Effects Of Coxsackievirus B₃ On The Function And Expression Of L-type Calcium Channels Of Rat Ventricular Myocytes

[Background and objective]Myocarditis is a myocardial inflammatory disease caused by many different causes, the most common of which is virus infection, especially the enterovirus group. It is reported that most of the patients with acute viral myocarditis had ECG abnormalities within 3 weeks after infection, including ST-T segments changes and many kinds of arrhythmias. To date, the underlying electrophysiological or molecular pathogenesis of these arrhythmias is unclear. The present study was therefore designed to: (1) establish a reliable cellular model of viral myocarditis; (2) investigate the effects of Coxsackie virus B₃ infection on the L-type calcium currents and calcium channel subunits expression in neonatal rat ventricular myocytes and (3) explore the possible ionic and molecular mechanism of ventricular arrhythmias in viral myocarditis.[Methods](1) To analyze the electrocardiograms of 127 cases of viral myocarditis retrospectively and make a statistic for the ratio of arrhythmias.(2) A primary culture of neonatal rat ventricular myocytes was prepared with 1-3-day-old Sprague-Dawlay rats. After 48 hours, the myocytes were infected with Coxsackie virus B₃. Cytopathic effect and ultrastructural changes of the myocytes were observed under inverted-microscope and electron microscope. RNA of Coxsackie virus B3 was detected in the infected ventricular myocytes and the medium with reverse transcription-polymerase chain reaction.(3) After 24 hours of infection, L-type calcium currents were recorded in normal and infected ventricular myocytes respectively using whole-cell patch clamp techniques.(4) The changes of L-type calcium channel subunits mRNA of the two groups were measured by reverse transcription-polymerase chain reaction.(5) Antibodies against subunits of L-type calcium channels were prepared and purified. Immunoblot was employed to determine the expression of the L-type calcium channel protein of the two groups.[Results](1) 79 cases were diagnosed positive for coxsackievirus-IgM in the total of 127 patients. The percentage was 62.20%. Among these positive patients, 64 cases (81.01%) were found to suffer from arrhythmias with Holter ECG monitoring. 8 cases were atrial premature beats or atrial tachycardia (12.5%); 2 cases were premature atrioventricular junctional beats (3.13%); 28 cases were ventricular premature beats or ventricular tachycardia (43.75%); 19 cases were atrioventricular block (29.69%).(2) The normal cultured neonatal rat ventricular myocytes beat regularly. After infected with Coxsackie virus B₃ for 48 hours, they began to beat more slowly and show obvious cytopathic effects gradually. Finally they stopped beating and detached from the coverlips and died. These external morphological abnormalities were accompanied by internal ultrastructural changes and evidence of a direct cytopathic effect in cardiac myocytes in which RNA of the structural protein VP1 of CVB₃ was positive. Moreover, CVB₃-RNA and increasing concentrations of CK-MB were detected in the medium of infected myocytes.(3) The results of the electrophysiology showed no significant differences in cell membrane capacitance between the two groups (43.30±5.08pF vs 44.50± 5.01pF, P=NS, n=20). But in contrast with the normal group, Coxsackie virus B₃ infection significantly increased the L-type calcium currents density at test potentials from -20 to ±10mV (P<0.05 vs control, n=7). At 0mV, the peak current density increased by 25.74%. No significant differences were observed for the voltage-dependent activation and inactivation of L-type calcium current between the two groups.(4) The α_1c and β₂ subunits mRNA of L-type calcium channels were increased in the infected group compared with the normal one (P<0.01 or P<0.05 vs control). The changes of the α₂/δ subunits have no statistical significance.(5) Immunofluorescent imaging showed that purified antibodies against α_1c, β₂ and α₂/δ subunits could combine with cultured rat ventricular myocytes specifically. Moreover, more protein for α_1c and β₂ subunits were probed with corresponding antibodies by immunoblots in the infected group. But no significant difference was observed for the α₂/δ subunits between the two groups.[Conclusion](1) Arrhythmias were frequently observed in viral myocarditis, the most common of which was ventricular ones.(2) This cellular model established here can be used as the basis for in vitro studies of viral myocarditis induced by Coxsackie virus B₃. Coxsackie virus B₃ infection increased L-type calcium currents of neonatal rat ventricular myocytes significantly. In accordance with this, the mRNA and protein expression of the channel were up-regulated in the infected group compared with those in the normal one. These may account for some of above abnormal electrophysiologies in viral myocarditis.