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

Significance Manipulating expression of proteins directly in isolated heart cells is an important method to study cardiac biology and disease. However, this approach is challenging for large genes (>6 kb) because these exceed the packaging capacity of commonly used viral vectors used to deliver genes into heart cells. Here, we used a split-intein–mediated protein transsplicing strategy to reconstitute the large pore-forming subunit of L-type calcium channels from two separate fragments in heart cells. This approach provides a general method to express different large proteins in heart cells and will help advance new insights into basic mechanisms underlying cardiac function and disease. 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.