RGS4 Mediates Catecholaminergic Inhibition of Short‐Chain Fatty Acid Receptor FFAR3 Signaling & Function in Cardiomyocytes
Authors: Carbone Alexandra M. , Cora Natalie , Suster Malka S. , Ferraino Krysten E. , Borges Jordana I. , Nagliya Deepika , Sizova Anastasiya , Wang Gaofeng , Iacobellis GIanluca , Goldberger Jeffrey J. , Lymperopoulos Anastasios 
Published: May 2022

BackgroundFree fatty acids (FFAs) are essential cell nutrients but also important signaling molecules regulating various cellular processes. Short‐chain fatty acids (SCFAs), like propionate and butyrate, activate two different types of G protein‐coupled receptors (GPCRs), FFAR2 and FFAR3. FFAR3 (also known as GPR41) couples to Gi/o proteins lowering cyclic 3’,5’‐adenosine monophosphate (cAMP) levels and activating mitogen‐activated protein kinases (MAPKs). In addition to adipose tissue, FFAR3 is highly expressed in peripheral sympathetic ganglia augmenting sympathetic nervous system (SNS) outflow. Regulator of G protein Signaling (RGS)‐4 belongs to the RGS superfamily of proteins and inactivates (terminates) both Gi/o‐ and Gq‐protein signaling. Cardiac RGS4 has been implicated in atrial fibrillation (AFib) prevention via calcium signaling attenuation. Finally, β‐adrenergic receptors (ARs), the major mediators of catecholaminergic and SNS actions in cardiac myocytes, stimulate cAMP synthesis and activate protein kinase A (PKA), which is known to activate RGS4 via phosphorylation.Study objectiveSince catecholamines/SNS and RGS4 play major roles in AFib pathophysiology and FFAR3 promotes SNS activity, we examined whether RGS4 regulates cardiac FFAR3 signaling.MethodsWe used H9c2 cardiomyocytes transfected to express the human FFAR3. After treatment with catecholaminergic agonists and/or propionic acid plus various pharmacological inhibitors and siRNA to deplete RGS4, we performed signaling assays (western blotting, cAMP accumulation, GTP binding, ELISA) to delineate the precise involvement of RGS4 in the cardiac βAR‐FFAR3 signaling crosstalk.ResultsRGS4 is essential for the dampening of propionate/FFAR3‐dependent signaling in H9c2 cardiomyocytes since siRNA‐mediated RGS4 depletion significantly enhances the effect of propionic acid on cAMP lowering, Gi/o activation, and p38 MAPK activation. Notably, concomitant isoproterenol or salbutamol treatment blocks propionic acid‐induced signaling via the FFAR3 in H9c2 cardiomyocytes, indicating negative regulation of FFAR3 by catecholamines via the β2AR. Mechanistically, this is mediated by RGS4, since neither isoproterenol, nor salbutamol can inhibit propionate signaling upon siRNA‐mediated RGS4 depletion. Finally, this β2AR trans‐inhibition of FFAR3 signaling via RGS4 is dependent on β2AR‐activated PKA, because pre‐treatment with the PKA inhibitor H89 abrogated the catecholaminergic inhibition of FFAR3 signaling (cAMP lowering, downstream p38 MAPK activation, interleukin (IL)‐6 production) in H9c2 cardiomyocytes.Conclusionsβ2AR‐activated RGS4 abrogates SCFA/FFAR3 signaling and function in cardiac myocytes. This may represent part of the mechanism via which RGS4 protects against both AFib pathogenesis and FFAR3‐promoted SNS hyperactivity and inflammation in the myocardium.