Structural remodeling of L-type calcium channel subunits in human and murine atherosclerosis

Abstract

The function of voltage-gated L-type calcium channels (LTCCs) has major impact on the function and contractility of vascular smooth muscle cells (VSMCs). The influx of external calcium regulates intracellular free calcium - thereby arterial tone - and is important for additional biological processes inside the cell. In atherosclerotic VSMCs, the function of LTCCs is shown to be reduced – but the molecular mechanism behind this has not yet been elucidated. It has been reported that in human atherosclerosis, vascular smooth muscle Ca_V1.2 isoforms containing exon 21 are replaced by one single isoform containing the alternative exon 22. Previous functional studies in our group reveal that this splice shift has major effects on LTCC function, depending on the β subunit co-expressed.<br /> The present work examined the LTCC subunit expression pattern in both human and murine atherosclerotic arterial tissues. Samples of human atherosclerotic arteries were obtained from patients undergoing carotid or femoral arterial bypass surgery. Human vascular smooth muscle tissue was isolated by laser microdissection or the whole arterial sample was examined. Mouse aorta was isolated from healthy wildtype and atherosclerotic apoE-knockout animals. LTCC subunit mRNA expression was quantified by qRT-PCR. Furthermore, the electrophysiology of the most abundant murine LTCC channel complexes was characterized at whole-cell level. <br /> he investigation of the recombinant murine channel complexes at whole-cell level demonstrates the functional relevance of the splice shift of Ca_V1.2 exon 21/22 in the context of the different β subunit isoforms (β₁, β₂ and β₃). The channel complexes containing β_2d and β₃ lead to a significantly reduced current density together with the exon 22 containing Ca_V1.2. In contrast channel complexes containing β_2b together with the exon 22 containing Ca_V1.2 cause an increased current density together with a shift of peak current density towards more hyperpolarizing potentials. The β₂ isoform in human arterial tissue and additionally β₃ in murine aortic tissue are the predominant β isoforms expressed on mRNA level. In murine aorta, β_2d is the predominant β₂ splice variant. Exon 21 as well as exon 22 are found in human and murine arterial tissue. There is no qualitative switch in human atherosclerotic tissue of LTCC subunits observed in this study. Additionally, no switch of LTCC subunits is observed in murine atherosclerosis. However, atherosclerosis is linked to a differential expression level of Ca_V1.2 and β subunit isoforms. This study do not hint at a clear cut “atherosclerotic” change of expression pattern, but indicates that the exact stage of atherosclerosis is a critical determinant of pathological LTCC remodeling.