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

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 alpha(1C)-subunit in adult cardiomyocytes. Here, we use split-intein-mediated protein transsplicing to reconstitute LTCC alpha(1C)-subunit from two distinct halves, overcoming the difficulty of expressing full-length alpha(1C) in cardiomyocytes. Split-intein-tagged alpha(1C) 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 beta-adrenergic regulation of excitation-contraction coupling. This approach lowers a longstanding technical hurdle to manipulating large proteins in cardiomyocytes.