Key points Prevailing dogma holds that activation of the β‐adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L‐type Ca V 1.2 channel activity, resulting in increased Ca 2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. Ca V 1.2 channel clusters decorate T‐tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca 2+ ‐dependent co‐operative gating behaviour mediated by physical interactions between adjacent channel C‐terminal tails. We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A‐dependent augmentation of Ca V 1.2 channel abundance along cardiomyocyte T‐tubules, resulting in the appearance of channel ‘super‐clusters’, and enhanced channel co‐operativity that amplifies Ca 2+ influx. On the basis of these data, we suggest a new model in which a sub‐sarcolemmal pool of pre‐synthesized Ca V 1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation–contraction coupling. Abstract Voltage‐dependent L‐type Ca V 1.2 channels play an indispensable role in cardiac excitation–contraction coupling. Activation of the β‐adrenergic receptor (βAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced Ca V 1.2 activity, resulting in increased Ca 2+ influx into ventricular myocytes and a positive inotropic response. Ca V 1.2 channels exhibit a clustered distribution along the T‐tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca 2+ ‐dependent co‐operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C‐terminal tails. This amplifies Ca 2+ influx and augments myocyte Ca 2+ transient and contraction amplitudes. We investigated whether βAR signalling could alter Ca V 1.2 channel clustering to facilitate co‐operative channel interactions and elevate Ca 2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the βAR agonist, isoproterenol (ISO), promotes enhanced Ca V 1.2–Ca V 1.2 physical interactions. Super‐resolution nanoscopy and dynamic channel tracking indicate tha...

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The human voltage-gated sodium (Nav) channel, hNav1.5, is predominantly expressed in cardiac myocytes and is responsible for the rapid upstroke of the cardiac action potential. hNav1.5 channel plays a central role in congenital and acquired cardiac arrhythmias and has been a key target for drug development. Mutagenesis studies have previously identified key residues in Nav channels S6 segments from central pore that form a receptor site for binding of local anesthetic and antiarrhythmic drugs. However, the structural details of how these drugs affect Nav channel function are not well understood. In this study, we used Rosetta computational modeling software to build a homology model of human Nav1.5 in open-inactivated and closed states based on the cryo-EM structures of electric eel Nav1.4 (PDB ID: 5XSY) and American cockroach NavPaS (PDB ID: 5X0M), respectively. We applied the RosettaLigand molecular docking program to study hNav1.5 channel interactions with local anesthetic and antiarrhythmic drugs, including lidocaine, etidocaine, QX-314, ranolazine, flecainide, and GS967. Our lowest energy models have shown that both local anesthetic and antiarrhythmic drugs bind to hNav1.5 via a common receptor site formed by S6 segments from domains III and IV in the central pore. Our results may further advance structural understanding for molecular mechanisms of local anesthetic and antiarrhythmic drug interaction with hNav1.5 and provide useful insights towards the rational design of novel modulators of ion channel activity for the treatment of cardiac arrhythmias.

Familial hypertrophic cardiomyopathy (FHC) patients are advised to avoid strenuous exercise due to increased risk of arrhythmias. Mice expressing the human FHC-causing mutation R403Q in the myosin heavy chain gene (MYH6) recapitulate the human phenotype, including cytoskeletal disarray and increased arrhythmia susceptibility. Following in vivo administration of isoproterenol, mutant mice exhibited tachyarrhythmias, poor recovery and fatigue. Arrhythmias were attenuated with the β-blocker atenolol and protein kinase A inhibitor PKI. Mutant cardiac myocytes had significantly prolonged action potentials and triggered automaticity due to reduced repolarization reserve and connexin 43 expression. Isoproterenol shortened cycle length, and escalated electrical instability. Surprisingly isoproterenol did not increase CaV1.2 current. We found alterations in CaV1.2-β1 adrenergic receptor colocalization assessed using super-resolution nanoscopy, and increased CaV1.2 phosphorylation in mutant hearts. Our results reveal for the first time that altered ion channel expression, co-localization and β-adrenergic receptor signaling associated with myocyte disarray contribute to electrical instability in the R403Q mutant heart.