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.

Understanding relationships between heart failure and arrhythmias, important causes of suffering and sudden death, remains an unmet goal for biomedical researchers and physicians. Evidence assembled over the last decade supports a view that activation of the multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) favors myocardial dysfunction and cell membrane electrical instability. CaMKII activation follows increases in intracellular Ca2+ or oxidation, upstream signals with the capacity to transition CaMKII into a Ca2+ and calmodulin-independeant, constitutively active enzyme. Constitutively active CaMKII appears poised to participate in disease pathways by catalyzing the phosphorylation of classes of protein targets important for excitation-contraction coupling and cell survival, including ion channels and Ca2+ homeostatic proteins, and transcription factors that drive hypertrophic and inflammatory gene expression. This rich diversity of downstream targets helps to explain the potential for CaMKII to simultaneously affect mechanical and electrical properties of heart muscle cells. Proof of concept studies from a growing number of investigators show that CaMKII inhibition is beneficial for improving myocardial performance and reducing arrhythmias. Here we review the molecular physiology of CaMKII, discuss CaMKII actions at key cellular targets and results of animal models of myocardial hypertrophy, dysfunction and arrhythmias that suggest CaMKII inhibition may benefit myocardial function while reducing arrhythmias.

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).

Rationale: Parasympathetic regulation of heart rate is mediated by acetylcholine binding to G protein-coupled muscarinic M2 receptors, which activate heterotrimeric G i/o proteins to promote G protein-coupled inwardly rectifying K ؉ (GIRK) channel activation. Regulator of G protein signaling (RGS) proteins, which function to inactivate G proteins, are indispensable for normal parasympathetic control of the heart. However, it is unclear which of the more than 20 known RGS proteins function to negatively regulate and thereby ensure normal parasympathetic control of the heart.Objective: To examine the specific contribution of RGS6 as an essential regulator of parasympathetic signaling in heart. Methods and Results: We developed RGS6 knockout mice to determine the functional impact of loss of RGS6 on parasympathetic regulation of cardiac automaticity. RGS6 exhibited a uniquely robust expression in the heart, particularly in sinoatrial and atrioventricular nodal regions. Loss of RGS6 provoked dramatically exaggerated bradycardia in response to carbachol in mice and isolated perfused hearts and significantly enhanced the effect of carbachol on inhibition of spontaneous action potential firing in sinoatrial node cells. Consistent with a role of RGS6 in G protein inactivation, RGS6-deficient atrial myocytes exhibited a significant reduction in the time course of acetylcholine-activated potassium current (I KACh ) activation and deactivation, as well as the extent of I KACh desensitization.Conclusions: RGS6 is a previously unrecognized, but essential, regulator of parasympathetic activation in heart, functioning to prevent parasympathetic override and severe bradycardia. These effects likely result from actions of RGS6 as a negative regulator of G protein activation of GIRK channels. (Circ Res. 2010;107:1345-1349.) Key Words: RGS6 Ⅲ SA node Ⅲ Heart rate Ⅲ K ϩ channel Ⅲ G proteins S ince the discovery that acetylcholine (ACh) release from the vagus produces bradycardia, key proteins and mechanisms underlying this action of ACh in heart have been identified. It is now known that ACh binds to muscarinic M2 receptors (M2Rs) that activate heterotrimeric G proteins (G i/o ) in key pacemaking regions of the heart. Activation of these G proteins causes release of G␤␥ subunits that bind to and activate G proteincoupled inwardly rectifying K ϩ (GIRK) channels, which results in a large K ϩ current (acetylcholine-activated potassium current [I KACh ]) and membrane hyperpolarization. 1 RGS proteins function as GTPase-activating proteins (GAPs) for G␣ subunits, accelerating their conversion to the inactive GDP-bound form. 2 This results in their reassembly with G␤␥ to form inactive G protein heterotrimers, thereby terminating signaling by both G␣ and G␤␥ proteins. Heterologous expression of various members of the RGS protein family with GIRK channels and M2Rs are required to reconstitute the normal activation and deactivation kinetics of native atrial GIRK channels. 3 In vivo evidence for this key role of RGS proteins in controlli...

The toll-like receptors (TLR) and myocardial infarction (MI) promote NF-κB-dependent inflammatory transcription and oxidative injury in myocardium. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by oxidation and contributes to NF-κB-dependent transcription, myocardial hypertrophy and post-MI death. The myeloid differentiation protein 88 (MyD88) is an adapter protein critical for many TLR functions, but downstream targets for TLR/MyD88 signaling in MI are not well understood. We asked if CaMKII and TLR/MyD88 pathways are interconnected and if TLR/MyD88 contributes to adverse outcomes after MI. Here we show that TLR-4 activation by lipopolysaccharide (LPS) induces CaMKII oxidation (ox-CaMKII) in cardiomyocytes. MI enhances ox-CaMKII in wild type (WT) hearts but not in MyD88−/− hearts that are defective in MyD88-dependent TLR signaling. In post-MI WT hearts expression of pro-inflammatory genes TNF-α (Tnfa), complement factor B (Cfb), myocyte death and fibrosis were significatly increased, but increases were significantly less in MyD88−/− hearts after MI. MyD88−/− cardiomyocytes were defective in NF-κB activation by LPS but not by the MyD88-independent TLR agonist poly(I:C). In contrast, TNF-α induced Cfb gene expression was not deficient in MyD88−/− cardiomyocytes. Several hypertrophy marker genes were upregulated in both WT and MyD88−/− hearts after MI, but Acta1 was significantly attenuated in MyD88−/− hearts, suggesting that MyD88 selectively affects expression of hypertrophic genes. Post-MI cardiac hypertrophy, inflammation, apoptosis, ox-CaMKII expression and mortality were significantly reduced in MyD88−/− compared to WT littermates. These data suggest that MyD88 contributes to CaMKII oxidation and is important for adverse hypertrophic and inflammatory responses to LPS and MI.

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