Systemic branched‐chain amino acid (BCAA) metabolism is dysregulated in cardiometabolic diseases. We previously demonstrated that upregulated AMP deaminase 3 (AMPD3) impairs cardiac energetics in a rat model of obese type 2 diabetes, Otsuka Long‐Evans‐Tokushima fatty (OLETF). Here, we hypothesized that the cardiac BCAA levels and the activity of branched‐chain α‐keto acid dehydrogenase (BCKDH), a rate‐limiting enzyme in BCAA metabolism, are altered by type 2 diabetes (T2DM), and that upregulated AMPD3 expression is involved in the alteration. Performing proteomic analysis combined with immunoblotting, we discovered that BCKDH localizes not only to mitochondria but also to the endoplasmic reticulum (ER), where it interacts with AMPD3. Knocking down AMPD3 in neonatal rat cardiomyocytes (NRCMs) increased BCKDH activity, suggesting that AMPD3 negatively regulates BCKDH. Compared with control rats (Long‐Evans Tokushima Otsuka [LETO] rats), OLETF rats exhibited 49% higher cardiac BCAA levels and 49% lower BCKDH activity. In the cardiac ER of the OLETF rats, BCKDH‐E1α subunit expression was downregulated, while AMPD3 expression was upregulated, resulting in an 80% lower AMPD3‐E1α interaction compared to LETO rats. Knocking down E1α expression in NRCMs upregulated AMPD3 expression and recapitulated the imbalanced AMPD3‐BCKDH expressions observed in OLETF rat hearts. E1α knockdown in NRCMs inhibited glucose oxidation in response to insulin, palmitate oxidation, and lipid droplet biogenesis under oleate loading. Collectively, these data revealed previously unrecognized extramitochondrial localization of BCKDH in the heart and its reciprocal regulation with AMPD3 and imbalanced AMPD3‐BCKDH interactions in OLETF. Downregulation of BCKDH in cardiomyocytes induced profound metabolic changes that are observed in OLETF hearts, providing insight into mechanisms contributing to the development of diabetic cardiomyopathy.
Aims The role of necroptosis in dilated cardiomyopathy (DCM) remains unclear. Here, we examined whether phosphorylation of mixed lineage kinase domain‐like protein (MLKL), an indispensable event for execution of necroptosis, is associated with the progression of DCM. Methods and results Patients with DCM (n = 56, 56 ± 15 years of age; 68% male) were enrolled for immunohistochemical analyses of biopsies. Adverse events were defined as a composite of death or admission for heart failure or ventricular arrhythmia. Compared with the normal myocardium, increased signals of MLKL phosphorylation were detected in the nuclei, cytoplasm, and intercalated discs of cardiomyocytes in biopsy samples from DCM patients. The phosphorylated MLKL (p‐MLKL) signal was increased in enlarged nuclei or nuclei with bizarre shapes in hypertrophied cardiomyocytes. Nuclear p‐MLKL level was correlated negatively with septal peak myocardial velocity during early diastole (r = −0.327, P = 0.019) and was correlated positively with tricuspid regurgitation pressure gradient (r = 0.339, P = 0.023), while p‐MLKL level in intercalated discs was negatively correlated with mean left ventricular wall thickness (r = −0.360, P = 0.014). During a median follow‐up period of 3.5 years, 10 patients (18%) had adverse events. To examine the difference in event rates according to p‐MLKL expression levels, patients were divided into two groups by using the median value of nuclear p‐MLKL or intercalated disc p‐MLKL. A group with high nuclear p‐MLKL level (H‐nucMLKL group) had a higher adverse event rate than did a group with low nuclear p‐MLKL level (L‐nucMLKL group) (32% vs. 4%, P = 0.012), and Kaplan–Meier survival curves showed that the adverse event‐free survival rate was lower in the H‐nucMLKL group than in the L‐nucMLKL group (P = 0.019 by the log‐rank test). Such differences were not detected between groups divided by a median value of intercalated disc p‐MLKL. In δ‐sarcoglycan‐deficient (Sgcd−/−) mice, a model of DCM, total p‐MLKL and nuclear p‐MLKL levels were higher than in wild‐type mice. Conclusion The results suggest that increased localization of nuclear p‐MLKL in cardiomyocytes is associated with left ventricular diastolic dysfunction and future adverse events in DCM.
Background A metabolomic study in the human heart suggested a pivotal role of amino acid (AA) metabolism in fatty acid oxidation, which is dysregulated in type 2 diabetes mellitus (T2DM) and heart failure. We previously reported that aberrant up-regulation of AMP deaminase 3 (AMPD3) impairs cardiac energetics in T2DM hearts, and AMPD3 was recently shown to be activated by fasting and to promote AA metabolism and fatty acid oxidation in skeletal muscle. A sodium glucose cotransporter 2 inhibitor (SGLT2i) has been shown to augment systemic AA metabolism, but its effect on cardiac AA metabolism remains unknown. Purpose We hypothesized that AMPD3 has a role in AA and lipid metabolism in cardiomyocytes and that the protective effect of an SGLT2i in diabetic hearts is mediated by modification of AA and lipid metabolism. Methods and results Proteomic analyses of AMPD3 immunoprecipitates in rat hearts revealed that AMPD3 interacted with the E1α and E2 components of the BCKDH complex, a rate-limiting enzyme of branched-chain AA (BCAA) catabolism. Immunoblotting using subcellular fractions revealed that BCKDH localized not only in the mitochondria matrix but also in the cytosol and endoplasmic reticulum (ER) and that AMPD3 interacted with BCKDH in the cytosol and ER. Despite comparable expression of BCKDH components and phosphorylation of E1α at Ser293, significant accumulation of BCAA was observed in T2DM rats (OLETF; 317±30 nmol/g) compared to that in control rats (LETO; 213±16 nmol/g), and the accumulation of BCAA was accompanied by up-regulation of AMPD3 in the cytosol and ER by 98% and 231%, respectively. In cardiomyocytes, disruption of BCAA catabolism by knockdown of BCKDH-E1α resulted in a 5.8-fold increase in AMPD3 at the transcriptional level and blunted lipid droplet biogenesis in response to a long-chain fatty acid challenge. Next, myocardial infarction (MI) was induced in LETO and OLETF pretreated with empagliflozin (10 mg/kg/day, 14 days) or a vehicle. Pathway analysis of cardiac metabolites revealed arginine biosynthesis and BCAA metabolism as the most significantly changed pathways with empagliflozin, with BCAA (791±187 nmol/g), glutamate, glutamine and urea being significantly increased. Empagliflozin restored myocardial ATP and survival after MI in OLETF to levels comparable to those in LETO. Electron microscopy showed a significantly higher prevalence of myocardium lipid droplets in OLETF, which was further increased by empagliflozin. Conclusions The results support the hypotheses that imbalance of extra-mitochondrial AMPD3-BCKDH interaction underlies dysregulated BCAA metabolism in T2DM hearts and that activation of cardiac AA metabolism by an SGLT2i normalizes fatty acid overload through sequestration into intracellular lipid droplets. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Boehringer Ingelheim
Background Dysregulation of branched-chain amino acid (BCAA) metabolism has been shown to be associated with type 2 diabetes mellitus (T2DM) and heart failure. BCAA reportedly protects cells from fatty acid-induced mitochondrial injury via sequestration of fatty acids in intracellular lipid droplets from mitochondria. We previously reported that up-regulation of AMP deaminase 3 (AMPD3) impairs cardiac energetics in T2DM hearts, and AMPD3 was recently shown to participate in regulation of amino acid metabolism in skeletal muscle. Purpose We hypothesized that AMPD3 regulates cardiac amino acid metabolism by interaction with branched-chain α-ketoacid dehydrogenase (BCKDH) complex and that cardioprotective effect of sodium glucose cotransporter 2 inhibitors is mediated by modification of BCAA metabolism in diabetic cardiomyocytes. Methods and results Proteomic analyses of immunoprecipitates with an anti-AMPD3 antibody in rat hearts revealed that AMPD3 interacted with the E1α component of BCKDH complex. Whereas BCKDH has been reported to localize in mitochondria matrix as a rate-limiting enzyme for BCAA catabolism, immunoblotting using subcellular fractions revealed that BCKDH E1α is present in cytosol and endoplasmic reticulum as well. AMPD3-BCKDH E1α interaction was decreased by 68% in T2DM rats (OLETF) compared to that in control rats (LETO), and significant accumulation of BCAAs was observed in OLETF hearts (317±30 vs. 213±16 nmol/g). Survival rate at 48 hours after myocardial infarction (MI) was significantly lower in OLETF than in LETO (40% vs 84%). Empagliflozin treatment (10 mg/kg/day, 14 days) before MI improved the survival rate in OLETF to 70%, increased BCAAs as the top of 92 detected metabolites (791±187 nmol/g) and significantly preserved tissue ATP in the non-MI remote region. Electron microscopy showed a significantly higher prevalence of myocardium lipid droplets in OLETF, which was further increased by empagliflozin. Conclusions Results of the present analyses support the hypotheses that conversion of BCAA-derived branched-chain α-ketoacid to branched-chain acyl-CoA is suppressed by reduced AMPD3-BCKDH interaction in the myocardium of T2DM and that empagliflozin induces compensation of the dysregulated cardiac BCAA metabolism by augmentation of BCAA influx and promotion of fatty acid sequestration in intracellular lipid droplets. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Boehringer Ingelheim
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