RESUMEN
Maternal hypothyroidism (MH) could adversely affect the cardiac disease responses of the progeny. This study tested the hypothesis that MH reduces early postnatal cardiomyocyte (CM) proliferation so that the adult heart of MH progeny has a smaller number of larger cardiac myocytes, which imparts adverse cardiac disease responses following injury. Thyroidectomy (TX) was used to establish MH. The progeny from mice that underwent sham or TX surgery were termed Ctrl (control) or MH (maternal hypothyroidism) progeny, respectively. MH progeny had similar heart weight (HW) to body weight (BW) ratios and larger CM size consistent with fewer CMs at postnatal day 60 (P60) compared with Ctrl (control) progeny. MH progeny had lower numbers of EdU+, Ki67+, and phosphorylated histone H3 (PH3)+ CMs, which suggests they had a decreased CM proliferation in the postnatal timeframe. RNA-seq data showed that genes related to DNA replication were downregulated in P5 MH hearts, including bone morphogenetic protein 10 (Bmp10). Both in vivo and in vitro studies showed Bmp10 treatment increased CM proliferation. After transverse aortic constriction (TAC), the MH progeny had more severe cardiac pathological remodeling compared with the Ctrl progeny. Thyroid hormone (T4) treatment for MH mothers preserved their progeny's postnatal CM proliferation capacity and prevented excessive pathological remodeling after TAC. Our results suggest that CM proliferation during early postnatal development was significantly reduced in MH progeny, resulting in fewer CMs with hypertrophy in adulthood. These changes were associated with more severe cardiac disease responses after pressure overload.NEW & NOTEWORTHY Our study shows that compared with Ctrl (control) progeny, the adult progeny of mothers who have MH (MH progeny) had fewer CMs. This reduction of CM numbers was associated with decreased postnatal CM proliferation. Gene expression studies showed a reduced expression of Bmp10 in MH progeny. Bmp10 has been linked to myocyte proliferation. In vivo and in vitro studies showed that Bmp10 treatment of MH progeny and their myocytes could increase CM proliferation. Differences in CM number and size in adult hearts of MH progeny were linked to more severe cardiac structural and functional remodeling after pressure overload. T4 (synthetic thyroxine) treatment of MH mothers during their pregnancy, prevented the reduction in CM number in their progeny and the adverse response to disease stress.
Asunto(s)
Cardiopatías , Hipotiroidismo , Embarazo , Femenino , Ratones , Animales , Miocitos Cardíacos/metabolismo , Cardiopatías/patología , Hipertrofia/metabolismo , Hipertrofia/patología , Hipotiroidismo/complicaciones , Hipotiroidismo/metabolismo , Hipotiroidismo/patología , Proteínas Morfogenéticas Óseas/metabolismo , Proliferación Celular , Cardiomegalia/metabolismoRESUMEN
Myeloid cells, including macrophages, play important roles as first responders to cardiac injury and stress. Epidermal growth factor receptor (EGFR) has been identified as a mediator of macrophage responsiveness to select diseases, though its impact on cardiac function or remodeling following acute ischemic injury is unknown. We aimed to define the role of myeloid cell-specific EGFR in the regulation of cardiac function and remodeling following acute myocardial infarction (MI)-induced injury. Floxed EGFR mice were bred with homozygous LysM-Cre (LMC) transgenic mice to yield myeloid-specific EGFR knockout (mKO) mice. Via echocardiography, immunohistochemistry, RNA sequencing and flow cytometry, the impact of myeloid cell-specific EGFR deletion on cardiac structure and function was assessed at baseline and following injury. Compared with LMC controls, myeloid cell-specific EGFR deletion led to an increase in cardiomyocyte hypertrophy at baseline. Bulk RNASeq analysis of isolated cardiac Cd11b+ myeloid cells revealed substantial changes in mKO cell transcripts at baseline, particularly in relation to predicted decreases in neovascularization. In response to myocardial infarction, mKO mice experienced a hastened decline in cardiac function with isolated cardiac Cd11b+ myeloid cells expressing decreased levels of the pro-reparative mediators Vegfa and Il10, which coincided with enhanced cardiac hypertrophy and decreased capillary density. Overall, loss of EGFR qualitatively alters cardiac resident macrophages that promotes a low level of basal stress and a more rapid decrease in cardiac function along with worsened repair following acute ischemic injury.
Asunto(s)
Receptores ErbB , Infarto del Miocardio , Ratones , Animales , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células Mieloides/metabolismo , Macrófagos/metabolismo , Corazón , Infarto del Miocardio/metabolismo , Ratones Transgénicos , Ratones Noqueados , Ratones Endogámicos C57BL , Remodelación Ventricular/genéticaRESUMEN
PURPOSE: ß-Adrenergic receptors (ßAR) are essential targets for the treatment of heart failure (HF); however, chronic use of ßAR agonists as positive inotropes to increase contractility in a Gs protein-dependent manner is associated with increased mortality. Alternatively, we previously reported that allosteric modulation of ß2AR with the pepducin intracellular loop (ICL)1-9 increased cardiomyocyte contractility in a ß-arrestin (ßarr)-dependent manner, and subsequently showed that ICL1-9 activates the Ras homolog family member A (RhoA). Here, we aimed to elucidate both the proximal and downstream signaling mediators involved in the promotion of cardiomyocyte contractility in response to ICL1-9. METHODS: We measured adult mouse cardiomyocyte contractility in response to ICL1-9 or isoproterenol (ISO, as a positive control) alone or in the presence of inhibitors of various potential components of ßarr- or RhoA-dependent signaling. We also assessed the contractile effects of ICL1-9 on cardiomyocytes lacking G protein-coupled receptor (GPCR) kinase 2 (GRK2) or 5 (GRK5). RESULTS: Consistent with RhoA activation by ICL1-9, both Rho-associated protein kinase (ROCK) and protein kinase D (PKD) inhibition were able to attenuate ICL1-9-mediated contractility, as was inhibition of myosin light chain kinase (MLCK). While neither GRK2 nor GRK5 deletion impacted ICL1-9-mediated contractility, pertussis toxin attenuated the response, suggesting that ICL1-9 promotes downstream RhoA-dependent signaling in a Gi protein-dependent manner. CONCLUSION: Altogether, our study highlights a novel signaling modality that may offer a new approach to the promotion, or preservation, of cardiac contractility during HF via the allosteric regulation of ß2AR to promote Gi protein/ßarr-dependent activation of RhoA/ROCK/PKD signaling.
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Insuficiencia Cardíaca , Miocitos Cardíacos , Ratones , Animales , Transducción de Señal , Proteína Quinasa C/metabolismo , Proteína Quinasa C/farmacología , Insuficiencia Cardíaca/metabolismo , Contracción MiocárdicaRESUMEN
G protein-coupled receptors (GPCRs) represent one of the most targeted drug classes in the human genome, accounting for greater than 40% of all Food and Drug Administration-approved drugs. However, the second-largest family of GPCRs, known as adhesion GPCRs (aGPCR), have yet to serve as a clinical target despite increasing evidence of their physiological and pathological functions, which suggests an opportunity toward the development of novel therapeutics. To date, the pathophysiological function of aGPCRs is associated with a plethora of diseases including cancer, central nervous system disorders, immunity and inflammation, and others. To highlight their potential as pharmacological targets, we will review three distinct aGPCR members (ADGRG1, ADGRE5, and ADGRF5), highlighting their molecular mechanisms of action and contributions to the development of pathophysiology.
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Neoplasias , Receptores Acoplados a Proteínas G , Sistemas de Liberación de Medicamentos , Humanos , Inflamación/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
ABSTRACT: Pepducins are small-lipidated peptides designed from the intracellular loops of G protein-coupled receptors (GPCRs) that act in an allosteric manner to modulate the activity of GPCRs. Over the past 2 decades, pepducins have progressed initially from pharmacologic tools used to manipulate GPCR activity in an orthosteric site-independent manner to compounds with therapeutic potential that have even been used safely in phase 1 and 2 clinical trials in human subjects. The effect of pepducins at their cognate receptors has been shown to vary between antagonist, partial agonist, and biased agonist outcomes in various primary and clonal cell systems, with even small changes in amino acid sequence altering these properties and their receptor selectivity. To date, pepducins designed from numerous GPCRs have been studied for their impact on pathologic conditions, including cardiovascular diseases such as thrombosis, myocardial infarction, and atherosclerosis. This review will focus in particular on pepducins designed from protease-activated receptors, C-X-C motif chemokine receptors, formyl peptide receptors, and the ß2-adrenergic receptor. We will discuss the historic context of pepducin development for each receptor, as well as the structural, signaling, pathophysiologic consequences, and therapeutic potential for each pepducin class.
Asunto(s)
Sistema Cardiovascular , Receptores Acoplados a Proteínas G , Secuencia de Aminoácidos , Sistema Cardiovascular/metabolismo , Humanos , Péptidos/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Cardiac rupture is a major lethal complication of acute myocardial infarction (MI). Despite significant advances in reperfusion strategies, mortality from cardiac rupture remains high. Studies suggest that cardiac rupture can be accelerated by thrombolytic therapy, but the relevance of this risk factor remains controversial. METHODS: We analyzed protease-activated receptor 4 (Par4) expression in mouse hearts with MI and investigated the effects of Par4 deletion on cardiac remodeling and function after MI by echocardiography, quantitative immunohistochemistry, and flow cytometry. RESULTS: Par4 mRNA and protein levels were increased in mouse hearts after MI and in isolated cardiomyocytes in response to hypertrophic and inflammatory stimuli. Par4-deficient mice showed less myocyte apoptosis, reduced infarct size, and improved functional recovery after acute MI relative to wild-type (WT). Conversely, Par4-/- mice showed impaired cardiac function, greater rates of myocardial rupture, and increased mortality after chronic MI relative to WT. Pathological evaluation of hearts from Par4-/- mice demonstrated a greater infarct expansion, increased cardiac hemorrhage, and delayed neutrophil accumulation, which resulted in impaired post-MI healing compared with WT. Par4 deficiency also attenuated neutrophil apoptosis in vitro and after MI in vivo and impaired inflammation resolution in infarcted myocardium. Transfer of Par4-/- neutrophils, but not of Par4-/- platelets, in WT recipient mice delayed inflammation resolution, increased cardiac hemorrhage, and enhanced cardiac dysfunction. In parallel, adoptive transfer of WT neutrophils into Par4-/- mice restored inflammation resolution, reduced cardiac rupture incidence, and improved cardiac function after MI. CONCLUSIONS: These findings reveal essential roles of Par4 in neutrophil apoptosis and inflammation resolution during myocardial healing and point to Par4 inhibition as a potential therapy that should be limited to the acute phases of ischemic insult and avoided for long-term treatment after MI.
Asunto(s)
Regulación de la Expresión Génica , Rotura Cardíaca , Infarto del Miocardio , Miocardio/metabolismo , Receptores de Trombina/deficiencia , Animales , Femenino , Rotura Cardíaca/etiología , Rotura Cardíaca/genética , Rotura Cardíaca/metabolismo , Rotura Cardíaca/prevención & control , Inflamación/genética , Inflamación/metabolismo , Inflamación/prevención & control , Masculino , Ratones , Ratones Noqueados , Infarto del Miocardio/clasificación , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/prevención & control , Receptores de Trombina/biosíntesisRESUMEN
Nitric oxide (NO) and S-nitrosothiol (SNO) are considered cardio- and vasoprotective substances. We now understand that one mechanism in which NO/SNOs provide cardiovascular protection is through their direct inhibition of cardiac G protein-coupled receptor (GPCR) kinase 2 (GRK2) activity via S-nitrosylation of GRK2 at cysteine 340 (C340). This maintains GPCR homeostasis, including ß-adrenergic receptors, through curbing receptor GRK2-mediated desensitization. Previously, we have developed a knockin mouse (GRK2-C340S) where endogenous GRK2 is resistant to dynamic S-nitrosylation, which led to increased GRK2 desensitizing activity. This unchecked regulation of cardiac GRK2 activity resulted in significantly more myocardial damage after ischemic injury that was resistant to NO-mediated cardioprotection. Although young adult GRK2-C340S mice show no overt phenotype, we now report that as these mice age, they develop significant cardiovascular dysfunction due to the loss of SNO-mediated GRK2 regulation. This pathological phenotype is apparent as early as 12 mo of age and includes reduced cardiac function, increased cardiac perivascular fibrosis, and maladaptive cardiac hypertrophy, which are common maladies found in patients with cardiovascular disease (CVD). There are also vascular reactivity and aortic abnormalities present in these mice. Therefore, our data demonstrate that a chronic and global increase in GRK2 activity is sufficient to cause cardiovascular remodeling and dysfunction, likely due to GRK2's desensitizing effects in several tissues. Because GRK2 levels have been reported to be elevated in elderly CVD patients, GRK2-C340 mice can give insight into the aged-molecular landscape leading to CVD.NEW & NOTEWORTHY Research on G protein-coupled receptor kinase 2 (GRK2) in the setting of cardiovascular aging is largely unknown despite its strong established functions in cardiovascular physiology and pathophysiology. This study uses a mouse model of chronic GRK2 overactivity to further investigate the consequences of long-term GRK2 on cardiac function and structure. We report for the first time that chronic GRK2 overactivity was able to cause cardiac dysfunction and remodeling independent of surgical intervention, highlighting the importance of GRK activity in aged-related heart disease.
Asunto(s)
Envejecimiento/fisiología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Cardiopatías/etiología , Corazón/fisiología , Miocardio/metabolismo , Óxido Nítrico/metabolismo , Envejecimiento/metabolismo , Animales , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Corazón/crecimiento & desarrollo , Corazón/fisiopatología , Cardiopatías/metabolismo , Homeostasis , Masculino , Ratones , MutaciónRESUMEN
Chronic pressure-overload (PO)- induced cardiomyopathy is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of the α isoform of glycogen synthase kinase-3 (GSK-3α) in PO-induced cardiac remodeling is unclear and its downstream molecular targets are largely unknown. To investigate the potential roles of GSK-3α, cardiomyocyte-specific GSK-3α conditional knockout (cKO) and control mice underwent trans-aortic constriction (TAC) or sham surgeries. Cardiac function in the cKOs and littermate controls declined equally up to 2â¯weeks of TAC. At 4â¯week, cKO animals retained concentric LV remodeling and showed significantly less decline in contractile function both at systole and diastole, vs. controls which remained same until the end of the study (6â¯wk). Histological analysis confirmed preservation of LV chamber and protection against TAC-induced cellular hypertrophy in the cKO. Consistent with attenuated hypertrophy, significantly lower level of cardiomyocyte apoptosis was observed in the cKO. Mechanistically, GSK-3α was found to regulate mitochondrial permeability transition pore (mPTP) opening and GSK-3α-deficient mitochondria showed delayed mPTP opening in response to Ca2+ overload. Consistently, overexpression of GSK-3α in cardiomyocytes resulted in elevated Bax expression, increased apoptosis, as well as a reduction of maximum respiration capacity and cell viability. Taken together, we show for the first time that GSK-3α regulates mPTP opening under pathological conditions, likely through Bax overexpression. Genetic ablation of cardiomyocyte GSK-3α protects against chronic PO-induced cardiomyopathy and adverse LV remodeling, and preserves contractile function. Selective inhibition of GSK-3α using isoform-specific inhibitors could be a viable therapeutic strategy to limit PO-induced heart failure.
Asunto(s)
Apoptosis , Cardiomegalia/enzimología , Glucógeno Sintasa Quinasa 3/metabolismo , Insuficiencia Cardíaca/enzimología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Miocitos Cardíacos/enzimología , Animales , Cardiomegalia/genética , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Glucógeno Sintasa Quinasa 3/genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Ratones , Ratones Noqueados , Proteínas de Transporte de Membrana Mitocondrial/genética , Poro de Transición de la Permeabilidad Mitocondrial , Contracción Miocárdica/genética , Miocitos Cardíacos/patología , Remodelación Ventricular/genéticaRESUMEN
Following cardiac injury, early immune cell responses are essential for initiating cardiac remodeling and tissue repair. We previously demonstrated the importance of ß2-adrenergic receptors (ß2ARs) in the regulation of immune cell localization following acute cardiac injury, with deficient leukocyte infiltration into the damaged heart. The purpose of this study was to investigate the mechanism by which immune cell-expressed ß2ARs regulate leukocyte recruitment to the heart following acute cardiac injury. Chemokine receptor 2 (CCR2) expression and responsiveness to C-C motif chemokine ligand 2 (CCL2)-mediated migration were abolished in ß2AR knockout (KO) bone marrow (BM), both of which were rescued by ß2AR reexpression. Chimeric mice lacking immune cell-specific CCR2 expression, as well as wild-type mice administered a CCR2 antagonist, recapitulated the loss of monocyte/macrophage and neutrophil recruitment to the heart following myocardial infarction (MI) observed in mice with immune cell-specific ß2AR deletion. Converse to ß2AR ablation, ß2AR stimulation increased CCR2 expression and migratory responsiveness to CCL2 in BM. Mechanistically, G protein-dependent ß2AR signaling was dispensable for these effects, whereas ß-arrestin2-biased ß2AR signaling was required for the regulation of CCR2 expression. Additionally, activator protein 1 (AP-1) was shown to be essential in mediating CCR2 expression in response to ß2AR stimulation in both murine BM and human monocytes. Finally, reconstitution of ß2ARKO BM with rescued expression of a ß-arrestin-biased ß2AR in vivo restored BM CCR2 expression as well as cardiac leukocyte infiltration following MI. These results demonstrate the critical role of ß-arrestin2/AP-1-dependent ß2AR signaling in the regulation of CCR2 expression and recruitment of leukocytes to the heart following injury.
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Leucocitos/citología , Miocardio/patología , Receptores Adrenérgicos beta/metabolismo , Receptores CCR2/metabolismo , Animales , Trasplante de Médula Ósea , Movimiento Celular , Quimiocinas/metabolismo , Vasos Coronarios/patología , Ecocardiografía , Insuficiencia Cardíaca/patología , Humanos , Antígenos Comunes de Leucocito/metabolismo , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/citología , Infarto del Miocardio/patología , Isquemia Miocárdica/patología , Miocardio/metabolismo , Infiltración Neutrófila , Transducción de SeñalRESUMEN
ß-adrenergic receptors (ßARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent ß1AR and Gi-dependent ß2AR pathways leads to enhanced cardiomyocyte death, reduced ß1AR expression, and decreased inotropic reserve. ß-blockers act to block excessive catecholamine stimulation of ßARs to decrease cellular apoptotic signaling and normalize ß1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, ß-arrestin-dependent signaling promotes cardiomyocyte survival. Given that ß2AR expression is unaltered in CHF, a ß-arrestin-biased agonist that operates through the ß2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective ß-blocker, has been classified as a ß-arrestin-biased agonist that can inhibit basal signaling from ßARs and also stimulate cell survival signaling pathways. To understand the relative contribution of ß-arrestin bias to the efficacy of select ß-blockers, a specific ß-arrestin-biased pepducin for the ß2AR, intracellular loop (ICL)1-9, was used to decouple ß-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote ß2AR phosphorylation, ß-arrestin recruitment, ß2AR internalization, and ß-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce ß2AR- and ß-arrestin-dependent and Ca(2+)-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the ß2AR and serves as a model for the next generation of cardiovascular drug development.
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Antagonistas Adrenérgicos beta/farmacología , Carbazoles/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Lipopéptidos/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Propanolaminas/farmacología , Antagonistas Adrenérgicos beta/uso terapéutico , Animales , Carbazoles/uso terapéutico , Carvedilol , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Lipopéptidos/uso terapéutico , Ratones , Cultivo Primario de Células , Propanolaminas/uso terapéutico , Conformación Proteica/efectos de los fármacos , beta-Arrestinas/agonistasRESUMEN
Increased G protein-coupled receptor kinase (GRK)2 is central to heart failure (HF) pathogenesis, via desensitization of ß-adrenergic receptors and loss of contractile reserve. Since GRK2 has been shown to compromise fatty acid (FA) oxidation, this kinase may link metabolic and contractile defects in HF. The aim of this study was to investigate the mechanistic role of GRK2 in FA metabolism and bioenergetics in the heart. For that purpose, we measured FA uptake and cluster of differentiation (CD)36 expression, phosphorylation, and ubiquitination in mice with cardiac-specific overexpression of GRK2 (TgGRK2) or expression of its c-terminus (GRK2 inhibitor- TgßARKct) or in global heterozygous GRK2 knockout (GRK2+/-) mice. Cellular bioenergetics were also measured in isolated cardiomyocytes following adenoviral delivery of exogenous GRK2, ßARKct, or short hairpin GRK2 (shGRK2). Additionally, CD36 expression and phosphorylation were evaluated following transverse aortic constriction (TAC) in wild type (WT) and GRK2+/- mice. Our results show a 33%⯱â¯0.81 reduction in FA uptake rate, accompanied by 51%⯱â¯0.17 lower CD36 protein, and 70%⯱â¯0.23 and 69%⯱â¯0.18 increases in CD36 phosphorylation and ubiquitination, respectively, in the TgGRK2 mice. Moreover, an in vitro kinase assay suggests that GRK2 directly phosphorylates CD36. In isolated cardiomyocytes, GRK2 overexpression induced a 26%⯱â¯2.21 decrease in maximal respiration, which was enhanced (20%⯱â¯4.02-5.14) with inhibition of the kinase. Importantly, in hearts with systolic dysfunction, notable reductions in CD36 mRNA and protein, as well as a significant increase in CD36 phosphorylation were normalized in the GRK2+/- mice post-TAC. Thus, we propose that GRK2 up-regulation in HF is, at least partly, responsible for reduced FA uptake and oxidation and may be a nodal link between metabolic and contractile defects.
Asunto(s)
Ácidos Grasos/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Insuficiencia Cardíaca/metabolismo , Metabolismo de los Lípidos , Animales , Biomarcadores , Antígenos CD36/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/fisiopatología , Ratones , Ratones Noqueados , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , FosforilaciónRESUMEN
BACKGROUND: Activated fibroblasts (myofibroblasts) play a critical role in cardiac fibrosis; however, their origin in the diseased heart remains unclear, warranting further investigation. Recent studies suggest the contribution of bone marrow fibroblast progenitor cells (BM-FPCs) in pressure overload-induced cardiac fibrosis. We have previously shown that interleukin-10 (IL10) suppresses pressure overload-induced cardiac fibrosis; however, the role of IL10 in inhibition of BM-FPC-mediated cardiac fibrosis is not known. We hypothesized that IL10 inhibits pressure overload-induced homing of BM-FPCs to the heart and their transdifferentiation to myofibroblasts and thus attenuates cardiac fibrosis. METHODS: Pressure overload was induced in wild-type (WT) and IL10 knockout (IL10KO) mice by transverse aortic constriction. To determine the bone marrow origin, chimeric mice were created with enhanced green fluorescent protein WT mice marrow to the IL10KO mice. For mechanistic studies, FPCs were isolated from mouse bone marrow. RESULTS: Pressure overload enhanced BM-FPC mobilization and homing in IL10KO mice compared with WT mice. Furthermore, WT bone marrow (from enhanced green fluorescent protein mice) transplantation in bone marrow-depleted IL10KO mice (IL10KO chimeric mice) reduced transverse aortic constriction-induced BM-FPC mobilization compared with IL10KO mice. Green fluorescent protein costaining with α-smooth muscle actin or collagen 1α in left ventricular tissue sections of IL10KO chimeric mice suggests that myofibroblasts were derived from bone marrow after transverse aortic constriction. Finally, WT bone marrow transplantation in IL10KO mice inhibited transverse aortic constriction-induced cardiac fibrosis and improved heart function. At the molecular level, IL10 treatment significantly inhibited transforming growth factor-ß-induced transdifferentiation and fibrotic signaling in WT BM-FPCs in vitro. Furthermore, fibrosis-associated microRNA (miRNA) expression was highly upregulated in IL10KO-FPCs compared with WT-FPCs. Polymerase chain reaction-based selective miRNA analysis revealed that transforming growth factor-ß-induced enhanced expression of fibrosis-associated miRNAs (miRNA-21, -145, and -208) was significantly inhibited by IL10. Restoration of miRNA-21 levels suppressed the IL10 effects on transforming growth factor-ß-induced fibrotic signaling in BM-FPCs. CONCLUSIONS: Our findings suggest that IL10 inhibits BM-FPC homing and transdifferentiation to myofibroblasts in pressure-overloaded myocardium. Mechanistically, we show for the first time that IL10 suppresses Smad-miRNA-21-mediated activation of BM-FPCs and thus modulates cardiac fibrosis.
Asunto(s)
Ecocardiografía/métodos , Fibroblastos/metabolismo , Fibrosis/metabolismo , Cardiopatías/complicaciones , Interleucina-10/genética , Interleucina-10/metabolismo , Miocardio/metabolismo , Animales , Médula Ósea , Femenino , Fibroblastos/patología , Humanos , Ratones , Ratones Transgénicos , Miocardio/patología , Transducción de SeñalRESUMEN
GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a ß2-adrenergic receptor (ß2AR) agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as ß2AR-induced hypertrophy.
Asunto(s)
Clenbuterol/efectos adversos , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/enzimología , Enfermedades Musculares/enzimología , Transducción de Señal/efectos de los fármacos , Animales , Clenbuterol/farmacocinética , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Hipertrofia/inducido químicamente , Hipertrofia/enzimología , Hipertrofia/genética , Hipertrofia/patología , Ratones , Ratones Noqueados , Contracción Muscular/genética , Músculo Esquelético/patología , Enfermedades Musculares/inducido químicamente , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Immune cell-mediated inflammation is an essential process for mounting a repair response after myocardial infarction (MI). The sympathetic nervous system is known to regulate immune system function through ß-adrenergic receptors (ßARs); however, their role in regulating immune cell responses to acute cardiac injury is unknown. METHODS: Wild-type (WT) mice were irradiated followed by isoform-specific ßAR knockout (ßARKO) or WT bone-marrow transplantation (BMT) and after full reconstitution underwent MI surgery. Survival was monitored over time, and alterations in immune cell infiltration after MI were examined through immunohistochemistry. Alterations in splenic function were identified through the investigation of altered adhesion receptor expression. RESULTS: ß2ARKO BMT mice displayed 100% mortality resulting from cardiac rupture within 12 days after MI compared with ≈20% mortality in WT BMT mice. ß2ARKO BMT mice displayed severely reduced post-MI cardiac infiltration of leukocytes with reciprocally enhanced splenic retention of the same immune cell populations. Splenic retention of the leukocytes was associated with an increase in vascular cell adhesion molecule-1 expression, which itself was regulated via ß-arrestin-dependent ß2AR signaling. Furthermore, vascular cell adhesion molecule-1 expression in both mouse and human macrophages was sensitive to ß2AR activity, and spleens from human tissue donors treated with ß-blocker showed enhanced vascular cell adhesion molecule-1 expression. The impairments in splenic retention and cardiac infiltration of leukocytes after MI were restored to WT levels via lentiviral-mediated re-expression of ß2AR in ß2ARKO bone marrow before transplantation, which also resulted in post-MI survival rates comparable to those in WT BMT mice. CONCLUSIONS: Immune cell-expressed ß2AR plays an essential role in regulating the early inflammatory repair response to acute myocardial injury by facilitating cardiac leukocyte infiltration.
Asunto(s)
Rotura Cardíaca/etiología , Leucocitos/metabolismo , Infarto del Miocardio/complicaciones , Receptores Adrenérgicos beta 2/fisiología , Anciano , Anciano de 80 o más Años , Animales , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/uso terapéutico , Humanos , Macrófagos/metabolismo , Masculino , Metoprolol/farmacología , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila , Quimera por Radiación , Receptores Adrenérgicos beta 2/deficiencia , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes de Fusión/metabolismo , Bazo/metabolismo , Bazo/patología , Esplenectomía , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Early reperfusion of ischemic cardiac tissue increases inflammatory cell infiltration which contributes to cardiomyocyte death and loss of cardiac function, referred to as ischemia/reperfusion (IR) injury. Neutrophil- and mast cell-derived proteases, cathepsin G (Cat.G) and chymase, are released early after IR, but their function is complicated by potentially redundant actions and targets. This study investigated whether a dual inhibition of Cat.G and chymase influences cardiomyocyte injury and wound healing after experimental IR in mice. Treatment with a dual Cat.G and chymase inhibitor (DCCI) immediately after reperfusion blocked cardiac Cat.G and chymase activity induced after IR, which resulted in decreased immune response in the infarcted heart. Mice treated with DCCI had less myocardial collagen deposition and showed preserved ventricular function at 1 and 7 days post-IR compared with vehicle-treated mice. DCCI treatment also significantly attenuated focal adhesion (FA) complex disruption and myocyte degeneration after IR. Treatment of isolated cardiomyocytes with Cat.G or chymase significantly promoted FA signaling downregulation, myofibril degeneration and myocyte apoptosis. Conversely, treatment of cardiac fibroblasts with Cat.G or chymase induced FA signaling activation and increased their migration and differentiation to myofibroblasts. These opposite responses in cardiomyocytes and fibroblasts were blocked by treatment with DCCI. These findings show that Cat.G and chymase are key mediators of myocyte apoptosis and fibroblast migration and differentiation that play a role in adverse cardiac remodeling and function post-IR. Thus, dual targeting of neutrophil- and mast cell-derived proteases could be used as a novel therapeutic strategy to reduce post-IR inflammation and improve cardiac remodeling.
Asunto(s)
Remodelación Atrial/fisiología , Catepsina G/antagonistas & inhibidores , Quimasas/antagonistas & inhibidores , Daño por Reperfusión Miocárdica/enzimología , Miocitos Cardíacos/patología , Animales , Apoptosis/fisiología , Inhibidores Enzimáticos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologíaRESUMEN
This commentary highlights the research presented by Li et al. [15]. In this issue of Clinical Science, which demonstrates that dual specific phosphatase 12 (DUSP12), through JNK1/2 inhibition, alleviates cardiac hypertrophy in response to pressure overload, making it a potential therapeutic target.
Asunto(s)
Cardiomegalia/enzimología , Fosfatasas de Especificidad Dual/metabolismo , Animales , Cardiomegalia/genética , Cardiomegalia/fisiopatología , Fosfatasas de Especificidad Dual/genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Noqueados , Miocitos Cardíacos/enzimología , Transducción de SeñalRESUMEN
G protein-coupled receptors (GPCRs) remain primary therapeutic targets for numerous cardiovascular disorders, including heart failure (HF), because of their influence on cardiac remodeling in response to elevated neurohormone signaling. GPCR blockers have proven to be beneficial in the treatment of HF by reducing chronic G protein activation and cardiac remodeling, thereby extending the lifespan of patients with HF. Unfortunately, this effect does not persist indefinitely, thus next-generation therapeutics aim to selectively block harmful GPCR-mediated pathways while simultaneously promoting beneficial signaling. Transactivation of epidermal growth factor receptor (EGFR) has been shown to be mediated by an expanding repertoire of GPCRs in the heart, and promotes cardiomyocyte survival, thus may offer a new avenue of HF therapeutics. However, GPCR-dependent EGFR transactivation has also been shown to regulate cardiac hypertrophy and fibrosis by different GPCRs and through distinct molecular mechanisms. Here, we discuss the mechanisms and impact of GPCR-mediated EGFR transactivation in the heart, focusing on angiotensin II, urotensin II, and ß-adrenergic receptor systems, and highlight areas of research that will help us to determine whether this pathway can be engaged as future therapeutic strategy.
Asunto(s)
Receptores ErbB/genética , Receptores ErbB/metabolismo , Miocitos Cardíacos/fisiología , Receptores Acoplados a Proteínas G/fisiología , Activación Transcripcional/fisiología , Animales , HumanosRESUMEN
Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na(+)-K(+)-ATPase and L-type Ca(2+) channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with ß1-adrenergic receptor, L-type Ca(2+) channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca(2+)]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca(2+) current (ICa) and sarcoplasmic reticulum (SR) Ca(2+) content but not Na(+)/Ca(2+) exchange current (INaCa) or SR Ca(2+) uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca(2+) entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the ß1-adrenergic receptor and L-type Ca(2+) channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Canales de Calcio Tipo L/metabolismo , Cardiomiopatía Dilatada/metabolismo , Insuficiencia Cardíaca/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Potenciales de Acción/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Animales , Proteínas Reguladoras de la Apoptosis/biosíntesis , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patología , Calcio/metabolismo , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Acoplamiento Excitación-Contracción , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Homeostasis , Humanos , Isoproterenol/administración & dosificación , Proteínas de la Membrana/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fosfoproteínas/metabolismo , ARN Interferente Pequeño/genética , Sarcolema/metabolismoRESUMEN
Vasopressin type 1A receptor (V1AR) expression is elevated in chronic human heart failure (HF) and contributes to cardiac dysfunction in animal models, in part via reduced ß-adrenergic receptor (ßAR) responsiveness. Although cardiac V1AR overexpression and V1AR stimulation are each sufficient to decrease ßAR activity, it is unknown whether V1AR inhibition conversely augments ßAR responsiveness. Further, although V1AR has been shown to contribute to chronic progression of HF, its impact on cardiac function following acute ischaemic injury has not been reported. Using V1AR knockout (V1AR KO) mice we assessed the impact of V1AR deletion on cardiac contractility at baseline and following ischaemic injury, ßAR sensitivity and cardiomyocyte responsiveness to ßAR stimulation. Strikingly, baseline cardiac contractility was enhanced in V1AR KO mice and they experienced a greater loss in contractile function than control mice following acute ischaemic injury, although the absolute levels of cardiac dysfunction and survival rates did not differ. Enhanced cardiac contractility in V1AR KO mice was associated with augmented ß-blocker sensitivity, suggesting increased basal ßAR activity, and indeed levels of left ventricular cAMP, as well as phospholamban (PLB) and cardiac troponin I (cTnI) phosphorylation were elevated compared with control mice. At the cellular level, myocytes isolated from V1AR KO mice demonstrated increased responsiveness to ßAR stimulation consistent with the finding that acute pharmacological V1AR inhibition enhanced ßAR-mediated contractility in control myocytes. Therefore, although V1AR deletion does not protect the heart from the rapid development of cardiac dysfunction following acute ischaemic injury, its effects on ßAR activity suggest that acute V1AR inhibition could be utilized to promote myocyte contractile performance.
RESUMEN
RATIONALE: G protein-coupled receptor kinases (GRKs) acting in the cardiomyocyte regulate important signaling events that control cardiac function. Both GRK2 and GRK5, the predominant GRKs expressed in the heart, have been shown to be upregulated in failing human myocardium. Although the canonical role of GRKs is to desensitize G protein-coupled receptors via phosphorylation, it has been demonstrated that GRK5, unlike GRK2, can reside in the nucleus of myocytes and exert G protein-coupled receptor-independent effects that promote maladaptive cardiac hypertrophy and heart failure. OBJECTIVE: To explore novel mechanisms by which GRK5 acting in the nucleus of cardiomyocytes participates in pathological cardiac hypertrophy. METHODS AND RESULTS: In this study, we have found that GRK5-mediated pathological cardiac hypertrophy involves the activation of the nuclear factor of activated T cells (NFAT) because GRK5 causes enhancement of NFAT-mediated hypertrophic gene transcription. Transgenic mice with cardiomyocyte-specific GRK5 overexpression activate an NFAT-reporter in mice basally and after hypertrophic stimulation, including transverse aortic constriction and phenylephrine treatment. Complimentary to this, GRK5 null mice exhibit less NFAT transcriptional activity after transverse aortic constriction. Furthermore, the loss of NFATc3 expression in the heart protected GRK5 overexpressing transgenic mice from the exaggerated hypertrophy and early progression to heart failure seen after transverse aortic constriction. Molecular studies suggest that GRK5 acts in concert with NFAT to increase hypertrophic gene transcription in the nucleus via GRK5's ability to bind DNA directly without a phosphorylation event. CONCLUSIONS: GRK5, acting in a kinase independent manner, is a facilitator of NFAT activity and part of a DNA-binding complex responsible for pathological hypertrophic gene transcription.