Diastolic dysfunction in familial hypertrophic cardiomyopathy transgenic model mice

Aims Several mutations in the ventricular myosin regulatory light chain (RLC) were identified to cause familial hypertrophic cardiomyopathy (FHC). Based on our previous cellular findings showing delayed calcium transients in electrically stimulated intact papillary muscle fibres from transgenic Tg-R58Q and Tg-N47K mice and, in addition, prolonged force transients in Tg-R58Q fibres, we hypothesized that the malignant FHC phenotype associated with the R58Q mutation is most likely related to diastolic dysfunction. Methods and results Cardiac morphology and in vivo haemodynamics by echocardiography as well as cardiac function in isolated perfused working hearts were assessed in transgenic (Tg) mutant mice. The ATPase-pCa relationship was determined in myofibrils isolated from Tg mouse hearts. In addition, the effect of both mutations on RLC phosphorylation was examined in rapidly frozen ventricular samples from Tg mice. Significantly, decreased cardiac function was observed in isolated perfused working hearts from both Tg-R58Q and Tg-N47K mice. However, echocardiographic examination showed significant alterations in diastolic transmitral velocities and deceleration time only in Tg-R58Q myocardium. Likewise, changes in Ca2+ sensitivity, cooperativity, and an elevated level of ATPase activity at low [Ca2+] were only observed in myofibrils from Tg-R58Q mice. In addition, the R58Q mutation and not the N47K led to reduced RLC phosphorylation in Tg ventricles. Conclusion Our results suggest that the N47K and R58Q mutations may act through similar mechanisms, leading to compensatory hypertrophy of the functionally compromised myocardium, but the malignant R58Q phenotype is most likely associated with more severe alterations in cardiac performance manifested as impaired relaxation and global diastolic dysfunction. At the molecular level, we suggest that by reducing the phosphorylation of RLC, the R58Q mutation decreases the kinetics of myosin cross-bridges, leading to an increased myofilament calcium sensitivity and to overall changes in intracellular Ca2+ homeostasis

Insulin directly stimulates mitochondrial glucose oxidation in the heart

Abstract Background Glucose oxidation is a major contributor to myocardial energy production and its contribution is orchestrated by insulin. While insulin can increase glucose oxidation indirectly by enhancing glucose uptake and glycolysis, it also directly stimulates mitochondrial glucose oxidation, independent of increasing glucose uptake or glycolysis, through activating mitochondrial pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. However, how insulin directly stimulates PDH is not known. To determine this, we characterized the impacts of modifying mitochondrial insulin signaling kinases, namely protein kinase B (Akt), protein kinase C-delta (PKC-δ) and glycogen synthase kinase-3 beta (GSK-3β), on the direct insulin stimulation of glucose oxidation. Methods We employed an isolated working mouse heart model to measure the effect of insulin on cardiac glycolysis, glucose oxidation and fatty acid oxidation and how that could be affected when mitochondrial Akt, PKC-δ or GSK-3β is disturbed using pharmacological modulators. We also used differential centrifugation to isolate mitochondrial and cytosol fraction to examine the activity of Akt, PKC-δ and GSK-3β between these fractions. Data were analyzed using unpaired t-test and two-way ANOVA. Results Here we show that insulin-stimulated phosphorylation of mitochondrial Akt is a prerequisite for transducing insulin’s direct stimulation of glucose oxidation. Inhibition of mitochondrial Akt completely abolishes insulin-stimulated glucose oxidation, independent of glucose uptake or glycolysis. We also show a novel role of mitochondrial PKC-δ in modulating mitochondrial glucose oxidation. Inhibition of mitochondrial PKC-δ mimics insulin stimulation of glucose oxidation and mitochondrial Akt. We also demonstrate that inhibition of mitochondrial GSK3β phosphorylation does not influence insulin-stimulated glucose oxidation. Conclusion We identify, for the first time, insulin-stimulated mitochondrial Akt as a prerequisite transmitter of the insulin signal that directly stimulates cardiac glucose oxidation. These novel findings suggest that targeting mitochondrial Akt is a potential therapeutic approach to enhance cardiac insulin sensitivity in condition such as heart failure, diabetes and obesity

Regulating cardiac energy metabolism and bioenergetics by targeting the DNA damage repair protein BRCA1

ObjectiveAlterations in cardiac energy and substrate metabolism play a critical role in the development and clinical course of heart failure. We hypothesized that the cardioprotective role of the breast cancer 1, early onset (BRCA1) gene might be mediated in part by alterations in cardiac bioenergetics.MethodsWe generated cardiomyocyte-specific BRCA1 homozygous and heterozygous knockout mice using the Cre-loxP technology and evaluated the key molecules and pathways involved in glucose metabolism, fatty acid metabolism, and mitochondrial bioenergetics.ResultsCardiomyocyte-specific BRCA1-deficient mice showed reduced cardiac expression of glucose and fatty acid transporters, reduced acetyl-coenzyme A carboxylase 2 and malonyl-coenzyme A decarboxylase (key enzymes that control malonyl coenzyme A, which in turn controls fatty acid oxidation), and reduced carnitine palmitoyltransferase I, a rate-limiting enzyme for mitochondrial fatty acid uptake. Peroxisome proliferator-activated receptor α and γ and carnitine palmitoyltransferase I levels were also downregulated in these hearts. Rates of glucose and fatty acid oxidation were reduced in the hearts of heterozygous cardiomyocyte-restricted BRCA1-deficient mice, resulting in a decrease in the rate of adenosine triphosphate production. This decrease in metabolism and adenosine triphosphate production occurred despite an increase in 5′-adenosine monophosphate-activated protein kinase and AKT activation in the heart.ConclusionsCardiomyocyte-specific loss of BRCA1 alters critical pathways of fatty acid and glucose metabolism, leading to an energy starved heart. BRCA1-based cell or gene therapy might serve as a novel target to improve cardiac bioenergetics in patients with heart failure

Targeting the glucagon receptor improves cardiac function and enhances insulin sensitivity following a myocardial infarction

Abstract Background In heart failure the myocardium becomes insulin resistant which negatively influences cardiac energy metabolism and function, while increasing cardiac insulin signalling improves cardiac function and prevents adverse remodelling in the failing heart. Glucagon’s action on cardiac glucose and lipid homeostasis counteract that of insulin’s action. We hypothesised that pharmacological antagonism of myocardial glucagon action, using a human monoclonal antibody (mAb A) against glucagon receptor (GCGR), a G-protein coupled receptor, will enhance insulin sensitivity and improve cardiac energy metabolism and function post myocardial infarction (MI). Methods Male C57BL/6 mice were subjected to a permanent left anterior descending coronary artery ligation to induce MI, following which they received either saline or mAb A (4 mg kg−1 week−1 starting at 1 week post-MI) for 3 weeks. Results Echocardiographic assessment at 4 weeks post-MI showed that mAb A treatment improved % ejection fraction (40.0 ± 2.3% vs 30.7 ± 1.7% in vehicle-treated MI heart, p < 0.05) and limited adverse remodelling (LV mass: 129 ± 7 vs 176 ± 14 mg in vehicle-treated MI hearts, p < 0.05) post MI. In isolated working hearts an increase in insulin-stimulated glucose oxidation was evident in the mAb A-treated MI hearts (1661 ± 192 vs 924 ± 165 nmol g dry wt−1 min−1 in vehicle-treated MI hearts, p < 0.05), concomitant with a decrease in ketone oxidation and fatty acid oxidation rates. The increase in insulin stimulated glucose oxidation was accompanied by activation of the IRS-1/Akt/AS160/GSK-3β pathway, an increase in GLUT4 expression and a reduction in pyruvate dehydrogenase phosphorylation. This enhancement in insulin sensitivity occurred in parallel with a reduction in cardiac branched chain amino acids content (374 ± 27 vs 183 ± 41 µmol g protein−1 in vehicle-treated MI hearts, p < 0.05) and inhibition of the mTOR/P70S6K hypertrophic signalling pathway. The MI-induced increase in the phosphorylation of transforming growth factor β-activated kinase 1 (p-TAK1) and p38 MAPK was also reduced by mAb A treatment. Conclusions mAb A-mediated cardioprotection post-myocardial infarction is associated with improved insulin sensitivity and a selective enhancement of glucose oxidation via, at least in part, enhancing branched chain amino acids catabolism. Antagonizing glucagon action represents a novel and effective pharmacological intervention to alleviate cardiac dysfunction and adverse remodelling post-myocardial infarction