Silencing of Cav1.2 gene in neonatal cardiomyocytes by lentiviral delivered shRNA

Abstract

Cav1.2 (α 1C) and Cav1.3 (α 1D) L-type Ca channels are co-expressed in the heart. To date, there are no pharmacological or biophysical tools to separate α 1D from α 1C Ca currents (I Ca-L) in cardiomyocytes. Here, we established a physiological model to study α 1D I Ca-L in native myocytes using RNA interference. Transfection of rat neonatal cardiomyocytes (RNC) with α 1C specific siRNA resulted in low silencing efficiency (50–60%) at the mRNA and protein levels. The use of lentivirus shRNA resulted in 100% transfection efficiency and 92% silencing of the α 1C gene by real-time PCR and Western blot. Electrophysiological experiments showed that the total I Ca-L was similarly reduced by 80% in lentivirus transfected cells. Both biochemical and functional data demonstrated high transfection and silencing efficiency in the cardiomyocytes using lentiviral shRNA. This novel approach allows for the assessments of the roles of α 1C and α 1D Ca channels in native myocytes and could be used to examine their roles in physiological and pathological settings.