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 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.

A powerful interaction between the autonomic and the immune systems plays a prominent role in the initiation and maintenance of hypertension and significantly contributes to cardiovascular pathology, end-organ damage and mortality. Studies have shown consistent association between hypertension, proinflammatory cytokines and the cells of the innate and adaptive immune systems. The sympathetic nervous system, a major determinant of hypertension, innervates the bone marrow, spleen and peripheral lymphatic system and is proinflammatory, whereas the parasympathetic nerve activity dampens the inflammatory response through α7-nicotinic acetylcholine receptors. The neuro-immune synapse is bidirectional as cytokines may enhance the sympathetic activity through their central nervous system action that in turn increases the mobilization, migration and infiltration of immune cells in the end organs. Kidneys may be infiltrated by immune cells and mesangial cells that may originate in the bone marrow and release inflammatory cytokines that cause renal damage. Hypertension is also accompanied by infiltration of the adventitia and perivascular adipose tissue by inflammatory immune cells including macrophages. Increased cytokine production induces myogenic and structural changes in the resistance vessels, causing elevated blood pressure. Cardiac hypertrophy in hypertension may result from the mechanical afterload and the inflammatory response to resident or migratory immune cells. Toll-like receptors on innate immune cells function as sterile injury detectors and initiate the inflammatory pathway. Finally, abnormalities of innate immune cells and the molecular determinants of their activation that include toll-like receptor, adrenergic, cholinergic and AT1 receptors can define the severity of inflammation in hypertension. These receptors are putative therapeutic targets.