Manipulating L-type calcium channels in cardiomyocytes using split-intein protein transsplicing

Abstract

Manipulating expression of large genes (>6 kb) in adult cardiomyocytes is challenging because these cells are only efficiently transduced by viral vectors with a 4-7 kb packaging capacity. This limitation impedes understanding structure-function mechanisms of important proteins in heart. L-type calcium channels (LTCCs) regulate diverse facets of cardiac physiology including excitation-contraction coupling, excitability, and gene expression. Many important questions about how LTCCs mediate such multidimensional signaling are best resolved by manipulating expression of the 6.6 kb pore-forming α1C-subunit in adult cardiomyocytes. Here, we use split-intein-mediated protein transsplicing to reconstitute LTCC α1C-subunit from two distinct halves, overcoming the difficulty of expressing full-length α1C in cardiomyocytes. Split-intein-tagged α1C fragments encoding dihydropyridine-resistant channels were incorporated into adenovirus and reconstituted in cardiomyocytes. Similar to endogenous LTCCs, recombinant channels targeted to dyads, triggered Ca(2+) transients, associated with caveolin-3, and supported β-adrenergic regulation of excitation-contraction coupling. This approach lowers a longstanding technical hurdle to manipulating large proteins in cardiomyocytes.

Keywords: CaV1.2; gene transfer; protein splicing; ventricular myocytes.

Fig. 1.

Model systems for structure–function studies of cardiac LTCCs. (A, Upper) Adult rat ventricular cardiomyocyte. (A, Bottom) Differential LTCC targeting to distinct subcellular microdomains in cardiomyocytes. (B) Model systems for LTCC structure–function studies. (Scale bar, 20 μm.) (C) Topology of LTCC pore-forming α_1C and auxiliary proteins (β, α2δ, and calmodulin).

Fig. 2.

Reconstituting LTCCs by split-intein–mediated protein transsplicing. (A) Strategy for reconstituting full-length α_1C using split-intein–mediated protein splicing. The α_1C subunit is split into two halves tagged with fluorophores and flanking N. punctiforme DnaE split inteins, creating _CFP[I–II]_N-intein and _C-intein[III–IV]_YFP, respectively. A 13-residue BBS tag is introduced to permit selective labeling of surface channels with quantum dot (QD₆₅₅). (B and C) Anti-GFP Western blot showing successful reconstitution of covalently linked full-length α_1C from intein-flanked α_1C moieties in HEK293 cells. Lane 1, full-length α_1C[BBS]–YFP (open circle) + β_2a-CFP (open square); lane 2, _CFP[I–II]_N-intein (inverted triangle); lane 3, _C-intein[III–IV]_YFP (open diamond); lane 4, _CFP[I–II]_N-intein + _C-intein[III–IV]_YFP (open triangle); lane 5, _CFP[I–II]_N-intein + _C-intein[III–IV^TQ/YM]_YFP (open triangle). Untagged β_2a is coexpressed in lanes 2–5. (C) Titration of _CFP[I–II]_N-intein to _C-intein[III–IV]_YFP transfection ratio (plus CFP-β_2a). (D) Confocal images showing CFP, YFP, and QD fluorescence in cells coexpressing _CFP[I–II]_N-intein + _C-intein[III–IV]_YFP ± β_2a. (Scale bar, 10 μm.) (E) Flow cytometry dot plot of YFP and QD fluorescence. (F) Quantification of QD₆₅₅ signal. *P = 0.0000427 compared with –β using two-tailed unpaired t test. n = 5, 50,000 cells per experiment.

Fig. 3.

Electrophysiological properties and DHP sensitivity of WT and split-intein–reconstituted LTCCs in HEK293 cells. (A) Exemplar Ba²⁺ currents from cells expressing full-length WT α_1C + β_2a in the absence (black trace) and presence of different nifedipine concentrations (1 μM, red; 5 μM, blue; 10 μM, cyan). (B) Population current-density versus voltage (J−V) relationship for cells expressing full-length WT α_1C + β_2a channels in the absence (black symbols) or presence (cyan symbols) of 10 μM nifedipine. n = 7 for each point. (C) Diary plots of nifedipine inhibition of full-length WT α_1C + β_2a channels. (D–F) Data for intein-spliced WT α_1C channels are in the same format as A–C. n = 9. (G–I) Data for intein-spliced DHP-resistant α_1C channels are in the same format as A–C. n = 10.

Fig. 4.

Expression and subcellular targeting of intein-spliced α_1C in cardiac myocytes. (A) Schematic showing incorporation of split-intein–tagged α_1C moieties into adenovirus and infection of cardiomyocytes. (B, Left) Confocal images showing high efficiency expression of CFP- and YFP-tagged split-intein α_1C moieties in adult cardiomyocytes. (Scale bar, 50 μm.) (B, Right) Western blot (anti-α_1C antibody) detection of endogenous (#) and intein-spliced (&) α_1C subunits. (C) Confocal images of CFP, YFP, and QD₆₅₅ fluorescence in a cardiomyocyte expressing intein-spliced α_1C. Control shows absence of QD₆₅₅ staining in an uninfected cell. (Scale bar, 20 μm.) (D, Upper) Immunostaining of endogenous α_1C (anti-α_1C, green) and RyR (anti-RyR, red) in an uninfected cardiomyocyte. (D, Lower) Immunostaining of intein-spliced α_1C (anti-GFP, green) and endogenous RYR (anti-RyR, red). (Scale bar, 20 μm.) (E) Colocalization analyses.

Fig. 5.

Electrophysiological characterization of intein-spliced WT and DHP-resistant α_1C in cardiac myocytes. Population J−V relationships in (A) uninfected cardiomyocytes (control), (B) myocytes expressing intein-spliced WT α_1C, or (C) intein-spliced DHP– α_1C. (D and E) Exemplar current traces and population J–V curves from cardiomyocytes expressing either intein-spliced WT α_1C (black trace and symbols, n = 9) or DHP– α_1C (red trace and symbols, n = 8) channels in the presence of 10 μM nifedipine. *P = 0.018 (–10 mV), *P = 0.0324 (0 mV), *P = 0.048 (10 mV), and *P = 0.04 (20 mV) using two-tailed unpaired t test.

Fig. 6.

Participation of intein-spliced WT and DHP– α_1C in CICR in cardiomyocytes. (A, Upper) The rhod-2-reported Ca²⁺ transients in an uninfected (UT) cardiomyocyte paced with 1 Hz field stimulation. (A, Lower) Ca²⁺ transients from UT myocyte in the presence of 1 μM nifedipine. (B and C) Ca²⁺ transients from cardiomyocytes expressing intein-spliced WT (α_1C) and DHP– α_1C, respectively. Same format as A. (D) Impact of 1 μM nifedipine on Ca²⁺ transient amplitudes. * P = 0.0014 (UT), * P = 0.0003 (α_1C), two-tailed unpaired t test. #, significantly different from control and intein-spliced WT α_1C +nifedipine using one-way ANOVA [F(2, 12) = 47.48, P = 2E-6] and Bonferroni pairwise comparisons, n = 5 cells. (E) CICR is abolished by 5 μM nifedipine in cardiomyocytes expressing intein-spliced WT α_1C. (F) Persistence of CICR in cardiomyocytes expressing intein-spliced DHP– α_1C in 5 μM nifedipine. (G) A total of 1 μM isoproterenol potentiates Ca²⁺ transients triggered by intein-spliced DHP– α_1C, *P = 0.018.