scholarly journals GRK5 is an Essential Co-repressor for Cardiac Mineralocorticoid Receptor Antagonism induced by Finerenone but not Eplerenone

2020 ◽  
Author(s):  
Victoria L. Desimine ◽  
Jennifer Ghandour ◽  
Natalie Cora ◽  
Celina M. Pollard ◽  
Rachel Valiente ◽  
...  
Keyword(s):  
Mineralocorticoid Receptor ◽  
Transcriptional Activity ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Human Heart Failure ◽  
Cardiac Mr ◽  
Inverse Agonism ◽  
Mineralocorticoid Receptor Antagonism ◽  
H9c2 Cardiomyocytes ◽  
Induced Apoptosis

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 (GPCR)-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. Herein, we sought to investigate potential differences between finerenone and eplerenone at engaging GRK5-dependent cardiac MR phosphorylation and subsequent blockade. Methods: We used the cardiomyocyte cell line H9c2 and neonatal rat ventricular myocytes (NRVMs). 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 in NRVMs, GRK5 is necessary for the anti-apoptotic 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 and fibrosis. Conclusions: 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.

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

2020 ◽  
Vol 21 (8) ◽  
pp. 2868
Author(s):  
Jennifer Maning ◽  
Katie A. McCrink ◽  
Celina M. Pollard ◽  
Victoria L. Desimine ◽  
Jennifer Ghandour ◽  
...  
Keyword(s):  
G Protein ◽  
Mineralocorticoid Receptor ◽  
Transcriptional Activity ◽  
Ventricular Myocytes ◽  
Cardiac Injury ◽  
Protective Effects ◽  
Cardiac Mr ◽  
Beneficial Effects ◽  
Adult Rat ◽  
G Protein Coupled

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 β2-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. β2AR-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 β2AR activation) might be of therapeutic value for heart disease treatment via boosting the efficacy of MR antagonists against Aldo-mediated cardiac injury.

scholarly journals The Ku Protein Complex Interacts with YY1, Is Up-Regulated in Human Heart Failure, and Represses α Myosin Heavy-Chain Gene Expression

2004 ◽  
Vol 24 (19) ◽  
pp. 8705-8715 ◽  
Author(s):  
Carmen C. Sucharov ◽  
Steve M. Helmke ◽  
Stephen J. Langer ◽  
M. Benjamin Perryman ◽  
Michael Bristow ◽  
...  
Keyword(s):  
Heart Failure ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Human Heart ◽  
Ventricular Myocytes ◽  
Major Effect ◽  
Neonatal Rat ◽  
Chain Gene ◽  
Human Heart Failure ◽  
Peptide Mass

ABSTRACT Human heart failure is accompanied by repression of genes such as α myosin heavy chain (αMyHC) and SERCA2A and the induction of fetal genes such as βMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human αMyHC promoter. We have now identified a region of the αMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human αMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases αMyHC mRNA expression and increases skeletal α-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the αMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human αMyHC promoter during heart failure.

Abstract 631 ADAM12 Is An Important Regulator Of Myocyte Apoptosis Induced By β-adrenergic Receptor Stimulation

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hang Xi ◽  
Khadija Rafiq ◽  
Marie Hanscom ◽  
Rachid Seqqat ◽  
Nikolay L Malinin ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Left Ventricular ◽  
Surface Proteins ◽  
Neonatal Rat ◽  
Human Heart Failure ◽  
Activation Mechanisms ◽  
Myocyte Apoptosis ◽  
Protease Deficient

ADAM (A Disintegrin And Metalloprotease)12 is a member of a family of cell surface proteins with protease and cell-binding activities. Recent work showed ADAM12 up-regulation in human heart failure. However, the activation mechanisms of ADAM12 in the heart are obscure. We hypothesized that β-adrenergic receptors (AR) stimulation regulates ADAM12 activation in neonatal rat ventricular myocytes (NRVMs) in-vitro and after injection of isoproterenol (ISO) in-vivo. Wistar rats received a single injection of ISO (5 mg/kg) and were sacrificed 6, 24 and 72 hrs later. In comparison with controls, left ventricular function was impaired in rats 24 hrs after ISO injection and started to improve at 72 hrs. The fraction of myocytes undergoing apoptosis peaked 24 hrs after ISO injection and declined thereafter. ADAM12 protein was reduced in hearts from ISO treated animals at 6 hrs, pointing to a possible increase in ADAM12 proteolytic activity. However, both ADAM12 expression and activation were significantly up-regulated at 24 and 72 hrs after ISO injection. We therefore assessed whether ADAM12 activation was involved in myocyte apoptosis secondary to excess exposure of catecholamine. Acute stimulation with ISO (10 μM, 30 min to 3 hrs) induced accumulation of ADAM12 N-terminal cleavage product in conditioned medium, demonstrating activation of the ADAM metalloprotease activity. However, chronic stimulation with ISO for 24 hrs and 48 hrs significantly increased both ADAM12 expression and secretion. This ISO-induced ADAM12 expression/activation was mediated through β 1 -AR stimulation and was dependent on intracellular calcium elevation and protein kinase C activation. Adenoviral expression of an ADAM12 protease-deficient mutant (ADAM12DeltaMP) blocks β-AR-induced myocyte apoptosis, while transduction of NRVMs with adenovirus harboring ADAM12 significantly increased myocyte apoptosis. These data suggest that ADAM12 is a regulator of myocyte apoptosis induced by β-AR in NRVMs and may play an important autocrine role in mediating the effects of β-AR on myocardial remodeling.

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

2018 ◽  
Author(s):  
Barbara M. Parker ◽  
Shelby L. Wertz ◽  
Celina M. Pollard ◽  
Victoria L. Desimine ◽  
Jennifer Maning ◽  
...  
Keyword(s):  
Heart Disease ◽  
G Protein ◽  
Mineralocorticoid Receptor ◽  
Animal Studies ◽  
Human Heart Failure ◽  
Gpcr Signaling ◽  
Cardiac Mr ◽  
Disease Therapy ◽  
Adverse Remodeling ◽  
G Protein Coupled

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.

H2 and H3 relaxin inhibit high glucose-induced apoptosis in neonatal rat ventricular myocytes

Biochimie ◽  
2015 ◽  
Vol 108 ◽  
pp. 59-67 ◽  
Author(s):  
Xiaohui Zhang ◽  
Xiao Ma ◽  
Meng Zhao ◽  
Bo Zhang ◽  
Jinyu Chi ◽  
...  
Keyword(s):  
High Glucose ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Rat Ventricular Myocytes ◽  
Induced Apoptosis ◽  
Neonatal Rat Ventricular Myocytes

Morphine mimics the antiapoptotic effect of preconditioning via an Ins(1,4,5)P3 signaling pathway in rat ventricular myocytes

2005 ◽  
Vol 288 (1) ◽  
pp. H83-H88 ◽  
Author(s):  
Stéphanie Barrère-Lemaire ◽  
Nicolas Combes ◽  
Catherine Sportouch-Dukhan ◽  
Sylvain Richard ◽  
Joël Nargeot ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Signaling Pathway ◽  
Dna Fragmentation ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Cytochrome C Release ◽  
Rat Cardiomyocytes ◽  
Rat Ventricular Myocytes ◽  
Antiapoptotic Effect ◽  
Induced Apoptosis

Morphine has cardioprotective effects against ischemic-reperfusion injuries. This study investigates whether morphine could mimic the antiapoptotic effect of preconditioning using a model of cultured neonatal rat cardiomyocytes subjected to metabolic inhibition (MI). To quantify MI-induced apoptosis, DNA fragmentation and mitochondrial cytochrome c release levels were measured by ELISA. MI-dependent DNA fragmentation was prevented by both Z-VAD-fmk (20 μM), a pan-caspase inhibitor, and cyclosporine A (CsA; 5 μM), a mitochondrial pore transition blocker, added during MI (36% and 54% decrease, respectively). MI-dependent cytochrome c release was not blocked by Z-VAD-fmk but was decreased (38%) by CsA during MI. Metabolic preconditioning (MIP) and preconditioning with morphine (1 μM) were also assessed. MI-dependent DNA fragmentation and cytochrome c release were prevented by MIP (40% and 45% decrease, respectively) and morphine (34% and 45%, respectively). The antiapoptotic effect of morphine was abolished by naloxone (10 nM), a nonselective opioid receptor antagonist, or xestospongin C (XeC, 400 nM), an inhibitor of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+ release. Ca2+ preconditioning, induced by increasing extracellular Ca2+ from 1.8 to 3.3 mM, mimicked the antiapoptotic effect of morphine on DNA fragmentation (24% decrease) and cytochrome c release (57% decrease). This effect mediated by extracellular Ca2+ was also abolished by XeC. Measurements of intracellular Ca2+ concentration using fura-2 microspectrofluorimetry showed that morphine induces Ins(1,4,5)P3-dependent Ca2+ transients abolished by 2-aminoethoxydiphenyl borate (2-APB), a cell-permeable Ins(1,4,5)P3 antagonist. These results suggest that morphine preconditioning prevents simulated ischemia-reperfusion-induced apoptosis via an Ins(1,4,5)P3 signaling pathway in rat ventricular myocytes.

CARP Plays a Key Role in Cellular Growth Under Hypertrophic Stimuli in Neonatal Rat Ventricular Myocytes

2013 ◽  
Vol 9 ◽  
Author(s):  
Tara A Shrout
Keyword(s):  
Gene Expression ◽  
Ventricular Myocytes ◽  
Cellular Growth ◽  
Neonatal Rat ◽  
Transcriptional Level ◽  
Dual Nature ◽  
Hypertrophic Growth ◽  
Rat Ventricular Myocytes ◽  
Neonatal Rat Ventricular Myocytes ◽  
Over Time

Cardiac hypertrophy is a growth process that occurs in response to stress stimuli or injury, and leads to the induction of several pathways to alter gene expression. Under hypertrophic stimuli, sarcomeric structure is disrupted, both as a consequence of gene expression and local changes in sarcomeric proteins. Cardiac-restricted ankyrin repeat protein (CARP) is one such protein that function both in cardiac sarcomeres and at the transcriptional level. We postulate that due to this dual nature, CARP plays a key role in maintaining the cardiac sarcomere. GATA4 is another protein detected in cardiomyocytes as important in hypertrophy, as it is activated by hypertrophic stimuli, and directly binds to DNA to alter gene expression. Results of GATA4 activation over time were inconclusive; however, the role of CARP in mediating hypertrophic growth in cardiomyocytes was clearly demonstrated. In this study, Neonatal Rat Ventricular Myocytes were used as a model to detect changes over time in CARP and GATA4 under hypertrophic stimulation by phenylephrine and high serum media. Results were detected by analysis of immunoblotting. The specific role that CARP plays in mediating cellular growth under hypertrophic stimuli was studied through immunofluorescence, which demonstrated that cardiomyocyte growth with hypertrophic stimulation was significantly blunted when NRVMs were co-treated with CARP siRNA. These data suggest that CARP plays an important role in the hypertrophic response in cardiomyocytes.

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