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

摘要

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 Ca²⁺ 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.