Efficient Biolistic Transfection of Fresh Adult Cardiac Myocytes with a Tagged Kv1.5 Channel

Modulation of ion channel trafficking is a potent means by which a cardiomyocyte can regulate its excitability. Much has been learned about the roles of motifs within K+ channels that affect their trafficking to the cell surface. However, by necessity, previous studies have relied on model expression systems, because the transfection of adult cardiomyocytes has, to date, proved intractable. Rat neonatal myocytes can be transfected but the currents expressed in these cells are quite different from those of adult cardiomyocytes. Viral transduction systems are effective in adult cells but require sophisticated containment facilities and prolonged culture of the myocytes, during which time substantial dedifferentiation generally occurs.We have developed a new method that, for the first time, allows the ready and convenient transfection of acutely isolated adult rat cardiac myocytes. Using a low pressure adaptation of a Bio-Rad Helios gene gun procedure, we have achieved efficient transfection of rat ventricular myocytes bombarded within two hours of myocyte isolation with gold particles coated with pcDNA3 constructs encoding tagged Kv1.5 constructs. Expression is rapid, robust, and detectable less than 24 hours post-transfection in myocytes retaining both current profiles and gross morphology comparable to freshly isolated cells. Using this system, we unequivocally demonstrate that tagged Kv1.5 is efficiently localized to the intercalated disk in ventricular myocytes and that it is expressed at the surface of that structure. We further demonstrate that Kv1.5 deletion mutations known to reduce the surface expression of the channel in heterologous cells similarly reduce the surface expression in transfected ventricular myocytes, although targeting to the intercalated disk per se, was generally unaffected. Thus, this new transfection method is an effective tool for the study of cardiac ion channel expression and targeting in a physiologically relevant system.