Dissecting the molecular mechanism of the cardiac fight-or-flight response via real-time light-controlled phosphorylation of Rad - Abstract: The fight-or-flight response is a critical regulator of heart function and major target of cardiovascular therapeutics for heart failure, arrhythmias, and ischemic heart disease. In response to stress, catecholamines released by the sympathetic nervous system stimulate β-adrenergic receptors on cardiomyocytes, resulting in increased heart rate and contractility. Although existing medications targeting upstream components of this pathway are effective therapeutics for cardiovascular disease, they often cause undesirable non-cardiac side effects. Rad, a small RGK-family GTPase and calcium channel inhibitor protein, has emerged as a downstream mediator of β-adrenergic signaling and a promising target for cardiac-specific therapeutics. In response to β-adrenergic stimulation, Rad is phosphorylated by protein kinase A (PKA), resulting in disinhibition of Cav1.2 and an increase in the L-type calcium current responsible for excitation-contraction coupling. However, the molecular mechanism by which Rad phosphorylation results in Cav1.2 current enhancement remains poorly understood. The objective of this proposal is to elucidate the physiological significance of Rad phosphorylation in regulating Cav1.2 function in the cardiac fight-or-flight response to inform the development of novel cardiac-specific therapeutics. To do this, we propose an innovative approach combining chemical biology and electrophysiology to achieve precise temporal and spatial control of Rad phosphorylation. By encoding the unnatural amino acid caged-serine, a derivative of serine that is “decaged” to natural serine by a brief pulse of near-UV light, at phosphorylation sites S273 and S301, we will achieve real-time, site-specific, light-mediated control of Rad phosphorylation during electrophysiological recordings of Cav1.2 currents. In Aim 1, we will employ a heterologous cell culture system to quantify the distinct contributions of S273 and S301 phosphorylation to Cav1.2 current enhancement upon forskolin-induced PKA activation. In Aim 2, we extend these studies to ex vivo cardiomyocytes isolated from Rad-knockout mice, enabling us to examine Rad phosphorylation in the context of native cardiac β-adrenergic signaling pathways. By elucidating the molecular mechanisms of Rad-mediated Cav1.2 regulation, this study will offer critical insights into the cardiac β-adrenergic response. These findings will lay the foundation for developing next- generation, cardiac-specific therapies targeting Rad phosphorylation for the treatment of cardiovascular diseases. The successful completion of this work will provide comprehensive training in biomedical research techniques, supporting the fellowship applicant's long-term goal of achieving a career as a physician-scientist dedicated to advancing the understanding and treatment of cardiovascular disorders.
