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 β-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)-1β and IL-6 production, and pro-fibrotic transforming growth factor (TGF)-β 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 βAR 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.


Introduction
The short-chain free fatty acid (SCFA) propionate stimulates cellular signaling primarily via two different G-protein-coupled receptors (GPCRs) and free fatty acid receptor (FFAR)-2 and -3 [1][2][3]. FFAR3, also known as GPR41, regulates cardiovascular function via effects in peripheral sympathetic neurons, wherein it promotes neuronal firing and norepinephrine (NE) synthesis and release [4,5]. FFAR3 couples to Gi/o proteins, which can activate phospholipase (PLC)-β 2/3 and downstream calcium signaling via their free Gβγ subunits [4]. Synapsin-2b is ultimately phosphorylated and activated to induce vesicle fusion with the neuronal plasma membrane and NE exocytosis/synaptic release from sympathetic nerve endings [5]. In addition, the stimulatory effect of propionate/FFAR3 in sympathetic neurons is directly regulated by β-adrenergic receptors (ARs), since FFAR3 seems to upregulate βAR function in sympathetic ganglia [4]. acid stimulation ( Figure 1B). Consistent with this, inhibition of forskolin-stimulated synthesis of the second messenger cyclic 3`, 5`-adenosine monophosphate (cAMP) was also significantly enhanced by propionate in RGS4-depleted cardiomyocytes ( Figure 1C). Taken together, these results suggest that indeed, RGS4 directly blocks Gi/o protein signaling stimulated by propionic acid via FFAR3 in H9c2 cardiac myocytes.

RGS4 Opposes Cardiac FFAR3 Pro-Inflammatory And Pro-Fibrotic Signaling
Next, we examined the functional consequences of RGS4-mediated inhibition of FFAR3 Gi/o protein signaling in cardiac myocytes. RGS4 depletion resulted in markedly enhanced activation of the pro-inflammatory p38 mitogen-activated protein kinase (MAPK) in response to propionic acid stimulation in H9c2 cardiomyocytes ( Figure 2A). Additionally, propionic acid-induced interleukin (IL)-1β and IL-6 synthesis and secretion were also increased in RGS4-knocked-down cardiomyocytes ( Figure 2B), and the same was observed for propionic acid-dependent production of transforming growth factor (TGF)-β1, a major pro-fibrotic stimulus [27] ( Figure 2C). Thus, it appears that propionate/FFAR3 promote cardiac inflammation and fibrosis, two major cardiac-adverse, remodeling-related processes, and this is blocked by RGS4 via FFAR3-coupled Gi/o protein inactivation in cardiac myocytes.

RGS4 Opposes Cardiac FFAR3 Pro-Inflammatory and Pro-Fibrotic Signaling
Next, we examined the functional consequences of RGS4-mediated inhibition of FFAR3 Gi/o protein signaling in cardiac myocytes. RGS4 depletion resulted in markedly enhanced activation of the pro-inflammatory p38 mitogen-activated protein kinase (MAPK) in response to propionic acid stimulation in H9c2 cardiomyocytes ( Figure 2A). Additionally, propionic acid-induced interleukin (IL)-1β and IL-6 synthesis and secretion were also increased in RGS4-knocked-down cardiomyocytes ( Figure 2B), and the same was observed for propionic acid-dependent production of transforming growth factor (TGF)-β 1 , a major pro-fibrotic stimulus [27] ( Figure 2C). Thus, it appears that propionate/FFAR3 promote cardiac inflammation and fibrosis, two major cardiac-adverse, remodeling-related processes, and this is blocked by RGS4 via FFAR3-coupled Gi/o protein inactivation in cardiac myocytes. MAPK phosphorylation/activation in the control (scrambled siRNA-transfected, Scr) or in RGS4depleted (RGS4 siRNA) H9c2 cells. A representative blot is shown on the left and the densitometric quantitation on the right. Prop: Propionate. *, p < .05; n = 3 independent experiments performed in triplicate. (B, C) A 1 mM propionate-induced IL-1β and IL-6 (B) or TGF-β1 (C) protein synthesis in these cells. *, p < .05, vs. respective Scr; n = 3 independent measurements per condition per cell clone.

β ARs Reduce Cardiac FFAR3 Signaling via PKA-Dependent RGS4 Stimulation
Protein kinase A (PKA), the major effector of cAMP, has been reported to directly phosphorylate and activate RGS4, by inducing its plasma membrane translocation, in cultured smooth muscle cells, with Ser52 of RGS4, being one of the major phosphorylation sites [13]. Since βARs activate PKA via coupling to Gs proteins, whose Gα subunits stimulate adenylyl cyclase-mediated cAMP synthesis [26,27], we next examined whether PKA activates RGS4 also in cardiomyocytes, thereby mediating a βAR-FFAR3 crosstalk in the heart. Indeed, pretreatment of H9c2 cardiomyocytes with either isoproterenol, which stimulates both β1and β2ARs, or salbutamol (albuterol), which is a β2AR-selective agonist [27][28][29], markedly reduced propionic acid-elicited Giα activity in control and wild-type H9c2 cardiomyocytes ( Figure 3A), but both catecholamines failed to do so in RGS4-depleted cells ( Figure 3B). Furthermore, in the presence of H89, a well-characterized PKA inhibitor [30], neither isoproterenol nor salbutamol, had any detectable effect on propionic acid-dependent Giα activation in the native, wild-type H9c2 cardiomyocytes ( Figure 3B). Taken together, these results strongly suggest that catecholamines via both β1and β2ARs induce PKA-dependent activation of RGS4, which subsequently impedes FFAR3 signaling in cardiac myocytes.

β ARs Reduce Cardiac FFAR3 Signaling via PKA-Dependent RGS4 Stimulation
Protein kinase A (PKA), the major effector of cAMP, has been reported to directly phosphorylate and activate RGS4, by inducing its plasma membrane translocation, in cultured smooth muscle cells, with Ser52 of RGS4, being one of the major phosphorylation sites [13]. Since βARs activate PKA via coupling to Gs proteins, whose Gα subunits stimulate adenylyl cyclase-mediated cAMP synthesis [26,27], we next examined whether PKA activates RGS4 also in cardiomyocytes, thereby mediating a βAR-FFAR3 crosstalk in the heart. Indeed, pretreatment of H9c2 cardiomyocytes with either isoproterenol, which stimulates both β 1 -and β 2 ARs, or salbutamol (albuterol), which is a β 2 AR-selective agonist [27][28][29], markedly reduced propionic acid-elicited Giα activity in control and wild-type H9c2 cardiomyocytes ( Figure 3A), but both catecholamines failed to do so in RGS4-depleted cells ( Figure 3B). Furthermore, in the presence of H89, a well-characterized PKA inhibitor [30], neither isoproterenol nor salbutamol, had any detectable effect on propionic acid-dependent Giα activation in the native, wild-type H9c2 cardiomyocytes ( Figure 3B). Taken together, these results strongly suggest that catecholamines via both β 1 -and β 2 ARs induce PKA-dependent activation of RGS4, which subsequently impedes FFAR3 signaling in cardiac myocytes.

RGS4 Inhibits FFAR3-Stimulated NE Release from Sympathetic Neurons
Since FFAR3 is also involved in sympathetic neuronal activation stimulating NE release [4], we also checked for a potential regulatory role of RGS4 in cardiac sympathetic neuronal FFAR3 signaling. To mimic the in vivo situation of cardiac sympathetic nerve terminals releasing NE directly into cardiac myocytes, we co-cultured H9c2 cardiomyocytes together with nerve growth factor (NGF)-differentiated mouse Neuro-2A cells, known to synthesize and release NE (as well as dopamine), thus, functioning, in essence, like sympathetic neurons [4,31]. Given that endogenous FFAR3 expression in Neuro-2A cells is equivocal (very low at best) [4], we transfected these neuroblastoma cells to express human FFAR3, prior to their NGF-elicited neuronal differentiation. Propionate treatment led to significant NE release from the FFAR3-expressing differentiated Neuro-2A cells, which was partially blocked by RGS4 lentiviral-mediated overexpression ( Figure 4A). We then co-cultured RGS4-overexpressing or control neuronal cells together with (native, wild-type) H9c2 cardiac myocytes, and, 24 h post-propionic acid treatment, we measured the βAR membrane density of the H9c2 cardiac cells. Propionate treatment of the control neuronal cells led to a significant βAR agonist-dependent downregulation in the co-cultured cardiomyocytes, as expected due to the propionate/FFAR3-elicited NE release into the cell co-culture medium ( Figure 4B). Specifically, normal (basal) total βAR membrane density of H9c2 cardiomyocytes was estimated at 21.1 + 1.05 fmol/mg of protein and it was reduced to 9.3 + 1.8 fmol/mg of protein upon propionate stimulation of co-cultured control Neuro-2A cells (n = 4 independent determinations per condition) ( Figure 4B). However, cardiomyocyte βAR membrane density was substantially higher 24 h post-propionate challenge, when co-cultured with RGS4-overexpressing neuronal cells (16.2 + 1.2 fmol/mg of protein, n = 4; Figure 4B). That was presumably because of the lower NE release from the RGS4-overexpressing neuronal cells, which reduced the levels of NE activating the H9c2 cardiomyocyte-residing βARs, causing their homologous downregulation. Taken together, these findings indicate that RGS4 opposes propionate/FFAR3 signaling also in sympathetic neurons, which may lower neuronal activity and NE release, thereby largely preserving βAR density/function of the innervated myocardium.

RGS4 Inhibits FFAR3-Stimulated NE Release from Sympathetic Neurons
Since FFAR3 is also involved in sympathetic neuronal activation stimulating NE release [4], we also checked for a potential regulatory role of RGS4 in cardiac sympathetic neuronal FFAR3 signaling. To mimic the in vivo situation of cardiac sympathetic nerve terminals releasing NE directly into cardiac myocytes, we co-cultured H9c2 cardiomyocytes together with nerve growth factor (NGF)-differentiated mouse Neuro-2A cells, known to synthesize and release NE (as well as dopamine), thus, functioning, in essence, like sympathetic neurons [4,31]. Given that endogenous FFAR3 expression in Neuro-2A cells is equivocal (very low at best) [4], we transfected these neuroblastoma cells to express human FFAR3, prior to their NGF-elicited neuronal differentiation. Propionate treatment led to significant NE release from the FFAR3-expressing differentiated Neuro-2A cells, which was partially blocked by RGS4 lentiviral-mediated overexpression ( Figure 4A). We then co-cultured RGS4-overexpressing or control neuronal cells together with (native, wild-type) H9c2 cardiac myocytes, and, 24 h post-propionic acid treatment, we measured the βAR membrane density of the H9c2 cardiac cells. Propionate treatment of the control neuronal cells led to a significant βAR agonist-dependent downregulation in the co-cultured cardiomyocytes, as expected due to the propionate/FFAR3-elicited NE release into the cell co-culture medium ( Figure 4B). Specifically, normal (basal) total βAR membrane density of H9c2 cardiomyocytes was estimated at 21.1 ± 1.05 fmol/mg of protein and it was reduced to 9.3 ± 1.8 fmol/mg of protein upon propionate stimulation of co-cultured control Neuro-2A cells (n = 4 independent determinations per condition) ( Figure 4B). However, cardiomyocyte βAR membrane density was substantially higher 24 h post-propionate challenge, when co-cultured with RGS4-overexpressing neuronal cells (16.2 ± 1.2 fmol/mg of protein, n = 4; Figure 4B). That was presumably because of the lower NE release from the RGS4-overexpressing neuronal cells, which reduced the levels of NE activating the H9c2 cardiomyocyte-residing βARs, causing their homologous downregulation. Taken together, these findings indicate that RGS4 opposes propionate/FFAR3 signaling also in sympathetic neurons, which may lower neuronal activity and NE release, thereby largely preserving βAR density/function of the innervated myocardium.

Discussion
In the present study, we have uncovered an important regulatory role for RGS4 at keeping propionic acid signaling through FFAR3/GPR41 in check in both cardiomyocytes and sympathetic neurons ( Figure 5). On the one hand, myocardial FFAR3 activated by SCFAs, such as propionic acid, upregulates several pro-inflammatory (IL-1β, IL-6, activated p38 MAPK) [32] and pro-fibrotic (e.g., TGFβ [26]) factors, which is highly likely to lead to exacerbated adverse remodeling in the presence of a cardiac insult, such as myocardial ischemia/infarction or pressure overload/severe hypertension ( Figure 5). Furthermore, TGFβ, but also inflammation and fibrosis in general, have been implicated in cardiac aging [33][34][35]. Thus, FFAR3 signaling may also promote cardiac aging and RGS4, and by opposing FFAR3 effects, may have anti-aging properties in the heart. On the other hand, FFAR3 present in post-ganglionic sympathetic neurons, including cardiac sympathetic nerve terminals, promotes neuronal firing and activity resulting in elevated NE release [4] (Figure 5). RGS4 terminates FFAR3 Gi/o protein-dependent signaling in both cell types (cardiomyocytes and sympathetic neurons), thereby lowering propionic acid-dependent cardiac inflammation/fibrosis and sympathetic activity/NE release at the same time ( Figure 5). The latter is particularly useful in the setting of chronic heart failure, during which the failing heart needs to be protected against the toxicity of elevated catecholamine levels [36][37][38].
Indeed, we found that inhibition of neuronal FFAR3-dependent NE release by RGS4 markedly reduced agonist (NE)-dependent downregulation of βARs in co-cultured cardiomyocytes, which suggests that RGS4 in sympathetic neurons has the potential of preserving, at least in part, the adrenergic and inotropic reserves of the failing heart. This is also consistent with the old studies showing a cardioprotective effect of RGS4 against pressure overload (increased afterload)-induced hypertrophy [16,18]. Indeed, apart from shielding the heart from pathological Gq protein-dependent pro-hypertrophic signaling that diminishes cardiac function [17,18], cardiac RGS4 overexpression was shown in that 1999 study [16] to preserve the positive inotropic response of transgenic mice to the

Discussion
In the present study, we have uncovered an important regulatory role for RGS4 at keeping propionic acid signaling through FFAR3/GPR41 in check in both cardiomyocytes and sympathetic neurons ( Figure 5). On the one hand, myocardial FFAR3 activated by SCFAs, such as propionic acid, upregulates several pro-inflammatory (IL-1β, IL-6, activated p38 MAPK) [32] and pro-fibrotic (e.g., TGFβ [26]) factors, which is highly likely to lead to exacerbated adverse remodeling in the presence of a cardiac insult, such as myocardial ischemia/infarction or pressure overload/severe hypertension ( Figure 5). Furthermore, TGFβ, but also inflammation and fibrosis in general, have been implicated in cardiac aging [33][34][35]. Thus, FFAR3 signaling may also promote cardiac aging and RGS4, and by opposing FFAR3 effects, may have anti-aging properties in the heart. On the other hand, FFAR3 present in post-ganglionic sympathetic neurons, including cardiac sympathetic nerve terminals, promotes neuronal firing and activity resulting in elevated NE release [4] (Figure 5). RGS4 terminates FFAR3 Gi/o protein-dependent signaling in both cell types (cardiomyocytes and sympathetic neurons), thereby lowering propionic aciddependent cardiac inflammation/fibrosis and sympathetic activity/NE release at the same time ( Figure 5). The latter is particularly useful in the setting of chronic heart failure, during which the failing heart needs to be protected against the toxicity of elevated catecholamine levels [36][37][38].
Indeed, we found that inhibition of neuronal FFAR3-dependent NE release by RGS4 markedly reduced agonist (NE)-dependent downregulation of βARs in co-cultured cardiomyocytes, which suggests that RGS4 in sympathetic neurons has the potential of preserving, at least in part, the adrenergic and inotropic reserves of the failing heart. This is also consistent with the old studies showing a cardioprotective effect of RGS4 against pressure overload (increased afterload)-induced hypertrophy [16,18]. Indeed, apart from shielding the heart from pathological Gq protein-dependent pro-hypertrophic signaling that diminishes cardiac function [17,18], cardiac RGS4 overexpression was shown in that 1999 study [16] to preserve the positive inotropic response of transgenic mice to the cate-cholaminergic agonist dobutamine post-pressure overload. Taken together, these findings strongly suggest that RGS4, in both the heart and the sympathetic nervous system, may serve as an important molecular brake for the cardiotoxic effects of propionic acid/FFAR3 signaling. Therefore, systemic RGS4 pharmacological stimulation might be of therapeutic value in heart disease, particularly in chronic human heart failure and in systemic hypertension. catecholaminergic agonist dobutamine post-pressure overload. Taken together, these findings strongly suggest that RGS4, in both the heart and the sympathetic nervous system, may serve as an important molecular brake for the cardiotoxic effects of propionic acid/FFAR3 signaling. Therefore, systemic RGS4 pharmacological stimulation might be of therapeutic value in heart disease, particularly in chronic human heart failure and in systemic hypertension. Our present study expands on the already established cardioprotective role of RGS4, which is upregulated, most likely as a compensatory mechanism, in the hearts of experimental animals of cardiac hypertrophy secondary to aortic or pulmonary artery banding in vivo [19] and in human failing hearts explanted from dilated or ischemic cardiomyopathy patients, or from end-stage or acute human heart failure patients [10,20,21]. Additionally, RGS4 exerts crucial cardioprotective effects in the atrial myocardium against AFib pathogenesis and pro-arrhythmogenic Ca 2+ transient generation [9,10,22,23].
In contrast to its well-documented actions in the myocardium, little (if anything at all) is known about the effects of RGS4 in sympathetic neurons. FFAR3 is robustly expressed in murine peripheral sympathetic neurons, including cardiac sympathetic nerve terminals, wherein it regulates not only cell metabolism but also neuronal activity via stimulation of NE release [4] (Figure 5). Although both NE and epinephrine mediate the effects of the sympathetic nervous system on all cells and tissues, NE is the neurotransmitter synthesized, stored, and released from sympathetic neurons, whereas epinephrine is a hormone synthesized in the adrenal medulla and secreted into the systemic circulation [39][40][41]. FFAR3 knockout mice display significantly lower catecholamine synthesis, as evidenced by tyrosine hydroxylase downregulation, the enzyme that catalyzes the rate-limiting step of catecholamine biosynthesis [42], as well as lower sympathetic neuronal firing rate and heart rate [1,4].
Mechanistically, FFAR3 stimulates NE release via Gi/o-derived free Gβγ subunit activation of PLCβ2/3 [4]. PLCβ2/3, in turn, induces Ca 2+ signaling that ultimately results in activation of extracellular signal-regulated kinase (ERK)1/2, which then has Our present study expands on the already established cardioprotective role of RGS4, which is upregulated, most likely as a compensatory mechanism, in the hearts of experimental animals of cardiac hypertrophy secondary to aortic or pulmonary artery banding in vivo [19] and in human failing hearts explanted from dilated or ischemic cardiomyopathy patients, or from end-stage or acute human heart failure patients [10,20,21]. Additionally, RGS4 exerts crucial cardioprotective effects in the atrial myocardium against AFib pathogenesis and pro-arrhythmogenic Ca 2+ transient generation [9,10,22,23].
In contrast to its well-documented actions in the myocardium, little (if anything at all) is known about the effects of RGS4 in sympathetic neurons. FFAR3 is robustly expressed in murine peripheral sympathetic neurons, including cardiac sympathetic nerve terminals, wherein it regulates not only cell metabolism but also neuronal activity via stimulation of NE release [4] (Figure 5). Although both NE and epinephrine mediate the effects of the sympathetic nervous system on all cells and tissues, NE is the neurotransmitter synthesized, stored, and released from sympathetic neurons, whereas epinephrine is a hormone synthesized in the adrenal medulla and secreted into the systemic circulation [39][40][41]. FFAR3 knockout mice display significantly lower catecholamine synthesis, as evidenced by tyrosine hydroxylase downregulation, the enzyme that catalyzes the rate-limiting step of catecholamine biosynthesis [42], as well as lower sympathetic neuronal firing rate and heart rate [1,4].
Mechanistically, FFAR3 stimulates NE release via Gi/o-derived free Gβγ subunit activation of PLCβ 2/3 [4]. PLCβ 2/3 , in turn, induces Ca 2+ signaling that ultimately results in activation of extracellular signal-regulated kinase (ERK)1/2, which then has phosphorylate synapsin-2b at Ser426 to induce vesicle fusion with the neuronal plasma membrane and subsequent NE release from sympathetic nerve endings [5]. RGS4 is known to directly bind not only Gα subunits (which it deactivates via its RGS function), but also Gi/o-derived free Gβγ subunits and PLCβ, thereby inhibiting PLCβ activation directly (independently of its RGS function) [12,13]. It is thus tempting to speculate that direct PLCβ binding and inhibition is the mechanism underlying RGS4's inhibitory effect on neuronal FFAR3 signaling towards NE release (and perhaps also some of its effects on cardiac FFAR3 signaling). However, this awaits verification in future studies and efforts to delineate the full signaling pathway(s) underlying the effects of RGS4 on cardiac and neuronal FFAR3 signaling are under way in our laboratory.
Finally, we have uncovered an important RGS4-centric regulatory negative feedback loop operating in cardiac myocytes between βARs (in particular, β 2 AR) and FFAR3: βARactivated PKA phosphorylates and stimulates RGS4 to abrogate SCFA/FFAR3 signaling and function in cardiac myocytes ( Figure 5). At the same time, FFAR3-activated Giα inhibits adenylyl cyclase and thus, cAMP synthesis, reducing PKA activation, which would indirectly decrease βAR-dependent RGS4 activity and enhance FFAR3 signaling. However, this may not be the case given a very recent study that suggests the existence of receptor-associated independent cAMP nanodomains operating as individual, independent cell signaling units inside the same cell; in other words, a GPCR may not interfere with the cAMP pool associated with a different receptor, despite residing at the same plasma membrane and perhaps even in close proximity with one another [43]. It should also be noted here that RGS4, like all other canonical RGS proteins identified to date, does not directly affect (i.e., inactivates) Gsα subunits, to which βARs normally couple [11]. The presence of this RGS4-mediated regulatory crosstalk between βARs and FFAR3 is entirely consistent with the finding by Kimura et al. that the effect of propionate/FFAR3 on heart rate was suppressed by pretreatment with the non-subtype selective β-blocker propranolol, which suggested reciprocal regulation of FFAR3 signaling by βARs [4]. It is also in line with the role of PKA in PLCβ blockade, (in part) via RGS4 activation, in gastric smooth muscle cells [13]. Thus, it seems that PKA activates RGS4 via phosphorylation in the heart, as well. Moreover, since β 2 AR-activated RGS4 obstructs cardiac FFAR3 proinflammatory and pro-fibrotic signaling ( Figure 5), these findings are also consistent with a potential beneficial role for the cardiac β 2 AR subtype in cardiac reverse remodeling [26,31], in direct juxtaposition to the much more abundant β 1 AR subtype [28,36,44]. Of note, in addition to the cardiac βAR-FFAR3 crosstalk via RGS4, a similar negative feedback crosstalk also exists between sympathetic neuronal FFAR3 and cardiac βARs ( Figure 5). More specifically, neuronal FFAR3 increases the amount of NE that activates cardiac βARs, promoting βAR homologous desensitization and downregulation in the myocardium that, over time, diminishes cardiac βAR function and number [36]. RGS4 again opposes neuronal FFAR3-dependent NE release, thereby helping to counteract βAR homologous desensitization/downregulation and to preserve myocardial βAR function ( Figure 5).
The present study has two major limitations: (a) Although it was done in physiologically relevant (cardiomyocyte-like and sympathetic neuron-like) cells, these cells were not, nonetheless, primary bona fide differentiated cells; and (b) Our findings obviously require confirmation in in vivo models and settings. Nevertheless, given that the cell lines we employed closely mimicked primary, differentiated cell cultures and were still physiologically relevant, our present findings are quite likely to hold true in the heart and in cardiac sympathetic neurons in vivo, as well.
In summary, we report here, for the first time to our knowledge, that: (a) Cardiac FFAR3 promotes inflammation and fibrosis via its classic Gi/o protein signaling, which is opposed by RGS4; (b) Catecholamines negatively modulate cardiac FFAR3 signaling via PKA-mediated RGS4 activation; and (c) RGS4 dampens FFAR3 signaling towards NE release in sympathetic neurons, thereby potentially affording sympatholysis that can help preserve cardiac βAR function, especially in the setting of chronic heart failure. Cardiac, as well as neuronal, RGS4 stimulation might, thus, be advantageous in helping the failing heart cope with the toxic effects, both direct and indirect (via sympathetic hyperactivation), of SCFAs that are aberrantly upregulated in human heart failure [45,46]. If confirmed in vivo, these findings will expand the growing list of beneficial effects of RGS4 in the myocardium, which already includes keeping heart rate and post-pressure overload cardiac hypertrophy in check. Although the focus of RGS protein-targeted drug discovery efforts has been almost exclusively on development of inhibitors, stimulation of specific RGS proteins is therapeutically desirable for certain conditions [10] and pharmacologically feasible, at least indirectly, by targeting RGS protein expression (i.e., inhibiting degradation/increasing stability of the RGS protein) [7]. Our present study provides substantial evidence that, in the case of cardiac FFAR3 signaling, development of an RGS4 activator or expression enhancer may be worth pursuing.

Materials
All chemicals and pharmacological agents, including propionic acid, forskolin, isoproterenol, and salbutamol, were from Sigma-Aldrich (St. Louis, MO, USA).

Cell Culture, Transfections, and Treatments
The H9c2 rat cardiomyoblast and Neuro-2A mouse neuroblastoma cell lines were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured as previously described [4,47]. For siRNA-mediated knockdown of RGS4 in H9c2 cells, an siRNA against rat RGS4 [Target RefSeqID: NM_017214, Rgs4 gene of the Rattus norvegicus species; Sequences: CCAAAUAUUGAUCUGUAUUdTdT (Sense); AAUACAGAU-CAAUAUUUGGdTdT (Antisense)] was designed and synthesized (Sigma-Aldrich), along with a negative control (scrambled siRNA oligo, Cat. #SIC001; also from Sigma-Aldrich). Cells were transfected with either the RGS4-specific siRNA or the negative control (Scrambled) using the Mission ® siRNA transfection reagent (Cat. #S1452; Sigma-Aldrich) and 48 h later, Western blotting for RGS4 protein levels was performed to ascertain the efficiency of the knockdown. For RGS4 overexpression in Neuro-2A cells, the cells were first transfected (via the Lipofectamine ® 3000 Transfection Reagent method; ThermoFisher Scientific, Waltham, MA, USA) with a human FFAR3-encoding cDNA (CloneID #OHu24442, RefSeq #NM_005304.4; GenScript Biotech, Piscataway, NJ, USA) to overexpress FFAR3, and then differentiated into sympathetic-like neurons via daily treatments with 50 ng/mL NGF (Sigma-Aldrich) for 5 consecutive days [48]. Subsequently, infections of the differentiated Neuro-2A cells were performed with either a lentiviral construct encoding for full-length RGS4 (Lenti-CMV-RGS4v1, Cat. #LH848503; Vigene Biosciences/Charles River Laboratories, Rockville, MD, USA) to overexpress RGS4, or a control construct encoding for GFP (Cat. #CV10002; also from Charles River Laboratories). RGS4 overexpression was verified via Western blotting 48 hrs post-infection. For the Giα activity assay, plasma membranes were prepared from the H9c2 cells, and the ligands/agonists were added directly into the assay's well plate immediately prior to the addition of the membrane suspension. For cAMP accumulation and p38 MAPK activation determinations, cells were harvested 30 min after agent/drug application, whereas for the ELISA measurements of IL-1β, Il-6, and TGFβ, cells were harvested 12 h post-treatment. Finally, NE release was measured 6 h post-propionate treatment, while H9c2 cardiomyocyte βAR density was assayed 24 h (the next day) post-propionic acid application to the H9c2-N2a cell co-culture.

Giα Activity Assay and cAMP Accumulation Determination
Giα activation was determined on isolated plasma membranes from H9c2 cells using a HTRF (homogeneous time-resolved fluorescence)-based GTP Gi binding assay kit (Cat. #62GTPPET; Cisbio-PerkinElmer, Inc., Waltham, MA, USA) and following the manufacturer's protocols/instructions [49]. In essence, this assay measures the level of Giα activation following agonist stimulation of a GPCR, thanks to the use of a europium-labeled GTP analog (Eu-GTP). During the activation process of the heterotrimeric Gi protein, GDP dissociates from the Giα subunit and gets replaced by the assay's fluorescent Eu-GTP analog that now binds to the Giα subunit, as the latter goes from its inactive to its active state. The fluorescent signal emitted by the Giα-bound Eu-GTP analog is detected via addition of an anti-Giα antibody, with the fluorescence intensity levels measured being directly proportional to the level of Giα activity. cAMP accumulation was measured with the "Direct cAMP ELISA kit" (Cat. #ADI-900-066; Enzo Life Sciences, Farmingdale, NY, USA), as described previously [27].

βAR Density Measurements
βAR density was measured in isolated plasma membranes using 125 I-CYP (Iodocyanopindolol; PerkinElmer, Inc.), as described [37]. Briefly, at 24 h post-propionate application, H9c2 cells were harvested, and plasma membrane fractions were prepared via lysis in a hypotonic buffer containing Tris . Cl (pH 7.5), EDTA (pH 8), EGTA, and protease inhibitors, followed by ultracentrifugation (62,000 rpm for 1 h at 4 • C). Protein content was determined and then incubations with 125 I-CYP were performed in the presence of 80 µM alprenolol (for non-specific binding determination). The reactions were terminated in a cell harvester, followed by sample loading onto a gamma counter (Beckman Coulter, Brea, CA, USA) for radioactivity counts determination. Receptor densities are expressed as fmol/mg of membrane protein.

Statistical Analysis
Data are generally expressed as mean ± SEM. Unpaired 2-tailed Student's t test and one-or two-way ANOVA with Bonferroni test were generally performed for statistical comparisons, unless otherwise indicated. For most 3-group statistical comparisons, Dunnett's test using SAS version 9 software (Cary, NC, USA) was used, as well. For all tests, a p value of <0.05 was generally considered to be significant.  Data Availability Statement: All data presented in this paper are available upon request to the correspondence author.