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.

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).

The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is now recognized to play a central role in pathological events in the cardiovascular system. CaMKII has diverse downstream targets that promote vascular disease, heart failure and arrhythmias, so improved understanding of CaMKII signaling has the potential to lead to new therapies for cardiovascular disease. CaMKII is a multimeric serine-threonine kinase that is initially activated by binding calcified calmodulin (Ca2+/CaM). Under conditions of sustained exposure to elevated Ca2+/CaM CaMKII transitions into a Ca2+/CaM-autonomous enzyme by two distinct but parallel processes. Autophosphorylation of threonine 287 in the CaMKII regulatory domain ‘traps’ CaMKII into an open configuration even after Ca2+/CaM unbinding. More recently, our group identified a pair of methionines (281/282) in the CaMKII regulatory domain that undergo a partially reversible oxidation which, like autophosphorylation, prevents CaMKII from inactivating after Ca2+/CaM unbinding. Here we review roles of CaMKII in cardiovascular disease with an eye to understanding how CaMKII may act as a transduction signal to connect pro-oxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of cardiovascular disease.

Extensive efforts have been made to understand genomic function through both experimental and computational approaches, yet proper annotation still remains challenging, especially in non-coding regions. In this manuscript, we introduce GenoSkyline, an unsupervised learning framework to predict tissue-specific functional regions through integrating high-throughput epigenetic annotations. GenoSkyline successfully identified a variety of non-coding regulatory machinery including enhancers, regulatory miRNA, and hypomethylated transposable elements in extensive case studies. Integrative analysis of GenoSkyline annotations and results from genome-wide association studies (GWAS) led to novel biological insights on the etiologies of a number of human complex traits. We also explored using tissue-specific functional annotations to prioritize GWAS signals and predict relevant tissue types for each risk locus. Brain and blood-specific annotations led to better prioritization performance for schizophrenia than standard GWAS p-values and non-tissue-specific annotations. As for coronary artery disease, heart-specific functional regions was highly enriched of GWAS signals, but previously identified risk loci were found to be most functional in other tissues, suggesting a substantial proportion of still undetected heart-related loci. In summary, GenoSkyline annotations can guide genetic studies at multiple resolutions and provide valuable insights in understanding complex diseases. GenoSkyline is available at http://genocanyon.med.yale.edu/GenoSkyline.

Aldosterone contributes to the endocrine basis of heart failure and studies on cardiac aldosterone signaling have reinforced its value as a therapeutic target. Recent focus has shifted to new roles of aldosterone that appear to depend on co-existing pathologic stimuli, cell type, and disease etiology. This review evaluates recent advances in mechanisms underlying aldosterone-induced cardiac disease and highlights the interplay between aldosterone and Ca2+ and calmodulin dependent protein kinase II, whose hyperactivity during heart failure contributes to disease progression. Increasing evidence implicates aldosterone in diastolic dysfunction, and there is need to develop more targeted therapeutics such as aldosterone synthase inhibitors and molecularly specific anti-oxidants. Despite accumulating knowledge, many questions still persist and will likely dictate areas of future research.

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His-Purkinje-related ventricular arrhythmias are a subset of ventricular tachycardias that use the specialized cardiac conduction system. These arrhythmias can occur in various different forms of structural heart disease. Here, we review the basic science discoveries and their analogous clinical observations that implicate the His-Purkinje system as a crucial component of the arrhythmia circuit. While mutations serve the molecular basis for arrhythmias in the heritable cardiomyopathies, transcriptional and posttranslational changes constitute the adverse remodeling leading to arrhythmias in acquired structural heart disease. Additional studies on the electrical properties of the His-Purkinje network and its interactions with the surrounding myocardium will improve the clinical diagnosis and treatment of these arrhythmias.