Atherosclerosis-related molecular alteration of the human CaV1.2 calcium channel alpha1C subunit

Abstract

Atherosclerosis is an inflammatory process characterized by proliferation and dedifferentiation of vascular smooth muscle cells (VSMC). Ca(v)1.2 calcium channels may have a role in atherosclerosis because they are essential for Ca(2+)-signal transduction in VSMC. The pore-forming Ca(v)1.2alpha1 subunit of the channel is subject to alternative splicing. Here, we investigated whether the Ca(v)1.2alpha1 splice variants are affected by atherosclerosis. VSMC were isolated by laser-capture microdissection from frozen sections of adjacent regions of arteries affected and not affected by atherosclerosis. In VSMC from nonatherosclerotic regions, RT-PCR analysis revealed an extended repertoire of Ca(v)1.2alpha1 transcripts characterized by the presence of exons 21 and 41A. In VSMC affected by atherosclerosis, expression of the Ca(v)1.2alpha1 transcript was reduced and the Ca(v)1.2alpha1 splice variants were replaced with the unique exon-22 isoform lacking exon 41A. Molecular remodeling of the Ca(v)1.2alpha1 subunits associated with atherosclerosis caused changes in electrophysiological properties of the channels, including the kinetics and voltage-dependence of inactivation, recovery from inactivation, and rundown of the Ca(2+) current. Consistent with the pathophysiological state of VSMC in atherosclerosis, cell culture data pointed to a potentially important association of the exon-22 isoform of Ca(v)1.2alpha1 with proliferation of VSMC. Our findings are consistent with a hypothesis that localized changes in cytokine expression generated by inflammation in atherosclerosis affect alternative splicing of the Ca(v)1.2alpha1 gene in the human artery that causes molecular and electrophysiological remodeling of Ca(v)1.2 calcium channels and possibly affects VSMC proliferation.

Fig. 1.

Representative immunohistochemical patterns of the vascular preparations used for LCM of VSMC and isolation of RNA from atherosclerotic (D) and adjacent nonatherosclerotic (N) regions of artery. Shown are photomicrographs of immunohistochemical staining of VSMC in serial sections of the same biopsy of arteries with antibodies against smooth muscle (SM) α-actin (A and F) (for the individual patient data, see Fig. 6), Ca_v1.2α1 (B and G), ubiquitous human nuclear protein Ki-67 (C and H), PDGF-BB (D and I), and PDGF-β receptor (E and J). (Scale bar: 50 μm.)

Fig. 2.

Identification of the Ca_v1.2α1 splice variants. (A) Hypothetical transmembrane topology of Ca_v1.2α1 (17, 18). Outlined are four internal repeats, I–IV, each composed of six transmembrane segments, S1–S6. Protein segments encoded by numbered exons are marked by bold lines. Arrows point to alternative exons (8/8A, 21/22, and 31/32) that are subject to mutually exclusive splicing. Constitutively spliced exons 1/1A, 9A, 33, 34A, 41A, and 45 are shown by white boxes. (B–G) Identification of alternative exons of the Ca_v1.2α1 transcript. (B) Exon 1a (lanes 1, 3, and 5) and exon 1 (lanes 2, 4, and 6). P283 is the exon 1a-specific primer 5′-tggatccgccaATGCTTCGAGCCTTTGTTCAGC-3′. (C) Exons 8A and 8. (D) Exon 9a and 9. (E) Exons 21 and 22. (F) Exons 31–34. Shown are RT-PCR products (lanes 1 and 2) and their analytical digestion with NsiI (lanes 3 and 4) and PvuII (lanes 5 and 6). Splice variants identified by numbers on the left side of the gel photograph correspond to α_1C,127 (1), α_1C,73 (2), α_1C,125 (3), α_1C,126 (4), α_1C,71 (5) and α_1C,77 (6) (for details, see Fig. 10). (G) Differential utilization of exon 41A and lack of alternative exons 40B, 43A, 44A, and 45. Schematic diagrams illustrate the arrangement of alternative exons (black boxes) in RT-PCR products amplified from human mRNA^Card (lanes C) and RNA extracted from VSMC^N (lanes N) and VSMC^D (lanes D), Exons (boxes) are numbered as in A. The missing exons are shown as gray boxes. Numerals separated by ≫ or ≪ indicate the sense and antisense amplification primers, respectively, defined by nucleotide positions relative to the ORF of pHLCC71. To the right of schematics are RT-PCR products identified on agarose gels and their size in base pairs (arrows).

Fig. 3.

Distribution of alternative exons in transcripts of the Ca_v1.2α1 splice isoforms identified in VSMC^N and VSMC^D. Amino acid sequences encoded in alternative exons 31–34 are shown (boxes) beneath the chart. The α_1C subunit isoforms indicated on the left correspond to electrophysiologically characterized variants lacking exon 9a.

Fig. 4.

Comparison of electrophysiological properties of I_Ca through the α_1C,77 and α_1C,127 channels coexpressed with the primary cardiac β_2a subunit and measured with 2.5 mM Ca²⁺ in the bath solution. (A) Representative traces of I_Ca evoked by 1-s step depolarizations to +20 mV from V_h = −90 mV and normalized to the same amplitude. (B) Averaged I–V curves (filled circles) and voltage-dependences of the time constant of fast inactivation, τ_f, (open circles) for I_Ca. A 1-s test pulse in the range of −40 to +50 mV (10-mV increments) was applied from V_h = −90 mV with 30-s intervals. (C and D) Ensembles of activation (G/G_max − V) curves (C) and steady-state inactivation curves (D) fit by Boltzmann function. (E) Fractional recovery of I_Ca from inactivation. (F) Run-down of I_Ca. Step depolarizations of 250 ms to +20 mV were applied from V_h = −90 mV every 30 s, and the maximum amplitude of the current was normalized to the initial value. ∗, P < 0.05; SD with α_1C,77 by ANOVA with Tukey's test.

Fig. 5.

Evidence that the exon-21 isoform of Ca_v1.2α1 is not expressed in proliferating human arterial smooth muscle cells. Primary smooth muscle cells were grown in sparse culture in 5% serum (S) before serum-deprivation for 48 h (SF). Total RNA was isolated and analyzed by RT-PCR and subsequent AvrII restriction analysis as described in Fig. 2E. Shown are gels before (Left) and after (Right) AvrII overdigestion.