Working Heart Perfusion Research Articles

Obesity Is a Major Determinant of Impaired Cardiac Energy Metabolism in Heart Failure with Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is a major health problem with limited treatment options. Although optimizing cardiac energy metabolism is a potential approach to treating heart failure, it is poorly understood what alterations in cardiac energy metabolism actually occur in HFpEF. To determine this, we used mice in which HFpEF was induced using an obesity and hypertension HFpEF protocol for 10 weeks. Next, carvedilol, a third-generation β-blocker and a biased agonist that exhibits agonist-like effects through β arrestins by activating extracellular signal-regulated kinase, was used to decrease one of these parameters, namely hypertension. Heart function was evaluated by invasive pressure-volume loops and echocardiography as well as by ex vivo working heart perfusions. Glycolysis and oxidation rates of glucose, fatty acids, and ketones were measured in the isolated working hearts. The development of HFpEF was associated with a dramatic decrease in cardiac glucose oxidation rates, with a parallel increase in palmitate oxidation rates. Carvedilol treatment decreased the development of HFpEF but had no major effect on cardiac energy substrate metabolism. Carvedilol treatment did increase the expression of cardiac β arrestin 2 and proteins involved in mitochondrial biogenesis. Decreasing bodyweight in obese HFpEF mice increased glucose oxidation and improved heart function. This suggests that the dramatic energy metabolic changes in HFpEF mice hearts are primarily due to the obesity component of the HFpEF model. SIGNIFICANCE STATEMENT: Metabolic inflexibility occurs in heart failure with preserved ejection fraction (HFpEF) mice hearts. Lowering blood pressure improves heart function in HFpEF mice with no major effect on energy metabolism. Between hypertension and obesity, the latter appears to have the major role in HFpEF cardiac energetic changes. Carvedilol increases mitochondrial biogenesis and overall energy expenditure in HFpEF hearts.

Abstract 13208: A Small Molecule Macrophage Migration Inhibitory Factor Agonist Ameliorates Age-Related Myocardial Intolerance to Ischemia-Reperfusion Insults via Metabolic Regulation

Introduction: The macrophage migration inhibitory factor (MIF) is emerged as a critical mediator during myocardia ischemia and reperfusion (I/R). An impaired MIF signaling is occurred in aging. MIF agonist, MIF20, can argument cardiac MIF signaling through increasing the binding affinity between MIF and its receptor, CD74/CD44. Hypothesis: Administration of MIF20 can increase the resistance of aged myocardium to ischemic insults caused by I/R via modulating the MIF signaling regulated energy metabolism. Methods: Young (3-5 months) and aged (24-26 months) wild-type C57BL/6J mice, cardiomyocyte specific MIF knock out C57BL/6J mice (cMIF-/-) and wild type littermates (MIF flox/flox ) were injected with MIF20 (0.15 μg/kg, I.V.) 5 minutes before reperfusion after 45 minutes of ischemia. The left ventricular myocardium was for immunoblotting, NAD/NADH assay, and metabolomics analysis. The working heart perfusion and the SeaHorse system were for metabolic measurements. Results: The small molecule MIF agonist, MIF20, which binds to MIF to increase signaling through its cognate receptor CD74/CD44 not only decreases infarct size but also preserves cardiac systolic function in the aged versus young hearts. MIF20 contributes to the maintenance of mitochondrial fitness and preserves the contractility of cardiomyocytes under hypoxia and reoxygenation stress. These beneficial effects of MIF20 are reduced in cardiomyocyte-specific MIF knock-out (cMIF -/- ) versus littermates (MIF flox/flox ) mice, supporting the autocrine role of MIF in mediating ischemic cardiac protection. The age-related disruption of metabolic homeostasis in aged versus young hearts was modulated by MIF20’s ability to upregulate glucose oxidation and downregulate the oxidation of fatty acid. These actions were associated with an upregulation of pyruvate dehydrogenase kinase 4 (PDK4) and downregulation of Long-chain acyl-CoA dehydrogenase (LCAD). MIF20 as MIF signaling agonism modulation of metabolic adaptation in aged hearts further led to reduced generation of reactive oxygen species (ROS) in the myocardium. Conclusions: MIF agonists such as MIF20 may have a therapeutic role by limiting cardiac damage and rescuing cardiac dysfunctions of senescent hearts to I/R stress.

Significant Reduction of Myocardial Injury Markers After Ischemia Minimization in the First Hour of an Ex Vivo Xeno Working Heart Model

<h3>Purpose</h3> Previous studies showed ischemia minimization (IM), accomplished by perfusing the heart xenograft during storage, prevented ‘initial cardiac xenograft dysfunction' (ICXD) and prolonged survival following <i>in vivo</i> orthotopic cardiac xenotransplantation. We report initial observations in a model designed to evaluate effects associated with IM and genetic modifications on heart performance in a working <i>ex vivo</i> model. <h3>Methods</h3> Hearts from genetically modified (gm) and wildtype (WT) pigs were procured and stored for 3 hours either in cold saline (cold storage (CS)) or were perfused with oxygenated Steen's solution with RBCs (IM). The gm hearts either had combined knockouts of 3 xenogenic carbohydrate genes (GTKO, CMAHKO, β4GALNT2KO: TKO) with variable expression of human transgenes (n=13); or GTKO with expression of hCD55 (GTKO.hCD55; n=2). Heart function and troponin I as a marker for myocardial injury were assessed on a working heart rig while perfused with whole human blood. <h3>Results</h3> 19 hearts were perfused <i>ex vivo</i>, ten with CS (TKO n=6, GTKO.hCD55 n=1, WT n=3), 9 with IM (TKO n=7, GTKO.hCD55 n=1, WT n=1). Mean troponin I levels were significantly reduced after 1 hour of <i>ex vivo</i> perfusion in the IM group (70.2 ng/mL vs. 279.5 ng/mL; p = .038), but not at final time points (see fig. 1a-c). Cardiac function (cardiac output in response to increased filling pressures) decreased over time after CS, whereas the IM-stored heart function tended to improve over time (Fig. 2). <h3>Conclusion</h3> IM significantly decreased myocardial injury during the first hour of <i>ex vivo</i> working heart perfusion relative to CS hearts: heart injury (troponin release) was attenuated, and graft failure was delayed in some TKO hearts treated with IM. Evaluation of whether variation in expression of human transgenes may have influenced TKO graft protection from ICXD is in progress. We provisionally conclude that IM attenuates ICXD of genetically modified pig hearts during initial exposure to human blood.

Stellaria media tea protects against diabetes-induced cardiac dysfunction in rats without affecting glucose tolerance

Background and aimCommon chickweed (Stellaria media) tea has traditionally been applied for treatment of various metabolic diseases including diabetes in folk medicine; however, experimental evidence to support this practice is lacking. Therefore, we aimed to assess the effect of Stellaria media tea on glucose homeostasis and cardiac performance in a rat model of diabetes. Experimental procedureHot water extract of Stellaria media herb were analyzed and used in this study, where diabetes was induced by fructose-enriched diet supplemented with a single injection of streptozotocin. Half of the animals received Stellaria media tea (100 mg/kg) by oral gavage. At the end of the 20-week experimental period, blood samples were collected and isolated working heart perfusions were performed. Results and conclusionCompared to the animals receiving standard chow, serum fasting glucose level was increased and glucose tolerance was diminished in diabetic rats. Stellaria media tea did not affect significantly fasting hyperglycemia and glucose intolerance; however, it attenuated diabetes-induced deterioration of cardiac output and cardiac work. Analysis of the chemical composition of Stellaria media tea suggested the presence of rutin and various apigenin glycosides which have been reported to alleviate diabetic cardiomyopathy. Moreover, Stellaria media prevented diabetes-induced increase in cardiac STAT3 phosphorylation. We demonstrated for the first time that Stellaria media tea may beneficially affect cardiac dysfunction induced by diabetes without improvement of glucose homeostasis. Rutin and/or apigenin glycosides as well as modulation of STAT3 signaling may be implicated in the protection of Stellaria media tea against diabetic cardiomyopathy.

Abstract 15649: Pharmacological Inhibition of Aldose Reductase by AT001 Prevents Abnormal Cardiac Energy Metabolism and Improves Heart Function in an Animal Model of Diabetic Cardiomyopathy

Introduction: Diabetic Cardiomyopathy (DCM) is a major cause of death in people with type 2 diabetes (T2D). Alterations in cardiac energy metabolism including increased fatty acid oxidation rates and reduced glucose oxidation rates are key contributing factors to the development of DCM. Studies have shown that Aldose Reductase (AR), an enzyme activated under hyperglycemic conditions, can modulate myocardial glucose and fatty acid oxidation, and promotes cardiac dysfunction. Hypothesis: Pharmacological inhibition of AR using a next-generation inhibitor AT-001, can mitigate DCM in mice by modulating cardiac energy metabolism and improving cardiac efficiency. Methods: Male human AR overexpressing (hAR-Tg) and C57BL/6J (Control) mice were subjected to experimental T2D (high-fat diet [60% kcal from lard] for 10-wk with a single intraperitoneal streptozotocin injection of 75 mg/kg) and treated for the last 3-wk with AT-001 (40mg/kg/day) or vehicle via oral gavage. Cardiac energy metabolism and in vivo cardiac function were assessed via isolated working heart perfusions and ultrasound echocardiography, respectively. Results: AT-001 treatment significantly improved cardiac energetics in a murine model of DCM (hAR-Tg mice with T2D). Particularly, AT-001-treated mice exhibited decreased cardiac fatty acid oxidation rates compared to the vehicle-treated mice (342 ± 53 vs 964 ± 130 nmol/min/g dry wt.). Concurrently, there was a significant decrease in cardiac oxygen consumption in the AT-001-treated compared to the vehicle-treated mice (41 ± 12 vs 60 ± 11 μmol/min/g dry wt.), suggesting increased cardiac efficiency. Furthermore, treatment with AT-001 prevented cardiac structural and functional abnormalities present in DCM, including diastolic dysfunction as reflected by an increase in the tissue Doppler E’/A’ ratio and decrease in E/E’ ratio. Moreover, AT-001 treatment prevented cardiac hypertrophy as reflected by a decrease in LV mass in AT-001-treated mice. Conclusions: AR inhibition with AT-001 prevents cardiac structural and functional abnormalities in a mouse model of DCM, and normalizes cardiac energetics by shifting cardiac metabolism towards a non-diabetic metabolic state.

The role of beta-hydroxybutyrate as inducer for adult cardiomyocyte proliferation during reprogramming in vivo

Abstract Background Regional loss of myocardium is the dominant cause of heart failure due to coronary artery disease. In mammals, the response to injury shows some similarities with border zone myocytes remodeling, but little proliferation and repair occur in adult mammalian hearts. Several studies have identified some cell cycle-related genes essential for dramatically inducing neonatal CM proliferation, but the same effects cannot be replicated in adult CMs. This implies that there is an underlying mechanism (an inducer) that is yet to be discovered, that should first be activated in order to subsequently increase adult CM proliferation efficiently in vivo. Purpose To determine the inducer for the purpose of efficiently increasing adult CM proliferation, and to examine its therapeutic potential for heart regeneration after injury. Methods Transgenic mice (αMHC-rtTA-OSKM), whose adult CMs can be specifically induced by Yamanaka's OSKM factors, were generated to systemically examine the early stage of iPSC reprogramming after tetracycline treatment. These induced reprogramming CMs were further isolated for gene ontology analysis by microarray to determine the specific inducer for the purpose of efficiently increasing CM proliferation. Finally, the inducer was applied to a mouse model of myocardial infarction (MI) to confirm its therapeutic potential for heart regeneration after injury. Results OSKM expressions of αMHC-rtTA-OSKM were confirmed to only increase in CMs after tetracycline treatment in vivo, and BrdU+ CM% was more than 2-times higher in isolated adult CMs from αMHC-rtTA-OSKM mice treated with tetracycline for 2 days compared to PBS-treated mice. More H3P+/cTnT+ population% was also confirmed in this CM-disorganized reprogramming hearts compared to normal hearts. Microarray data revealed that the morphological changes of the reprogrammed CMs were due to a metabolic switch shown in gene ontology analysis. Thus, we profiled the metabolic switch in normal and CM reprogramming hearts by perfusing them with 13C-labeled metabolic substrates through the Langendorff ex-vivo working heart perfusion system. Through 13C NMR spectroscopy we determined that TCA cycle metabolites were 2-times lower while β-hydroxybutyrate were 2-times higher in the reprogrammed hearts compared to that in control hearts. Next, we surprisingly found that β-hydroxybutyrate supplement in MI mice at post-MI day 3 improved the heart function but showed the opposite effects when supplement was administered at post-MI day 7. Thus, the timing of β-hydroxybutyrate supplement is important for increasing adult CM proliferation for effectiveness of heart regeneration after MI. Conclusion This is the first study to identify the metabolism shift to increase β-hydroxybutyrate as the inducer for the purpose of efficiently increasing adult CM proliferation, which supports heart function improvement when treating at short period immediately after MI. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): 2020 Academia Sinica Young Scholarship Travel Award

Kolaviron offered a novel cardioprotection against ischemic/reperfusion injury by modulation of Akt/protein kinase B (PKB)/p38MAPK/Caspase 3/poly‐ADP‐ribose polymerase signaling pathway

Free radical generation and oxidative stress have been implicated in the pathogenesis and pathophysiology of ischemic/reperfusion injury. Kolaviron is a bioflavonoid with arrays of biological and pharmacological activities. The study was designed to investigate the modulatory role of Kolaviron on ischemic/reperfusion injury in experimental animal models. Twenty male Wistar albino rats were randomly divided into two groups of ten rats per group: Group 1 received corn oil as a vehicle (Control) and rats in Group 2 were pre‐treated with Kolaviron at 200 mg/kg for 4 weeks. After 4 weeks of Kolaviron administration, hearts were excised and mounted on the working heart perfusion system. Western blot analysis for protein expressions was carried out on frozen heart samples. Results revealed significant (p &lt; 0.05) reduction in the activity of cardiac catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)with concomitant reduction in oxygen radical absorbance capacity (ORAC) in ischemic rat heart of Control compared to group pre‐treated with Kolaviron. Similarly, intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) contents were significantly elevated in control compared to Kolaviron pre‐treated rats. Western blot showed that pre‐treatment with Kolaviron significantly increased total Akt/protein kinase B (PKB), phosphorylated Akt/PKB at serine 473 with concomitant significant reduction in the expressions of p38 mitogen‐activated protein kinase (p38MAPK), Caspase 3, and cleaved poly adenosine diphosphate ribose polymerase (PARP). Taken together, Kolaviron offered significant cardioprotection via free radical scavenging activity and upregulation of pro‐survival signaling pathway.

Cytochrome P450‐Derived Epoxy Lipids of N‐3 PUFAs Protect the Heart From Ischemia‐Reperfusion Injury by Regulating Mitochondrial Sirtuin 3

ObjectiveCytochrome P450 epoxygenases can metabolize dietary n‐3 polyunsaturated fatty acids (n‐3 PUFAs) to epoxy lipids, which have known cardioprotective effects; however, it remains unclear how this cardioprotection occurs. Our recent data demonstrate that cytochrome P450 epoxygenase‐derived metabolites of the n‐3 PUFA docosahexaenoic acid (DHA), 19,20‐epoxydocosapentaenoic acid (19,20‐EDP), exhibit protective effects toward cardiac mitochondria. Maintaining mitochondrial quality is essential to limit myocardial damage following ischemia‐reperfusion (IR) injury. The mitochondrial deacetylase sirtuin 3 (SIRT3) plays a pivotal role in the maintenance of mitochondrial function and cardiac energy metabolism. We hypothesize that the cardioprotective mechanism(s) of 19,20‐EDP against IR injury involve(s) stimulating mitochondrial SIRT3.MethodsLangendorff and isolated working heart perfusions were performed in male and female C57BL/6 mice to assess changes in cardiac function and energy metabolism in response to IR injury. Hearts were perfused with either vehicle, 19,20‐EDP (1 μM) and/or the SIRT3 inhibitor nicotinamide (30 μM) for a 20 min baseline perfusion, following which hearts were subjected to 30 min of global no flow ischemia and 40 min of reperfusion. Glucose and palmitate oxidation rates were measured to detect alterations in cardiac energy metabolism. Mitochondrial fractions were assessed for changes in SIRT3 activity and total lysine acetylation levels. Mitochondrial SIRT3 downstream targets were assessed in human ventricular tissues obtained from individuals with ischemic heart disease (IHD) included in the Human Explanted Heart Program and compared to non‐failing control hearts (NFC) collected from unused transplant donors through the Human Organ Procurement and Exchange Program at the University of Alberta. In addition, mitochondrial respiration was assessed in cardiac fibers isolated from the human tissues.ResultsHearts perfused with 19,20‐EDP demonstrated improved post‐ischemic functional recovery, glucose oxidation and cardiac efficiency compared to control groups. The cardioprotective effects were associated with enhanced mitochondrial SIRT3 activity and reduced global protein acetylation. We observed increased acetylation of the mitochondrial antioxidant superoxide dismutase 2 in tissues obtained from IHD human myocardium suggesting decreased antioxidant capacity. Treatment with 19,20‐EDP also markedly improved mitochondrial respiration in permeabilized human fibers obtained from IHD hearts. Importantly, the cardioprotective effects of 19,20‐EDP was abolished by the SIRT3 inhibitor nicotinamide.ConclusionWe demonstrate that 19,20‐EDP‐mediated cardioprotective mechanisms against IR injury involve preservation of mitochondrial SIRT3 activity, which results in improved cardiac efficiency.

Prediabetes Induced by Fructose-Enriched Diet Influences Cardiac Lipidome and Proteome and Leads to Deterioration of Cardiac Function prior to the Development of Excessive Oxidative Stress and Cell Damage.

Prediabetes is a condition affecting more than 35% of the population. In some forms, excessive carbohydrate intake (primarily refined sugar) plays a prominent role. Prediabetes is a symptomless, mostly unrecognized disease which increases cardiovascular risk. In our work, we examined the effect of a fructose-enriched diet on cardiac function and lipidome as well as proteome of cardiac muscle. Male Wistar rats were divided into two groups. The control group received a normal diet while the fructose-fed group received 60% fructose-supplemented chow for 24 weeks. Fasting blood glucose measurement and oral glucose tolerance test (OGTT) showed slightly but significantly elevated values due to fructose feeding indicating development of a prediabetic condition. Both echocardiography and isolated working heart perfusion performed at the end of the feeding protocol demonstrated diastolic cardiac dysfunction in the fructose-fed group. Mass spectrometry-based, high-performance lipidomic and proteomic analyses were executed from cardiac tissue. The lipidomic analysis revealed complex rearrangement of the whole lipidome with special emphasis on defects in cardiolipin remodeling. The proteomic analysis showed significant changes in 75 cardiac proteins due to fructose feeding including mitochondria-, apoptosis-, and oxidative stress-related proteins. Nevertheless, just very weak or no signs of apoptosis induction and oxidative stress were detected in the hearts of fructose-fed rats. Our results suggest that fructose feeding induces marked alterations in the cardiac lipidome, especially in cardiolipin remodeling, which leads to mitochondrial dysfunction and impaired cardiac function. However, at the same time, several adaptive responses are induced at the proteome level in order to maintain a homeostatic balance. These findings demonstrate that even very early stages of prediabetes can impair cardiac function and can result in significant changes in the lipidome and proteome of the heart prior to the development of excessive oxidative stress and cell damage.

Trabecular cutting: a novel surgical therapy to increase diastolic compliance.

Heart failure with preserved ejection fraction (HFpEF) is a common cause of hospital admission in patients over 65 yr old and has high mortality. HFpEF is characterized by left ventricular (LV) hypertrophy that reduces compliance. Current HFpEF therapies control symptoms, but no existing medications or therapies can sustainably increase LV compliance. LV trabeculae develop hypertrophy and fibrosis that contribute to reduced LV compliance. This study expands our previous results in ex vivo human hearts to show that severing LV trabeculae increases diastolic compliance in an ex vivo working rabbit heart model. Trabecular cutting was performed in ex vivo rabbit hearts set up in a working heart perfusion system perfused with oxygenated Krebs-Henseleit buffer. A hook was inserted in the LV to cut trabeculae. End-systolic and end-diastolic pressure-volume relationships during transient preload reduction were recorded using an admittance catheter in the following three groups: control (no cutting; n = 9), mild cutting (15 cuts; n = 5), and aggressive cutting (30 cuts; n = 5). In a second experiment, each heart served as its own control. Hemodynamic data were recorded before and after trabecular cutting (n = 10) or sham cutting (n = 5) within the same heart. In the first experiments, trabecular cutting did not affect systolic function (P > 0.05) but significantly increased overall diastolic compliance (P = 0.009). Greater compliance was seen as trabecular cutting increased (P = 0.002, r2 = 0.435). In the second experiment, significant increases in systolic function (P = 0.048) and diastolic compliance (P = 0.002) were seen after trabecular cutting compared with baseline. In conclusion, trabecular cutting significantly increases diastolic compliance without reducing systolic function.NEW & NOTEWORTHY We postulate that, in mammalian hearts, free-running trabeculae carneae exist to provide tensile support to the left ventricle and minimize diastolic wall stress. Because of hypertrophy and fibrosis of trabeculae in patients with left ventricular hypertrophy, this supportive role can become pathologic, worsening diastolic compliance. We demonstrate a novel operation involving cutting trabeculae as a method to acutely increase diastolic compliance in patients presenting with heart failure and diastolic dysfunction to improve their left ventricle compliance.

Kolaviron abrogates ischaemic heart reperfusion injury through enhancement of Akt/PKB and down‐regulation of p38MAPK/PARP/Caspase 3 signalling pathways

The study was designed to investigate the ameliorative effect of Kolaviron (KV) on ischaemic‐reperfusion injury in experimental rat models. Twenty male Wistar rats were randomly divided into 2 groups: Group one received corn oil as a vehicle and rats in group two were orally administered KV at 200 mg/kg for four weeks. After four weeks of KV pre‐treatment, hearts were excised and mounted on the Working heart perfusion system. Western blot analysis was carried out on frozen heart samples. From this study, there was a significant (p&lt;0.05) reduction in the activity of catalase, superoxide dismutase and glutathione peroxidase with concomitant reduction in oxygen radical absorbance capacity (ORAC) in ischaemic rat heart of animals that received corn oil without KV pre‐treatment. Similarly, pre‐treatment of rats with KV led to a significant (p&lt;0.05) decline in intracellular reactive oxygen species (ROS) and malondialdehyde. Western blots analyses revealed that KV significantly increased the expressions of total Akt/PKB, phosphorylated Akt/PKB at serine 473 and also caused a significant reduction in p38 MAPK, Caspase 3, and cleaved PARP. Combining all, we propose that KV offered notable cardioprotection via quenching of ROS and up‐regulation of pro‐survival sinalling pathways. KV may therefore offer a novel therepeutic window for cardiovascular and other heart related conditionsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardiac-Specific Deletion of Pyruvate Dehydrogenase Impairs Glucose Oxidation Rates and Induces Diastolic Dysfunction.

Obesity and type 2 diabetes (T2D) increase the risk for cardiomyopathy, which is the presence of ventricular dysfunction in the absence of underlying coronary artery disease and/or hypertension. As myocardial energy metabolism is altered during obesity/T2D (increased fatty acid oxidation and decreased glucose oxidation), we hypothesized that restricting myocardial glucose oxidation in lean mice devoid of the perturbed metabolic milieu observed in obesity/T2D would produce a cardiomyopathy phenotype, characterized via diastolic dysfunction. We tested our hypothesis via producing mice with a cardiac-specific gene knockout for pyruvate dehydrogenase (PDH, gene name Pdha1), the rate-limiting enzyme for glucose oxidation. Cardiac-specific Pdha1 deficient (Pdha1Cardiac−/−) mice were generated via crossing a tamoxifen-inducible Cre expressing mouse under the control of the alpha-myosin heavy chain (αMHC-MerCreMer) promoter with a floxed Pdha1 mouse. Energy metabolism and cardiac function were assessed via isolated working heart perfusions and ultrasound echocardiography, respectively. Tamoxifen administration produced an ~85% reduction in PDH protein expression in Pdha1Cardiac−/− mice versus their control littermates, which resulted in a marked reduction in myocardial glucose oxidation and a corresponding increase in palmitate oxidation. This myocardial metabolism profile did not impair systolic function in Pdha1Cardiac−/− mice, which had comparable left ventricular ejection fractions and fractional shortenings as their αMHC-MerCreMer control littermates, but did produce diastolic dysfunction as seen via the reduced mitral E/A ratio. Therefore, it does appear that forced restriction of glucose oxidation in the hearts of Pdha1Cardiac−/− mice is sufficient to produce a cardiomyopathy-like phenotype, independent of the perturbed metabolic milieu observed in obesity and/or T2D.

Cardiomyocyte-specific deletion of Sirt1 gene sensitizes myocardium to ischaemia and reperfusion injury.

A longevity gene, Sirtuin 1 (SIRT1) and energy sensor AMP-activated protein kinase (AMPK) have common activators such as caloric restriction, oxidative stress, and exercise. The objective of this study is to characterize the role of cardiomyocyte SIRT1 in age-related impaired ischemic AMPK activation and increased susceptibility to ischemic insults. Mice were subjected to ligation of left anterior descending coronary artery for in vivo ischemic models. The glucose and fatty acid oxidation were measured in a working heart perfusion system. The cardiac functions by echocardiography show no difference in young wild-type C57BL/6 J (WT, 4-6 months), aged WT C57BL/6 J (24-26 months), and young inducible cardiomyocyte-specific SIRT1 knockout (icSIRT1 KO) (4-6 months) mice under physiological conditions. However, after 45 mins ischaemia and 24-h reperfusion, the ejection fraction of aged WT and icSIRT1 KO mice was impaired. The aged WT and icSIRT1 KO hearts vs. young WT hearts also show an impaired post-ischemic contractile function in a Langendorff perfusion system. The infarct size of aged WT and icSIRT1 KO hearts was larger than that of young WT hearts. The immunoblotting data demonstrated that aged WT and icSIRT1 KO hearts vs. young WT hearts had impaired phosphorylation of AMPK and downstream acetyl-CoA carboxylase during ischaemia. Intriguingly, AMPK upstream LKB1 is hyper-acetylated in both aged WT and icSIRT1 KO hearts; this could blunt activation of LKB1, leading to an impaired AMPK activation. The working heart perfusion results demonstrated that SIRT1 deficiency significantly impaired substrate metabolism in the hearts; fatty acid oxidation is augmented and glucose oxidation is blunted during ischaemia and reperfusion. Adeno-associated virus (AAV9)-Sirt1 was delivered into the aged hearts via a coronary delivery approach, which significantly rescued the protein level of SIRT1 and the ischemic tolerance of aged hearts. Furthermore, AMPK agonist can rescue the tolerance of aged heart and icSIRT1 KO heart to ischemic insults. Cardiac SIRT1 mediates AMPK activation via LKB1 deacetylation, and AMPK modulates SIRT1 activity via regulation of NAD+ level during ischaemia. SIRT1 and AMPK agonists have therapeutic potential for treatment of aging-related ischemic heart disease.

FoxO1 regulates myocardial glucose oxidation rates via transcriptional control of pyruvate dehydrogenase kinase 4 expression.

Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation and a critical regulator of metabolic flexibility during the fasting to feeding transition. PDH is regulated via both PDH kinases (PDHK) and PDH phosphatases, which phosphorylate/inactivate and dephosphorylate/activate PDH, respectively. Our goal was to determine whether the transcription factor forkhead box O1 (FoxO1) regulates PDH activity and glucose oxidation in the heart via increasing the expression of Pdk4, the gene encoding PDHK4. To address this question, we differentiated H9c2 myoblasts into cardiac myocytes and modulated FoxO1 activity, after which Pdk4/PDHK4 expression and PDH phosphorylation/activity were assessed. We assessed binding of FoxO1 to the Pdk4 promoter in cardiac myocytes in conjunction with measuring the role of FoxO1 on glucose oxidation in the isolated working heart. Both pharmacological (1 µM AS1842856) and genetic (siRNA mediated) inhibition of FoxO1 decreased Pdk4/PDHK4 expression and subsequent PDH phosphorylation in H9c2 cardiac myocytes, whereas 10 µM dexamethasone-induced Pdk4/PDHK4 expression was abolished via pretreatment with 1 µM AS1842856. Furthermore, transfection of H9c2 cardiac myocytes with a vector expressing FoxO1 increased luciferase activity driven by a Pdk4 promoter construct containing the FoxO1 DNA-binding element region, but not in a Pdk4 promoter construct lacking this region. Finally, AS1842856 treatment in fasted mice enhanced glucose oxidation rates during aerobic isolated working heart perfusions. Taken together, FoxO1 directly regulates Pdk4 transcription in the heart, thereby controlling PDH activity and subsequent glucose oxidation rates.NEW & NOTEWORTHY Although studies have shown an association between FoxO1 activity and pyruvate dehydrogenase kinase 4 expression, our study demonstrated that pyruvate dehydrogenase kinase 4 is a direct transcriptional target of FoxO1 (but not FoxO3/FoxO4) in the heart. Furthermore, we report here, for the first time, that FoxO1 inhibition increases glucose oxidation in the isolated working mouse heart.

Abstract 12526: Snf1-related Kinase Improves Cardiac Mitochondrial Efficiency by Decreasing Mitochondrial Uncoupling

Introduction: Ischemic heart disease limits oxygen and metabolic substrate availability to the heart, resulting in cardiac tissue death. Snf1-related Kinase (SNRK) is a serine/threonine kinase with sequence similarity to AMP-activated protein kinase, but its function is unknown. We found that SNRK is increased in hearts from patients with ischemic cardiomyopathy, and our gene array data suggested that SNRK alters metabolic genes. Here, we assessed the hypothesis that SNRK improves cardiac metabolic efficiency and mitochondrial uncoupling, and protects against ischemia/reperfusion (I/R) injury. Results: Cardiac-specific SNRK transgenic (TG) mice displayed decreased glycolysis, glucose oxidation, and palmitate oxidation compared to wild type littermate controls in perfused working hearts. However, cardiac power, contractility, ATP, and triglyceride and glycogen stores were maintained, while oxygen consumption was reduced, demonstrating that SNRK TG mice have increased cardiac metabolic efficiency. Consistent with improved cardiac metabolic efficiency, SNRK TG mice exhibited decreased infarct size after I/R. Mitochondria from SNRK TG mice also had reduced respiration, and increased respiratory control ratio and mitochondrial membrane potential, indicating reduced mitochondrial uncoupling. Uncoupling protein 3 (UCP3) levels were also decreased. Conversely, hearts from heterozygous SNRK knockout mice had increased glucose and palmitate oxidation and UCP3. SNRK knockdown in cardiac cells decreased mitochondrial efficiency, which was abolished with UCP3 knockdown. To determine the mechanism for the regulation of UCP3 by SNRK, we performed yeast two-hybrid assay, and identified tribbles homolog 3 (Trib3) to bind to SNRK. Furthermore, Trib3 enabled downregulation of UCP3 by downregulating the expression of PPARα. Conclusions: Our results demonstrate that overexpression of SNRK decreases cardiac metabolic substrate usage and oxygen consumption but maintains cardiac function and enhanced mitochondrial coupling. We also identify a binding partner of SNRK, Trib3, which is upregulated by SNRK in mice and mediates the regulation of PPARα and UCP3 by SNRK.

Abstract 12050: Cardiomyocyte Specific Deletion of Sirt1 Gene Alters Substrate Metabolism and Sensitizes Myocardium to Ischemia and Reperfusion Injury

Introduction: A longevity gene, sirtuin 1 (SIRT1) is a conserved NAD + -dependent protein deacetylase. AMP-activated protein kinase (AMPK) is an energy sensor. AMPK and SIRT1 signaling pathways have common activators such as caloric restriction, oxidative stress and exercise. Hypothesis: Aging-related impaired ischemic AMPK activation and increased susceptibility to ischemic insults are due to a decreased SIRT1 levels in aging. Methods: Mice were subjected to ligation of left anterior descending coronary artery for in vivo ischemic models. The glucose and fatty acid oxidation were measured in a working heart perfusion system. Results: The cardiac functions by echocardiography show no difference in young (SIRT1 flox/flox ), aged (SIRT1 flox/flox ) and young inducible cardiomyocyte specific SIRT1 knockout (icSIRT1 KO) mice under physiological conditions, but after 45 mins ischemia and 24 hours reperfusion, the ejection fraction of aged and icSIRT1 KO mice were impaired. The aged and icSIRT1 KO hearts versus young hearts also show an impaired post-ischemic contractile function in Langendorff perfusion system. The infarct size of aged and icSIRT1 KO hearts was larger than that of young hearts (p&lt;0.05). The immunoblotting data demonstrated that aged and icSIRT1 KO hearts versus young hearts had impaired phosphorylation of AMPK and downstream acetyl-CoA carboxylase during ischemia. Intriguingly, AMPK upstream LKB1 is hyper-acetylated in both aged and icSIRT1 KO hearts that could blunt activation of LKB1, leading to an impaired AMPK activation. The working heart perfusion results demonstrated that SIRT1 deficiency significantly impair substrate metabolism in the hearts, fatty acid oxidation is augmented and glucose oxidation is blunted during ischemia and reperfusion. Aged and icSIRT1 KO hearts versus young hearts demonstrated significant cardiac fibrosis and macrophage infiltration during ischemia and reperfusion. Furthermore, AMPK agonist A769662 treatment can rescue the tolerance of aged heart but not icSIRT1 KO hearts to ischemic insults. Conclusions: Cardiac SIRT1 mediates AMPK activation via LKB1 deacetylation during ischemia. SIRT1 and AMPK agonists have therapeutic potential for treatment of aging-related ischemic heart disease.

Differential stress response mechanisms in right and left ventricles

Right ventricular (RV) failure is the major cause of death among patients with pulmonary hypertension. However, differences between the RV and left ventricle (LV) of the adult heart have not been defined, despite myocytes from these two ventricles originate from different progenitor cells. The lack of such knowledge interferes with developing therapeutic strategies to protect the RV. The goal of this study was to identify possible differences between stress responses in the RV and LV free walls of adult rats. We found that levels of angiogenesis and autophagy/mitophagy proteins are higher in the LV than in the RV. Thus, the LV may be more resistant to stress-induced damage. To test this, isolated rat hearts were subjected to biventricular working heart perfusion and ischemia/reperfusion (I/R) injury. However, I/R was found to cause apoptosis in both LV and RV to a similar extent. One mechanism of cardiac apoptosis involves downregulation of GATA4 transcription factor that controls gene transcription of anti-apoptotic Bcl-xL. Interestingly, only in the RV, I/R caused downregulation of GATA4 and Bcl-xL, suggesting that mechanisms of apoptosis may be different between the two ventricles. Levels of tropomyosin and troponin T were also found to be decreased in response to I/R only in the RV, but not in the LV. Downregulation of the GATA4/Bcl-xL axis and the reduction of tropomyosin and troponin T are RV-specific events that occur in response to stress. This information may be useful for designing RV-specific therapeutic strategies to treat RV failure in pulmonary hypertension patients.

Kolaviron, a biflavonoid of Garcinia kola seed mitigates ischemic/reperfusion injury by modulation of pro-survival and apoptotic signaling pathways.

The study was designed to investigate the ameliorative effect of Kolaviron (KV) on ischemic/reperfusion injury in experimental animal models. Male Wistar rats were randomly divided into two groups: Group 1 received corn oil as a vehicle and rats in Group 2 were administered KV at 200 mg/kg for 4 weeks. The rats were fed with rat standard chow pellet and water administered ad libitum. After 4 weeks of KV administration, hearts were excised and mounted on the working heart perfusion system. Western blot analysis for protein expression was carried out on frozen heart samples. There was significant (P < 0.05) reduction in the activity of catalase, superoxide dismutase, and glutathione peroxidase with concomitant reduction in oxygen radical absorbance capacity in ischemic rat heart of control compared to group pre-treated with KV, respectively. Similarly, intracellular reactive oxygen species and malondialdehyde were significantly elevated in control compared to KV pre-treated rats. KV significantly increased total Akt/protein kinase B (PKB), phosphorylated Akt/PKB at serine 473 and also caused a significant reduction in p38 mitogen-activated protein kinase, Caspase 3, and cleaved poly adenosine diphosphate ribose polymerase. Taken together, KV offered significant cardioprotection via free radical scavenging activity and upregulation of pro-survival pathway.

Cardiac-Specific Deletion of the Pdha1 Gene Sensitizes Heart to Toxicological Actions of Ischemic Stress.

Pyruvate dehydrogenase (PDH) plays a key role in aerobic energy metabolism and occupies a central crossroad between glycolysis and the tricarboxylic acid cycle. We generated inducible cardiac-specific PDH E1α knockout (CreER(T2)-PDH(flox/flox)) mice that demonstrated a high mortality rate. It was hypothesized that PDH modulating cardiac glucose metabolism is crucial for heart functions under normal physiological and/or stress conditions. The myocardial infarction was conducted by a ligation of the left anterior descending coronary arteries. Cardiac PDH E1α deficiency caused large myocardial infarcts size and macrophage infiltration in the hearts (P < .01 vs wild-type [WT]). Wheat germ agglutinin and Masson trichrome staining revealed significantly increased hypertrophy and fibrosis in PDH E1α-deficient hearts (P < .05 vs WT). Measurements of heart substrate metabolism in an ex vivo working heart perfusion system demonstrated a significant impairment of glucose oxidation in PDH E1α-deficient hearts during ischemia/reperfusion (P < .05 vs WT). Dichloroacetate, a PDH activator, increased glucose oxidation in WT hearts during ischemia/reperfusion and reduced myocardial infarct size in WT, but not in PDH E1α-deficient hearts. Immunoblotting results demonstrated that cardiac PDH E1α deficiency leads to an impaired ischemic AMP-activated protein kinase activation through Sestrin2-liver kinase B1 interaction which is responsible for an increased susceptibility of PDH E1α-deficient heart to ischemic insults. Thus, cardiac PDH E1α deficiency impairs ischemic AMP-activated protein kinase signaling and sensitizes hearts to the toxicological actions of ischemic stress.

Comprehensive metabolic modeling of multiple 13C-isotopomer data sets to study metabolism in perfused working hearts.

In many forms of cardiomyopathy, alterations in energy substrate metabolism play a key role in disease pathogenesis. Stable isotope tracing in rodent heart perfusion systems can be used to determine cardiac metabolic fluxes, namely those relative fluxes that contribute to pyruvate, the acetyl-CoA pool, and pyruvate anaplerosis, which are critical to cardiac homeostasis. Methods have previously been developed to interrogate these relative fluxes using isotopomer enrichments of measured metabolites and algebraic equations to determine a predefined metabolic flux model. However, this approach is exquisitely sensitive to measurement error, thus precluding accurate relative flux parameter determination. In this study, we applied a novel mathematical approach to determine relative cardiac metabolic fluxes using 13C-metabolic flux analysis (13C-MFA) aided by multiple tracer experiments and integrated data analysis. Using 13C-MFA, we validated a metabolic network model to explain myocardial energy substrate metabolism. Four different 13C-labeled substrates were queried (i.e., glucose, lactate, pyruvate, and oleate) based on a previously published study. We integrated the analysis of the complete set of isotopomer data gathered from these mouse heart perfusion experiments into a single comprehensive network model that delineates substrate contributions to both pyruvate and acetyl-CoA pools at a greater resolution than that offered by traditional methods using algebraic equations. To our knowledge, this is the first rigorous application of 13C-MFA to interrogate data from multiple tracer experiments in the perfused heart. We anticipate that this approach can be used widely to study energy substrate metabolism in this and other similar biological systems.