Calcium/calmodulin dependent protein kinase II (CaMKII) is a key regulator of a wide range of functions in cardiac myocytes including Ca regulation, contraction, and transcriptional control. When CaM binds and activates CaMKII, the low basal Ca/CaM affinity allows for CaMKII to be turned on and off rapidly. CaMKII autophosphorylation at the well-known T287 site, however, can prolong the active state by slowing the off-rate ~100-fold and ?trapping? Ca/CaM in the bound state, or by making CaMKII ?autonomous? ? or partially active even after Ca/CaM dissociation. This prolonged ?memory? effect of CaMKII activity and chronic autonomous CaMKII over-activation are implicated in cardiac pathologies such as heart failure (HF) and arrhythmias. Recently, we and others have discovered four novel post-translational modifications (PTMs) (oxidation at MM281/282, O-GlcNAcylation at S280, and S-nitrosylation at C273 and C290) that prolong CaMKII activity and are likely to be activated by a broader range of pathological stressors such as oxidative stress (ROS), nitric oxide synthase (NOS) activation, and diabetic hyperglycemia. Despite the recognized importance and potentially significant clinical implications, little is known about these new PTMs with respect to their effects on CaMKII autonomy or their specific influence on cardiac myocyte physiology. Moreover, the key functional role that CaMKII plays in numerous cardiac pathologies makes it essential to understand exactly how CaMKII is really regulated in adult cardiac myocytes. The overall goal of this proposed study, therefore, is to investigate how these four novel PTMs integrate to modulate CaMKII memory and affect cardiac myocyte physiology.
Aim 1 will test the hypothesis that all four regulatory domain PTMs would comparably increase CaM affinity. All of the PTMs are predicted to promote autonomous activation, though S-nitrosylation at C273 is predicted to inhibit activation by Ca/CaM.
Aim 2 will test the hypothesis that PTMs that impute greater CaM affinity and longer autonomous CaMKII activation durations would have greater impact on cardiac myocyte physiology. To achieve these ends, experiments using molecular imaging techniques (e.g. FRET) involving novel fluorescently-labeled proteins (e.g. GFP-CaMKII, AF-CaM, CFP-CaMKII-YFP (Camui)) and their PTM-resistant mutant variants, patch clamp electrophysiology, and biochemical approaches will be utilized in permeabilized and intact ventricular myocytes from rabbits or PTM-resistant mutant mice. The proposed studies are anticipated to provide tremendous and highly original insights into fundamental mechanisms of CamKII activation and memory in myocytes, and into the synergy among clinically relevant PTMs. Clarifying these mechanisms of CaMKII activity is critical and timely, and may yield new therapeutic targets for treating cardiac disease.
The pivotal role that CaMKII plays in an array of molecular pathways in the cardiac myocyte can be a double- edged sword: while normal levels of CaMKII activation is responsible for the fine regulation of a wide range of essential cardiac functions, over-activation of CaMKII can lead to a widespread development of functional abnormalities underlying cardiac disease. The recent discovery of four novel CaMKII post-translational modifications has revealed that CaMKII may be susceptible to an even wider range of pathological stressors than once thought, such as oxidative stress, nitric oxide synthase activation, and diabetic hyperglycemia, which are associated with morbidity, mortality, and health care costs involving cardiac disease. This study, which proposes to investigate how these four novel post-translational modifications integrate to modulate CaMKII memory and affect cardiac myocyte physiology, will yield valuable mechanistic insights that can lead to the development of therapeutic strategies for treating cardiac disease.
