HCN channels sense temperature and determine heart rate responses to heat.

Heart rate increases with heat, [1-3] constituting a fundamental physiological relationship in vertebrates. Each normal heartbeat is initiated by an action potential generated in a sinoatrial nodal pacemaker cell. Pacemaker cells are enriched with hyperpolarization activated cyclic nucleotide-gated ion channels (HCN) that deliver cell membrane depolarizing inward current that triggers action potentials. HCN channel current increases due to cAMP binding, a mechanism coupling adrenergic tone to physiological 'fight or flight' heart rate acceleration. However, the mechanism(s) for heart rate response to thermal energy is unknown. We used thermodynamical and homology computational modeling, site-directed mutagenesis and mouse models to identify a concise motif on the S4-S5 linker of the cardiac pacemaker HCN4 channels (M407/Y409) that determines HCN4 current (If) and cardiac pacemaker cell responses to heat. This motif is required for heat sensing in cardiac pacemaker cells and in isolated hearts. In contrast, the cyclic nucleotide binding domain is not required for heat induced HCN4 current increases. However, a loss of function M407/Y409 motif mutation prevented normal heat and cAMP responses, suggesting that heat sensing machinery is essential for operating the cAMP allosteric pathway and is central to HCN4 modulation. The M407/Y409 motif is conserved across all HCN family members suggesting that HCN channels participate broadly in coupling heat to changes in cell membrane excitability.

An improved reporter identifies ruxolitinib as a potent and cardioprotective CaMKII inhibitor.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) hyperactivity causes cardiac arrhythmias, a major source of morbidity and mortality worldwide. Despite proven benefits of CaMKII inhibition in numerous preclinical models of heart disease, translation of CaMKII antagonists into humans has been stymied by low potency, toxicity, and an enduring concern for adverse effects on cognition due to an established role of CaMKII in learning and memory. To address these challenges, we asked whether any clinically approved drugs, developed for other purposes, were potent CaMKII inhibitors. For this, we engineered an improved fluorescent reporter, CaMKAR (CaMKII activity reporter), which features superior sensitivity, kinetics, and tractability for high-throughput screening. Using this tool, we carried out a drug repurposing screen (4475 compounds in clinical use) in human cells expressing constitutively active CaMKII. This yielded five previously unrecognized CaMKII inhibitors with clinically relevant potency: ruxolitinib, baricitinib, silmitasertib, crenolanib, and abemaciclib. We found that ruxolitinib, an orally bioavailable and U.S. Food and Drug Administration-approved medication, inhibited CaMKII in cultured cardiomyocytes and in mice. Ruxolitinib abolished arrhythmogenesis in mouse and patient-derived models of CaMKII-driven arrhythmias. A 10-min pretreatment in vivo was sufficient to prevent catecholaminergic polymorphic ventricular tachycardia, a congenital source of pediatric cardiac arrest, and rescue atrial fibrillation, the most common clinical arrhythmia. At cardioprotective doses, ruxolitinib-treated mice did not show any adverse effects in established cognitive assays. Our results support further clinical investigation of ruxolitinib as a potential treatment for cardiac indications.

Genome-wide CRISPR screen reveals genetic modifiers of Ca 2+ -mediated cell death.

Ca 2+ is a fundamental determinant of survival in living cells. Excessive intracellular Ca 2+ causes cellular toxicity and death but the genetic pathways contributing to Ca 2+ induced cell death are incompletely understood. Here, we performed genome-wide CRISPR knock-out screening in human cells challenged with the Ca 2+ ionophore ionomycin and identified genes and pathways essential for cell death after Ca 2+ overload. We discovered 115 protective gene knockouts, 82 of which are non-essential genes and 21 of which belong to the druggable genome. Notably, members of store operated Ca 2+ entry (SOCE), very long-chain fatty acid synthesis, and SWItch/Sucrose Non-Fermentable (SWI/SNF) pathways provided marked protection against Ca 2+ toxicity. These results reveal pathways previously unknown to mediate Ca 2+ -induced cell death and provide a resource for the development of pharmacotherapies against the sequelae of Ca 2+ overload in disease.

CaMKII and reactive oxygen species contribute to early reperfusion arrhythmias, but oxidation of CaMKIIδ at methionines 281/282 is not a determining factor.

BACKGROUND: Available evidence suggest that Ca2+/calmodulin-dependent protein kinase type IIδ (CaMKIIδ) and reactive oxygen species (ROS) are important in early ischemia-reperfusion arrhythmias (IRA). Since ROS can activate CaMKIIδ by oxidation of two methionines at positions 281/282, oxidized-CaMKIIδ (Ox-CaMKIIδ) has been proposed to be important for IRA. However, direct evidence for this is missing. METHODS: We exposed Langendorff-perfused hearts and ventricular cardiomyocytes from C57BL/6 mice to global and simulated ischemia, respectively, and recorded arrhythmic events during early reperfusion. Hearts were collected for immunoblotting of key phosphoproteins. We evaluated the effects of beta-adrenoceptor stimulation, inhibition of CaMKII, and reduced ROS levels with isoprenaline, KN93/AIP and N-acetylcysteine (NAC), respectively. We further tested the importance of Ox-CaMKIIδ by using hearts and cardiomyocytes from mice with CaMKIIδ resistant to oxidation of methionines 281 and 282 (MMVV). RESULTS: Hearts treated with KN93, AIP or NAC had lower incidence of early IRA, and NAC-treated cardiomyocytes had lower incidence of arrhythmogenic events. However, hearts from MMVV mice had a similar incidence of early IRA to wild type mice (WT), and MMVV and WT cardiomyocytes had a similar frequency of Ca2+ waves and Ca2+ sparks. Immunoblotting confirmed high levels of oxidation in early reperfusion, but revealed no significant differences in the phosphorylation levels of Ca2+-handling proteins in MMVV and WT hearts. CONCLUSIONS: Although CaMKII and ROS both contribute to early IRA, hearts from mice with CaMKII resistant to oxidation at methionines 281/282 were not protected from such arrhythmias, suggesting that oxidation at these sites is not a determining factor.

CaMKII as a Therapeutic Target in Cardiovascular Disease.

CaMKII (the multifunctional Ca2+ and calmodulin-dependent protein kinase II) is a highly validated signal for promoting a variety of common diseases, particularly in the cardiovascular system. Despite substantial amounts of convincing preclinical data, CaMKII inhibitors have yet to emerge in clinical practice. Therapeutic inhibition is challenged by the diversity of CaMKII isoforms and splice variants and by physiological CaMKII activity that contributes to learning and memory. Thus, uncoupling the harmful and beneficial aspects of CaMKII will be paramount to developing effective therapies. In the last decade, several targeting strategies have emerged, including small molecules, peptides, and nucleotides, which hold promise in discriminating pathological from physiological CaMKII activity. Here we review the cellular and molecular biology of CaMKII, discuss its role in physiological and pathological signaling, and consider new findings and approaches for developing CaMKII therapeutics.

Oxidative stress-induced autonomous activation of the calcium/calmodulin-dependent kinase II involves disulfide formation in the regulatory domain.

Calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ) has a pivotal role in cardiac signaling. Constitutive and deleterious CaMKII "autonomous" activation is induced by oxidative stress, and the previously reported mechanism involves oxidation of methionine residues in the regulatory domain. Here, we demonstrate that covalent oxidation leads to a disulfide bond with Cys273 in the regulatory domain causing autonomous activity. Autonomous activation was induced by treating CaMKII with diamide or histamine chloramine, two thiol-oxidizing agents. Autonomy was reversed when the protein was incubated with DTT or thioredoxin to reduce disulfide bonds. Tryptic mapping of the activated CaMKII revealed formation of a disulfide between Cys273 and Cys290 in the regulatory domain. We determined the apparent pKa of those Cys and found that Cys273 had a low pKa while that of Cys290 was elevated. The low pKa of Cys273 facilitates oxidation of its thiol to the sulfenic acid at physiological pH. The reactive sulfenic acid then attacks the thiol of Cys290 to form the disulfide. The previously reported CaMKII mutant in which methionine residues 281 and 282 were mutated to valine (MMVV) protects mice and flies from cardiac decompensation induced by oxidative stress. Our initial hypothesis was that the MMVV mutant underwent a conformational change that prevented disulfide formation and autonomous activation. However, we found that the thiol-oxidizing agents induced autonomy in the MMVV mutant and that the mutant undergoes rapid degradation by the cell, potentially preventing accumulation of the injurious autonomous form. Together, our results highlight additional mechanistic details of CaMKII autonomous activation.

Myocardial ATP depletion detected noninvasively predicts sudden cardiac death risk in patients with heart failure.

BACKGROUNDSudden cardiac death (SCD) remains a worldwide public health problem in need of better noninvasive predictive tools. Current guidelines for primary preventive SCD therapies, such as implantable cardioverter defibrillators (ICDs), are based on left ventricular ejection fraction (LVEF), but these guidelines are imprecise: fewer than 5% of ICDs deliver lifesaving therapy per year. Impaired cardiac metabolism and ATP depletion cause arrhythmias in experimental models, but to our knowledge a link between arrhythmias and cardiac energetic abnormalities in people has not been explored, nor has the potential for metabolically predicting clinical SCD risk.METHODSWe prospectively measured myocardial energy metabolism noninvasively with phosphorus magnetic resonance spectroscopy in patients with no history of significant arrhythmias prior to scheduled ICD implantation for primary prevention in the setting of reduced LVEF (≤35%).RESULTSBy 2 different analyses, low myocardial ATP significantly predicted the composite of subsequent appropriate ICD firings for life-threatening arrhythmias and cardiac death over approximately 10 years. Life-threatening arrhythmia risk was approximately 3-fold higher in patients with low ATP and independent of established risk factors, including LVEF. In patients with normal ATP, rates of appropriate ICD firings were several-fold lower than reported rates of ICD complications and inappropriate firings.CONCLUSIONTo the best of our knowledge, these are the first data linking in vivo myocardial ATP depletion and subsequent significant arrhythmic events in people, suggesting an energetic component to clinical life-threatening ventricular arrhythmogenesis. The findings support investigation of metabolic strategies that limit ATP loss to treat or prevent life-threatening cardiac arrhythmias and herald noninvasive metabolic imaging as a complementary SCD risk stratification tool.TRIAL REGISTRATIONClinicalTrials.gov NCT00181233.FUNDINGThis work was supported by the DW Reynolds Foundation, the NIH (grants HL61912, HL056882, HL103812, HL132181, HL140034), and Russell H. Morgan and Clarence Doodeman endowments at Johns Hopkins.

PDE1 Inhibition Modulates Cav1.2 Channel to Stimulate Cardiomyocyte Contraction.

[Figure: see text].

The oxidation-resistant CaMKII-MM281/282VV mutation does not prevent arrhythmias in CPVT1.

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited arrhythmogenic disorder caused by missense mutations in the cardiac ryanodine receptors (RyR2), that result in increased ß-adrenoceptor stimulation-induced diastolic Ca2+ leak. We have previously shown that exercise training prevents arrhythmias in CPVT1, potentially by reducing the oxidation of Ca2+ /calmodulin-dependent protein kinase type II (CaMKII). Therefore, we tested whether an oxidation-resistant form of CaMKII protects mice carrying the CPVT1-causative mutation RyR2-R2474S (RyR2-RS) against arrhythmias. Antioxidant treatment (N-acetyl-L-cysteine) reduced the frequency of ß-adrenoceptor stimulation-induced arrhythmogenic Ca2+ waves in isolated cardiomyocytes from RyR2-RS mice. To test whether the prevention of CaMKII oxidation exerts an antiarrhythmic effect, mice expressing the oxidation-resistant CaMKII-MM281/282VV variant (MMVV) were crossed with RyR2-RS mice to create a double transgenic model (RyR2-RS/MMVV). Wild-type mice served as controls. Telemetric ECG surveillance revealed an increased incidence of ventricular tachycardia and an increased arrhythmia score in both RyR2-RS and RyR2-RS/MMVV compared to wild-type mice, both following a ß-adrenoceptor challenge (isoprenaline i.p.), and following treadmill exercise combined with a ß-adrenoceptor challenge. There were no differences in the incidence of arrhythmias between RyR2-RS and RyR2-RS/MMVV mice. Furthermore, no differences were observed in ß-adrenoceptor stimulation-induced Ca2+ waves in RyR2-RS/MMVV compared to RyR2-RS. In conclusion, antioxidant treatment reduces ß-adrenoceptor stimulation-induced Ca2+ waves in RyR2-RS cardiomyocytes. However, oxidation-resistant CaMKII-MM281/282VV does not protect RyR2-RS mice from ß-adrenoceptor stimulation-induced Ca2+ waves or arrhythmias. Hence, alternative oxidation-sensitive targets need to be considered to explain the beneficial effect of antioxidant treatment on Ca2+ waves in cardiomyocytes from RyR2-RS mice.

CaMKII oxidation is a critical performance/disease trade-off acquired at the dawn of vertebrate evolution.

Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca2+ and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates.

Excessive O-GlcNAcylation Causes Heart Failure and Sudden Death.

BACKGROUND: Heart failure is a leading cause of death worldwide and is associated with the rising prevalence of obesity, hypertension, and diabetes. O-GlcNAcylation (the attachment of O-linked ß-N-acetylglucosamine [O-GlcNAc] moieties to cytoplasmic, nuclear, and mitochondrial proteins) is a posttranslational modification of intracellular proteins and serves as a metabolic rheostat for cellular stress. Total levels of O-GlcNAcylation are determined by nutrient and metabolic flux, in addition to the net activity of 2 enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Failing myocardium is marked by increased O-GlcNAcylation, but whether excessive O-GlcNAcylation contributes to cardiomyopathy and heart failure is unknown. METHODS: We developed 2 new transgenic mouse models with myocardial overexpression of OGT and OGA to control O-GlcNAcylation independent of pathologic stress. RESULTS: We found that OGT transgenic hearts showed increased O-GlcNAcylation and developed severe dilated cardiomyopathy, ventricular arrhythmias, and premature death. In contrast, OGA transgenic hearts had lower O-GlcNAcylation but identical cardiac function to wild-type littermate controls. OGA transgenic hearts were resistant to pathologic stress induced by pressure overload with attenuated myocardial O-GlcNAcylation levels after stress and decreased pathologic hypertrophy compared with wild-type controls. Interbreeding OGT with OGA transgenic mice rescued cardiomyopathy and premature death, despite persistent elevation of myocardial OGT. Transcriptomic and functional studies revealed disrupted mitochondrial energetics with impairment of complex I activity in hearts from OGT transgenic mice. Complex I activity was rescued by OGA transgenic interbreeding, suggesting an important role for mitochondrial complex I in O-GlcNAc-mediated cardiac pathology. CONCLUSIONS: Our data provide evidence that excessive O-GlcNAcylation causes cardiomyopathy, at least in part, attributable to defective energetics. Enhanced OGA activity is well tolerated and attenuation of O-GlcNAcylation is beneficial against pressure overload-induced pathologic remodeling and heart failure. These findings suggest that attenuation of excessive O-GlcNAcylation may represent a novel therapeutic approach for cardiomyopathy.

Loss of CASK Accelerates Heart Failure Development.

[Figure: see text].

Oxidized CaMKII and O-GlcNAcylation cause increased atrial fibrillation in diabetic mice by distinct mechanisms.

Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.

Mitochondrial CaMKII causes adverse metabolic reprogramming and dilated cardiomyopathy.

Despite the clear association between myocardial injury, heart failure and depressed myocardial energetics, little is known about upstream signals responsible for remodeling myocardial metabolism after pathological stress. Here, we report increased mitochondrial calmodulin kinase II (CaMKII) activation and left ventricular dilation in mice one week after myocardial infarction (MI) surgery. By contrast, mice with genetic mitochondrial CaMKII inhibition are protected from left ventricular dilation and dysfunction after MI. Mice with myocardial and mitochondrial CaMKII overexpression (mtCaMKII) have severe dilated cardiomyopathy and decreased ATP that causes elevated cytoplasmic resting (diastolic) Ca2+ concentration and reduced mechanical performance. We map a metabolic pathway that rescues disease phenotypes in mtCaMKII mice, providing insights into physiological and pathological metabolic consequences of CaMKII signaling in mitochondria. Our findings suggest myocardial dilation, a disease phenotype lacking specific therapies, can be prevented by targeted replacement of mitochondrial creatine kinase or mitochondrial-targeted CaMKII inhibition.