Differential Modulation of Catecholamine and Adipokine Secretion by the Short Chain Fatty Acid Receptor FFAR3 and α2-Adrenergic Receptors in PC12 Cells.

Sympathetic nervous system (SNS) hyperactivity is mediated by elevated catecholamine (CA) secretion from the adrenal medulla, as well as enhanced norepinephrine (NE) release from peripheral sympathetic nerve terminals. Adrenal CA production from chromaffin cells is tightly regulated by sympatho-inhibitory α2-adrenergic (auto)receptors (ARs), which inhibit both epinephrine (Epi) and NE secretion via coupling to Gi/o proteins. α2-AR function is, in turn, regulated by G protein-coupled receptor (GPCR)-kinases (GRKs), especially GRK2, which phosphorylate and desensitize them, i.e., uncouple them from G proteins. On the other hand, the short-chain free fatty acid (SCFA) receptor (FFAR)-3, also known as GPR41, promotes NE release from sympathetic neurons via the Gi/o-derived free Gßγ-activated phospholipase C (PLC)-ß/Ca2+ signaling pathway. However, whether it exerts a similar effect in adrenal chromaffin cells is not known at present. In the present study, we examined the interplay of the sympatho-inhibitory α2A-AR and the sympatho-stimulatory FFAR3 in the regulation of CA secretion from rat adrenal chromaffin (pheochromocytoma) PC12 cells. We show that FFAR3 promotes CA secretion, similarly to what GRK2-dependent α2A-AR desensitization does. In addition, FFAR3 activation enhances the effect of the physiologic stimulus (acetylcholine) on CA secretion. Importantly, GRK2 blockade to restore α2A-AR function or the ketone body beta-hydroxybutyrate (BHB or 3-hydroxybutyrate), via FFAR3 antagonism, partially suppress CA production, when applied individually. When combined, however, CA secretion from PC12 cells is profoundly suppressed. Finally, propionate-activated FFAR3 induces leptin and adiponectin secretion from PC12 cells, two important adipokines known to be involved in tissue inflammation, and this effect of FFAR3 is fully blocked by the ketone BHB. In conclusion, SCFAs can promote CA and adipokine secretion from adrenal chromaffin cells via FFAR3 activation, but the metabolite/ketone body BHB can effectively inhibit this action.

Angiotensin II-dependent aldosterone production in the adrenal cortex.

The adrenal cortex is responsible for production of adrenal steroid hormones and is anatomically divided into three distinct zones: zona glomerulosa secreting mineralocorticoids (mainly aldosterone), zona fasciculata secreting glucocorticoids (cortisol), and zona reticularis producing androgens. Importantly, due to their high lipophilicity, no adrenal steroid hormone (including aldosterone) is stored in vesicles but rather gets synthesized and secreted instantly upon cell stimulation with specific stimuli. Aldosterone is the most potent mineralocorticoid hormone produced from the adrenal cortex in response to either angiotensin II (AngII) or elevated K+ levels in the blood (hyperkalemia). AngII, being a peptide, cannot cross cell membranes and thus, uses two distinct G protein-coupled receptor (GPCR) types, AngII type 1 receptor (AT1R) and AT2R to exert its effects inside cells. In zona glomerulosa cells, AT1R activation by AngII results in aldosterone synthesis and secretion via two main pathways: (a) Gq/11 proteins that activate phospholipase C ultimately raising intracellular free calcium concentration; and (b) ßarrestin1 and -2 (also known as Arrestin-2 and -3, respectively) that elicit sustained extracellular signal-regulated kinase (ERK) activation. Both pathways induce upregulation and acute activation of StAR (steroidogenic acute regulatory) protein, the enzyme that catalyzes the rate-limiting step in aldosterone biosynthesis. This chapter describes these two salient pathways underlying AT1R-induced aldosterone production in zona glomerulosa cells. We also highlight some pharmacologically important notions pertaining to the efficacy of the currently available AT1R antagonists, also known as angiotensin receptor blockers (ARBs) or sartans at suppressing both pathways, i.e., their inverse agonism efficacy at G proteins and ßarrestins.

Nicotine Diminishes Alpha2-Adrenergic Receptor-Dependent Protection Against Oxidative Stress in H9c2 Cardiomyocytes.

Introduction: Nicotine is a major component of cigarette smoke with various detrimental cardiovascular effects, including increased oxidative stress in the heart. Agonism of α2-adrenergic receptors (ARs), such as with dexmedetomidine, has been documented to exert cardioprotective effects against oxidative stress and related apoptosis and necroptosis. α2-ARs are membrane-residing G protein-coupled receptors (GPCRs) that primarily activate Gi/o proteins. They are also subjected to GPCR-kinase (GRK)-2-dependent desensitization, which entails phosphorylation of the agonist-activated receptor by GRK2 to induce its decoupling from G proteins, thus terminating α2AR-mediated G protein signaling. Objective: In the present study, we sought to examine the effects of nicotine on α2AR signaling and effects in H9c2 cardiomyocytes exposed to H2O2 to induce oxidative cellular damage. Methods and Results: As expected, treatment of H9c2 cardiomyocytes with H2O2 significantly decreased cell viability and increased oxidative stress, as assessed by reactive oxygen species (ROS)-associated fluorescence levels (DCF assay) and superoxide dismutase activity. Both H2O2 effects were partly rescued by α2AR activation with brimonidine in control cardiomyocytes but not in cells pretreated with nicotine for 24 hours, in which brimonidine was unable to reduce H2O2-induced cell death and oxidative stress. This was due to severe α2AR desensitization, manifested as very low Gi protein activation by brimonidine, and accompanied by GRK2 upregulation in nicotine-treated cardiomyocytes. Finally, pharmacological inhibition of adenylyl cyclase (AC) blocked H2O2-dependent oxidative damage in nicotine-pretreated H9c2 cardiomyocytes, indicating that α2AR activation protects against oxidative injury via its classic coupling to Gai-mediated AC inhibition. Discussion/Conclusions: Nicotine can negate the cardioprotective effects of α2AR agonists against oxidative injury, which may have important implications for patients treated with this class of drugs that are chronic tobacco smokers.

Cardiac RGS Proteins in Human Heart Failure and Atrial Fibrillation: Focus on RGS4.

The regulator of G protein signaling (RGS) proteins are crucial for the termination of G protein signals elicited by G protein-coupled receptors (GPCRs). This superfamily of cell membrane receptors, by far the largest and most versatile in mammals, including humans, play pivotal roles in the regulation of cardiac function and homeostasis. Perturbations in both the activation and termination of their G protein-mediated signaling underlie numerous heart pathologies, including heart failure (HF) and atrial fibrillation (AFib). Therefore, RGS proteins play important roles in the pathophysiology of these two devasting cardiac diseases, and several of them could be targeted therapeutically. Although close to 40 human RGS proteins have been identified, each RGS protein seems to interact only with a specific set of G protein subunits and GPCR types/subtypes in any given tissue or cell type. Numerous in vitro and in vivo studies in animal models, and also in diseased human heart tissue obtained from transplantations or tissue banks, have provided substantial evidence of the roles various cardiomyocyte RGS proteins play in cardiac normal homeostasis as well as pathophysiology. One RGS protein in particular, RGS4, has been reported in what are now decades-old studies to be selectively upregulated in human HF. It has also been implicated in protection against AFib via knockout mice studies. This review summarizes the current understanding of the functional roles of cardiac RGS proteins and their implications for the treatment of HF and AFib, with a specific focus on RGS4 for the aforementioned reasons but also because it can be targeted successfully with small organic molecule inhibitors.

Cardiovascular GPCR regulation by regulator of G protein signaling proteins.

G protein-coupled receptors (GPCRs) play pivotal roles in regulation of cardiovascular homeostasis across all vertebrate species, including humans. In terms of normal cellular function, termination of GPCR signaling via the heterotrimeric G proteins is equally (if not more) important to its stimulation. The Regulator of G protein Signaling (RGS) protein superfamily are indispensable for GPCR signaling cessation at the cell membrane, and thus, for cellular control of GPCR signaling and function. Perturbations in both activation and termination of G protein signaling underlie many examples of cardiovascular dysfunction and heart disease pathogenesis. Despite the plethora of over 30 members comprising the mammalian RGS protein superfamily, each member interacts with a specific set of second messenger pathways and GPCR types/subtypes in a tissue/cell type-specific manner. An increasing number of studies over the past two decades have provided compelling evidence for the involvement of various RGS proteins in physiological regulation of cardiovascular GPCRs and, consequently, also in the pathophysiology of several cardiovascular ailments. This chapter summarizes the current understanding of the functional roles of RGS proteins as they pertain to cardiovascular, i.e., heart, blood vessel, and platelet GPCR function, with a particular focus on their implications for chronic heart failure pathophysiology and therapy.

GRK5 is an essential co-repressor of the cardiac mineralocorticoid receptor and is selectively induced by finerenone.

BACKGROUND: In the heart, aldosterone (Aldo) binds the mineralocorticoid receptor (MR) to exert damaging, adverse remodeling-promoting effects. We recently showed that G protein-coupled receptor-kinase (GRK)-5 blocks the cardiac MR by directly phosphorylating it, thereby repressing its transcriptional activity. MR antagonist (MRA) drugs block the cardiac MR reducing morbidity and mortality of advanced human heart failure. Non-steroidal MRAs, such as finerenone, may provide better cardio-protection against Aldo than classic, steroidal MRAs, like spironolactone and eplerenone. AIM: To investigate potential differences between finerenone and eplerenone at engaging GRK5-dependent cardiac MR phosphorylation and subsequent blockade. METHODS: We used H9c2 cardiomyocytes, which endogenously express the MR and GRK5. RESULTS: GRK5 phosphorylates the MR in H9c2 cardiomyocytes in response to finerenone but not to eplerenone. Unlike eplerenone, finerenone alone potently and efficiently suppresses cardiac MR transcriptional activity, thus displaying inverse agonism. GRK5 is necessary for finerenone's inverse agonism, since GRK5 genetic deletion renders finerenone incapable of blocking cardiac MR transcriptional activity. Eplerenone alone does not fully suppress cardiac MR basal activity regardless of GRK5 expression levels. Finally, GRK5 is necessary for the anti-apoptotic, anti-oxidative, and anti-fibrotic effects of both finerenone and eplerenone against Aldo, as well as for the higher efficacy and potency of finerenone at blocking Aldo-induced apoptosis, oxidative stress, and fibrosis. CONCLUSION: Finerenone, but not eplerenone, induces GRK5-dependent cardiac MR inhibition, which underlies, at least in part, its higher potency and efficacy, compared to eplerenone, as an MRA in the heart. GRK5 acts as a co-repressor of the cardiac MR and is essential for efficient MR antagonism in the myocardium.

Regulator of G-Protein Signaling-4 Attenuates Cardiac Adverse Remodeling and Neuronal Norepinephrine Release-Promoting Free Fatty Acid Receptor FFAR3 Signaling.

Propionic acid is a cell nutrient but also a stimulus for cellular signaling. Free fatty acid receptor (FFAR)-3, also known as GPR41, is a Gi/o protein-coupled receptor (GPCR) that mediates some of the propionate's actions in cells, such as inflammation, fibrosis, and increased firing/norepinephrine release from peripheral sympathetic neurons. The regulator of G-protein Signaling (RGS)-4 inactivates (terminates) both Gi/o- and Gq-protein signaling and, in the heart, protects against atrial fibrillation via calcium signaling attenuation. RGS4 activity is stimulated by ß-adrenergic receptors (ARs) via protein kinase A (PKA)-dependent phosphorylation. Herein, we examined whether RGS4 modulates cardiac FFAR3 signaling/function. We report that RGS4 is essential for dampening of FFAR3 signaling in H9c2 cardiomyocytes, since siRNA-mediated RGS4 depletion significantly enhanced propionate-dependent cAMP lowering, Gi/o activation, p38 MAPK activation, pro-inflammatory interleukin (IL)-1ß and IL-6 production, and pro-fibrotic transforming growth factor (TGF)-ß synthesis. Additionally, catecholamine pretreatment blocked propionic acid/FFAR3 signaling via PKA-dependent activation of RGS4 in H9c2 cardiomyocytes. Finally, RGS4 opposes FFAR3-dependent norepinephrine release from sympathetic-like neurons (differentiated Neuro-2a cells) co-cultured with H9c2 cardiomyocytes, thereby preserving the functional ßAR number of the cardiomyocytes. In conclusion, RGS4 appears essential for propionate/FFAR3 signaling attenuation in both cardiomyocytes and sympathetic neurons, leading to cardioprotection against inflammation/adverse remodeling and to sympatholysis, respectively.

Short-Chain Fatty Acid Receptors and Cardiovascular Function.

Increasing experimental and clinical evidence points toward a very important role for the gut microbiome and its associated metabolism in human health and disease, including in cardiovascular disorders. Free fatty acids (FFAs) are metabolically produced and utilized as energy substrates during almost every biological process in the human body. Contrary to long- and medium-chain FFAs, which are mainly synthesized from dietary triglycerides, short-chain FFAs (SCFAs) derive from the gut microbiota-mediated fermentation of indigestible dietary fiber. Originally thought to serve only as energy sources, FFAs are now known to act as ligands for a specific group of cell surface receptors called FFA receptors (FFARs), thereby inducing intracellular signaling to exert a variety of cellular and tissue effects. All FFARs are G protein-coupled receptors (GPCRs) that play integral roles in the regulation of metabolism, immunity, inflammation, hormone/neurotransmitter secretion, etc. Four different FFAR types are known to date, with FFAR1 (formerly known as GPR40) and FFAR4 (formerly known as GPR120) mediating long- and medium-chain FFA actions, while FFAR3 (formerly GPR41) and FFAR2 (formerly GPR43) are essentially the SCFA receptors (SCFARs), responding to all SCFAs, including acetic acid, propionic acid, and butyric acid. As with various other organ systems/tissues, the important roles the SCFARs (FFAR2 and FFAR3) play in physiology and in various disorders of the cardiovascular system have been revealed over the last fifteen years. In this review, we discuss the cardiovascular implications of some key (patho)physiological functions of SCFAR signaling pathways, particularly those regulating the neurohormonal control of circulation and adipose tissue homeostasis. Wherever appropriate, we also highlight the potential of these receptors as therapeutic targets for cardiovascular disorders.

Adrenal G Protein-Coupled Receptors and the Failing Heart: A Long-distance, Yet Intimate Affair.

ABSTRACT: Systolic heart failure (HF) is a chronic clinical syndrome characterized by the reduction in cardiac function and still remains the disease with the highest mortality worldwide. Despite considerable advances in pharmacological treatment, HF represents a severe clinical and social burden. Chronic human HF is characterized by several important neurohormonal perturbations, emanating from both the autonomic nervous system and the adrenal glands. Circulating catecholamines (norepinephrine and epinephrine) and aldosterone elevations are among the salient alterations that confer significant hormonal burden on the already compromised function of the failing heart. This is why sympatholytic treatments (such as ß-blockers) and renin-angiotensin system inhibitors or mineralocorticoid receptor antagonists, which block the effects of angiotensin II (AngII) and aldosterone on the failing heart, are part of the mainstay HF pharmacotherapy presently. The adrenal gland plays an important role in the modulation of cardiac neurohormonal stress because it is the source of almost all aldosterone, of all epinephrine, and of a significant amount of norepinephrine reaching the failing myocardium from the blood circulation. Synthesis and release of these hormones in the adrenals is tightly regulated by adrenal G protein-coupled receptors (GPCRs), such as adrenergic receptors and AngII receptors. In this review, we discuss important aspects of adrenal GPCR signaling and regulation, as they pertain to modulation of cardiac function in the context of chronic HF, by focusing on the 2 best studied adrenal GPCR types in that context, adrenergic receptors and AngII receptors (AT 1 Rs). Particular emphasis is given to findings from the past decade and a half that highlight the emerging roles of the GPCR-kinases and the ß-arrestins in the adrenals, 2 protein families that regulate the signaling and functioning of GPCRs in all tissues, including the myocardium and the adrenal gland.