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

Abstract

Cardiac voltage-gated Ca 2+ channels regulate the intracellular Ca 2+ 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 Ca 2+ channel in the heart, we have expressed a dihydropyridine (DHP)-insensitive Ca V1.2 channel in rat ventricular myocytes and fibroblasts. Following isolation and culture, cells were infected with adenovirus expressing either LacZ or a mutant Ca V1.2 channel (Ca V1.2DHP i) containing the double mutation (T1039Y & Q1043M). This mutation renders the channel insensitive to neutral DHP compounds such as nisoldipine. The whole-cell, L-type Ca 2+ current ( I Ca) measured in control myocytes was inhibited in a concentration-dependent manner by nisoldipine with an IC 50 of 66 nM and complete block at 250 nM. In contrast, I Ca in cells infected with AdCa V1.2DHP i was inhibited by only 35% by 500 nM nisoldipine but completely blocked by 50 μM diltiazem. In order to study Ca V1.2DHP i in isolation, myocytes infected with AdCa V1.2DHP i were incubated with nisoldipine. Under this condition the cells expressed a large I Ca (12 pA/pF) and displayed Ca 2+ transients during field stimulation. Furthermore, addition of 2 μM forskolin and 100 μM 3-isobutyl-1-methylxanthine (IBMX), to stimulate protein kinase A, strongly increased I Ba in the AdCa V1.2DHP i-infected cells. A Cd 2+-sensitive I Ba was also recorded in cardiac fibroblasts infected with AdCa V1.2DHP i. Thus, expression of Ca V1.2DHP i will provide an important tool in studies of cardiac myocyte and fibroblast function.