COMMENTARIES

Remodeling of Human CaV1.2 Calcium Channels in Atherosclerosis

This commentary is based on Tiwari S, Zhang Y, Heller J, Abernethy DR, Soldatov NM. Atherosclerosis-related molecular alteration of the human Cav1.2 calcium channel α1C subunit. Proc Natl Acad Sci U S A 2006;103:17024-29.

Nikolai M. Soldatov, Ph.D.
National Institute on Aging
National Institutes of Health
5600 Nathan Shock Drive
Baltimore, MD 21224
U.S.A.

Atherosclerosis is an inflammatory process characterized by endothelial perturbation, local release of cytokines, as well as proliferation, dedifferentiation, and migration of vascular smooth muscle cells (VSMC) [1]. The role of voltage-gated L-type Cav1.2 calcium channels in these processes is not yet understood, but the critical importance of these channels for Ca2+ signal transduction is well known. Cav1.2 channels are composed of the pore-forming Cav1.2α1C subunits that require close association with auxiliary β and α2-d subunits for functioning [2]. An increasing body of data shows that genetic variability of auxiliary subunits, which is further complicated by alternative splicing, strongly affects expression, Ca2+-sensitivity, kinetics, and voltage-dependence of the Ca2+ current through the α1C subunit. Even a greater diversity of the Cav1.2 channel emerges from alternative splicing of the α1C subunit that spans an estimated 150 kb of the human genome and contains 58 identified exons [3]. Twenty exons of Cav1.2α1C are spliced in an alternative manner generating impressive molecular array of the channel isoforms with altered electrophysiological and pharmacological properties. Given that complex transcriptome of the Cav1.2α1C subunit gene may be tightly regulated, as in the case of the T-type Cav3.1 calcium channel [4], it is reasonable to suggest that atherosclerosis, and the associated local exposure to cytokines (primarily PDGF BB), orderly reprogram alternative splicing of the Cav1.2α1C subunit primary transcript with pathophysiological consequences.

Approach to Cell-specific Splice Variation of Cav1.2α1C in Atherosclerosis

There are a number of elegant studies that laid the foundation for our understanding of the molecular diversity of the vascular tissue Cav1.2α1C subunit [5-8]. However, when we decided to investigate whether the Cav1.2α1C splice variation in VSMC is affected by atherosclerosis, the complex organization of the vascular tissue challenging, as was its local deterioration in the atherosclerotic plaque region. The analysis was even more complicated by the presence of fibroblasts that are known to express Cav1.2 channels [9]. To resolve the problem of arterial tissue heterogeneity, we used laser-assisted microdissection of VSMC immunostained against specific marker. Arterial biopsy samples (3 carotid and 3 femoral) were obtained at surgery from six patients of an average age of 75 years old with severe atherosclerotic vascular disease. Small areas not visually affected by atherosclerosis (“N”, non-atherosclerotic) and those occluded with heavy plaque burden (“D”, diseased) were identified and dissected. VSMC were quickly immunostained with anti-SM α-actin antibody in 5-7 µm frozen sections of adjacent regions of arteries not affected and affected by the disease, and then isolated by laser-capture microdissection. RNA was extracted from 200-300 collected cells for subsequent RT-PCR analysis.

Expression of Cav1.2α1C in VSMC Is Reduced in Atherosclerosis

By quantifying the Cav1.2 transcript in cells dissected from 6 different preparations with quantitative real-time PCR, we determined the relative Cav1.2α1C mRNA level in VSMC (normalized to 18S RNA) to be reduced approximately 4 fold in the plaque region as compared to adjacent non-diseased tissue. Previously we found that Cav1.2 expression in normal diploid human fibroblasts is strongly suppressed on induction of cell proliferation by mitogens [10]. This finding is in agreement with the reduction in Cav1.2 expression at atherosclerosis that stimulated PDGF BB expression and caused a large increase in proliferating cells in atherosclerotic artery.

Isoform Switch in VSMC Cav1.2α1C Associated with Atherosclerosis

To determine whether change in Cav1.2 expression in VSMC of atherosclerotic regions of the artery is associated with specific splice isoforms of Cav1.2, RNA extracted from the laser-microdissected cells was analyzed by RT-PCR and all alternative exons of the Cav1.2α1C subunit were identified. In spite of the differences in gender, ethnicity, drug exposure and other co-morbid conditions, all of the six donors showed the same pattern of alternative splicing of Cav1.2α1 in non-affected VSMC and its change in diseased cells. In VSMC from non-atherosclerotic regions, alternative splicing affected only a small subset of exons. RT-PCR analysis revealed an extended repertoire of Cav1.2α1C transcripts all characterized by the presence of alternative exons 21 and 41A. Another hallmark of normal VSMC Cav1.2α1C transcripts is alternative splicing of exons 9 and 9a, as well as exons 31-34, and lack of cardiac-specific exons (1a, 8a, 45). In atherosclerotic VSMC all these normally occurring Cav1.2α1C splice variants were replaced with the unique exon-22 isoform that lacks exon 41A. The Cav1.2 remodeling in atherosclerosis caused changes in electrophysiological properties of the identified Cav1.2α1C isoforms including the kinetics and voltage-dependence of inactivation, and recovery from inactivation. Consistent with the pathophysiological state of VSMC in atherosclerosis, cell culture data pointed to a potentially important association of the exon-22 isoform of Cav1.2α1C with proliferation of VSMC.

Atherosclerosis may Change Pharmacological Properties of VSMC

The exon 21 to exon 22 isoform switch changes the structure of one of the transmembrane segments of the Cav1.2α1C molecule and affects voltage-dependent inhibition of the Cav1.2 channel by dihydropyridine blockers. In recombinant expression system it was shown earlier that the exon-22 splice variant exhibits higher sensitivity to isradipine at negative membrane potentials [11]. Thus, distinctly different expression of exon 21/22 Cav1.2α1C splice isoforms in atherosclerosis points to a possibility that response of VSMC to dihydropyridines should be changed in regions of atherosclerosis suggesting that the disease may locally alter pharmacological properties of VSMC.

Questions and Hypotheses

Physiological role of Cav1.2 variability in the maintenance of VSMC remains unknown. It is not clear why the exon-21 isoform of Cav1.2α1C is present in quiescent VSMC while the exon 22-isoform is not. It is possible that the structural changes in the Cav1.2α1C transmembrane segment IIIS2 encoded in exon 21 may affect whole multitude of the Cav1.2α1C voltage-gated rearrangements, some of which are directly linked to Ca2+-signal transduction and are crucial, for example, for CREB-dependent transcription [12]. We are now trying to find whether snRNA-mediated skipping of exon 22 in Cav1.2α1C would rescue VSMC from atherosclerosis.
In conclusion, we found that localized changes in cytokine expression generated by inflammation in atherosclerosis affect alternative splicing of the Cav1.2α1 gene in VSMC of the human artery that causes molecular and electrophysiological remodeling of Cav1.2 calcium channels and possibly affects VSMC proliferation.

References

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