The role of endogenous RGS proteins in regulation of cardiac automaticity.

摘要

Regulator of G protein Signaling (RGS) proteins modulate G protein-mediated signaling by accelerating the GTPase activity of Galpha subunits, thereby shortening the lifetime of active signaling molecules, i.e. Galpha-GTP and free Gbetagamma complexes. Despite extensive studies for a decade, our understanding of the physiological functions of RGS proteins is still in its infancy. The lack of pharmacological inhibitors for RGS proteins and functional redundancy among multiple family members (>30) imposes tremendous challenges for the application of antisense or genetic knockout strategies in assessing the role of RGS proteins in physiological processes. Moreover, another level of complexity lies in the interaction of RGS proteins and multiple isoforms of G proteins. Therefore, we employed RGS-insensitive mutations (RGSi) in Galpha subunits that disrupt the Galpha-RGS interaction and introduced them into embryonic stem (ES) cells by homologous recombination. This approach allowed me to study the role of endogenous RGS proteins as a class in a Galpha isoform-specific manner without alterations in expression of mutant proteins. Cardiac automaticity was examined in spontaneously contracting ES cell-derived cardiocytes (ESDC) in vitro, in an isolated heart perfusion system, and in vivo in intact animals. I demonstrate that endogenous RGS proteins potently modulate the chronotropic control by adenosine A, and muscarinic M2 receptors, which differentially utilize Galpha o and Galphai2 and associated downstream effectors. The G-protein-coupled inward rectifying K+ currents are strongly modulated by RGS proteins and play an essential role in M2 receptor-induced bradycardia, whereas A1, receptors seem to preferentially couple to Galphao and other downstream effectors. Telemetry recording in conscious, unrestrained mice revealed hyperactivity, disrupted thermoregulation, and enhanced basal cardiac function, which strongly suggest alterations in the central nervous system (CNS) of Galphai2RGSi mutant mice. Using an isolated heart perfusion system, I confirmed that RGS proteins modulate intrinsic cardiac automaticity independent of CNS and vascular inputs. Furthermore, enhanced Galphai2 signaling by blocking RGS actions also results in the development of atrioventricular block. Thus, endogenous RGS proteins play an important role in regulation of cardiovascular and neurobehavioral function and may also be implicated in pathophysiological processes.