Ca2+/Calmodulin-dependent protein kinase II (CaMKII) is a key regulator of cardiac function and dysfunction in pathological states via its influence on ion channels, Ca2+ balance, Ca2+ handling proteins involved in cell death, transcriptional activation of hypertrophy, inflammation, and arrhythmias. CaMKII signaling regulates diverse cardiac cellular processes including excitation-contraction coupling, excitation-transcription coupling, mechanics and energetics. Chronic activation of CaMKII results in significant cellular remodeling and alterations in Ca2+ handling, ion channel function, cell-to-cell coupling and metabolism leading to increased risk of atrial and ventricular arrhythmias. The prominent role of CaMKII is also well-established in the pathophysiology of several common heart diseases, including cardiac hypertrophy, heart failure, ischemia-reperfusion injury and post-myocardial infarction (MI) remodeling, atrial fibrillation and ventricular arrhythmias. Pharmacological or genetic inhibition of CaMKII limits arrhythmias and progression of HF, so CaMKII is widely considered a bona fide drug target for heart disease. Post-translational modifications of CaMKII include S-nitrosylation of Cys273 and Cys290. Notably, while S-Nitrosylation of Cys290 on CaMKII promotes autonomous kinase activity, S-Nitrosylation of Cys273 suppresses activation by Ca-CaM.Mechanical stress can affect Ca2+ dynamics in cardiomyocytes and lead to cardiac remodeling, hypertrophy, arrhythmias, and heart failure. Mechanical loading can also evoke physiological adaptations that help the heart cope with increased preload or end-diastolic volume (via the Frank-Starling effect) and with afterload or aortic pressure (via the Anrep effect). Both nitric oxide (NO) produced by NOS1 and CaMKII signaling are required mediators of mechano-chemo-transduction (MCT) whereby mechanical afterload promotes enhanced Ca2+ transients and stronger contraction. To test whether Cys290 in CaMKII mediates the acute S-nitrosylation of CaMKII that promotes increased sarcoplasmic reticulum (SR) Ca release, we developed a novel CaMKIIδ knock-in mouse (C290A substitution). This knock-in mouse exhibited normal cardiac size and function, myocyte Ca transients and -adrenergic responses. However, compared to wildtype (WT) littermates the C290A myocytes failed to exhibit an increase in spontaneous SR Ca2+ release events (Ca2+ sparks) in response to S-nitrosylating agent, GSNO. Next, we tested whether this single amino acid on CaMKII is necessary for afterload-induced increase in Ca2+ sparks and Ca2+ transients, using our cell-in-gel system to exert multiaxial 3D mechanical stress during contraction. In WT myocytes, mechanical afterload increased Ca2+ spark frequency, Ca2+ transient amplitude and SR Ca uptake vs. load-free WT myocytes, which enhances contractility to better meet mechanical afterload against which the myocyte or heart has to work. All of these MCT effects were abolished in either cardiac-specific CaMKII knockout mice, or the point mutant C290A CaMKII knock-in myocytes. Thus, data here shows that CaMKIIδ activation by nitrosylation at the Cys290 site is essential to acute mechanical-stress induced Ca2+ upregulation in cardiomyocytes. They also suggest that NOS1 activation is upstream of S-nitrosylation at Cys290 of CaMKII and enhanced SR Ca uptake and release in this intrinsic MCT autoregulatory pathway in the heart. Since NO and CaMKII signaling are altered in disease, this may raise novel therapeutic strategies for treating cardiac disease.
I also investigate the effect of the post translational modification - nitrosylation - on CaMKII Cys273 via Fluorescence Lifetime Imaging Microscopy (FLIM) and Fluorescence Recovery After Photobleaching (FRAP). Results show that nitric oxide (NO) alters CaMKIIδ activation state and mobility in adult cardiomyocytes. I then investigate the relevance of the Cys273 site on CaMKIIδ for NO-induced suppression of CaMKIIδ activation and mobility in adult ventricular myocytes. The data derived shows for the first time in ventricular cardiomyocytes, that nitrosylation of CaMKII at Cys273 prior to electrical field stimulation and activation by Ca2+ or β-adrenergic agonists, suppresses CaMKIIδ activity and mobility – whereas mutation of the Cysteine residue at 273 to Serine, eliminates this effect.
The multifunctional signaling molecule CaMKII has received considerable attention over recent years for its central role in maladaptive remodeling and arrhythmias in the setting of chronic disease. Hopefully, with continued research, these basic science discoveries will contribute to a greater understanding of cardiac function and heart disease and ultimately translate into new therapies for human patients.
