Dysfunction of cardiac ryanodine receptors in the metabolic syndrome

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a Department of Physiology, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112, USA b Department of Pharmacology ...
Journal of Molecular and Cellular Cardiology 41 (2006) 108 – 114 www.elsevier.com/locate/yjmcc

Original article

Dysfunction of cardiac ryanodine receptors in the metabolic syndrome U. Deniz Dincer a,⁎, Alberto Araiza a , Jarrod D. Knudson a , Chun Hong Shao b , Keshore R. Bidasee b , Johnathan D. Tune a a

Department of Physiology, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112, USA b Department of Pharmacology, University of Nebraska Medical Center, Omaha, NE 68198-6260, USA Received 14 December 2005; received in revised form 20 April 2006; accepted 25 April 2006

Abstract This study examined the hypothesis that the prediabetic metabolic syndrome alters expression, phosphorylation state and binding affinity of cardiac RyR2. Real-time PCR and Western blot analysis were used to assess mRNA and protein expression in the left ventricle, right ventricle and right atrium from control (n = 5) and chronically high-fat-fed (n = 5) dogs with the metabolic syndrome. Functional integrity of RyR2 was assessed by RyR2-Ser2809 phosphorylation and the receptor's ability to bind [3H]ryanodine. We found that RyR2 phosphorylation at Ser2809 was significantly elevated in right and left ventricle from high-fat-fed dogs compared to normal control dogs. This hyperphosphorylation was associated with a decrease in RyR2 binding affinity in right and left ventricle (high-fat diet = 80.2 and 90.5 fmol/mg protein vs. control = 243.6 and 200.9 fmol/mg protein, respectively) and a decrease in cardiac index in exercising dogs. RyR2 phosphorylation at Ser2809 and RyR2 binding affinity were not altered in the right atria of high-fat-fed dogs. In addition, no significant differences in cardiac RyR2 mRNA or protein expression were noted between groups. These data suggest that alterations in RyR2 could be an important mechanism of early cardiac dysfunction in obesity and insulin resistance. © 2006 Elsevier Inc. All rights reserved. Keywords: Ryanodine receptor; Heart; Prediabetic metabolic syndrome; RyR2-Ser2809 phosphorylation; “Leaky” RyR2

The metabolic syndrome is known by a variety of names, including syndrome X, dysmetabolic syndrome and insulin resistance syndrome [1,2]. In the late 1980s, Reaven characterized a multifactorial syndrome that consists of a combination of obesity, hypertension, insulin resistance, dyslipidemia and impaired glucose metabolism [2,3]. Importantly, patients with this prediabetic syndrome have significantly elevated rates of cardiovascularrelated morbidity and mortality [4–6]. However, the mechanisms underlying obesity-related cardiovascular disease have not been fully elucidated. Obesity and the metabolic syndrome can result in a variety of cardiac and hemodynamic alterations that ultimately result in development of congestive heart failure [7–10]. This obesityinduced cardiomyopathy typically occurs in patients with severe

⁎ Corresponding author. Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr. Indianapolis, IN 46202, USA. Tel.: +1 317 274 4466; fax: +1 317 274 3318. E-mail address: [email protected] (U.D. Dincer). 0022-2828/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2006.04.018

or long-term obesity; however, our laboratory as well as others has observed alterations in cardiac function and control of blood pressure and heart rate following only a few weeks of high-fat feeding in animals [11–14]. The mechanisms responsible for this dysfunction are unclear but could be related to alterations in type 2 ryanodine receptors (RyR2), i.e. sarcoplasmic reticular (SR) Ca++ release channels. RyR2 is a key component of cardiac excitation–contraction coupling and may play a role in the pathogenesis of human cardiac disease [15,16]. In particular, recent experimental evidence indicates that hyperphosphorylation of RyR2 contributes to impaired contraction, generation of ventricular arrhythmias and the development of heart failure [16]. In many cases, alterations in intracellular Ca++ cycling precede overt depression of myocardial contractile performance [15]; therefore, determining the effects of the prediabetic metabolic syndrome on cardiac RyR2/SR Ca++ release channels holds the potential for novel therapeutic strategies against obesity-induced cardiomyopathy. The purpose of the study was to examine the hypothesis that the prediabetic metabolic syndrome alters expression, phosphorylation

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state and binding affinity of cardiac RyR2. Real-time PCR and Western blot analysis were used to assess mRNA and protein expression in the left ventricle, right ventricle and right atrium from control (n = 5) and chronically high-fat-fed (n = 5) dogs with the metabolic syndrome [14,17,18]. The functional integrity of RyR2 was assessed by RyR2-Ser2809 phosphorylation and the receptor's ability to bind [3H]ryanodine. 1. Materials and methods 1.1. Induction and validation of the high-fat diet This investigation was approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85–23, Revised 1996). Animals of either sex were fed either normal dog chow (Teklad, ∼13% calories from fat; n = 5) or a high-fat diet that provided ∼60% of calories from fat (n = 5). The high-fat diet was administered in the morning and afternoon for ∼6 weeks [14,17,18]. The morning feeding consisted of a homogenous

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mixture of canned dog food (748 g, Alpo), dry dog food (227 g, Teklad) and lard (57 g, Morrell). The afternoon feeding consisted of a homogenous mixture of canned dog food (748 g, Alpo), lard (454 g, Morrell) and chicken or beef baby food (71 g). Following ∼6 weeks administration of the abovementioned dietary protocols, animals were euthanized with sodium pentabarbitol (30 mg/kg, iv). Cardiac tissues were harvested, flash frozen in liquid nitrogen and stored at −80 °C until molecular and functional analyses were performed. 1.2. Preparation of first-strand cDNA via reverse transcriptase reactions RNA samples were used as templates for synthesis of firststrand cDNAs as described previously [19]. Briefly, 1 μl of oligo (dT)15 primer (Promega) was added to equivalent amounts of total RNA from right atria and left and right ventricles obtained from control (n = 5) and chronically high-fat-fed (n = 5) dogs. The mixtures were placed into a thermocycler (My Cycler, Bio-Rad) and held at 70 °C for 5 min. The samples were then transferred into an ice bath for 5 min to permit selective binding of the oligo(dT)15

Fig. 1. Real-time PCR reactions performed for RyR2 and β-actin expressions in duplicate with SYBR green. The PCR Amp/Cycle Graph and melt curve for RyR2 and β-actin are shown in panels A and B, respectively. No significant differences in RyR2 mRNA expression were detected between normal control and chronically highfat-fed dog right atria, right ventricle or left ventricle (C).

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to the poly(A) tail of the mRNA. First-strand cDNA was then synthesized with an ImProm-II Reverse Transcriptase kit (Promega). 1.3. Amplification of cDNA Real-time PCR reactions were performed for RyR2 and βactin in duplicate with a custom designed SYBR Green mix [2.4 μl of 25 mM MgCl2, 5 μl of 1:10 000 dilution SYBR Green I (Molecular Probes), 5 μl of 1 nM/l Fluorescein Calibration Dye (1 mM/l in DMSO, Bio-Rad) for 50 μl of total reaction using Taq DNA polymerase (Promega)] [17]. Primers were designed based on published sequences in the National Center for Biotechnology Information GenBank database http://www3.ncbi.nlm.nih.gov/entrez/ [RyR2 receptor, sense 115ATATGCTTTGGGAACCAGCA134 and antisense 321GGQ GCAAACATCTTCCACATT302 (207 bp) (accession number AF440217); β-actin, sense 21GACATCCGCAAGGACCTQ CTA40 and antisense 176CACAGAGTACTTGCGCT CAG157 (accession number U67202)]. Amplification was carried out with iCyclerÌQ Multicolor real-time PCR detection system (Bio-Rad) as follows: 45-s denaturation (95 °C) followed by 45-s annealing and 1-min extension (72 °C), repeated for a total of 33 cycles. β-actin was amplified in each set of PCR reactions and served as an internal reference during quantitation to correct for operator and/or experimental variations. The data were analyzed in duplicate using the 2−ΔΔCT equation [20]. DDCT ¼ ðCTRyR2  CTbactin Þ

ryanodine [21,22]. For these experiments, 100 μg/ml of membrane vesicle protein was incubated in binding buffer containing 200 μmol/l free calcium (500 mmol/l KCl, 20 mmol/l Tris–HCl, 300 μmol/l CaCl2, 0.1 mmol/l EGTA, 6.7 nmol/l [3H]ryanodine, pH 7.4) for 2 h at 37 °C. After incubation, vesicles were filtered and washed, and the amount of [3H]ryanodine bound to RyR2 was determined by liquid scintillation counting. Nonspecific binding was determined simultaneously by incubating vesicles with 1 μmol/l unlabeled ryanodine. 1.7. Surgical instrumentation and exercise protocol To further assess the effects of our high-fat feeding protocol on cardiac function, we pooled data from our recent publications (to increase the sample size) [14,18]. The surgical procedures and exercise protocol performed were previously described by our laboratory [14,18]. Briefly, catheters were placed in the aorta to measure blood pressure and to obtain arterial blood samples, and coronary sinus for coronary venous blood sampling. Flow transducers (Transonic Systems) were also placed around the circumflex coronary artery and aorta to measure coronary blood flow and cardiac output (minus coronary flow), respectively. Coronary blood flow, aortic pressure, cardiac output and heart rate were continuously measured while the dogs were resting in a sling and then during three levels of treadmill exercise: (1) 2 mph, 0% grade; (2) 3 mph, 5% grade; (3) 4 mph, 10% grade. Data were collected when hemodynamic variables were stable at each exercise level [14,18]. 1.8. Statistical analyses

high fat diet  ðCTRyR2  CTbactin Þ control The mean threshold cycle (CT) values for both the target (RyR2) and internal control (β-actin) genes were determined in each sample.

Data are expressed as means ± SE. An unpaired t test was used to compare differences in left ventricle, right ventricle and right atrial RyR2 gene expression, protein expression, phosphorylation state and binding affinity between the control and

1.4. Determination of amount of RyR2 protein Membrane vesicles were prepared from left ventricle, right ventricle and right atria from control (n = 5) and high-fat-fed (n = 5) dogs as previously described [21,22]. Protein content was determined using the method of Lowry et al. [23]. Western blot analyses were also performed to confirm relative levels of RyR2 protein in each vesicle preparation [21,22]. For these experiments, β-actin served as an internal control to correct for sample loading error. 1.5. RyR2 phosphorylation at Ser2809 Extent of RyR2 phosphorylation at Ser2809 in right and left ventricles from control and high-fat-fed dogs was determined employing Ser2809 phospho-specific antibodies using Western blot analyses as previously described [22,24]. 1.6. Ability of RyR2 to bind [3H]ryanodine The functional integrity of RyR2 in left ventricle, right ventricle and right atria obtained from control (n = 5) and highfat-fed (n = 5) dogs was assessed by its ability to bind [3H]

Fig. 2. Western blot analysis revealed no significant changes in RyR2 protein expression from left ventricle, right ventricle and right atria between normal control and chronically high-fat-fed dogs. Signal intensities were normalized to concomitant β-actin (means ± SE obtained from five hearts analyzed in duplicate).

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2.2. Gene and protein expression of cardiac RyR2 in normal control and chronically high-fat-fed dogs Real-time PCR revealed no significant alterations in cardiac RyR2 mRNA expression in right atria, right ventricle and left ventricle in high-fat-fed dogs relative to normal control dogs (Fig. 1). Western blot analysis also demonstrated no significant changes in RyR2 protein expression (Fig. 2).

Fig. 3. RyR2 phosphorylation at Ser2809 was significantly elevated in right and left ventricles from high-fat dogs relative to normal control dogs. Upper panel shows a typical autoradiogram from Western blot analysis for Ser2809 phosphorylation in ventricular tissues. Lower panel shows autoradiogram from Western blot analysis for β-actin, which was used to correct for sample load. Data shown represent average data (means ± SE) from four experimental animals in each group. * denotes P b 0.05 vs. control.

high-fat dogs. A two-way repeated measures analysis of variance was used to compare the effects of diet and exercise on cardiovascular hemodynamics and cardiac index. Significance was accepted at P b 0.05. 2. Results 2.1. Chronic high-fat diet Chronic high-fat feeding increased body weight from 22.1 ± 2.1 kg at baseline to 27.3 ± 1.9 kg following ∼6 weeks of high-fat diet (P b 0.01). The average body weight of normal, control dogs was 23.9 ± 1.5 kg (P b 0.05 vs. high-fat diet). These findings are consistent with earlier studies in which our laboratory has demonstrated that the chronic high-fat feeding protocol used in this investigation induces many common features of the metabolic syndrome in that these animals have increased body weight, dyslipidemia, hyperinsulinemia with normoglycemia (i.e. insulin resistance), impaired control of blood pressure and hyperleptinemia [14,17,18].

Fig. 4. [3H]ryanodine binding was significantly depressed in membrane vesicles from left ventricle and right ventricle obtained from chronically high-fat-fed dogs relative to normal control dogs. Data shown are means ± SE from five experimental animals in each group. * denotes P b 0.05 vs. control.

Fig. 5. Mean aortic blood pressure (A), heart rate (B) and cardiac index (C— cardiac output/body weight) from normal control and chronically high-fat-fed dogs at rest and during graded treadmill exercise. Aortic pressure and heart rate were significantly elevated at rest and during exercise in high-fat-fed dogs while cardiac index was depressed at the highest level of exercise. Data pooled from earlier studies from our laboratory [14,18].

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2.3. RyR2 phosphorylation at Ser2809 RyR2 phosphorylation at Ser2809 was significantly elevated in right and left ventricle from high-fat-fed dogs compared to normal control dogs (Fig. 3). No significant differences in right atrial Ser2809 phosphorylation were detected (data not shown). 2.4. [3H]ryanodine binding to membrane vesicles from left ventricle, right atria and right ventricle obtained from control and chronically high-fat-fed dogs Chronic high-fat feeding significantly decreased the ability of cardiac RyR2 to bind the specific ligand [3H]ryanodine in right ventricle (high-fat diet = 80.2 fmol/mg protein vs. control = 243.6 fmol/mg protein) and left ventricle (high-fat diet = 90.5 fmol/mg protein vs. control = 200.9 fmol/mg protein; Fig. 4). No significant differences in right atrial RyR2 binding affinity were detected (high-fat diet = 70.7 fmol/mg protein vs. control = 98.6 fmol/mg protein). 2.5. Hemodynamics at rest and during exercise in control and chronically high-fat-fed dogs To assess the effects of our high-fat feeding protocol on global cardiac function, we pooled data from our previous publications [14,18]. Pooling these data increased the sample size from our earlier publications from six to nine. This increase in sample size demonstrates the dietary-induced increases in aortic pressure (Fig. 5A) and heart rate (Fig. 5B) at rest and during exercise in our chronic high-fat-fed dog model. In addition, cardiac index (cardiac output/animal body weight) was significantly depressed at high levels of exercise (Fig. 5C). 3. Discussion The purpose of the present investigation was to examine the hypothesis that the prediabetic metabolic syndrome alters expression, phosphorylation state and binding affinity of cardiac RyR2. This hypothesis is relevant especially in light of the recent investigations which indicate that hyperphosphorylation of cardiac RyR2 is an important mechanism which contributes to impaired contraction and the generation of ventricular arrhythmias [16]. We found that RyR2 mRNA and protein expression (Figs. 1 and 2) were unaltered by chronic high-fat feeding. Alternatively, RyR2 phosphorylation at Ser2809 was significantly elevated in right and left ventricle from high-fat-fed dogs compared to normal control dogs (Fig. 3). This specific increase in RyR2 phosphorylation resulted in decreased RyR2 binding affinity (Fig. 4). These data indicate that alterations in RyR2 could be an important mechanism of early cardiac dysfunction (Fig. 5C) in obesity and insulin resistance. 3.1. Obesity, cardiac function and RyR2 Obesity is associated with a hyperdynamic circulatory state characterized by increased blood pressure, total blood volume, cardiac output and tissue metabolic demand. As a result, obesity is

often associated with decrements in systolic and diastolic function [7,25,26], cardiac β-adrenoceptor responsiveness [11,12,27] and control of blood pressure and heart rate [13,14] despite overall increases in sympathetic activation [13,28–30]. These alterations have been shown to be related to a defect in post β-adrenoceptor signaling [31] and depressed SR Ca++ handling [27]. To our knowledge, the present study is the first investigation to examine the effects of obesity and insulin resistance on cardiac RyR2. Our laboratory has previously demonstrated that the high-fat feeding protocol used in this investigation induces many common features of the prediabetic metabolic syndrome [14,17,18]. In addition, we have examined the effects of this high-fat diet on cardiac function and systemic hemodynamic parameters. Earlier, we reported that chronic high-fat feeding modestly increases blood pressure, heart rate and cardiac output under baseline conditions; however, no significant differences in cardiac contractile function were noted [14,18]. However, it should be pointed out that there was a trend for lower cardiac index during exercise in the high-fat-fed dogs. To further examine the effects of our high-fat feeding protocol on cardiac function, we pooled experiments from these previous publications (to increase the sample size) [14,18]. Pooling these experiments now more clearly demonstrates the dietary-induced increases in aortic pressure and heart rate at rest and during exercise as well as diminished cardiac function (cardiac index, i.e. cardiac output normalized to animal body weight) at high levels of exercise (Fig. 5). This finding is the first to suggest that cardiac function is depressed in our dog model of the prediabetic metabolic syndrome. However, the impairment is relatively modest and likely represents an early stage of cardiac dysfunction that is not accompanied by significant alterations in the rate of ventricular pressure development, i.e. dP/dt [14]. Taken together, our findings suggest that diminished functional integrity of cardiac RyR2 could be an important early mechanism of obesity-related cardiac dysfunction. However, it must be acknowledged that these data do not provide a direct causal link between altered ryanodine receptor function and diminished cardiac function in the metabolic syndrome. 3.2. Potential mechanisms for dysfunction of cardiac RyR2 Our present results suggest that increased phosphorylation at Ser2809 decreases function of cardiac (ventricular) RyR2 as evidenced by a significant decrease in RyR2 binding affinity and diminished cardiac function when the sympathetic nervous system is activated during exercise in high-fat-fed dogs. Our findings are in line with those of Marks et al. and others who have demonstrated that chronic activation of the sympathetic nervous system, as occurs in obesity and the metabolic syndrome [13,28–30], leads to increased PKA-mediated phosphorylation of cardiac RyR2 and a “leaky” RyR2 which promotes cardiac arrhythmias and contractile dysfunction [32–36]. In contrast, other studies are inconsistent with critical aspects of the RyR2-PKA/diastolic Ca+ leak hypothesis [37]. The discrepancy between these investigations is related primarily to differences in the involvement of PKA and phosphorylation of RyR2 in mediating Ca++ sparks and SR Ca++ content [37–44]. However, it should be noted that this controversy deals exclusively with alterations in cardiac RyR2 in heart failure

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as no study has examined the effects of the metabolic syndrome on the functional integrity of cardiac RyR2. We hypothesize that the increased phosphorylation of Ser2809 documented in the present study is mediated by protein kinase A (PKA). Our laboratory recently found that basal epinephrine and norepinephrine levels are elevated in our chronically high-fat-fed dogs [45]. This finding is supported by other studies demonstrating that the metabolic syndrome is associated with a state of sympathetic hyperactivity [13,28–30]. We propose that this increase in circulating catecholamines will stimulate G-protein-coupled β-adrenoceptors, elevating intracellular cyclic adenosine monophosphate (cAMP) thereby activating mainly cAMP-dependent PKA [33,35]. Additional mechanisms that could be involved/associated with the impaired functional integrity of cardiac RyR2 in the metabolic syndrome include the altered dissociation of FK 506-binding protein (channel-stabilizing subunit of calstabine 2, FKBP 12.6) from RyR2 [35,46] and/or deficiency in cAMP-specific type 4 phoshodiesterase 3 (PDE4D3) [33,35,47]. In addition, a growing body of literature suggests that nonenzymatic reactions, such as peroxidative degradation of polyunsaturated fatty acids (lipid peroxidation) and oxidative modifications of amino acids can cause reactive aldehydic intermediates that can form intra- and intermolecular covalent adducts that can significantly impair receptor function [48]. Clearly, many additional studies are needed to address the precise cellular and molecular mechanisms by which the metabolic syndrome impairs the functional integrity of ventricular RyR2. Findings from this investigation agree with our earlier study, which demonstrated that early stage diabetes mellitus compromised functional integrity of cardiac RyR2 without alterations in receptor expression [21]. We hypothesized that diabetes mellitus could affect expression and/or function of specific proteins involved in regulating/maintaining intracellular ionic homeostasis in the heart [18]. Additional studies revealed that the dysfunction of RyR2 in streptozotocin-diabetic rat hearts results from formation of noncross-linking advanced glycation end products (AGE)s on RyR2 and sarco(endo)plasmic reticulum Ca2+-ATPase [22,49]. Further studies revealed that the dysfunction of RyR2 induced in diabetes may be due, at least in part, to formation of disulfide bonds between adjacent sulfhydryl groups [24]. Since chronic high-fat feeding does not significantly alter plasma glucose levels [14], it is unlikely that the alterations in cardiac RyR2 in the present study are specifically related to glycation of the receptor. However, formation of disulfide bonds is a potential mechanism that should be further explored. 3. Conclusion The present study demonstrates that the functional integrity of ventricular RyR2 is compromised by induction of the prediabetic metabolic syndrome in the heart; as evidenced by diminished binding affinity of RyR2 with no changes in mRNA or protein expression. Interestingly, no significant changes in RyR2 binding affinity, phosphorylation or receptor expression were noted in right atria. These alterations directly correlate with elevated aortic pressure and heart rate as well as diminished cardiac index during

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sympathetic activation with exercise in the same high-fat-fed dog model. These findings are important since they are the first to suggest that the metabolic syndrome induces significant alterations in cardiac RyR2 phosphorylation and activity. Since, alterations in intracellular Ca++ cycling have been shown to precede overt depression of myocardial function [15], we propose that cardiac RyR2 channels hold novel therapeutic potential against early stages of obesity-induced cardiomyopathy. Acknowledgments This work was funded by grants from the National Heart Lung and Blood Institute HL67804 (JDT). Phospho-RyR2 (Ser2809) antibodies were kindly provided by Drs. Andrew Marks and Steve Reiken (Columbia University, New York). References [1] DeFronzo RA, Ferrannini E. Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 1991;14:173–94. [2] Reaven GM. Banting lecture 1988. Role of insulin resistance in human disease. Diabetes 1988;37:1595–607. [3] Hansen BC. The metabolic syndrome X. Ann N YAcad Sci 1999;892:1–24. [4] Ford ES, Giles WH, Mokdad AH. Increasing prevalence of the metabolic syndrome among U.S. adults. Diabetes Care 2004;27:2444–9. [5] Henry P, Thomas F, Benetos A, Guize L. Impaired fasting glucose, blood pressure and cardiovascular disease mortality. Hypertension 2002;40:458–63. [6] Lakka HM, Laaksonen DE, Lakka TA, Niskanen LK, Kumpusalo E, Tuomilehto J, et al. The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men. Jama 2002;288:2709–16. [7] Alpert MA. Obesity cardiomyopathy: pathophysiology and evolution of the clinical syndrome. Am J Med Sci 2001;321:225–36. [8] Alpert MA, Fraley MA, Birchem JA, Senkottaiyan N. Management of obesity cardiomyopathy. Expert Rev Cardiovasc Ther 2005;3:225–30. [9] Kasper EK, Hruban RH, Baughman KL. Cardiomyopathy of obesity: a clinicopathologic evaluation of 43 obese patients with heart failure. Am J Cardiol 1992;70:921–4. [10] Koch R, Sharma AM. Obesity and cardiovascular hemodynamic function. Curr Hypertens Rep 1999;1:127–30. [11] Cabrol P, Galinier M, Fourcade J, Verwaerde P, Massabuau P, Tran MA, et al. Functional decoupling of left ventricular beta-adrenoceptor in a canine model of obesity-hypertension. Arch Mal Coeur Vaiss 1998;91:1021–4. [12] Carroll JF, Jones AE, Hester RL, Reinhart GA, Cockrell K, Mizelle HL. Reduced cardiac contractile responsiveness to isoproterenol in obese rabbits. Hypertension 1997;30:1376–81. [13] Hall JE, Brands MW, Zappe DH, Dixon WN, Mizelle HL, Reinhart GA, et al. Hemodynamic and renal responses to chronic hyperinsulinemia in obese, insulin-resistant dogs. Hypertension 1995;25:994–1002. [14] Setty S, Sun W, Tune JD. Coronary blood flow regulation in the prediabetic metabolic syndrome. Basic Res Cardiol 2003;98:416–23. [15] Houser SR, Margulies KB. Is depressed myocyte contractility centrally involved in heart failure? Circ Res 2003;92:350–8. [16] Scoote M, Williams AJ. The cardiac ryanodine receptor (calcium release channel): emerging role in heart failure and arrhythmia pathogenesis. Cardiovasc Res 2002;56:359–72. [17] Knudson JD, Dincer UD, Dick GM, Shibata H, Akahane R, Saito M, et al. Leptin resistance extends to the coronary vasculature in prediabetic dogs and provides a protective adaptation against endothelial dysfunction. Am J Physiol, Heart Circ Physiol 2005;289:H1038–46. [18] Zhang C, Knudson JD, Setty S, Araiza A, Dincer UD, Kuo L, et al. Coronary arteriolar vasoconstriction to angiotensin II is augmented in prediabetic metabolic syndrome via activation of AT1 receptors. Am J Physiol, Heart Circ Physiol 2005;288:H2154–62.

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