Background- m6A methylation is the most prevalent internal post-transcriptional modification on mammalian mRNA. The role of m6A mRNA methylation in the heart is not known. Methods- To determine the role of m6A methylation in the heart we isolated primary cardiomyocytes and performed m6A immunoprecipitation followed by RNA sequencing. We then generated genetic tools to modulate m6A levels in cardiomyocytes by manipulating the levels of the m6A RNA methylase METTL3 both in culture and in vivo. We generated cardiac-restricted gain and loss of function mouse models to allow assessment of the METTL3-m6A pathway in cardiac homeostasis and function. Results- We measured the level of m6A methylation on cardiomyocyte mRNA, and found a significant increase in response to hypertrophic stimulation, suggesting a potential role for m6A methylation in the development of cardiomyocyte hypertrophy. Analysis of m6A methylation showed significant enrichment in genes that regulate kinases and intracellular signaling pathways. Inhibition of METTL3 completely abrogated the ability of cardiomyocytes to undergo hypertrophy when stimulated to grow, while increased expression of the m6A RNA methylase METTL3 was sufficient to promote cardiomyocyte hypertrophy both in vitro and in vivo. Finally, cardiac-specific METTL3 knockout mice exhibit morphological and functional signs of heart failure with aging and stress, showing the necessity of RNA methylation for maintenance of cardiac homeostasis. Conclusions- Our study identified METTL3-mediated methylation of mRNA on N6-adenosines as a dynamic modification that is enhanced in response to hypertrophic stimuli and is necessary for a normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA methylation results in compensated cardiac hypertrophy whereas diminished m6A drives eccentric cardiomyocyte remodeling and dysfunction, highlighting the critical importance of this novel stress-response mechanism in the heart for maintaining normal cardiac function.

Ion channel function is fundamental to the existence of life. In metazoans, the coordinate activities of voltagegated Na + channels underlie cellular excitability and control neuronal communication, cardiac excitationcontraction coupling, and skeletal muscle function. However, despite decades of research and linkage of Na + channel dysfunction with arrhythmia, epilepsy, and myotonia, little progress has been made toward understanding the fundamental processes that regulate this family of proteins. Here, we have identified β IV -spectrin as a multifunctional regulatory platform for Na + channels in mice. We found that β IV -spectrin targeted critical structural and regulatory proteins to excitable membranes in the heart and brain. Animal models harboring mutant β IV -spectrin alleles displayed aberrant cellular excitability and whole animal physiology. Moreover, we identified a regulatory mechanism for Na + channels, via direct phosphorylation by β IV -spectrin-targeted calcium/calmodulin-dependent kinase II (CaMKII). Collectively, our data define an unexpected but indispensable molecular platform that determines membrane excitability in the mouse heart and brain.

Background-Computational biology is a powerful tool for elucidating arrhythmogenic mechanisms at the cellular level, where complex interactions between ionic processes determine behavior. A novel theoretical model of the canine ventricular epicardial action potential and calcium cycling was developed and used to investigate ionic mechanisms underlying Ca 2ϩ transient (CaT) and action potential duration (APD) rate dependence. Methods and Results-The Ca 2ϩ /calmodulin-dependent protein kinase (CaMKII) regulatory pathway was integrated into the model, which included a novel Ca 2ϩ -release formulation, Ca 2ϩ subspace, dynamic chloride handling, and formulations for major ion currents based on canine ventricular data. Decreasing pacing cycle length from 8000 to 300 ms shortened APD primarily because of I Ca(L) reduction, with additional contributions from I to1 , I NaK , and late I Na . CaT amplitude increased as cycle length decreased from 8000 to 500 ms. This positive rate-dependent property depended on CaMKII activity. Key Words: electrophysiology Ⅲ action potentials Ⅲ calcium Ⅲ ion channels T he dependence of action potential duration (APD) and the Ca 2ϩ transient (CaT) on pacing rate is a fundamental property of cardiac myocytes that, when altered, may promote life-threatening cardiac arrhythmias. We have developed a detailed and physiologically based mathematical canine ventricular cell model (Hund-Rudy dynamic [HRd] cell model) that simulates rate-dependent phenomena associated with ion-channel kinetics, AP properties, and Ca handling. The dog is commonly used to investigate cardiac electrophysiology, making it a logical choice for modeling. An epicardial myocyte was chosen rather than endocardial or midmyocardial myocytes because epicardial cells contain the highest density of I to1 (transient outward K ϩ current), producing a unique and complex AP morphology. Conclusions-CaMKII MethodsComplete HRd equations, definitions, and detailed comments appear in the online-only Data Supplement. Important model properties (schematic in Figure 1A) are summarized here. Figure 1B) is introduced through a multiplicative factor dependent on the I Ca(L) driving force. Though controversial (online-only Data Supplement section J), CaMKII phosphorylation is thought to promote RyR channel opening. 5,14,20 Accordingly, the I rel inactivation time constant ( ri ) depends on CaMKII activity. A 10-ms maximal CaMKII-dependent increase in ri yields a steady-state CaT amplitude (CaT amp ) 95% 20 greater for control than with CaMKII suppressed at rapid pacing (CLϭ300 ms). Variable 1-second prepulse (V pre ) was followed by 10-ms holding interval at Ϫ50 mV and ϩ80 mV test pulse. Right, I Ca(L) recovery from voltage-dependent inactivation compared with canine ventricular data. 52 Prepulse of 350 ms to ϩ20 mV was followed by varying interpulse interval at Ϫ40 mV and ϩ20 mV test pulse. Model Ca 2ϩ -dependent inactivation gates were held constant to isolate voltage-dependent inactivation. E, Peak I Ks and I Kr tail currents on repolariz...

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.

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.