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.

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.

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.

CaMKII exacerbates heart failure progression by activating class I HDACs.

BACKGROUND: Persistent cardiac Ca2+/calmodulin dependent Kinase II (CaMKII) activation plays an essential role in heart failure development. However, the molecular mechanisms underlying CaMKII induced heart failure progression remains incompletely understood. Histone deacetylases (HDACs) are critical for transcriptional responses to stress, and contribute to expression of pathological genes causing adverse ventricular remodeling. Class I HDACs, including HDAC1, HDAC2 and HDAC3, promote pathological cardiac hypertrophy, whereas class IIa HDACs suppress cardiac hypertrophy. While it is known that CaMKII deactivates class IIa HDACs to enhance cardiac hypertrophy, the role of CaMKII in regulating class I HDACs during heart failure progression is unclear. METHODS AND RESULTS: CaMKII increases the deacetylase activity of recombinant HDAC1, HDAC2 and HDAC3 via in vitro phosphorylation assays. Phosphorylation sites on HDAC1 and HDAC3 are identified with mass spectrometry. HDAC1 activity is also increased in cardiac-specific CaMKIIδC transgenic mice (CaMKIIδC-tg). Beyond post-translational modifications, CaMKII induces HDAC1 and HDAC3 expression. HDAC1 and HDAC3 expression are significantly increased in CaMKIIδC-tg mice. Inhibition of CaMKII by overexpression of the inhibitory peptide AC3-I in the heart attenuates the upregulation of HDAC1 after myocardial infarction surgery. Importantly, a potent HDAC1 inhibitor Quisinostat improves downregulated autophagy genes and cardiac dysfunction in CaMKIIδC-tg mice. In addition to Quisinostat, selective class I HDACs inhibitors, Apicidin and Entinostat, HDAC3 specific inhibitor RGFP966, as well as HDAC1 and HDAC3 siRNA prevent CaMKII overexpression induced cardiac myocyte hypertrophy. CONCLUSION: CaMKII activates class I HDACs in heart failure, which may be a central mechanism for heart failure progression. Selective class I HDACs inhibition may be a novel therapeutic avenue to alleviate CaMKII hyperactivity induced cardiac dysfunction.

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.

MICAL1 constrains cardiac stress responses and protects against disease by oxidizing CaMKII.

Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell-derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.

Myocardial death and dysfunction after ischemia-reperfusion injury require CaMKIIδ oxidation.

Reactive oxygen species (ROS) contribute to myocardial death during ischemia-reperfusion (I/R) injury, but detailed knowledge of molecular pathways connecting ROS to cardiac injury is lacking. Activation of the Ca2+/calmodulin-dependent protein kinase II (CaMKIIδ) is implicated in myocardial death, and CaMKII can be activated by ROS (ox-CaMKII) through oxidation of regulatory domain methionines (Met281/282). We examined I/R injury in mice where CaMKIIδ was made resistant to ROS activation by knock-in replacement of regulatory domain methionines with valines (MMVV). We found reduced myocardial death, and improved left ventricular function 24 hours after I/R injury in MMVV in vivo and in vitro compared to WT controls. Loss of ATP sensitive K+ channel (KATP) current contributes to I/R injury, and CaMKII promotes sequestration of KATP from myocardial cell membranes. KATP current density was significantly reduced by H2O2 in WT ventricular myocytes, but not in MMVV, showing ox-CaMKII decreases KATP availability. Taken together, these findings support a view that ox-CaMKII and KATP are components of a signaling axis promoting I/R injury by ROS.