Myocardial mitochondrial Ca 2+ entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca 2+ are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca 2+ uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca 2+ entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O 2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU-permeabilized myocardial fibers or isolated mitochondria in low Ca 2+ were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca 2+ homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca 2+ ] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca 2+ homeostasis. Mitochondrial Ca 2+ overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca 2+ homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.myocardium | mitochondrial calcium uniporter | ischemia-reperfusion injury E ntry of Ca 2+ into the mitochondrial matrix is a central event for Ca 2+ homeostasis in cardiomyocytes (1) as well as for coordinating fundamental and diverse responses to physiological (2) and pathological stress (3). The paradigm for Ca 2+ as a physiological second messenger that enhances oxidative phosphorylation to enable fight-or-flight responses but in excess contributes to disease and dysfunction is well established in myocardium (4). The molecular identity of the mitochondrial Ca 2+ uniporter (MCU) was recently discovered, enabling development of new genetic models to understand the role of MCU in vivo. MCU is an ion channel protein that acts as the primary pathway for Ca 2+ entry into the mitochondrial matrix (5, 6). Recent findings in global Mcu −/− mice (7) suggest that the MCU pathway is dispensable for regulating cellular energy production, except under extreme physiological stress, and for activation of pathways leading to cell death; however, the effect of selective myocardial MCU inhibition is unknown. We developed a new transgenic mouse model with myocardial delimited dominant negative (DN)-MCU protein overexpressio...

Heart rate increases are a fundamental adaptation to physiological stress, while inappropriate heart rate increases are resistant to current therapies. However, the metabolic mechanisms driving heart rate acceleration in cardiac pacemaker cells remain incompletely understood. The mitochondrial calcium uniporter (MCU) facilitates calcium entry into the mitochondrial matrix to stimulate metabolism. We developed mice with myocardial MCU inhibition by transgenic expression of a dominant negative (DN) MCU. Here we show that DN-MCU mice had normal resting heart rates but were incapable of physiological fight or flight heart rate acceleration. We found MCU function was essential for rapidly increasing mitochondrial calcium in pacemaker cells and that MCU enhanced oxidative phoshorylation was required to accelerate reloading of an intracellular calcium compartment prior to each heartbeat. Our findings show the MCU is necessary for complete physiological heart rate acceleration and suggest MCU inhibition could reduce inappropriate heart rate increases without affecting resting heart rate.

The role of the mitochondrial Ca2+ uniporter (MCU) in physiologic cell proliferation remains to be defined. Here, we demonstrated that the MCU was required to match mitochondrial function to metabolic demands during cell cycling. During the G1/S transition (the cycle phase with the highest mitochondrial ATP output), mitochondrial fusion, oxygen consumption and Ca2+ uptake increased in wild-type cells, but not in cells lacking MCU. In proliferating wild-type control cells, the addition of the growth factors promoted the activation of the Ca2+/calmodulin-dependent kinase II (CaMKII) and the phosphorylation of the mitochondrial fission factor Drp1 at Ser616. The lack of the MCU was associated with baseline activation of CaMKII, mitochondrial fragmentation due to increased Drp1 phosphorylation, and impaired mitochondrial respiration and glycolysis. The mitochondrial fission/fusion ratio and proliferation in MCU-deficient cells recovered after MCU restoration or inhibition of mitochondrial fragmentation or of CaMKII in the cytosol. Our data highlight a key function for the MCU in mitochondrial adaptation to the metabolic demands during cell cycle progression. Cytosolic CaMKII and the MCU participate in a regulatory circuit whereby mitochondrial Ca2+ uptake affects cell proliferation through Drp1.

Rationale The sodium-calcium exchanger 1 (NCX1) is predominantly expressed in the heart and is implicated in controlling automaticity in isolated sinoatrial nodal (SAN) pacemaker cells, but the potential role of NCX1 in determining heart rate in vivo is unknown. Objective Determine the role of Ncx1 in heart rate. Methods and Results We employed global myocardial and SAN-targeted conditional Ncx1 knockout (Ncx1−/−) mice to measure the effect of the NCX current (INCX) in pacemaking activity in vivo, ex vivo and in isolated SAN cells. We induced conditional Ncx1−/− using a Cre/loxP system. Unexpectedly, in vivo and ex vivo hearts and isolated SAN cells showed that basal rates in Ncx1−/− (retaining ~20% of control level INCX) and control mice were similar, suggesting that physiological NCX1 expression is not required for determining resting heart rate. However, heart rate and SAN cell automaticity increases in response to isoproterenol or the dihydropyridine Ca2+ channel agonist BayK8644 were significantly blunted or eliminated in Ncx1−/− mice, indicating that NCX1 is important for fight or flight heart rate responses. In contrast the ‘pacemaker’ current (If) and L-type Ca2+ currents were equivalent in control and Ncx1−/− SAN cells under resting and isoproterenol-stimulated conditions. Ivabradine, an If antagonist with clinical efficacy, reduced basal SAN cell automaticity similarly in control and Ncx1−/− mice. However, ivabradine decreased automaticity in SAN cells isolated from Ncx1−/− mice more effectively than in control SAN cells after isoproterenol, suggesting that the importance of INCX in fight or flight rate increases is enhanced after If inhibition. Conclusion Physiological Ncx1 expression is required for increasing sinus rates in vivo, ex vivo and in isolated SAN cells but not for maintaining resting heart rate.