Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.

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.

The best understood ''fight or flight'' mechanism for increasing heart rate (HR) involves activation of a cyclic nucleotide-gated ion channel (HCN4) by ␤-adrenergic receptor (␤AR) agonist stimulation. HCN4 conducts an inward ''pacemaker'' current (If) that increases the sinoatrial nodal (SAN) cell membrane diastolic depolarization rate (DDR), leading to faster SAN action potential generation. Surprisingly, HCN4 knockout mice were recently shown to retain physiological HR increases with isoproterenol (ISO), suggesting that other I f-independent pathways are critical to SAN fight or flight responses. Here, we show that CaMKII plays a previously unanticipated but decisive role to increase SAN rates during ␤AR stimulation. Studies in hearts from mice with SAN cell CaMKII inhibition suggest that CaMKII activity is required for chronotropic responses to ISO. CaMKII is selectively engaged in SAN cells during ␤AR stimulation and leads to coordinated enhancement of SR Ca 2ϩ filling, greater diastolic SR Ca 2ϩ release, and an increased diastolic depolarization rate (DDR) to increase HRs, independent of I f . In contrast, CaMKII inhibition does not slow HRs or SAN cell action potential (AP) frequency in the absence of ␤AR stimulation or when SR Ca 2ϩ release is disabled. These studies define a novel, CaMKII-dependent cellular mechanism for SAN fight or flight physiology.

Myocardial Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) inhibition improves cardiac function following myocardial infarction (MI), but the CaMKII-dependent pathways that participate in myocardial stress responses are incompletely understood. To address this issue, we sought to determine the transcriptional consequences of myocardial CaMKII inhibition after MI. We performed gene expression profiling in mouse hearts with cardiomyocyte-delimited transgenic expression of either a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C) following MI. Of the 8,600 mRNAs examined, 156 were substantially modulated by MI, and nearly half of these showed markedly altered responses to MI with CaMKII inhibition. CaMKII inhibition substantially reduced the MI-triggered upregulation of a constellation of proinflammatory genes. We studied 1 of these proinflammatory genes, complement factor B (Cfb), in detail, because complement proteins secreted by cells other than cardiomyocytes can induce sarcolemmal injury during MI. CFB protein expression in cardiomyocytes was triggered by CaMKII activation of the NF-κB pathway during both MI and exposure to bacterial endotoxin. CaMKII inhibition suppressed NF-κB activity in vitro and in vivo and reduced Cfb expression and sarcolemmal injury. The Cfb -/-mice were partially protected from the adverse consequences of MI. Our findings demonstrate what we believe is a novel target for CaMKII in myocardial injury and suggest that CaMKII is broadly important for the genetic effects of MI in cardiomyocytes.

Background-Activation of cellular Ca2ϩ signaling molecules appears to be a fundamental step in the progression of cardiomyopathy and arrhythmias. Myocardial overexpression of the constitutively active Ca 2ϩ -dependent phosphatase calcineurin (CAN) causes severe cardiomyopathy marked by left ventricular (LV) dysfunction, arrhythmias, and increased mortality rate, but CAN antagonist drugs primarily reduce hypertrophy without improving LV function or risk of death. Methods and Results-We found that activity and expression of a second Ca

The voltage-gated cardiac Na+ channel (Nav1.5), encoded by the SCN5A gene, conducts the inward depolarizing cardiac Na+ current (INa) and is vital for normal cardiac electrical activity. Inherited loss-of-function mutations in SCN5A lead to defects in the generation and conduction of the cardiac electrical impulse and are associated with various arrhythmia phenotypes1. Here we show that sirtuin 1 deacetylase (Sirt1) deacetylates Nav1.5 at lysine 1479 (K1479) and stimulates INa via lysine-deacetylation-mediated trafficking of Nav1.5 to the plasma membrane. Cardiac Sirt1 deficiency in mice induces hyperacetylation of K1479 in Nav1.5, decreases expression of Nav1.5 on the cardiomyocyte membrane, reduces INa and leads to cardiac conduction abnormalities and premature death owing to arrhythmia. The arrhythmic phenotype of cardiac-Sirt1-deficient mice recapitulated human cardiac arrhythmias resulting from loss of function of Nav1.5. Increased Sirt1 activity or expression results in decreased lysine acetylation of Nav1.5, which promotes the trafficking of Nav1.5 to the plasma membrane and stimulation of INa. As compared to wild-type Nav1.5, Nav1.5 with K1479 mutated to a nonacetylatable residue increases peak INa and is not regulated by Sirt1, whereas Nav1.5 with K1479 mutated to mimic acetylation decreases INa. Nav1.5 is hyperacetylated on K1479 in the hearts of patients with cardiomyopathy and clinical conduction disease. Thus, Sirt1, by deacetylating Nav1.5, plays an essential part in the regulation of INa and cardiac electrical activity.