Myocardial cell death is initiated by excessive mitochondrial Ca2+ entry, causing Ca2+ overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm)1,2. However, the signaling pathways that control mitochondrial Ca2+ entry through the inner membrane mitochondrial Ca2+ uniporter (MCU)3–5 are not known. The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is activated in ischemia reperfusion (I/R), myocardial infarction (MI) and neurohumoral injury, common causes of myocardial death and heart failure, suggesting CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (IMCU). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A (CsA), an mPTP antagonist with clinical efficacy in I/R injury6, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to I/R injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition are resistant to I/R injury, MI and neurohumoral injury, suggesting pathological actions of CaMKII are substantially mediated by increasing IMCU. Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca2+ entry and suggest mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure dysfunction in response to common experimental forms of pathophysiological stress.

Excessive activation of β-adrenergic, angiotensin II, and aldosterone (Aldo) signaling pathways promotes mortality after myocardial infarction (MI), while antagonist drugs targeting these pathways are core therapies for treating post-MI patients. Catecholamines and angiotensin II activate the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII), and CaMKII inhibition prevents isoproterenol- and angiotensin II-mediated cardiomyopathy. Here we show that Aldo exerts direct toxic actions on myocardium by oxidative activation of CaMKII, causing cardiac rupture and increased mortality in mice after MI. Aldo oxidizes CaMKII by recruiting NADPH oxidase, and oxidized CaMKII promotes matrix metalloproteinase 9 (Mmp9) expression in cardiomyocytes. Myocardial CaMKII inhibition, over-expression of methionine sulfoxide reductase A, an enzyme that reduces oxidized CaMKII, or NADPH oxidase inhibition prevented Aldo-enhanced post-MI cardiac rupture. These findings show oxidized myocardial CaMKII mediates cardiotoxic effects of Aldo on cardiac matrix and establish CaMKII as a nodal signal for the neurohumoral pathways associated with poor outcomes after MI.

Background Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ(ox-CaMKII) could contribute to AF. Methods and Results We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion. Conclusions Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.

Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca 2+ /calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.

Sinus node dysfunction (SND) is a major public health problem that is associated with sudden cardiac death and requires surgical implantation of artificial pacemakers. However, little is known about the molecular and cellular mechanisms that cause SND. Most SND occurs in the setting of heart failure and hypertension, conditions that are marked by elevated circulating angiotensin II (Ang II) and increased oxidant stress. Here, we show that oxidized calmodulin kinase II (ox-CaMKII) is a biomarker for SND in patients and dogs and a disease determinant in mice. In wild-type mice, Ang II infusion caused sinoatrial nodal (SAN) cell oxidation by activating NADPH oxidase, leading to increased ox-CaMKII, SAN cell apoptosis, and SND. p47 --mice lacking functional NADPH oxidase and mice with myocardial or SAN-targeted CaMKII inhibition were highly resistant to SAN apoptosis and SND, suggesting that ox-CaMKII-triggered SAN cell death contributed to SND. We developed a computational model of the sinoatrial node that showed that a loss of SAN cells below a critical threshold caused SND by preventing normal impulse formation and propagation. These data provide novel molecular and mechanistic information to understand SND and suggest that targeted CaMKII inhibition may be useful for preventing SND in high-risk patients. IntroductionEach normal heart beat is initiated as an electrical impulse from a small number of highly specialized sinoatrial node (SAN) pacemaker cells that reside in the lateral right atrium. There is now general agreement that physiological SAN function requires a pacemaker current (I f ) (1) and spontaneous release of sarcoplasmic reticulum (SR) intracellular Ca 2+ that triggers depolarizing current through the Na + /Ca 2+ exchanger (I NCX ) (2, 3). The multifunctional Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is essential for increasing SR Ca 2+ release in SAN cells in response to stress to cause physiological "fight-or-flight" heart rate (HR) increases (4). Although the physiological basis for SAN behavior is increasingly understood, very little is known about SAN disease. Severe SAN dysfunction (SND) is marked by irregular prolonged pauses between heart beats, pathologically slow HRs at rest, and inadequate activity-related increases in HR. At present, surgical implantation of permanent pacemakers is required for treatment of SND and costs $2 billion annually in the United States (5). SND commonly occurs in the setting of heart failure and hypertension (6-8), conditions characterized by excessive activation of renin-Ang II signaling (9) and elevated levels of ROS (10). Ang II increases ROS in ventricular myocardium by stimulating NADPH oxidase to cause activation of CaMKII (ox-CaMKII) by oxidation of Met281/282 in the CaMKII regulatory domain (11).

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...

Excessive activation of calmodulin kinase II (CaMKII) causes arrhythmias and heart failure, but the cellular mechanisms for CaMKII-targeted proteins causing disordered cell membrane excitability and myocardial dysfunction remain uncertain. Failing human cardiomyocytes exhibit increased CaMKII and voltagegated Ca 2þ channel (Ca V 1.2) activity, and enhanced expression of a specific Ca V 1.2 β-subunit protein isoform (β 2a ). We recently identified Ca V 1.2 β 2a residues critical for CaMKII phosphorylation (Thr 498) and binding (Leu 493), suggesting the hypothesis that these amino acids are crucial for cardiomyopathic consequences of CaMKII signaling. Here we show WT β 2a expression causes cellular Ca 2þ overload, arrhythmia-triggering cell membrane potential oscillations called early afterdepolarizations (EADs), and premature death in paced adult rabbit ventricular myocytes. Prevention of intracellular Ca 2þ release by ryanodine or global cellular CaMKII inhibition reduced EADs and improved cell survival to control levels in WT β 2a -expressing ventricular myocytes. In contrast, expression of β 2a T498A or L493A mutants mimicked the protective effects of ryanodine or global cellular CaMKII inhibition by reducing Ca 2þ entry through Ca V 1.2 and inhibiting EADs. Furthermore, Ca V 1.2 currents recorded from cells overexpressing CaMKII phosphorylation-or binding-incompetent β 2a subunits were incapable of entering a CaMKII-dependent high-activity gating mode (mode 2), indicating that β 2a Thr 498 and Leu 493 are required for Ca V 1.2 activation by CaMKII in native cells. These data show that CaMKII binding and phosphorylation sites on β 2a are concise but pivotal components of a molecular and biophysical and mechanism for EADs and impaired survival in adult cardiomyocytes.arrhythmias | calcium | calcium channel | calmodulin kinase | cardiac myocytes T he multifunctional Ca 2þ and calmodulin-dependent protein kinase II (CaMKII) is a proarrhythmic (1) and proapoptotic (2) signaling molecule activated in failing human myocardium and in animal models of heart failure (3). The Ca 2þ homeostatic proteins involved in excitation-contraction coupling (ECC) are CaMKII targets (4), and excessive CaMKII-mediated phosphorylation of ECC proteins has recently emerged as a critical transition event leading to myocardial dysfunction and arrhythmias (5). The L-type Ca 2þ channel (LTCC) protein complex is the predominant entry point for Ca 2þ that supplies intracellular sarcoplasmic reticulum (SR) Ca 2þ stores and is an important source of inward current (I Ca ) for prolonging the action potential duration (APD) (6). CaMKII drives LTCCs into an active gating mode (mode 2) with frequent, prolonged openings, and mode 2 gating occurs together with an I Ca property called facilitation (7). Mode 2 gating and I Ca facilitation are part of a hypothesized mechanism favoring SR Ca 2þ overload and early afterdepolarizations (EADs), arrhythmia-initiating oscillations in cell membrane potential (8). LTCCs contain a pore forming α-subunit (Ca V 1....

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.