Vehicle-treated Hearts Research Articles

Empagliflozin improves cardiac energetics during ischaemia/reperfusion by directly increasing cardiac ketone utilization.

Empagliflozin (EMPA), a potent inhibitor of the renal sodium-glucose cotransporter 2 and an effective treatment for Type 2 diabetes, has been shown to have cardioprotective effects, independent of improved glycaemic control. Several non-canonical mechanisms have been proposed to explain these cardiac effects, including increasing circulating ketone supply to the heart. This study aims to test whether EMPA directly alters cardiac ketone metabolism independent of supply. The direct effects of EMPA on cardiac function and metabolomics were investigated in Langendorff rat heart perfused with buffer containing 5 mM glucose, 4 mM β-hydroxybutyrate (βHb) and 0.4 mM intralipid, subject to low flow ischaemia/reperfusion. Cardiac energetics were monitored in situ using 31P NMR spectroscopy. Steady-state 13C labelling was performed by switching 12C substrates for 13C1 glucose or 13C4 βHb and 13C incorporation into metabolites determined using 2D 1H-13C HSQC NMR spectroscopy. EMPA treatment improved left ventricular-developed pressure during ischaemia and reperfusion compared to vehicle-treated hearts. In EMPA-treated hearts, total adenosine triphosphate (ATP) and phosphocreatine (PCr) levels, and Gibbs free energy for ATP hydrolysis were significantly higher during ischaemia and reperfusion. EMPA treatment did not alter the incorporation of 13C from glucose into glycolytic products lactate or alanine neither during ischaemia nor reperfusion. In ischaemia, EMPA led to a decrease in 13C1 glucose incorporation and a concurrent increase in 13C4 βHb incorporation into tricarboxylic acid (TCA) cycle intermediates succinate, citrate, and glutamate. During reperfusion, the concentration of metabolites originating from 13C1 glucose was similar to vehicle but those originating from 13C4 βHb remained elevated in EMPA-treated hearts. Our findings indicate that EMPA causes a switch in metabolism away from glucose oxidation towards increased ketone utilization in the rat heart, thereby improving function and energetics both during ischaemia and recovery during reperfusion. This preference of ketone utilization over glucose was observed under conditions of constant supply of substrate, suggesting that EMPA acts directly by modulating cardiac substrate preference, independent of substrate availability. The mechanisms underlying our findings are currently unknown, warranting further study.

Abstract 847: Adipocyte-specific Pharmacological Inhibition of Adipose Triglyceride Lipase (ATGL) Ameliorates Cardiac Fibrosis in Heart Failure

Objectives: ATGL is a key enzyme that regulates triglyceride lipolysis in most cells, including the adipocyte and the cardiomyocyte. Recent studies indicate that accelerated adipose tissue lipolysis contributes to worsening heart failure (HF), suggesting that ATGL inhibition specifically in the adipocyte may prevent worsening HF. Indeed, we have shown that the ATGL inhibitor, Atglistatin, which appears to be largely adipocyte-specific, ameliorates cardiac dysfunction and hypertrophy in HF induced by transverse aortic constriction (TAC). However, the mechanisms by which Atglistatin treatment prevents worsening HF in mice remains unknown. Methods: Eight week-old C57BL/6N mice underwent TAC or sham surgery. After 1 week, mice with HF were assigned into 4 groups: 1) vehicle (standard chow)-sham, 2) Atglistain-sham, 3) vehicle-TAC and 4) Atglistatin-TAC. Atglistain was administered in food pellets containing 2mmol Atglistatin/kg for 2 weeks. Thereafter, mice were euthanized and hearts were used for subsequent histological and quantitative PCR analysis. Results: Picro-sirius red staining demonstrated that hearts from the Atglistatin TAC mice had significantly less cardiac fibrosis than the vehicle TAC mice (1.6±0.4 vs 5.3±1.1%, in Atglistatin TAC vs vehicle TAC; p<0.01). Consistent with this finding, Atglistatin treatment of the TAC group reduced transcript levels of fibrosis-related genes, including Col1a1 (-58.7%, p=0.03) and Postn (-54.7%, p<0.01), compared to vehicle-treated hearts. Furthermore, Atglistatin decreased the transcript levels of the macrophage polarization-related genes, including Lgals3 (-55.3%, p<0.01) and Spp1 (-38.7%, p=0.03) , in the hearts from TAC mice compared to controls. Moreover, immunofluorescent analysis showed that Atglistatin reduced infiltration of Galectin-3 positive macrophages in the heart compared to vehicle-TAC mice, suggesting reduced macrophage infiltration and activation and less cardiac fibrosis. Conclusion: Our data indicate that reducing triglyceride lipolysis in the adipose tissue via ATGL inhibition may ameliorate HF via modulation of HF-induced macrophage and fibroblast activation in the heart.

Abstract 856: Mitochondrial Protein Kinase B (akt) Translocation Mediates Insulin-stimulated Cardiac Glucose Oxidation

Introduction: Insulin stimulates glucose oxidation, an effect which is associated with simulating pyruvate dehydrogenase (PDH). However, how the insulin signal is transduced from the cell membrane to the mitochondria to stimulate PDH is not known. Protein kinase B (Akt), protein kinase C-delta (PKCδ) and glycogen synthase kinase-3beta (GSK-3β) are main components of the cytosolic insulin signalling pathway and it has been suggested that they can be translocated to the mitochondria following insulin receptor activation in noncardiac tissue. Therefore, we investigated whether any of these kinases has a role mediated cardiac insulin-stimulated glucose oxidation in the heart. Methods and Results: Male and female C57BL/6 mice were anesthetized and hearts were collected and perfused in the isolated working heart mode. Hearts were perfused with [5- 3 H] glucose and [U- 14 C] glucose to simultaneously measure glycolysis and glucose oxidation rates, respectively, in the presence and absence of insulin. Insulin enhanced the phosphorylation and translocation of Akt Ser473 , PKCδ Tyr311 and GSK-3β Ser9 to the cardiac mitochondria along with enhancing PDH activity by decreasing its phosphorylation. Pharmacological inhibition of Akt using AktiVIII completely abolish the stimulatory effect of insulin on cardiac glucose oxidation rates (354 ± 145 vs 2307 ± 185 nmol. g dry wt -1 . min -1 in vehicle-treated hearts, p<0.05). However, pharmacological inhibition of either PKCδ or GSK-3β using Bisindolylmaleimide I or 3F8, respectively, did not have a significant effect on insulin-stimulated glucose oxidation rates. None of the pharmacological inhibitors had any significant effect on cardiac glycolysis. Conclusion: Insulin mediates its stimulatory effect on cardiac glucose oxidation via a mechanism which involve the activation and translocation of Akt to the mitochondria.

Adropin regulates cardiac energy metabolism and improves cardiac function and efficiency

BackgroundImpaired cardiac insulin signalling and high cardiac fatty acid oxidation rates are characteristics of conditions of insulin resistance and diabetic cardiomyopathies. The potential role of liver-derived peptides such as adropin in mediating these changes in cardiac energy metabolism is unclear, despite the fact that in skeletal muscle adropin can preferentially promote glucose metabolism and improve insulin sensitivity. ObjectivesTo determine the influence of adropin on cardiac energy metabolism, insulin signalling and cardiac efficiency. MethodsC57Bl/6 mice were injected with either vehicle or a secretable form of adropin (450 nmol/kg, i.p.) three times over a 24-h period. The mice were fasted to accentuate the differences between animals in adropin plasma levels before their hearts were isolated and perfused using a working heart system. In addition, direct addition of adropin to the perfusate of ex vivo hearts isolated from non-fasting mice was utilized to investigate the acute effects of the peptide on heart metabolism and ex vivo function. ResultsIn contrast to the observed fasting-induced predominance of fatty acid oxidation as a source of ATP production in control hearts, insulin inhibition of fatty acid oxidation was preserved by adropin treatment. Adropin-treated mouse hearts also showed a higher cardiac work, which was accompanied by improved cardiac efficiency and enhanced insulin signalling compared to control hearts. Interestingly, acute adropin administration to isolated working hearts also resulted in an inhibition of fatty acid oxidation, accompanied by a robust stimulation of glucose oxidation compared to vehicle-treated hearts. Adropin also increased activation of downstream cardiac insulin signalling. Moreover, both in vivo and ex vivo treatment protocols induced a reduction in the inhibitory phosphorylation of pyruvate dehydrogenase (PDH), the major enzyme of glucose oxidation, and the protein levels of the responsible kinase PDH kinase 4 and the insulin-signalling inhibitory phosphorylation of JNK (p-T183/Y185) and IRS-1 (p-S307), suggesting acute receptor- and/or post-translational modification-mediated mechanisms. ConclusionsThese results demonstrate that adropin has important effects on energy metabolism in the heart and may be a putative candidate for the treatment of cardiac disease associated with impaired insulin sensitivity.

Sensory Neuropathy Affects Cardiac miRNA Expression Network Targeting IGF-1, SLC2a-12, EIF-4e, and ULK-2 mRNAs.

Background: Here we examined myocardial microRNA (miRNA) expression profile in a sensory neuropathy model with cardiac diastolic dysfunction and aimed to identify key mRNA molecular targets of the differentially expressed miRNAs that may contribute to cardiac dysfunction. Methods: Male Wistar rats were treated with vehicle or capsaicin for 3 days to induce systemic sensory neuropathy. Seven days later, diastolic dysfunction was detected by echocardiography, and miRNAs were isolated from the whole ventricles. Results: Out of 711 known miRNAs measured by miRNA microarray, the expression of 257 miRNAs was detected in the heart. As compared to vehicle-treated hearts, miR-344b, miR-466b, miR-98, let-7a, miR-1, miR-206, and miR-34b were downregulated, while miR-181a was upregulated as validated also by quantitative real time polymerase chain reaction (qRT-PCR). By an in silico network analysis, we identified common mRNA targets (insulin-like growth factor 1 (IGF-1), solute carrier family 2 facilitated glucose transporter member 12 (SLC2a-12), eukaryotic translation initiation factor 4e (EIF-4e), and Unc-51 like autophagy activating kinase 2 (ULK-2)) targeted by at least three altered miRNAs. Predicted upregulation of these mRNA targets were validated by qRT-PCR. Conclusion: This is the first demonstration that sensory neuropathy affects cardiac miRNA expression network targeting IGF-1, SLC2a-12, EIF-4e, and ULK-2, which may contribute to cardiac diastolic dysfunction. These results further support the need for unbiased omics approach followed by in silico prediction and validation of molecular targets to reveal novel pathomechanisms.

Ischemic Preconditioning Efficacy Following Anabolic Steroid Usage: A Clear Difference Between Sedentary and Exercise-Trained Rat Hearts.

Previous studies show that anabolic steroids impair innate cardioprotective mechanisms. Here, we investigated the effect of supraphysiological doses of nandrolone on ischemic preconditioning (IPC) as a potent cardioprotective tool against ischemia reperfusion (IR) injury in rat hearts. Male Wistar rats in two experimental settings of sedentary and exercise-trained (60min/day swimming, 5days/week, for 8weeks) were either pretreated with intramuscular injections of arachis oil (Arach, n = 16) as vehicle or nandrolone decanoate (ND, n = 8), 10mg/kg/week, for 8weeks. At the end, the hearts were excised and perfused in a Langendorff system. Then, the vehicle-treated hearts subdivided into the IR (30min of LAD coronary artery occlusion and 120min reperfusion, n = 8) and IPC (three cycles of 3-min ischemia and 3-min reperfusion before test ischemia, n = 8) groups and nandrolone-treated hearts served as ND + IPC (nandrolone pretreatment before IR and IPC protocols, n = 8) group. Post-ischemic cardiac function and infarct size were assessed. Reperfusion arrhythmias were analyzed using a standard scoring system. In sedentary hearts, ND slightly increased heart-to-body weight ratio and increased baseline cardiac contractile function. In trained hearts, ND markedly increased heart-to-body weight ratio which was also associated with enhanced baseline cardiac function. ND pretreatment enhanced protective effects of IPC in sedentary group; however, abolished these effects in exercise-trained group. The arrhythmia score was not significantly different between nandrolone-treated groups vs. respective preconditioned groups. Our findings show that ND impairs IPC-induced cardioprotection in exercise-trained rat hearts. Cardiac hypertrophy seems to play a crucial role in this response.

Empagliflozin Increases Cardiac Energy Production in Diabetes: Novel Translational Insights Into the Heart Failure Benefits of SGLT2 Inhibitors

SGLT2 inhibitors have profound benefits on reducing heart failure and cardiovascular mortality in individuals with type 2 diabetes, although the mechanism(s) of this benefit remain poorly understood. Because changes in cardiac bioenergetics play a critical role in the pathophysiology of heart failure, the authors evaluated cardiac energy production and substrate use in diabetic mice treated with the SGTL2 inhibitor, empagliflozin. Empagliflozin treatment of diabetic db/db mice prevented the development of cardiac failure. Glycolysis, and the oxidation of glucose, fatty acids and ketones weremeasured in the isolated working heart perfused with 5 mmol/l glucose, 0.8 mmol/l palmitate, 0.5 mmol/l ß-hydroxybutyrate (ßOHB), and 500 μU/ml insulin. In vehicle-treated db/db mice, cardiac glucose oxidation rates were decreased by 61%, compared with control mice, but only by 43% in empagliflozin-treated diabetic mice. Interestingly, cardiac ketone oxidation rates in db/db mice decreased to 45% of the rates seen in control mice, whereas a similar decrease (43%) was seen in empagliflozin-treated db/db mice. Overall cardiac adenosine triphosphate (ATP) production rates decreased by 36% in db/db vehicle-treated hearts compared with control mice, with fatty acid oxidation providing 42%, glucose oxidation 26%, ketone oxidation 10%, and glycolysis 22% of ATP production in db/db mouse hearts. In empagliflozin-treated db/db mice, cardiac ATP production rates increased by 31% compared with db/db vehicle-treated mice, primarily due to a 61% increase in the contribution of glucose oxidation to energy production. Cardiac efficiency (cardiac work/O2 consumed) decreased by 28% in db/db vehicle-treated hearts, compared with control hearts, and empagliflozin did not increase cardiac efficiency per se. Because ketone oxidation was impaired in db/db mouse hearts, the authors determined whether this contributed to the decrease in cardiac efficiency seen in the db/db mouse hearts.Addition of 600 μmol/l ßOHB to db/db mouse hearts perfused with5mmol/l glucose, 0.8 mmol/l palmitate, and100 μU/ml insulin increased ketone oxidation rates, butdidnotdecrease either glucose oxidation or fatty acid oxidation rates. The presence of ketones did not increase cardiac efficiency, but did increase ATP production rates, due tothe additional contribution ofketoneoxidation to energy production. The authors conclude that empagliflozin treatment is associatedwithanincrease in ATP production, resulting in an enhanced energy status of the heart.

Cardiac-specific inducible overexpression of human plasma membrane Ca2+ ATPase 4b is cardioprotective and improves survival in mice following ischemic injury.

Background: Heart failure (HF) is associated with reduced expression of plasma membrane Ca2+-ATPase 4 (PMCA4). Cardiac-specific overexpression of human PMCA4b in mice inhibited nNOS activity and reduced cardiac hypertrophy by inhibiting calcineurin. Here we examine temporally regulated cardiac-specific overexpression of hPMCA4b in mouse models of myocardial ischemia reperfusion injury (IRI) ex vivo, and HF following experimental myocardial infarction (MI) in vivoMethods and results: Doxycycline-regulated cardiomyocyte-specific overexpression and activity of hPMCA4b produced adaptive changes in expression levels of Ca2+-regulatory genes, and induced hypertrophy without significant differences in Ca2+ transients or diastolic Ca2+ concentrations. Total cardiac NOS and nNOS-specific activities were reduced in mice with cardiac overexpression of hPMCA4b while nNOS, eNOS and iNOS protein levels did not differ. hMPCA4b-overexpressing mice also exhibited elevated systolic blood pressure vs. controls, with increased contractility and lusitropy in vivo In isolated hearts undergoing IRI, hPMCA4b overexpression was cardioprotective. NO donor-treated hearts overexpressing hPMCA4b showed reduced LVDP and larger infarct size versus vehicle-treated hearts undergoing IRI, demonstrating that the cardioprotective benefits of hPMCA4b-repressed nNOS are lost by restoring NO availability. Finally, both pre-existing and post-MI induction of hPMCA4b overexpression reduced infarct expansion and improved survival from HF.Conclusions: Cardiac PMCA4b regulates nNOS activity, cardiac mass and contractility, such that PMCA4b overexpression preserves cardiac function following IRI, heightens cardiac performance and limits infarct progression, cardiac hypertrophy and HF, even when induced late post-MI. These data identify PMCA4b as a novel therapeutic target for IRI and HF.

Non-nuclear estrogen receptor alpha activation in endothelium reduces cardiac ischemia-reperfusion injury in mice

Steroid hormone receptors including estrogen receptors (ER) classically function as ligand-regulated transcription factors. However, estrogens also elicit cellular effects through binding to extra-nuclear ER (ERα, ERβ, and G protein-coupled ER or GPER) that are coupled to kinases. How extra-nuclear ER actions impact cardiac ischemia-reperfusion (I/R) injury is unknown. We treated ovariectomized wild-type female mice with estradiol or an estrogen-dendrimer conjugate (EDC), which selectively activates extra-nuclear ER, or vehicle interventions for two weeks. I/R injury was then evaluated in isolated Langendorff perfused hearts. Two weeks of treatment with estradiol significantly decreased infarct size and improved post-ischemic contractile function. Similarly, EDC treatment significantly decreased infarct size and increased post-ischemic functional recovery compared to vehicle-treated hearts. EDC also caused an increase in myocardial protein S-nitrosylation, consistent with previous studies showing a role for this post-translational modification in cardioprotection. In further support of a role for S-nitrosylation, inhibition of nitric oxide synthase, but not soluble guanylyl cyclase blocked the EDC mediated protection. The administration of ICI182,780, which is an agonist of G-protein coupled estrogen receptor (GPER) and an antagonist of ERα and ERβ, did not result in protection; however, ICI182,780 significantly blocked EDC-mediated cardioprotection, indicating participation of ERα and/or ERβ. In studies determining the specific ER subtype and cellular target involved, EDC decreased infarct size and improved functional recovery in mice lacking ERα in cardiomyocytes. In contrast, protection was lost in mice deficient in endothelial cell ERα. Thus, extra-nuclear ERα activation in endothelium reduces cardiac I/R injury in mice, and this likely entails increased protein S-nitrosylation. Since EDC does not stimulate uterine growth, in the clinical setting EDC-like compounds may provide myocardial protection without undesired uterotrophic and cancer-promoting effects.

Abstract 282: Adropin Enhancement of Cardiac Insulin Sensitivity and Inhibition of Fatty Acid Oxidation are Associated With Improvement of Cardiac Function and Efficiency in Hearts From Fasted Mice

Impaired cardiac insulin signaling and high cardiac fatty acid oxidation rates are characteristics of diabetic cardiomyopathy. Potential roles for liver-derived metabolic factors in mediating cardiac energy homeostasis are underappreciated. Plasma levels of adropin, a liver secreted peptide, increase during feeding and decrease during fasting and diabetes. In skeletal muscle, adropin preferentially promotes glucose over fatty acid oxidation. We therefore determined what effect adropin has on cardiac energy metabolism, insulin signaling and cardiac efficiency. C57Bl/6 mice were fasted to accentuate the differences in adropin plasma levels between animals injected 3 times over 24 hr with either vehicle or adropin (450 nmol/kg i.p.). Despite fasting-induced predominance of fatty acid oxidation measured in isolated working hearts, insulin inhibition of fatty acid oxidation was re-established in adropin-treated mice (from 1022±143 to 517±56 nmol. g dry wt -1 . min -1 , p <0.05) compared to vehicle-treated mice (from 757±104 to 818±103 nmol. g dry wt -1 . min -1 ). Adropin-treated mice hearts showed higher cardiac work over the course of perfusion (p<0.05 vs. vehicle), which was accompanied by improved cardiac efficiency and enhanced phosphorylation of insulin signaling enzymes (tyrosine-IRS-1, AS160, p<0.05). Acute addition of adropin (2nM) to isolated working hearts from non-fasting mice showed a robust stimulation of glucose oxidation compared to vehicle-treated hearts (3025±401 vs 1708±292 nmol. g dry wt -1 . min -1 , p<0.05, respectively) with a corresponding inhibition of palmitate oxidation (325±61 vs 731±160 nmol. g dry wt -1 . min -1 , p<0.05, respectively), even in the presence of insulin. Acute adropin addition to hearts also increased IRS-1 tyrosine-phosphorylation as well as Akt, and GSK3β phosphorylation (p<0.05), suggesting acute receptor- and/or post-translational modification-mediated mechanisms. These results suggest adropin as a putative candidate for the treatment of diabetic cardiomyopathy.

Abstract 037: Transient Cardiac Expression Of Constitutively Active G alpha Q Activates Renin-angiotensin System, Leading To Progressive Heart Failure And Ventricular Arrhythmias In Transgenic Mice

Introduction: Transgenic mice with transient cardiac expression of constitutively active Galpha q (Gαq-TG) caused progressive heart failure and ventricular arrhythmias after the initiating stimulus becomes undetectable. However, the mechanisms are still unknown. Renin-angiotensin system plays a critical role in the development of cardiac hypertrophy and heart failure. We examined the effects of chronic administration of olmesartan on ventricular function, the number of premature ventricular contractions (PVC), and ventricular remodeling in Gαq-TG mice. Methods and Results: Olmesartan (1 mg/kg/day) or vehicle was chronically administered to Gαq-TG from 6 to 32 weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic olmesartan treatment prevented the severe reduction of left ventricular fractional shortening and inhibited ventricular interstitial fibrosis and ventricular myocyte hypertrophy in Gαq-TG. Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min) observed in 9 of 10 vehicle-treated Gαq-TG but in none of 10 olmesartan -treated Gαq-TG. The QT interval was significantly shorter in olmesartan-treated Gαq-TG than vehicle-treated Gαq-TG. CTGF, collagen type 1, ANP, BNP, and β-MHC gene expression was increased in vehicle-treated Gαq-TG. Olmesartan significantly decreased these gene expressions in Gαq-TG. Moreover, protein expressions of canonical transient receptor potential (TRPC) channels 3 and 6 increased in vehicle-treated Gαq-TG hearts. Olmesartan significantly decreased TRPC6 expressions in Gαq-TG. Angiotensin converting enzyme (ACE) 1 and 2 gene expressions were also increased in vehicle-treated Gαq-TG and was not decreased to the control level in olmesartan-treated Gαq-TG. Conclusions: These findings suggest that renin-angiotensin system has an important role in the development of cardiac hypertrophy and heart failure even if the initiating stimulus is different from the activation of renin-angiotensin system.

Impairment of Endothelial-Myocardial Interaction Increases the Susceptibility of Cardiomyocytes to Ischemia/Reperfusion Injury

Endothelial-myocardial interactions may be critically important for ischemia/reperfusion injury. Tetrahydrobiopterin (BH4) is a required cofactor for nitric oxide (NO) production by endothelial NO synthase (eNOS). Hyperglycemia (HG) leads to significant increases in oxidative stress, oxidizing BH4 to enzymatically incompetent dihydrobiopterin. How alterations in endothelial BH4 content impact myocardial ischemia/reperfusion injury remains elusive. The aim of this study was to examine the effect of endothelial-myocardial interaction on ischemia/reperfusion injury, with an emphasis on the role of endothelial BH4 content. Langendorff-perfused mouse hearts were treated by triton X-100 to produce endothelial dysfunction and subsequently subjected to 30 min of ischemia followed by 2 h of reperfusion. The recovery of left ventricular systolic and diastolic function during reperfusion was impaired in triton X-100 treated hearts compared with vehicle-treated hearts. Cardiomyocytes (CMs) were co-cultured with endothelial cells (ECs) and subsequently subjected to 2 h of hypoxia followed by 2 h of reoxygenation. Addition of ECs to CMs at a ratio of 1∶3 significantly increased NO production and decreased lactate dehydrogenase activity compared with CMs alone. This EC-derived protection was abolished by HG. The addition of 100 µM sepiapterin (a BH4 precursor) or overexpression of GTP cyclohydrolase 1 (the rate-limiting enzyme for BH4 biosynthesis) in ECs by gene trasfer enhanced endothelial BH4 levels, the ratio of eNOS dimer/monomer, eNOS phosphorylation, and NO production and decreased lactate dehydrogenase activity in the presence of HG. These results demonstrate that increased BH4 content in ECs by either pharmacological or genetic approaches reduces myocardial damage during hypoxia/reoxygenation in the presence of HG. Maintaining sufficient endothelial BH4 is crucial for cardioprotection against hypoxia/reoxygenation injury.

Abstract 419: Two Models of Endothelial Dysfunction: the Importance of Intrinsic Cardioprotective Properties of the Coronary-endothelium

The coronary endothelium plays a major role in protecting cardiomyocytes (CMs) from hypoxia/reoxygenation injury. We are describing two models of endothelial dysfunction compromising the intrinsic cardioprotective properties of endothelial cells (ECs). Tetrahydrobiopterin (BH4) is an essential cofactor for coupling of endothelial nitric oxide synthase (eNOS) to produce the cardioprotective mediator nitric oxide (NO). However, hyperglycemia (HG) leads to significant increases in oxidative stress, oxidizing BH4 to dihydrobiopterin and thereby causing endothelial dysfunction. Additionally, Langendorff-perfused mouse hearts were treated by triton X-100 to produce endothelial dysfunction and subsequently subjected to 30 min of ischemia followed by 2 h of reperfusion. The recovery of left ventricular systolic and diastolic function during reperfusion was impaired in triton X-100 treated hearts compared with vehicle-treated hearts. CMs were co-cultured with ECs and subsequently subjected to 2 h of hypoxia followed by 2 h of reoxygenation. Addition of ECs to CMs at a ratio of 1:3 significantly increased NO production and decreased lactate dehydrogenase activity compared with CMs alone. This EC-derived protection was abolished by HG. The addition of 100 μM sepiapterin (a BH4 precursor) or overexpression of GTP cyclohydrolase 1 (the rate-limiting enzyme for BH4 biosynthesis) in ECs by gene trasfer enhanced endothelial BH4 levels, the ratio of eNOS dimer/monomer, eNOS phosphorylation, and NO production and decreased lactate dehydrogenase activity in the presence of HG. These results demonstrate the important intrinsic cardioprotective properties of the coronary endothelium, more specifically increased BH4 content in ECs by either pharmacological or genetic approaches reduces myocardial damage during hypoxia/reoxygenation in the presence of HG. Maintaining sufficient endothelial BH4 is crucial for cardioprotection against hypoxia/reoxygenation injury.

ANG II causes insulin resistance and induces cardiac metabolic switch and inefficiency: a critical role of PDK4

The renin-angiotensin system (RAS) may alter cardiac energy metabolism in heart failure. Angiotensin II (ANG II), the main effector of the RAS in heart failure, has emerged as an important regulator of cardiac hypertrophy and energy metabolism. We studied the metabolic perturbations and insulin response in an ANG II-induced hypertrophy model. Ex vivo heart perfusion showed that hearts from ANG II-treated mice had a lower response to insulin with significantly reduced rates of glucose oxidation in association with increased pyruvate dehydrogenase kinase 4 (PDK4) levels. Palmitate oxidation rates were significantly reduced in response to insulin in vehicle-treated hearts but remained unaltered in ANG II-treated hearts. Furthermore, phosphorylation of Akt was also less response to insulin in ANG II-treated wild-type (WT) mice, suggestive of insulin resistance. We evaluated the role of PDK4 in the ANG II-induced pathology and showed that deletion of PDK4 prevented ANG II-induced diastolic dysfunction and normalized glucose oxidation to basal levels. ANG II-induced reduction in the levels of the deacetylase, SIRT3, was associated with increased acetylation of pyruvate dehydrogenase (PDH) and a reduced PDH activity. In conclusion, our findings show that a combination of insulin resistance and decrease in PDH activity are involved in ANG II-induced reduction in glucose oxidation, resulting in cardiac inefficiency. ANG II reduces PDH activity via acetylation of PDH complex, as well as increased phosphorylation in response to increased PDK4 levels.

Abstract P301: Investigation of the Mechanism of the Cardioprotective Effects of Flavonols in Langendorff Perfused Rat Hearts

We investigated the mechanism(s) of flavonol-induced cardioprotection using a flavonol which inhibits calcium utilization and has antioxidant activity 3’,4’-dihydroxylflavonol, a flavonol which only affects calcium activity 4’-OH-3’-OCH3 flavonol and a water-soluble flavonol with selective antioxidant activity DiOHF-6-succinamic acid in rat isolated hearts. Hearts were perfused with physiological solution using a Langendorff apparatus and subjected to global, no-flow 20-min ischaemia followed by 30-min reperfusion. Hearts (n = 6–8 per group) were randomly treated with vehicle (0.05% DMSO), DiOHF, 4’-OH-3’-OCH3 flavonol or DiOHF-6-succinamic acid (all 10 µM). Flavonols were included in the perfusate for 10 min before ischaemia and during the entire duration of reperfusion. In vehicle treated hearts, LV+dP/dt was significantly reduced at the end of reperfusion (60±8% decrease in LV+dP/dt) compared to the pre-ischaemic level. Furthermore, LDH release (895±121 U/L) was significantly elevated during reperfusion compared to shams (70±10 U/L) and total eNOS expression was significantly lower in vehicle-treated hearts. In comparison, DiOHF treatment significantly improved LV function upon reperfusion (35±6% reduction in LV+dP/dt), significantly decreased the level of LDH (265±66 U/L) and preserved total eNOS expression. Hearts treated with the antioxidant DiOHF-6-succinamic acid had a similar preservation of contractility, resulting in a 36±5% reduction in LV+dP/dt, and a significantly lower release of LDH (388±57 U/L) and preserved total eNOS expression. In contrast, hearts treated with 4’-OH-3’-OCH3 showed similar impairment of contractility to the vehicle group. DiOHF-6-succinamic acid also exerted cardioprotection when given only during reperfusion but not when administered only prior to ischaemia. This study demonstrated that flavonol-induced cardioprotection relies on the antioxidant activity and is mainly exerted during the reperfusion phase. The water-soluble DiOHF-6-succinamic acid with selective antioxidant activity may lead to the development of a new cardioprotective agent effective when applied at reperfusion and therefore appropriate for clinical use.

Abstract P170: Nicorandil Inhibits Gáq-Induced Chronic Heart Failure in Transgenic Mice

Introduction: Beneficial effects of nicorandil on the treatment of hypertensive heart failure (HF) and ischemic heart disease have been suggested. However, whether nicorandil has inhibitory effects on HF and ventricular arrhythmias caused by the activation of G protein alpha q (Gαq) -coupled receptor (GPCR) signaling pathway still remains unknown. We examined effects of chronic and acute administration of nicorandil on the development of HF and ventricular action potential (VAP) in transgenic mice with transient cardiac expression of activated Gαq (Gαq-TG), respectively. Method and Results: Nicorandil (6 mg/kg/day) or vehicle was chronically administered in Gαq-TG mice for 24 weeks from 8 weeks of age, and then ventricular function, and electrical and structural changes were investigated in the hearts. Chronic nicorandil administration improved the reduction of left ventricular fractional shortening (p < 0.001) in Gαq-TG hearts. During 10 min of electrocardiogram recording, premature ventricular contractions (more than 20 beats/min) were observed in 7 of 10 vehicle-treated Gαq-TG but in none of 10 nicorandil-treated Gαq-TG hearts (p < 0.01). QT interval was significantly shorter in nicorandil-treated Gαq-TG than in vehicle-treated Gαq-TG hearts (p < 0.05). Chronic nicorandil administration improved the increased ventricular interstitial fibrosis (p < 0.05) but not cardiac hypertrophy in Gαq-TG left ventricles. Real time RT-PCR revealed that mRNA expression levels of s sulfonylurea receptor 2B (SUR-2B) were decreased in vehicle-treatd Gαq-TG but not in nicorandil-treated Gαq-TG. In addition, chronic nicorandil increased endotherial nitric oxide syntheses gene expression in Gαq-TG hearts (p < 0.05). Acute nicorandil administration (1 microM) significantly shortened the prolonged VAP duration and reduced the number of PVCs in vehicle treated Gαq-TG hearts. Conclusions: These findings suggest that nicorandil inhibits ventricular electrical and structural remodeling and arrhythmias through the shortening of VAP duration and the increased expression of SUR-2B and eNOS in a mouse model of HF.

Antioxidant activity contributes to flavonol cardioprotection during reperfusion of rat hearts

The mechanism of flavonol-induced cardioprotection is unclear. We compared the protective actions of a flavonol that inhibits calcium utilization and has antioxidant activity, 3′,4′-dihydroxyflavonol (DiOHF); a flavonol that affects only calcium activity, 4′-OH-3′-OCH3-flavonol (4′-OH-3′-OCH3F); and a water-soluble flavonol with selective antioxidant activity, DiOHF-6-succinamic acid (DiOHF-6-SA), in isolated, perfused rat hearts. Hearts were subjected to global ischemia for 20min followed by 30min reperfusion and were treated with vehicle (0.05% DMSO), DiOHF, 4′-OH-3′-OCH3F, or DiOHF-6-SA (all 10μM, n=5–8 per group). Flavonols were infused for 10min before ischemia and during reperfusion. In vehicle-treated hearts, left-ventricular (LV) +dP/dt was reduced by 60% at the end of reperfusion compared to the preischemic level. Lactate dehydrogenase (LDH) release was elevated and endothelial NO synthase (eNOS) expression was lower in vehicle-treated hearts compared to shams. In comparison, DiOHF treatment improved LV function upon reperfusion, decreased LDH, and preserved eNOS expression. The antioxidant DiOHF-6-SA also preserved contractility, reduced LDH, and preserved eNOS expression. In contrast, hearts treated with 4′-OH-3′-OCH3F showed a degree of contractile impairment similar to that of the vehicle group. DiOHF and DiOHF-6-SA also exerted cardioprotection when given only during reperfusion and not when administered only before ischemia. Flavonol-induced cardioprotection relies on antioxidant activity and is mainly exerted during reperfusion.

Intracoronary Mesenchymal Stem Cells Promote Postischemic Myocardial Functional Recovery, Decrease Inflammation, and Reduce Apoptosis via a Signal Transducer and Activator of Transcription 3 Mechanism

Signal transducer and activator of transcription 3 (STAT3) regulates myocardial apoptosis, cellular proliferation, and the immune response after ischemia/reperfusion (I/R). STAT3 is also necessary for the production of vascular endothelial growth factor (VEGF) by mesenchymal stem cells (MSCs), which are known to reduce myocardial injury after I/R. However, it remains unknown whether STAT3 is an important mediator of MSC-based cardioprotection. We hypothesized that knockout of stem cell STAT3 would reduce MSC-derived myocardial functional recovery and increase myocardial inflammatory and apoptotic signaling. With a Langendorff apparatus, male rat hearts were subjected to 15 minutes of equilibration and 25 minutes of ischemia, followed by 40 minutes of reperfusion. Immediately before ischemia, hearts received intracoronary infusions of vehicle, wild-type MSCs (WT MSCs) or STAT3 knockout MSCs (STAT3KO MSCs). Heart function was measured continuously. Myocardial homogenates were analyzed for production of interleukin (IL)-1, IL-6, and tumor necrosis factor-α (TNF-α). Additionally, MSC production of hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) were measured in vitro. Hearts treated with WT MSCs exhibited the greatest functional recovery, and those treated with STAT3KO MSCs had equivalent recovery to vehicle. The highest proinflammatory cytokine levels were seen in vehicle-treated hearts, and the lowest in the WT MSC group. STAT3KO MSCs produced less IGF-1, but more HGF than WT MSCs. Finally, hearts treated with STAT3KO MSCs or vehicle had significantly higher caspase-3 levels than those treated with WT MSCs. Intracoronary infusions of MSCs improve postischemic left ventricular function and reduce proapoptotic and proinflammatory signaling via a STAT3-dependent mechanism.

Estrogen Receptor-β Activation Results in S-Nitrosylation of Proteins Involved in Cardioprotection

It has been shown that the activation of estrogen receptor-beta (ER-beta) plays an important cardioprotective role against ischemia/reperfusion injury. However, the mechanism for this protection is not clear. We hypothesize that estrogen protects by ER-beta activation, which leads to S-nitrosylation (SNO) of key cardioprotective proteins. We treated ovariectomized C57BL/6J mice with the ER-beta selective agonist 2,2-bis(4-hydroxyphenyl)-proprionitrile (DPN), 17beta-estradiol (E2), or vehicle using Alzet minipumps for 2 weeks. Isolated hearts were Langendorff perfused and subjected to ischemia and reperfusion. Compared with vehicle-treated hearts, DPN- and E2-treated hearts had significantly better postischemic functional recovery and decreased infarct size. To test the specificity of DPN, we treated ER-beta-knockout mice with DPN. However, no cardioprotective effect of DPN was found in ER-beta-knockout mice, indicating that the DPN-induced cardioprotection occurs through the activation of ER-beta. Using DyLight-maleimide fluors and a modified biotin switch method, we used a 2-dimensional DyLight fluorescence difference gel electrophoresis proteomic method to quantify differences in SNO of proteins. DPN- and E2-treated hearts showed an increase in SNO of a number of proteins. Interestingly, many of these proteins also had been shown to have increased SNO in preconditioned hearts. In addition, the DPN-induced cardioprotection and increased SNO were abolished by treatment with a nitric oxide synthase inhibitor. The activation of ER-beta by DPN treatment leads to increased protein SNO and cardioprotection against ischemia/reperfusion injury, suggesting that long-term estrogen exposure protects hearts largely via activation of ER-beta and nitric oxide/SNO signaling.

Over-expression of calpastatin inhibits calpain activation and attenuates myocardial dysfunction during endotoxaemia

Lipopolysaccharide (LPS) induces cardiomyocyte caspase-3 activation and proinflammatory factors, in particular tumour necrosis factor-alpha (TNF-alpha) production, both of which contribute to myocardial dysfunction during sepsis. The present study was to investigate the roles of calpain/calpastatin system in cardiomyocyte caspase-3 activation, TNF-alpha expression, and myocardial dysfunction during LPS stimulation. In cultured adult rat cardiomyocytes, LPS (1 microg/mL) induced calpain and caspase-3 activity, and up-regulated TNF-alpha expression. These effects of LPS were abrogated by over-expression of calpastatin, an endogenous calpain inhibitor, transfection of calpain-1 siRNA, or various pharmacological calpain inhibitors. Furthermore, blocking gp91(phox)-NADPH oxidase prevented calpain and caspase-3 activation and decreased TNF-alpha expression in LPS-stimulated cardiomyocytes. To investigate the role of calpastatin in endotoxaemia, transgenic mice with calpastatin over-expression (CAST-Tg) and wild-type mice were treated with LPS (4 mg/kg, i.p.) or saline in the presence of calpain inhibitor-III (10 mg/kg, i.p.) for 4 h, and their heart function was measured with a Langendorff system. Over-expression of calpastatin significantly attenuated myocardial dysfunction (P < 0.05). Consistently, calpain activity, caspase-3 activity, and TNF-alpha expression were also reduced in CAST-Tg and calpain inhibitor-III compared with wild-type and vehicle-treated hearts, respectively. gp91(phox)-NADPH oxidase-mediated calpain-1 activation induces caspase-3 activation and TNF-alpha expression in cardiomyocytes during LPS stimulation. Over-expression of calpastatin inhibits calpain activation and improves myocardial function in endotoxaemia. The present study suggests that targeting calpain/calpastatin system may be a potential therapeutic intervention for septic hearts.