Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes

The functional consequences of overexpression of rat heart Na+/Ca2+ exchang er (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, culture d adult rat myocytes retained normal contractile function compared with fre shly isolated myocytes for at least 48 h. Infection of myocytes by adenovir us expressing green fluorescent protein (GFP) resulted in 95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48- h postinfection was not affected. When they were examined 48 h after in fection, myocytes infected by adenovirus expressing both GFP and NCX1 had s imilar cell sizes but exhibited significantly altered contraction amplitude s and intracellular Ca2+ concentration ([Ca2+](i)) transients, and lower re sting and diastolic [Ca2+](i) when compared with myocytes infected by the a denovirus expressing GFP alone. The effects of NCX1 overexpression on sarco plasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentr ation ([ Ca2+](o)), with a decrease at low [Ca2+](o) and an increase at hig h [Ca2+](o). The half-times for [Ca2+](i) transient decline were similar, s uggesting little to no changes in SR Ca2+-ATPase activity. Western blots de monstrated a significant (P less than or equal to0.02) threefold increase i n NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myoc ytes 48 h after infection by adenovirus expressing both GFP and NCX1 compar ed with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+](o) r esulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+](i) transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting an d diastolic [Ca2+](i).