Alvarado, F. J. | bioRxiv Science

bioRxiv2026

Rad and Phospholamban are Key Drivers of the Ventricular Adrenergic Response and Stress-Induced Arrhythmia

The adrenergic response is a fundamental mechanism that regulates heart rate (chronotropy), cardiac contractility (inotropy) and relaxation (lusitropy). Adrenergic stress is also a recognized trigger of arrhythmia in disease. Yet, our understanding of the underlying molecular basis remains incomplete. Protein kinase A (PKA) and the calcium/calmodulin-dependent kinase II (CaMKII) phosphorylate multiple targets proposed to participate in the adrenergic response, including the GTP-binding protein Rad, phospholamban (PLB) and ryanodine receptor 2 (RyR2). Here we demonstrate that phosphorylation of both Rad and PLB is necessary for inotropy and lusitropy. We show that changes in cardiac contractility and relaxation are primarily dependent on intracellular calcium handling. Finally, we report that Rad and PLB control stress-induced arrhythmogenesis, despite the phosphorylation of other pro-arrhythmic targets. We have identified the essential molecular components of the adrenergic response, resolving a long-standing debate in cardiac excitation-contraction coupling and refining current models of sympathetic regulation in health and disease.

physiology↗

bioRxiv2024

Ablation of three major phospho-sites in RyR2 preserves the global adrenergic response but creates an arrhythmogenic substrate

BackgroundRyanodine receptor 2 (RyR2) is one of the first substrates undergoing phosphorylation upon catecholaminergic stimulation. Yet, the role of RyR2 phosphorylation in the adrenergic response remains debated. To date, three residues in RyR2 are known to undergo phosphorylation upon adrenergic stimulation. We generated a model of RyR2 phospho-ablation of all three canonical phospho-sites (RyR2-S2031A/S2808A/S2814A, triple phospho-mutant, TPM) to elucidate the role of phosphorylation at these residues in the adrenergic response. MethodsCardiac structure and function, cellular Ca2+ dynamics and electrophysiology, and RyR2 channel activity both under basal conditions and under isoproterenol (Iso) stimulation were systematically evaluated. We used echocardiography and electrocardiography in anesthetized mice, single-cell Ca2+ imaging and whole-cell patch clamp in isolated adult cardiomyocytes, and biochemical assays. ResultsIso stimulation produced normal chronotropic and inotropic responses in TPM mice as well as an increase in the global Ca2+ transients in isolated cardiomyocytes. Functional studies revealed fewer Ca2+ sparks in permeabilized TPM myocytes, and reduced RyR2-mediated Ca2+ leak in intact myocytes under Iso stimulation, suggesting that the canonical sites may regulate RyR2-mediated Ca2+ leak. TPM mice also displayed increased propensity for arrhythmia. TPM myocytes were prone to develop early afterdepolarizations (EADs), which were abolished by chelating intracellular Ca2+ with EGTA, indicating that EADs require SR Ca2+ release. EADs were also blocked by a low concentration of tetrodotoxin, further suggesting reactivation of the sodium current (INa) as the underlying cause. ConclusionPhosphorylation of the three canonical residues on RyR2 may not be essential for the global adrenergic responses. However, these sites play a vital role in maintaining electrical stability during catecholamine stimulation by fine-tuning RyR2-mediated Ca2+ leak. These findings underscore the importance of RyR2 phosphorylation and a finite diastolic Ca2+ leak in maintaining electrical stability during catecholamine stimulation.

physiology↗