2024 Theses Doctoral

Elucidating the Trafficking and Regulation of CaV1.2 in Adult Mouse Cardiomyocytes

Borowik, Sergej

Calcium (Ca²⁺) influx through Caᵥ1.2 channels mediates cardiac excitation-contraction coupling, tunes cardiac action potential duration and excitability, and regulates cardiomyocytes’ (CM) gene expression. Mechanisms regulating the sub-cellular localization, trafficking, and dynamics of surface Caᵥ1.2 in ventricular CMs are poorly understood though these are critical determinants of cardiac function.

To gain new insights into Caᵥ1.2 organization, dynamics, and regulation at the CM surface we generated transgenic mice expressing an αMHC controlled cardiac-specific, dihydropyridine (DHP)- resistant α₁_ᴄ construct, tagged at the N-terminus with FLAG and HA epitopes, at the C- terminus with YFP, a 13-residue bungarotoxin binding site (BBS) inserted into in the third extracellular loop of domain II, and mutations that prevent cleavage of the C-terminus. We found robust inducible expression of DHP-resistant FLAG-HA-BBS-α₁_ᴄ-YFP in the heart that targeted to dyadic junctions, generated nisoldipine-resistant Ca²⁺ currents, supported cardiac excitation-contraction coupling, and was normally up-regulated by β-adrenergic activation with isoproterenol. Incubating transgenic CMs with AlexaFluor₆₄₇-conjugated α- bungarotoxin (BTX₆₄₇) enabled selective labeling of surface BBS-tagged Caᵥ1.2 channels.

We used total internal fluorescence (TIRF) microscopy to investigate the spatiotemporal organization and dynamics of surface Caᵥ1.2 channels. Similar to endogenous Caᵥ1.2, transgenic α1C-YFP forms clusters with exponentially distributed sizes at the cell surface. A flow cytometry-based optical pulse-chase assay revealed surface Caᵥ1.2 channels in adult cardiomyocytes fully turn over within two hours. Application of angiotensin II (Ang II) decreased transgenic Caᵥ1.2 surface density and this effect was blocked by the selective Ang II receptor type I (AT1R) blocker losartan. Application of losartan by itself increased Caᵥ1.2 surface density, suggesting the potential presence of constitutively active Ang II receptors in adult CMs. Our results provide new insights into spatiotemporal organization, dynamics, and regulation of Caᵥ1.2 channels in adult CMs and introduce an approach that can be widely applied to elucidate spatiotemporal dynamics of cardiac ion channels and membrane proteins.