MICU1 and MICU2 control mitochondrial calcium signaling in the mammalian heart.

Activating Ca2+-sensitive enzymes of oxidative metabolism while preventing calcium overload that leads to mitochondrial and cellular injury requires dynamic control of mitochondrial Ca2+ uptake. This is ensured by the mitochondrial calcium uptake (MICU)1/2 proteins that gate the pore of the mitochondrial calcium uniporter (mtCU). MICU1 is relatively sparse in the heart, and recent studies claimed the mammalian heart lacks MICU1 gating of mtCU. However, genetic models have not been tested. We find that MICU1 is present in a complex with MCU in nonfailing human hearts. Furthermore, using murine genetic models and pharmacology, we show that MICU1 and MICU2 control cardiac mitochondrial Ca2+ influx, and that MICU1 deletion alters cardiomyocyte mitochondrial calcium signaling and energy metabolism. MICU1 loss causes substantial compensatory changes in the mtCU composition and abundance, increased turnover of essential MCU regulator (EMRE) early on and, later, of MCU, that limit mitochondrial Ca2+ uptake and allow cell survival. Thus, both the primary consequences of MICU1 loss and the ensuing robust compensation highlight MICU1's relevance in the beating heart.

Pharmacological inhibition of the mitochondrial Ca2+ uniporter: Relevance for pathophysiology and human therapy.

Mitochondrial Ca2+ uptake has long been considered crucial for meeting the fluctuating energy demands of cells in the heart and other tissues. Increases in mitochondrial matrix [Ca2+] drive mitochondrial ATP production via stimulation of Ca2+-sensitive dehydrogenases. Mitochondria-targeted sensors have revealed mitochondrial matrix [Ca2+] rises that closely follow the cytoplasmic [Ca2+] signals in many paradigms. Mitochondrial Ca2+ uptake is mediated by the Ca2+ uniporter (mtCU). Pharmacological manipulation of the mtCU is potentially key to understanding its physiological significance, but no specific, cell-permeable inhibitors were identified. In the past decade, as the molecular identity of the mtCU was brought to light, efforts have focused on genetic targeting. However, in the cells/animals that are able to survive impaired mtCU function, robust compensatory changes were found in the mtCU as well as other mechanisms. Thus, the discovery, through chemical library screens on normal and mtCU-deficient cells, of new small-molecule inhibitors with improved cell permeability and specificity might offer a better chance to test the relevance of mitochondrial Ca2+ uptake. Success with the development of small molecule mtCU inhibitors is also expected to have clinical impact, considering the growing evidence for the role of mitochondrial Ca2+ uptake in a variety of diseases, including heart attack, stroke and various neurodegenerative disorders. Here, we review the progress in pharmacological targeting of mtCU and illustrate the challenges in this field using data obtained with MCU-i11, a new small molecule inhibitor.

Cardiac Insulin Resistance in Heart Failure: The Role of Mitochondrial Dynamics.

Heart failure (HF) frequently coexists with conditions associated with glucose insufficiency, such as insulin resistance and type 2 diabetes mellitus (T2DM), and patients with T2DM have a significantly high incidence of HF. These two closely related diseases cannot be separated on the basis of their treatment. Some antidiabetic drugs failed to improve cardiac outcomes in T2DM patients, despite lowering glucose levels sufficiently. This may be, at least in part, due to a lack of understanding of cardiac insulin resistance. Basic investigations have revealed the significant contribution of cardiac insulin resistance to the pathogenesis and progression of HF; however, there is no clinical evidence of the definition or treatment of cardiac insulin resistance. Mitochondrial dynamics play an important role in cardiac insulin resistance and HF because they maintain cellular homeostasis through energy production, cell survival, and cell proliferation. The innovation of diagnostic tools and/or treatment targeting mitochondrial dynamics is assumed to improve not only the insulin sensitivity of the myocardium and cardiac metabolism, but also the cardiac contraction function. In this review, we summarized the current knowledge on the correlation between cardiac insulin resistance and progression of HF, and discussed the role of mitochondrial dynamics on the pathogenesis of cardiac insulin resistance and HF. We further discuss the possibility of mitochondria-targeted intervention to improve cardiac metabolism and HF.

Mitochondrial fission protein, dynamin-related protein 1, contributes to the promotion of hypertensive cardiac hypertrophy and fibrosis in Dahl-salt sensitive rats.

BACKGROUND: Hypertension promotes cardiac hypertrophy which finally leads to cardiac dysfunction. Although aberrant mitochondrial dynamics is known to be a relevant contributor of pathogenesis in heart disease, little is known about the relationship between mitochondrial dynamics and cardiac hypertrophy. We investigated the pathophysiological roles of Dynamin-related protein1 (Drp1, a mitochondrial fission protein) on the hypertensive cardiac hypertrophy. METHODS & RESULTS: Dahl salt-sensitive rats were fed with a low-salt (0.3% NaCl) or a high-salt (8% NaCl) chow to promote hypertension with and without administration of mdivi1 (an inhibitor of Drp1: 1 mg/kg/every alternative day), and then the hypertensive cardiac hypertrophy was assessed. High-salt fed rats exhibited left ventricular hypertrophy (LVH), myocytes hypertrophy, and cardiac fibrosis, and mdivi-1 suppressed them without alteration of the blood pressure. Mdivi1 also reduced ROS production by hypertension, which subsequently suppressed the Ca2+-activated protein phosphatase calcineurin and Ca2+/calmodulin-dependent kinase II (CaMKII). CONCLUSIONS: Our results suggest that Drp1 contributes to the pathogenesis of hypertensive cardiac hypertrophy via ROS production and the Drp1 suppression may be effective to prevent the hypertensive cardiac hypertrophy.

Eicosapentaenoic acid ameliorates palmitate-induced lipotoxicity via the AMP kinase/dynamin-related protein-1 signaling pathway in differentiated H9c2 myocytes.

BACKGROUND: Emerging evidence suggested the preferable effects of eicosapentaenoic acid (EPA; n-3 polyunsaturated fatty acid) against cardiac lipotoxicity, which worsens cardiac function by means of excessive serum free fatty acids due to chronic adrenergic stimulation under heart failure. Nonetheless, the precise molecular mechanisms remain elusive. In this study, we focused on dynamin-related protein-1 (Drp1) as a possible modulator of the EPA-mediated cardiac protection against cardiac lipotoxicity, and investigated the causal relation between AMP-activated protein kinase (AMPK) and Drp1. METHODS AND RESULTS: When differentiated H9c2 myocytes were exposed to palmitate (PAL; saturated fatty acid, 400µM) for 24h, these myocytes showed activation of caspases 3 and 7, enhanced caspase 3 cleavage, depolarized mitochondrial membrane potential, depleted intracellular ATP, and enhanced production of intracellular reactive oxygen species. These changes suggested lipotoxicity due to excessive PAL. PAL enhanced mitochondrial fragmentation with increased Drp1 expression, as well. EPA (50µM) restored the PAL-induced apoptosis, mitochondrial dysfunction, and mitochondrial fragmentation with increased Drp1 expression by PAL. EPA activated phosphorylation of AMPK, and pharmacological activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleotide ameliorated the PAL-induced apoptosis, mitochondrial dysfunction, and downregulated Drp1. An AMPK knockdown via RNA interference enhanced Drp1 expression and attenuated the protective effects of EPA against the PAL-induced lipotoxicity. CONCLUSION: EPA ameliorates the PAL-induced lipotoxicity via AMPK activation, which subsequently suppresses mitochondrial fragmentation and Drp1 expression. Our findings may provide new insights into the molecular mechanisms of EPA-mediated myocardial protection in heart failure.

Intracellular Renin Inhibits Mitochondrial Permeability Transition Pore via Activated Mitochondrial Extracellular Signal-Regulated Kinase (ERK) 1/2 during Ischemia in Diabetic Hearts.

Although beneficial effects of non-secreting intracellular renin (ns-renin) against ischemia have been reported, the precise mechanism remains unclear. In this study, we investigated the roles of ns-renin and mitochondrial extracellular signal-related kinase (ERK) 1/2 on mitochondrial permeability transition pore (mPTP) opening during ischemia in diabetes mellitus (DM) hearts. When isolated hearts from Wistar rats (non-DM hearts) and Goto-Kakizaki rats (DM hearts) were subjected to ischemia for 70 min by left anterior descending coronary artery ligation, DM hearts exhibited higher left ventricular (LV) developed pressure and lower LV end-diastolic pressure than non-DM hearts, suggesting ischemic resistance. In addition, DM hearts showed increased intracellular renin (int-renin, including secreting and non-secreting renin) in the ischemic area, and a direct renin inhibitor (DRI; aliskiren) attenuated ischemic resistance in DM hearts. ERK1/2 was significantly phosphorylated after ischemia in both whole cell and mitochondrial fractions in DM hearts. In isolated mitochondria from DM hearts, rat recombinant renin (r-renin) significantly phosphorylated mitochondrial ERK1/2, and hyperpolarized mitochondrial membrane potential (ΔΨm) in a U0126 (an inhibitor of mitogen-activated protein kinases/ERK kinases)-sensitive manner. R-renin also attenuated atractyloside (Atr, an mPTP opener)-induced ΔΨm depolarization and Atr-induced mitochondrial swelling in an U0126-sensitive manner in isolated mitochondria from DM hearts. Furthermore, U0126 attenuated ischemic resistance in DM hearts, whereas it did not alter the hemodynamics in non-DM hearts. Our results suggest that the increased int-renin during ischemia may inhibit mPTP opening through activation of mitochondrial ERK1/2, which may be involved in ischemic resistance in DM hearts.

Mitochondrial membrane potential regulates nuclear DNA methylation and gene expression through phospholipid remodeling.

Maintenance of the mitochondrial inner membrane potential (ΔΨM) is critical for many aspects of mitochondrial function, including mitochondrial protein import and ion homeostasis. While ΔΨM loss and its consequences are well studied, little is known about the effects of increased ΔΨM. In this study, we used cells deleted of ATPIF1, a natural inhibitor of the hydrolytic activity of the ATP synthase, as a genetic model of mitochondrial hyperpolarization. Our data show that chronic ΔΨM increase leads to nuclear DNA hypermethylation, regulating transcription of mitochondria, carbohydrate and lipid metabolism genes. Surprisingly, remodeling of phospholipids, but not metabolites or redox changes, mechanistically links the ΔΨM to the epigenome. These changes were also observed upon chemical exposures and reversed by decreasing the ΔΨM, highlighting them as hallmark adaptations to chronic mitochondrial hyperpolarization. Our results reveal the ΔΨM as the upstream signal conveying the mitochondrial status to the epigenome to regulate cellular biology, providing a new framework for how mitochondria can influence health outcomes in the absence of canonical dysfunction.

Pinacidil, a KATP channel opener, stimulates cardiac Na+/Ca2+ exchanger function through the NO/cGMP/PKG signaling pathway in guinea pig cardiac ventricular myocytes.

Pinacidil, a nonselective ATP-sensitive K+ (KATP) channel opener, has cardioprotective effects for hypertension, ischemia/reperfusion injury, and arrhythmia. This agent abolishes early afterdepolarizations, delayed afterdepolarizations (DADs), and abnormal automaticity in canine cardiac ventricular myocytes. DADs are well known to be caused by the Na+/Ca2+ exchange current (INCX). In this study, we used the whole-cell patch-clamp technique and Fura-2/AM (Ca2+-indicator) method to investigate the effect of pinacidil on INCX in isolated guinea pig cardiac ventricular myocytes. In the patch-clamp study, pinacidil enhanced INCX in a concentration-dependent manner. The half-maximal effective concentration values were 23.5 and 23.0 µM for the Ca2+ entry (outward) and Ca2+ exit (inward) components of INCX, respectively. The pinacidil-induced INCX increase was blocked by L-NAME, a nitric oxide (NO) synthase inhibitor, by ODQ, a soluble guanylate cyclase inhibitor, and by KT5823, a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor, but not by N-2-mercaptopropyonyl glycine (MPG), a reactive oxygen species (ROS) scavenger. Glibenclamide, a nonselective KATP channel inhibitor, blocked the pinacidil-induced INCX increase, while 5-HD, a selective mitochondria KATP channel inhibitor, did not. In the Fura-2/AM study pinacidil also enhanced intracellular Ca2+ concentration, which was inhibited by L-NAME, ODQ, KT5823, and glibenclamide, but not by MPG and 5-HD. Sildenafil, a phosphodiesterase 5 inhibitor, increased further the pinacidil-induced INCX increase. Sodium nitroprusside, a NO donor, also increased INCX. In conclusion, pinacidil may stimulate cardiac Na+/Ca2+ exchanger (NCX1) by opening plasma membrane KATP channels and activating the NO/cGMP/PKG signaling pathway.

Glycogen synthase kinase-3ß opens mitochondrial permeability transition pore through mitochondrial hexokinase II dissociation.

Accumulating evidence has revealed pivotal roles of glycogen synthase kinase-3ß (GSK3ß) inactivation on cardiac protection. Because the precise mechanisms of cardiac protection against ischemia/reperfusion (I/R) injury by GSK3ß-inactivation remain elusive, we investigated the relationship between GSK3ß-mediated mitochondrial hexokinase II (mitoHK-II; a downstream target of GSK3ß) dissociation and mitochondrial permeability transition pore (mPTP) opening. In Langendorff-perfused hearts, GSK3ß inactivation by SB216763 improved the left ventricular-developed pressure and retained mitoHK-II binding after I/R. In permeabilized myocytes, GSK3ß depolarized mitochondrial membrane potential with accelerated mitochondrial calcein release (suggesting GSK3ß-mediated mPTP opening) and decreased mitoHK-II bindings. GSK3ß-mediated mPTP opening depended on mitoHK-II binding, i.e., it was accelerated by dissociation of mitoHK-II (dicyclohexylcarbodiimide) and attenuated by enhancement of mitoHK-II binding (dextran). However, inactivation of mitoHK-II by glucose-depletion or glucose-6-phosphate inhibited the GSK3ß-mediated mPTP opening. We conclude that GSK3ß-mediated mPTP opening may be involved in I/R injury and regulated by mitoHK-II binding and activity.