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.

Low STAT3 expression sensitizes to toxic effects of ß-adrenergic receptor stimulation in peripartum cardiomyopathy.

Aims: The benefit of the ß1-adrenergic receptor (ß1-AR) agonist dobutamine for treatment of acute heart failure in peripartum cardiomyopathy (PPCM) is controversial. Cardiac STAT3 expression is reduced in PPCM patients. Mice carrying a cardiomyocyte-restricted deletion of STAT3 (CKO) develop PPCM. We hypothesized that STAT3-dependent signalling networks may influence the response to ß-AR agonist treatment in PPCM patients and analysed this hypothesis in CKO mice. Methods and Results: Follow-up analyses in 27 patients with severe PPCM (left ventricular ejection fraction ≤25%) revealed that 19 of 20 patients not obtaining dobutamine improved cardiac function. All seven patients obtaining dobutamine received heart transplantation (n = 4) or left ventricular assist devices (n = 3). They displayed diminished myocardial triglyceride, pyruvate, and lactate content compared with non-failing controls. The ß-AR agonist isoproterenol (Iso) induced heart failure with high mortality in postpartum female, in non-pregnant female and in male CKO, but not in wild-type mice. Iso induced heart failure and high mortality in CKO mice by impairing fatty acid and glucose uptake, thereby generating a metabolic deficit. The latter was governed by disturbed STAT3-dependent signalling networks, microRNA-199a-5p, microRNA-7a-5p, insulin/glucose transporter-4, and neuregulin/ErbB signalling. The resulting cardiac energy depletion and oxidative stress promoted dysfunction and cardiomyocyte loss inducing irreversible heart failure, which could be attenuated by the ß1-AR blocker metoprolol or glucose-uptake-promoting drugs perhexiline and etomoxir. Conclusions: Iso impairs glucose uptake, induces energy depletion, oxidative stress, dysfunction, and death in STAT3-deficient cardiomyocytes mainly via ß1-AR stimulation. These cellular alterations may underlie the dobutamine-induced irreversible heart failure progression in PPCM patients who frequently display reduced cardiac STAT3 expression.

Mst1 inhibition rescues ß1-adrenergic cardiomyopathy by reducing myocyte necrosis and non-myocyte apoptosis rather than myocyte apoptosis.

It is generally held that inhibition of mammalian sterile 20-like kinase 1 (Mst1) protects the heart through reducing myocyte apoptosis. We determined whether inhibition with a dominant-negative Mst1 (DN-Mst1) would protect against the cardiomyopathy induced by chronic ß1-adrenergic receptor (ß1-AR) stimulation by preventing myocyte apoptosis. DN-Mst1 mice were mated with ß1-AR transgenic (Tg) mice and followed for 20 months. ß1-AR Tg mice developed cardiomyopathy as they aged, as reflected by premature mortality and depressed cardiac function, which were rescued in ß1-AR × DN-Mst1 bigenic mice. Surprisingly, myocyte apoptosis did not significantly decrease with Mst1 inhibition. Instead, Mst1 inhibition predominantly reduced non-myocyte apoptosis, e.g., fibroblasts, macrophages, neutrophils and endothelial cells. Fibrosis in the hearts with cardiomyopathy increased fivefold and this increase was nearly abolished in the bigenic mice with Mst1 inhibition. Regression analysis showed no correlation between myocyte apoptosis and cardiac function or myocyte number, whereas the latter two correlated significantly, p < 0.05, with fibrosis, which generally results from necrosis. To examine the role of myocyte necrosis, chronic ß-AR stimulation with isoproterenol was induced for 24 h and myocyte necrosis was assessed by 1% Evans blue dye. Compared to WT, DN-Mst1 mice showed significant inhibition, p < 0.05, of myocyte necrosis. We confirmed this result in Mst1-knockout mice, which also showed significant protection, p < 0.05, against myocyte necrosis compared to WT. These data indicate that Mst1 inhibition rescued cardiac fibrosis and myocardial dysfunction in ß1-AR cardiomyopathy. However, this did not occur through Mst1 inhibition of myocyte apoptosis but rather by inhibition of cardiomyocyte necrosis and non-myocyte apoptosis, features of Mst1 not considered previously.