Increased phosphorylation of Ca2+ handling protein as a proarrhythmic mechanism in myocarditis

Abstract

Fatal arrhythmia is an important cause of death in patients with myocarditis. This study investigated the proarrhythmic mechanisms of experimental autoimmune myocarditis (EAM). EAM was induced by the injection of porcine cardiac myosin of 2 mg into footpads of adult Lewis rats on day 1 and 8 (Myo, n=15) and compared with control rats (Control, n=15). In additional rats, corticosteroid of 6 mg was injected into the gluteus muscle before the injection of porcine cardiac myosin on day 1 and 8 (MyoS, n=15). Myocytes with myocarditis produced by TNF-α treatment used in intracellular calcium measurement. Myocarditis hearts had longer action potential duration (APD), slower conduction velocity (CV; p<0.01 versus control), steeper CV restitution kinetics, greater fibrosis, higher levels of transcripts for HMGB1, IL-6, and TNF-α and phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CAMKII), ryanodine receptor type 2 (RyR2) and phospholamban(PLB). Steroid treatment reversed the transcripts for myocarditis, flattened CV restitution kinetics, reduced APD at 90% recovery to baseline, increased CV (p<0.01), and reversed fibrosis and phosphorylation of CaMKII, RyR2 and PLB (p<0.05). Programmed stimulation triggered sustained VT in Myo (n=4/5), but not in control (n=0/5), and steroid (n=0/5, p<0.001) and CaMKII inhibitor (KN93) pretreatment (n=0/4, p=0.001). Myocytes with myocarditis had increased intracellular calcium (p<0.01 versus control), which was reversed by pretreatment with steroid and KN93. The electrophysiologic characteristics of myocarditis were increased APD, slower CV and steeper CV restitution kinetics. These changes might be related with calcium overloading by increased phosphorylation of CaMKII.