions by the statin in

CLP mice. This is, to our knowledge, the first study to dem-

onstrate that a statin such as ATR can preserve myocardial

"1-AR signaling in sepsis. We found that ATR prevented sepsis-

induced decrease in "1-AR protein expression, which was

paralleled by a reduction in "1-AR mRNA level. The parallel

behavior of the receptor mRNA level and receptor expression

suggests that decreased mRNA level may be responsible for

the downregulation of "1-AR in sepsis. This observation is

consistent with the concept of agonist-dependent process lead-

ing to decreased mRNA level (22). Although there is limited

information on the influence of sepsis on cardiac "1-AR mRNA

levels, reduction in the "1-AR transcript in human heart fail-

ure has been reported (23, 24). During sepsis, various studies

have demonstrated elevated catecholamine levels in patients

(25) and animals (26). We also observed increased plasma

catecholamine levels in CLP mice. It is therefore possible that

excess stimulation of myocardial "1-AR by catecholamines

resulted in decreased receptor mRNA expression, followed by

downregulation of the receptors. According to our study, ATR

completely prevented increase in plasma catecholamine level

in CLP mice. This observation provides an explanation how

the statin might attenuate "1-AR downregulation in sepsis. Our

study is consistent with a previous study that demonstrated that

FIG. 3. Basal and dobutamine-stimulated cAMP content in mice heartfrom SO, ATR + SO, CLP, and ATR + CLP. The hearts were challenged with1 HM of dobutamine for 1 min, and the cAMP content was measured. A, Thebasal cAMP level was decreased ((P G 0.01, n = 5) in CLP mice as comparedwith SO, but ATR prevented (P G 0.05, n = 5) the decrease in cAMP content insepsis. In comparison to the basal level of cAMP, dobutamine significantlyincreased the tissue cAMP level in all the groups (SO, ATR + SO, and ATR +CLP) except CLP mice. Results are expressed as mean T SEM. Data wereanalyzed using two-way ANOVA followed by multiple comparisons withBonferroni post hoc test. *P G 0.05 CLP versus SO; #P G 0.05 ATR + CLPversus CLP, aP G 0.001 (SO), bP G 0.01 (ATR + SO), cP G 0.05 (ATR + CLP),n = 5 in each group.

410 SHOCK VOL. 41, NO. 5 THANGAMALAI ET AL.

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another statin simvastatin decreased central sympathetic out-

flow, indicated by reduced plasma norepinephrine level in

the rabbit model of congestive heart failure (27). It is believed

that statins reduce adrenergic outflow by attenuating oxidative

stress in central brain regions involved in sympathetic modula-

tion (28). Further studies are, however, required to delineate

the mechanism of statin-induced decrease in plasma catechol-

amines in sepsis.

Stimulation of "1-AR induces downstream signals such as

cAMP and PKA that are able to promote PKA phosphorylation

of phospholamban and troponin I leading to myocardial con-

traction (29). In accordance with decreased expression "1-AR,

we found that sepsis profoundly decreased basal cAMP accu-

mulation in the heart tissue. Notably, the cAMP response was

responsive to dobutamine stimulation in SO and ATR-treated

CLP mice, but not in the untreated CLP animals. Atorvastatin,

however, not only preserved the basal cAMP level but also

restored its sensitivity to dobutamine stimulation. These find-

ings are consistent with the functional studies done earlier,

which demonstrated that refractoriness of cardiac output and

blood pressure to dobutamine in CLP mice was restored by

statins (10).

Receptor desensitization is a common phenomenon of re-

fractoriness to agonists. Several studies show that desensiti-

zation of the myocardial "-AR can be caused by the two

kinases, such as GRK2 and PKA, which are known to phos-

phorylate the receptor protein. G proteinYcoupled receptor ki-

nase 2 mediates homologous desensitization through "-AR

uncoupling from Gs protein in response to high concentrations

of agonists. According to a recent report, GRK2-mediated

desensitization/downregulation of "1-AR was found to de-

crease inotropic response of dobutamine in a rat model of right

ventricular hypertrophy associated with pulmonary hyperten-

sion (30). We found that GRK2 protein is overexpressed in the

heart from untreated CLP mice, but it was at the control level

in ATR-treated CLP mice. This observation suggests that

GRK2 may play a critical role in "1-AR desensitization in

sepsis, and ATR can prevent the desensitization phenomenon

by attenuating the GRK2 protein expression in CLP mice. In

contrast to a marked decline in the cardiac tissue levels of

cAMP in sepsis, we observed that basal PKA activity was

significantly increased. In accordance with our findings, the

cAMP-independent increase in PKA activity in the rat heart

during hypodynamic phase of sepsis has been reported earlier

by other workers (31). Although the mechanism of cAMP-

independent elevation of PKA activity in sepsis is not clear,

peroxynitrite has been shown to directly activate PKA inde-

pendent of cAMP in cardiomyocytes (32). A previous study

has demonstrated an increase in the myocardial peroxynitrite

level in the CLP model of sepsis in mice (33). Therefore, it is

reasonable to believe that a rise in PKA level in sepsis may be

related to tissue peroxynitrite levels. An acute increase in

PKA activity is known to increase myocardial contraction

through phosphorylation of different target proteins involved

in Ca2+ homeostasis and contractile mechanisms. However, a

sustained increase in PKA activity may lead to "-AR desen-

sitization and hence depressing myocardial contractility in

sepsis (31). We found that ATR markedly attenuated increase

FIG. 4. A, G proteinYcoupled receptor kinase 2 protein expression.Upper and middle panels show the representative blots of GRK2 and GAPDH.Lower panel illustrates relative expression of GRK2. G proteinYcoupled receptorkinase 2 protein expression was significantly (#P G 0.05) increased in untreatedCLP versus ATR-pretreated CLP mice (ATR + CLP vs. CLP). In comparison toSO, CLP significantly (*P G 0.05) increased the GRK2 protein expression. n = 4for each group. The ratio of GRK2/GAPDH was set as 1 for the SO group, andthe relative density of other groups was calculated, accordingly B, Basal PKAactivity was significantly increased (*P G 0.05) in CLP mice in comparison toSO. Atorvastatin significantly (#P G 0.05) attenuated CLP-induced increase inthe PKA activity. Results are expressed as mean T SEM. Data were analyzedusing one-way ANOVA followed by Newman-Keuls post hoc test.

FIG. 5. Plasma catecholamine level in mice. In comparison to SO, CLPsignificantly (*P G 0.01; n = 6) increased plasma catecholamine level. However,in CLPmice treated with ATR, there was no increase in plasma catecholamines(#P G 0.01; n = 6). Results are expressed as mean T SEM. Data were analyzedusing one-way ANOVA followed by Newman-Keuls post hoc test.

SHOCK MAY 2014 STATIN & "1-ADRENOCEPTOR SIGNALING IN SEPSIS 411

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in the basal PKA activity in CLP mice. The results of "1-AR

protein expression, together with GRK2 protein expression

and PKA activity suggest that ATR could preserve both basal

cAMP and its response to dobutamine stimulation through at-

tenuation of "1-AR downregulation/desensitization. These bio-

chemical changes of "1-AR signaling very well corroborate

with ATR-mediated cardioprotection in sepsis.

One of the key limitations of the present study is that CLP

mice were pretreated with ATR to assess its effect on sepsis-

induced myocardial dysfunctions and the mechanisms in-

volved in the preservation of cardiac functions. Therefore, it

yields little information on its therapeutic benefit after induc-

tion of sepsis. Nevertheless, evidence from observational studies

and basic science research suggests that statins might be asso-

ciated with a reduced mortality in sepsis. For instance, according

to a recent report, prior ATR use was associated with lower level

of basal IL-6 and improved survival in patients of sepsis (34).

According to some unpublished data from our laboratory, we

observed that posttreatment of mice with a combination of ATR

and imipenem significantly increased the survival time. Further

studies are, however, required to elucidate the mechanisms,

including hemodynamic functions involved in the survival ben-

efit offered by the posttreatment with the statin in combination

with an antibiotic in sepsis.

In conclusion, the results of the present study suggest that

ATR pretreatment can improve left ventricular systolic and

diastolic functions in vitro through the preservation of "1-AR

signaling in the mouse model of sepsis.

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