Investigating Cardiac Ion Channels by Novel Methods

In patients with heart failure (HF), the continual activation of the sympathetic nervous system, which compensates for lower cardiac output, harms the heart. This not only worsens HF but also increases the risk of life-threatening arrhythmias. Although β-adrenergic receptor agonists and phosphodiesterase inhibitors were commonplace, seminal studies revealed reduced survival. β-adrenergic receptor agonists and PDE inhibitors are still utilized, although their use is limited by the need for intravenous administration and close monitoring due to potential arrhythmias. A notable gap exists in our understanding and treatment approaches for the short- and long-term management of HF, particularly regarding the safe, direct targeting of myocyte contractility by enhancing calcium influx and transient levels. Our long-term aim is to deploy innovative strategies to identify and evaluate targets for targeted therapies for arrhythmias and HF. Over the last five years, we have uncovered the mechanisms behind the adrenergic regulation of cardiac calcium channels. We discovered that the RGK G-protein Rad, which inhibits high-voltage activated calcium channels, is a crucial PKA target within the CaV1.2 complex. PKA phosphorylation at two Ser residues on Rad's C-terminus causes Rad to detach from the membrane, decreasing its affinity for CaVβ and relieving CaV1.2 inhibition. Unlike the pro-arrhythmic effects seen with the inotropic drug BayK 8644—which increases calcium influx by slowing the inactivation properties of CaV1.2—up-regulating calcium influx through the expression of a CaVβ2B subunit that does not interact with Rad does not appear to have any detrimental effects or induce arrhythmias. These results challenge the prevailing belief that all increased calcium influx is harmful or that all animal models featuring elevated calcium influx will inevitably suffer from HF or arrhythmias. Enhancing calcium influx, like adrenergic stimulation, may be more precise and effective than traditional methods, as it avoids harmful SR calcium leak and overload associated with adrenergic agonists. To this end, we have created gene and base editors capable of introducing either a frame-shift insertion/deletion (indel) in Rad or a mutation in the calcium channel β-subunit to prevent Rad binding in cardiomyocytes. This editing is achieved through AAV9 injections subcutaneously in young pups or retro-orbitally in adult mice. Additionally, we have devised a base-editing strategy to significantly reduce adrenergic regulation of calcium channels while sparing the basal function of CaV1.2 channels, which may help prevent arrhythmias induced by adrenergic agonists. We propose three specific aims: Aim 1: To determine whether enhancing calcium influx through Rad or CaVβ2 gene editing can slow the decline of cardiac function in non-ischemic HF models. Aim 2: To determine if increased calcium influx can restore cardiac function after HF onset. Aim 3: Investigate whether restricting the sympathetic nervous system’s enhancement of Ca2+ influx can attenuate the cardiac dysfunctions associated with catecholaminergic polymorphic VT (CPVT) and hypertrophic cardiomyopathy (HCM).