Adenoviral-mediated expression of dihydropyridine-insensitive L-type calcium channels in cardiac ventricular myocytes and fibroblasts

Cardiac voltage-gated Ca2+ channels regulate the intracellular Ca2+ concentration and are therefore essential for muscle contraction, second messenger activation, gene expression and electrical signaling. As a first step in accessing the structural versus functional properties of the L-type Ca2+ channel in the heart, we have expressed a dihydropyridine (DHP)-insensitive Ca(V)1.2 channel in rat ventricular myocytes and fibroblasts. Following isolation and culture, cells were infected with adenovirus expressing either LacZ or a mutant Ca(V)1.2 channel (CaV1.2DHPi) containing the double mutation (T1039Y & Q1043M). This mutation renders the channel insensitive to neutral DHP compounds such as nisoldipine. The whole-cell, L-type Ca2+ current (I-Ca) measured in control myocytes was inhibited in a concentration-depen dent manner by nisoldipine with an IC50 of 66 nM and complete block at 250 nM. In contrast, I-Ca in cells infected with AdCa(V)1.2DHP(i) was inhibited by only 35% by 500 nM nisoldipine but completely blocked by 50 mu M diltiazem. In order to study Ca(V)1.2DHP(i) in isolation, myocytes infected with AdCa(V)1.2DHP(i) were incubated with nisoldipine. Under this condition the cells expressed a large I-Ca (12 pA/pF) and displayed Ca2+ transients during field stimulation. Furthermore, addition of 2 mu M forskolin and 100 mu M 3-isobutyl-1-methylxanthine (IBMX), to stimulate protein kinase A, strongly increased I-Ba in the AdCa(V)1.2DHP(i)-infected cells. A Cd2+-sensitive I-Ba was also recorded in cardiac fibroblasts infected with AdCa(V)1.2DHP(i). Thus, expression of Ca(v)1.2DHP(i) will provide an important tool in studies of cardiac myocyte and fibroblast function. (c) 2007 Elsevier B.V All rights reserved.