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.

Editorial to the IJMS Special Issue on Sglt2 Inhibitors Vol. 1.

The goal of this Special Issue is to highlight the ever-increasing progress in pharmacological research on sodium-glucose co-transporter (SGLT) type 2 (SGLT2) inhibitors or gliflozins [...].

Editorial for the IJMS Special Issue on Sglt2 Inhibitors (Volume 2).

The goal of the second volume of this Special Issue was to build upon the success of the first one and to continue to highlight the ever-expanding list of pharmacological properties of the sodium/glucose co-transporter (SGLT) type 2 (SGLT2) inhibitor (SGLT2i) drug class (also known as gliflozins) [...].

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.

Autonomic Nervous System Regulation of Epicardial Adipose Tissue: Potential Roles for Regulator of G Protein Signaling-4.

The epicardial adipose tissue (EAT) or epicardial fat is a visceral fat depot in the heart that contains intrinsic adrenergic and cholinergic nerves, through which it interacts with the cardiac sympathetic (adrenergic) and parasympathetic (cholinergic) nervous systems. These EAT nerves represent a significant source of several adipokines and other bioactive molecules, including norepinephrine, epinephrine, and free fatty acids. The production of these molecules is biologically relevant for the heart, since abnormalities in EAT secretion are implicated in the development of pathological conditions, including coronary atherosclerosis, atrial fibrillation, and heart failure. Sympathetic hyperactivity and parasympathetic (cholinergic) derangement are associated with EAT dysfunction, leading to a variety of adverse cardiac conditions, such as heart failure, diastolic dysfunction, atrial fibrillation, etc.; therefore, several studies have focused on exploring the autonomic regulation of EAT as it pertains to heart disease pathogenesis and progression. In addition, Regulator of G protein Signaling (RGS)-4 is a protein with significant regulatory roles in both adrenergic and muscarinic receptor signaling in the heart. In this review, we provide an overview of the autonomic regulation of EAT, with a specific focus on cardiac RGS4 and the potential roles this protein plays in this regulation.

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.

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.

Carvedilol Selectively Stimulates ßArrestin2-Dependent SERCA2a Activity in Cardiomyocytes to Augment Contractility.

Heart failure (HF) carries the highest mortality in the western world and ß-blockers [ß-adrenergic receptor (AR) antagonists] are part of the cornerstone pharmacotherapy for post-myocardial infarction (MI) chronic HF. Cardiac ß1AR-activated ßarrestin2, a G protein-coupled receptor (GPCR) adapter protein, promotes Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a SUMO (small ubiquitin-like modifier)-ylation and activity, thereby directly increasing cardiac contractility. Given that certain ß-blockers, such as carvedilol and metoprolol, can activate ßarrestins and/or SERCA2a in the heart, we investigated the effects of these two agents on cardiac ßarrestin2-dependent SERCA2a SUMOylation and activity. We found that carvedilol, but not metoprolol, acutely induces ßarrestin2 interaction with SERCA2a in H9c2 cardiomyocytes and in neonatal rat ventricular myocytes (NRVMs), resulting in enhanced SERCA2a SUMOylation. However, this translates into enhanced SERCA2a activity only in the presence of the ß2AR-selective inverse agonist ICI 118,551 (ICI), indicating an opposing effect of carvedilol-occupied ß2AR subtype on carvedilol-occupied ß1AR-stimulated, ßarrestin2-dependent SERCA2a activation. In addition, the amplitude of fractional shortening of NRVMs, transfected to overexpress ßarrestin2, is acutely enhanced by carvedilol, again in the presence of ICI only. In contrast, metoprolol was without effect on NRVMs' shortening amplitude irrespective of ICI co-treatment. Importantly, the pro-contractile effect of carvedilol was also observed in human induced pluripotent stem cell (hIPSC)-derived cardiac myocytes (CMs) overexpressing ßarrestin2, and, in fact, it was present even without concomitant ICI treatment of human CMs. Metoprolol with or without concomitant ICI did not affect contractility of human CMs, either. In conclusion, carvedilol, but not metoprolol, stimulates ßarrestin2-mediated SERCA2a SUMOylation and activity through the ß1AR in cardiac myocytes, translating into direct positive inotropy. However, this unique ßarrestin2-dependent pro-contractile effect of carvedilol may be opposed or masked by carvedilol-bound ß2AR subtype signaling.

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.

Cardiovascular angiotensin II type 1 receptor biased signaling: Focus on non-Gq-, non-ßarrestin-dependent signaling.

The physiological and pathophysiological roles of the angiotensin II type 1 (AT1) receptor, a G protein-coupled receptor ubiquitously expressed throughout the cardiovascular system, have been the focus of intense investigations for decades. The success of angiotensin converting enzyme inhibitors (ACEIs) and of angiotensin receptor blockers (ARBs), which are AT1R-selective antagonists/inverse agonists, in the treatment of heart disease is a testament to the importance of this receptor for cardiovascular homeostasis. Given the pleiotropic signaling of the cardiovascular AT1R and, in an effort to develop yet better drugs for heart disease, the concept of biased signaling has been exploited to design and develop biased AT1R ligands that selectively activate ß-arrestin transduction pathways over Gq protein-dependent pathways. However, by focusing solely on Gq or ß-arrestins, studies on AT1R "biased" signaling & agonism tend to largely ignore other non-Gq-, non ß-arrestin-dependent signaling modalities the very versatile AT1R employs in cardiovascular tissues, including two very important types of signal transducers/regulators: other G protein types (e.g., Gi/o, G12/13) & the Regulator of G protein Signaling (RGS) proteins. In this review, we provide a brief overview of the current state of cardiovascular AT1R biased signaling field with a special focus on the non-Gq-, non ß-arrestin-dependent signaling avenues of this receptor in the cardiovascular system, which usually get left out of the conversation of "biased" AT1R signal transduction.

Sympatholytic Mechanisms for the Beneficial Cardiovascular Effects of SGLT2 Inhibitors: A Research Hypothesis for Dapagliflozin's Effects in the Adrenal Gland.

Heart failure (HF) remains the leading cause of morbidity and death in the western world, and new therapeutic modalities are urgently needed to improve the lifespan and quality of life of HF patients. The sodium-glucose co-transporter-2 (SGLT2) inhibitors, originally developed and mainly indicated for diabetes mellitus treatment, have been increasingly shown to ameliorate heart disease, and specifically HF, in humans, regardless of diabetes co-existence. Indeed, dapagliflozin has been reported to reduce cardiovascular mortality and hospitalizations in patients with HF and reduced ejection fraction (HFrEF). This SGLT2 inhibitor demonstrates these benefits also in non-diabetic subjects, indicating that dapagliflozin's efficacy in HF is independent of blood glucose control. Evidence for the effectiveness of various SGLT2 inhibitors in providing cardiovascular benefits irrespective of their effects on blood glucose regulation have spurred the use of these agents in HFrEF treatment and resulted in FDA approvals for cardiovascular indications. The obvious question arising from all these studies is, of course, which molecular/pharmacological mechanisms underlie these cardiovascular benefits of the drugs in diabetics and non-diabetics alike. The fact that SGLT2 is not significantly expressed in cardiac myocytes (SGLT1 appears to be the dominant isoform) adds even greater perplexity to this answer. A variety of mechanisms have been proposed over the past few years and tested in cell and animal models and prominent among those is the potential for sympatholysis, i.e., reduction in sympathetic nervous system activity. The latter is known to be high in HF patients, contributing significantly to the morbidity and mortality of the disease. The present minireview first summarizes the current evidence in the literature supporting the notion that SGLT2 inhibitors, such as dapagliflozin and empagliflozin, exert sympatholysis, and also outlines the main putative underlying mechanisms for these sympatholytic effects. Then, we propose a novel hypothesis, centered on the adrenal medulla, for the sympatholytic effects specifically of dapagliflozin. Adrenal medulla is responsible for the production and secretion of almost the entire amount of circulating epinephrine and of a significant percentage of circulating norepinephrine in the human body. If proven true experimentally, this hypothesis, along with other emerging experimental evidence for sympatholytic effects in neurons, will shed new light on the pharmacological effects that mediate the cardiovascular benefits of SGLT2 inhibitor drugs, independently of their blood glucose-lowering effects.

Correction: Maning et al. Antagonistic Roles of GRK2 and GRK5 in Cardiac Aldosterone Signaling Reveal GRK5-Mediated Cardioprotection via Mineralocorticoid Receptor Inhibition. Int. J. Mol. Sci. 2020, 21, 2868.

The authors wish to make the following correction to this paper [...].

Antagonistic Roles of GRK2 and GRK5 in Cardiac Aldosterone Signaling Reveal GRK5-Mediated Cardioprotection via Mineralocorticoid Receptor Inhibition.

Aldosterone (Aldo), when overproduced, is a cardiotoxic hormone underlying heart failure and hypertension. Aldo exerts damaging effects via the mineralocorticoid receptor (MR) but also activates the antiapoptotic G protein-coupled estrogen receptor (GPER) in the heart. G protein-coupled receptor (GPCR)-kinase (GRK)-2 and -5 are the most abundant cardiac GRKs and phosphorylate GPCRs as well as non-GPCR substrates. Herein, we investigated whether they phosphorylate and regulate cardiac MR and GPER. To this end, we used the cardiomyocyte cell line H9c2 and adult rat ventricular myocytes (ARVMs), in which we manipulated GRK5 protein levels via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and GRK2 activity via pharmacological inhibition. We report that GRK5 phosphorylates and inhibits the cardiac MR whereas GRK2 phosphorylates and desensitizes GPER. In H9c2 cardiomyocytes, GRK5 interacts with and phosphorylates the MR upon ß2-adrenergic receptor (AR) activation. In contrast, GRK2 opposes agonist-activated GPER signaling. Importantly, GRK5-dependent MR phosphorylation of the MR inhibits transcriptional activity, since aldosterone-induced gene transcription is markedly suppressed in GRK5-overexpressing cardiomyocytes. Conversely, GRK5 gene deletion augments cardiac MR transcriptional activity. ß2AR-stimulated GRK5 phosphorylates and inhibits the MR also in ARVMs. Additionally, GRK5 is necessary for the protective effects of the MR antagonist drug eplerenone against Aldo-induced apoptosis and oxidative stress in ARVMs. In conclusion, GRK5 blocks the cardiotoxic MR-dependent effects of Aldo in the heart, whereas GRK2 may hinder beneficial effects of Aldo through GPER. Thus, cardiac GRK5 stimulation (e.g., via ß2AR activation) might be of therapeutic value for heart disease treatment via boosting the efficacy of MR antagonists against Aldo-mediated cardiac injury.

GRK2-Mediated Crosstalk Between ß-Adrenergic and Angiotensin II Receptors Enhances Adrenocortical Aldosterone Production In Vitro and In Vivo.

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone via both G proteins and the adapter protein ßarrestin1, which binds the receptor following its phosphorylation by GPCR-kinases (GRKs) to initiate G protein-independent signaling. ß-adrenergic receptors (ARs) also induce aldosterone production in AZG cells. Herein, we investigated whether GRK2 or GRK5, the two major adrenal GRKs, is involved in the catecholaminergic regulation of AngII-dependent aldosterone production. In human AZG (H295R) cells in vitro, the ßAR agonist isoproterenol significantly augmented both AngII-dependent aldosterone secretion and synthesis, as measured by the steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA inductions. Importantly, GRK2, but not GRK5, was indispensable for the ßAR-mediated enhancement of aldosterone in response to AngII. Specifically, GRK2 inhibition with Cmpd101 abolished isoproterenol's effects on AngII-induced aldosterone synthesis/secretion, whereas the GRK5 knockout via CRISPR/Cas9 had no effect. It is worth noting that these findings were confirmed in vivo, since rats overexpressing GRK2, but not GRK5, in their adrenals had elevated circulating aldosterone levels compared to the control animals. However, treatment with the ß-blocker propranolol prevented hyperaldosteronism in the adrenal GRK2-overexpressing rats. In conclusion, GRK2 mediates a ßAR-AT1R signaling crosstalk in the adrenal cortex leading to elevated aldosterone production. This suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems at the level of the adrenal cortex and that its inhibition might be therapeutically advantageous in hyperaldosteronism-related conditions.

Chemically synthesized Secoisolariciresinol diglucoside (LGM2605) improves mitochondrial function in cardiac myocytes and alleviates septic cardiomyopathy.

Sepsis is the overwhelming systemic immune response to infection, which can result in multiple organ dysfunction and septic shock. Myocardial dysfunction during sepsis is associated with advanced disease and significantly increased in-hospital mortality. Our group has shown that energetic failure and excess reactive oxygen species (ROS) generation constitute major components of myocardial dysfunction in sepsis. Because ROS production is central to cellular metabolic health, we tested if the synthetic anti-oxidant lignan secoisolariciresinol diglucoside (SDG; LGM2605) would alleviate septic cardiac dysfunction and investigated the underlying mechanism. Using the cecal ligation and puncture (CLP) mouse model of peritonitis-induced sepsis, we observed impairment of cardiac function beginning at 4 h post-CLP surgery. Treatment of mice with LGM2605 (100 mg/kg body weight, i.p.) 6 h post-CLP surgery reduced cardiac ROS accumulation and restored cardiac function. Assessment of mitochondrial respiration (Seahorse XF) in primary cardiomyocytes obtained from adult C57BL/6 mice that had undergone CLP and treatment with LGM2605 showed restored basal and maximal respiration, as well as preserved oxygen consumption rate (OCR) associated with spare capacity. Further analyses aiming to identify the cellular mechanisms that may account for improved cardiac function showed that LGM2605 restored mitochondria abundance, increased mitochondrial calcium uptake and preserved mitochondrial membrane potential. In addition to protecting against cardiac dysfunction, daily treatment with LGM2605 and antibiotic ertapenem (70 mg/kg) protected against CLP-associated mortality and reversed hypothermia when compared against mice receiving ertapenem and saline. Therefore, treatment of septic mice with LGM2605 emerges as a novel pharmacological approach that reduces cardiac ROS accumulation, protects cardiac mitochondrial function, alleviates cardiac dysfunction, and improves survival.

Role of ßarrestin1 in AT1 R-mediated mitogen-activated protein kinase activation in Wistar and SHR brainstem astrocytes.

ßarrestin (ßarr)-1 and -2 are ubiquitously (outside the retina) expressed G-protein-coupled receptor adapter proteins. They uncouple G-protein-coupled receptors from G proteins, internalize the receptor, and subsequently initiate their own wave of signaling independently of G proteins. Angiotensin (Ang) II type 1 receptor (AT1 R) is a well-established example of a receptor signaling through ßarrs. Despite the pivotal role of brain AT1 Rs in the regulation of blood pressure, the involvement of ßarr-dependent signaling, mediated by AT1 Rs is not well studied. Particularly, in brain astrocytes very little is known about the effects of ßarrs in AT1 R signaling. Herein, we utilized a combination of pharmacological and gene manipulation approaches to investigate the role of ßarrs in AT1 R-mediated signaling in isolated brainstem astrocytes from spontaneously hypertensive rats (SHRs) and Wistar rats. We observed that ßarr1 is the predominant arrestin isoform at the protein level in these cells. Its expression was down-regulated in SHR astrocytes compared to Wistar rat astrocytes. Ang II, contrary to observations in SHR astrocytes where it had no effect, up-regulates ßarr1 protein in Wistar rat astrocytes. We observed differential involvement of ßarr1 in MAPK activation in brainstem astrocytes of SHR versus Wistar rats. The ßarr-biased agonist peptide [Sar1 , Il4 , Il8] Ang-II (SII), induced AT1 R-mediated ERK and p38 activation in Wistar rat astrocytes. SII had no effect on ERK and p38 activation in SHRs brainstem astrocytes. Our results indicate, reduced involvement of ßarr1 in dampening Ang II-induced MAPKs activation and diminished ßarr1-mediated ERK and p38 activation in SHR brainstem astrocytes. These findings might be mechanistically related to the development of the brain renin-angiotensin-aldosterone system hyperactivity, which leads to pathogenesis of the hypertensive state of the SHR model.

A case report: Pharmacology and resistance patterns of three generations of ALK inhibitors in metastatic inflammatory myofibroblastic sarcoma.

BACKGROUND: Little exists currently in research about the mechanisms of resistance of ALK inhibitors in inflammatory myofibroblastic sarcoma. It is known, however, that ALK gene rearrangements are common in inflammatory myofibroblastic tumors, similar to non-small cell lung cancer. In roughly 50% of inflammatory myofibroblastic tumors, gene rearrangement has been found to occur on chromosome 2 at band 2p23. In non-small cell lung cancer, it has been shown that about a third of patients who progress on the first generation ALK inhibitor, crizotinib develops mutations in the ALK kinase domain. The remaining two-thirds of patients tend to develop amplification of ALK or activation of alternative signaling pathways. Chromoplexy has also been described as a mechanism of resistance, where multiple closed chain rearrangements cause loss-of-function of tumor suppressor genes and gain-in-function of oncogenic fusions. Partner genes that have been identified in IMTs are tropomyosin 3 (TPM3), tropomyosin 4 (TPM4), clathrin heavy chain (CLTC), Ran-binding protein 2 (RANBP2), cysteinyl-tRNA synthetase (CARS), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), and SEC31L1. All are active promoters for the fusion gene, in response to NPM binding. Several inflammatory myofibroblastic tumor case reports indicated that fusion of ALK and RANBP2 led to a more aggressive clinical course. Although the majority of inflammatory myofibroblastic tumor case reports have utilized first and second generation ALK inhibitors, all generations of ALK inhibitors have demonstrated some ability to impair disease progression and extend life expectancy. However, at some point in the course of therapy with each generation of ALK inhibitor, resistance ultimately developed. In order to better understand the pharmacology and resistance patterns behind three generations of ALK inhibitors, we sought to examine a patient with metastatic anaplastic lymphoma kinase-1-rearranged inflammatory myofibroblastic sarcoma to the brain. We also explored the similarities and differences of this clinical case to other inflammatory myofibroblastic sarcoma case reports involving the use of ALK inhibitors. CASE REPORT: A rare case of pulmonary IMS with ALK-1 gene rearrangement and multiple brain metastases responded to three generations of ALK inhibitors. However, similar to other case reports, due to the development of resistance and recurrence, the patient eventually succumbed to the disease. CONCLUSIONS: ALK inhibitors are beneficial in the temporary prevention of progression of disease in patients with inflammatory myofibroblastic tumors. In this case, due to the inability to reveal the fusion partner in this patient via DNA sequencing, it is unknown exactly if that partner was RANBP2 or another ALK partner gene. Brain biopsy tissue was also unobtainable during sequence of ALK due to risk versus benefit, which would have provided insight as which type of ALK resistance mutations the patient was developing. It is likely that this patient had some form of chromoplexy occurring.

Deletion of Osteopontin Enhances ß2-Adrenergic Receptor-Dependent Anti-Fibrotic Signaling in Cardiomyocytes.

Cardiac ß2-adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. The ß2AR is a Gs protein-coupled receptor (GPCR) and, upon its activation, stimulates the generation of cyclic 3',5'-adenosine monophosphate (cAMP). cAMP has two effectors: protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac). Epac1 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro-inflammatory cytokine, which also mediates fibrosis in several tissues, including the heart. OPN underlies several cardiovascular pathologies, including atherosclerosis and cardiac adverse remodeling. We found that the cardiotoxic hormone aldosterone transcriptionally upregulates OPN in H9c2 rat cardiac myoblasts-an effect prevented by endogenous ß2AR activation. Additionally, CRISPR-mediated OPN deletion enhanced cAMP generation in response to both ß1AR and ß2AR activation in H9c2 cardiomyocytes, leading to the upregulation of Epac1 protein levels. These effects rendered ß2AR stimulation capable of completely abrogating transforming growth factor (TGF)-ß-dependent fibrosis in OPN-lacking H9c2 cardiomyocytes. Finally, OPN interacted constitutively with Gαs subunits in H9c2 cardiac cells. Thus, we uncovered a direct inhibitory role of OPN in cardiac ß2AR anti-fibrotic signaling via cAMP/Epac1. OPN blockade could be of value in the treatment and/or prevention of cardiac fibrosis.

Novel Insights into the Crosstalk between Mineralocorticoid Receptor and G Protein-Coupled Receptors in Heart Adverse Remodeling and Disease.

The mineralocorticoid hormone aldosterone regulates sodium and potassium homeostasis but also adversely modulates the maladaptive process of cardiac adverse remodeling post-myocardial infarction. Through activation of its mineralocorticoid receptor (MR), a classic steroid hormone receptor/transcription factor, aldosterone promotes inflammation and fibrosis of the heart, the vasculature, and the kidneys. This is why MR antagonists reduce morbidity and mortality of heart disease patients and are part of the mainstay pharmacotherapy of advanced human heart failure. A plethora of animal studies using cell type⁻specific targeting of the MR gene have established the importance of MR signaling and function in cardiac myocytes, vascular endothelial and smooth muscle cells, renal cells, and macrophages. In terms of its signaling properties, the MR is distinct from nuclear receptors in that it has, in reality, two physiological hormonal agonists: not only aldosterone but also cortisol. In fact, in several tissues, including in the myocardium, cortisol is the primary hormone activating the MR. There is a considerable amount of evidence indicating that the effects of the MR in each tissue expressing it depend on tissue- and ligand-specific engagement of molecular co-regulators that either activate or suppress its transcriptional activity. Identification of these co-regulators for every ligand that interacts with the MR in the heart (and in other tissues) is of utmost importance therapeutically, since it can not only help elucidate fully the pathophysiological ramifications of the cardiac MR's actions, but also help design and develop novel better MR antagonist drugs for heart disease therapy. Among the various proteins the MR interacts with are molecules involved in cardiac G protein-coupled receptor (GPCR) signaling. This results in a significant amount of crosstalk between GPCRs and the MR, which can affect the latter's activity dramatically in the heart and in other cardiovascular tissues. This review summarizes the current experimental evidence for this GPCR-MR crosstalk in the heart and discusses its pathophysiological implications for cardiac adverse remodeling as well as for heart disease therapy. Novel findings revealing non-conventional roles of GPCR signaling molecules, specifically of GPCR-kinase (GRK)-5, in cardiac MR regulation are also highlighted.

Biased agonism/antagonism at the AngII-AT1 receptor: Implications for adrenal aldosterone production and cardiovascular therapy.

Many of the effects of angiotensin II (AngII), including adrenocortical aldosterone release, are mediated by the AngII type 1 receptor (AT1R), a receptor with essential roles in cardiovascular homeostasis. AT1R belongs to the G protein-coupled receptor (GPCR) superfamily, mainly coupling to the Gq/11 type of G proteins. However, it also signals through ßarrestins, oftentimes in parallel to eliciting G protein-dependent signaling. This has spurred infinite possibilities for cardiovascular pharmacology, since various beneficial effects are purportedly exerted by AT1R via ßarrestins, unlike AT1R-induced G protein-mediated pathways that usually result in damaging cardiovascular effects, including hypertension and aldosterone elevation. Over the past decade however, a number of studies from our group and others have suggested that AT1R-induced ßarrestin signaling can also be damaging for the heart, similarly to the G protein-dependent one, with regard to aldosterone regulation. Additionally, AT1R-induced ßarrestin signaling in astrocytes from certain areas of the brain may also play a significant role in central regulation of blood pressure and hypertension pathogenesis. These findings have provided the impetus for testing available angiotensin receptor blockers (ARBs) in their efficacy towards blocking both routes (i.e. both G protein- and ßarrestin-dependent) of AT1R signaling in vitro and in vivo and also have promoted structure-activity relationship (SAR) studies for the AngII molecule in terms of ßarrestin signaling to certain cellular effects, e.g. adrenal aldosterone production. In the present review, we will recount all of these recent studies on adrenal and astrocyte AT1R-dependent ßarrestin signaling while underlining their implications for cardiovascular pathophysiology and therapy.