RESUMEN
G protein-coupled receptors that signal through Gαq (GqPCRs), like α1-adrenergic and angiotensin receptors (α1-AR, AT-R), are traditionally thought to mediate pathologic remodeling in heart failure, including cardiac myocyte death. However, we previously demonstrated that α1- ARs are cardioprotective and identified an α1A-subtype-ERK survival-signaling pathway in adult cardiac myocytes. Recently, we demonstrated that α1-ARs localize to and signal from the nucleus, whereas AT-R localize to and signal from the sarcolemma in adult cardiac myocytes. Thus, we proposed a novel paradigm, predicated on compartmentalization of GqPCR signaling, to explain the phenotypic diversity of GqPCRs. Here, we tested the hypothesis that differential subcellular compartmentalization of α1-AR and AT-R mediated activation of ERK might explain the differential effects of these receptors on cardiac myocyte survival. Using a fluorescent ERK activity FRET-based biosensor, EKAR, to measure subcellular localization and extent of receptor-mediated ERK activation in single adult cardiac myocytes, we found that α1-ARs induced ERK activity at the nucleus and in the cytosol in 60% of cardiac myocytes, whereas AT-Rs showed no consistent ERK activation. The cell-specific α1-mediated activation of ERK in 60% of adult cardiac myocytes showed concordance with previous studies indicating that the α1A-subtype is expressed in only 60% of cardiac myocytes. Consistent with the ability to activate ERK, we found that only α1-ARs induced phosphorylation of Bcl-2 family member Bad, improved mitochondrial membrane stability, and promoted cardiac myocyte survival. In summary, our results suggest that compartmentalization of GqPCRs dictate activation of ERK and cardiac myocyte survival in adult cardiac myocytes.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Sistema de Señalización de MAP Quinasas , Miocitos Cardíacos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Envejecimiento , Animales , Muerte Celular , Núcleo Celular/metabolismo , Supervivencia Celular , Citosol/metabolismo , Femenino , Potencial de la Membrana Mitocondrial , Ratones Endogámicos C57BL , Fosforilación , Fracciones Subcelulares/metabolismo , Proteína Letal Asociada a bcl/metabolismoRESUMEN
G protein-coupled receptors that signal through Gαq (Gq receptors), such as α1-adrenergic receptors (α1-ARs) or angiotensin receptors, share a common proximal signaling pathway that activates phospholipase Cß1 (PLCß1), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. Despite these common proximal signaling mechanisms, Gq receptors produce distinct physiological responses, yet the mechanistic basis for this remains unclear. In the heart, Gq receptors are thought to induce myocyte hypertrophy through a mechanism termed excitation-transcription coupling, which provides a mechanistic basis for compartmentalization of calcium required for contraction versus IP3-dependent intranuclear calcium required for hypertrophy. Here, we identified subcellular compartmentalization of Gq-receptor signaling as a mechanistic basis for unique Gq receptor-induced hypertrophic phenotypes in cardiac myocytes. We show that α1-ARs co-localize with PLCß1 and PIP2 at the nuclear membrane. Further, nuclear α1-ARs induced intranuclear PLCß1 activity, leading to histone deacetylase 5 (HDAC5) export and a robust transcriptional response (i.e. significant up- or down-regulation of 806 genes). Conversely, we found that angiotensin receptors localize to the sarcolemma and induce sarcolemmal PLCß1 activity, but fail to promote HDAC5 nuclear export, while producing a transcriptional response that is mostly a subset of α1-AR-induced transcription. In summary, these results link Gq-receptor compartmentalization in cardiac myocytes to unique hypertrophic transcription. They suggest a new model of excitation-transcription coupling in adult cardiac myocytes that accounts for differential Gq-receptor localization and better explains distinct physiological functions of Gq receptors.
Asunto(s)
Cardiomegalia/patología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Miocitos Cardíacos/patología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipasa C beta/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular , Animales , Cardiomegalia/genética , Cardiomegalia/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/patología , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/análisis , Histona Desacetilasas/análisis , Histona Desacetilasas/metabolismo , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/patología , Fenotipo , Fosfatidilinositol 4,5-Difosfato/análisis , Fosfolipasa C beta/análisis , Receptores Adrenérgicos alfa 1/análisis , Sarcolema/metabolismo , Sarcolema/patología , Activación TranscripcionalRESUMEN
Heart failure with preserved ejection fraction (HFpEF) is half of all HF, but standard HF therapies are ineffective. Diastolic dysfunction, often secondary to interstitial fibrosis, is common in HFpEF. Previously, we found that supra-physiologic levels of ω3-PUFAs produced by 12 weeks of ω3-dietary supplementation prevented fibrosis and contractile dysfunction following pressure overload [transverse aortic constriction (TAC)], a model that resembles aspects of remodeling in HFpEF. This raised several questions regarding ω3-concentration-dependent cardioprotection, the specific role of EPA and DHA, and the relationship between prevention of fibrosis and contractile dysfunction. To achieve more clinically relevant ω3-levels and test individual ω3-PUFAs, we shortened the ω3-diet regimen and used EPA- and DHA-specific diets to examine remodeling following TAC. The shorter diet regimen produced ω3-PUFA levels closer to Western clinics. Further, EPA, but not DHA, prevented fibrosis following TAC. However, neither ω3-PUFA prevented contractile dysfunction, perhaps due to reduced uptake of ω3-PUFA. Interestingly, EPA did not accumulate in cardiac fibroblasts. However, FFA receptor 4, a G protein-coupled receptor for ω3-PUFAs, was sufficient and required to block transforming growth factor ß1-fibrotic signaling in cultured cardiac fibroblasts, suggesting a novel mechanism for EPA. In summary, EPA-mediated prevention of fibrosis could represent a novel therapy for HFpEF.
Asunto(s)
Ácidos Docosahexaenoicos/uso terapéutico , Ácido Eicosapentaenoico/uso terapéutico , Ácidos Grasos no Esterificados/uso terapéutico , Fibrosis/prevención & control , Insuficiencia Cardíaca/prevención & control , Animales , Suplementos Dietéticos , Ratones , Distribución Aleatoria , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Although convention dictates that G protein-coupled receptors localize to and signal at the plasma membrane, accumulating evidence suggests that G protein-coupled receptors localize to and signal at intracellular membranes, most notably the nucleus. In fact, there is now significant evidence indicating that endogenous alpha-1 adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. Cumulatively, the data suggest that α1-ARs localize to the inner nuclear membrane, activate intranuclear signaling, and regulate physiologic function in adult cardiac myocytes. Although α1-ARs signal through Gαq, unlike other Gq-coupled receptors, α1-ARs mediate important cardioprotective functions including adaptive/physiologic hypertrophy, protection from cell death (survival signaling), positive inotropy, and preconditioning. Also unlike other Gq-coupled receptors, most, if not all, functional α1-ARs localize to the nuclei in adult cardiac myocytes, as opposed to the sarcolemma. Together, α1-AR nuclear localization and cardioprotection might suggest a novel model for compartmentalization of Gq-coupled receptor signaling in which nuclear Gq-coupled receptor signaling is cardioprotective.
Asunto(s)
Miocitos Cardíacos/fisiología , Membrana Nuclear/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Compartimento Celular/fisiología , Núcleo Celular/fisiología , Humanos , Transducción de Señal/fisiologíaRESUMEN
Aims: Free fatty acid receptor 4 (Ffar4) is a receptor for long-chain fatty acids that attenuates heart failure driven by increased afterload. Recent findings suggest that Ffar4 prevents ischemic injury in brain, liver, and kidney, and therefore, we hypothesized that Ffar4 would also attenuate cardiac ischemic injury. Methods and Results: Using a mouse model of ischemia-reperfusion (I/R), we found that mice with systemic deletion of Ffar4 (Ffar4KO) demonstrated impaired recovery of left ventricular systolic function post-I/R with no effect on initial infarct size. To identify potential mechanistic explanations for the cardioprotective effects of Ffar4, we performed bulk RNAseq to compare the transcriptomes from wild-type (WT) and Ffar4KO infarcted myocardium 3-days post-I/R. In the Ffar4KO infarcted myocardium, gene ontology (GO) analyses revealed augmentation of glycosaminoglycan synthesis, neutrophil activation, cadherin binding, extracellular matrix, rho signaling, and oxylipin synthesis, but impaired glycolytic and fatty acid metabolism, cardiac repolarization, and phosphodiesterase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated impaired AMPK signaling and augmented cellular senescence in the Ffar4KO infarcted myocardium. Interestingly, phosphodiesterase 6c (PDE6c), which degrades cGMP, was the most upregulated gene in the Ffar4KO heart. Further, the soluble guanylyl cyclase stimulator, vericiguat, failed to increase cGMP in Ffar4KO cardiac myocytes, suggesting increased phosphodiesterase activity. Finally, cardiac myocyte-specific overexpression of Ffar4 prevented systolic dysfunction post-I/R, defining a cardioprotective role of Ffa4 in cardiac myocytes. Conclusions: Our results demonstrate that Ffar4 in cardiac myocytes attenuates systolic dysfunction post-I/R, potentially by attenuating oxidative stress, preserving mitochondrial function, and modulation of cGMP signaling.
RESUMEN
Following myocardial infarction, the prognosis for females is better than males. Estrogen is thought to be protective, but clinical trials with hormone replacement failed to show protection. Here, we sought to identify novel mechanisms that might explain this sex-based difference. By diverging from the traditional focus on sex hormones, we employed a conceptually novel approach to this question by using a non-biased approach to measure global changes in gene expression following infarction. We hypothesized that specific gene programs are initiated in the heart following infarction that might account for this sex-based difference. We induced small, medium, and large infarcts in male and female mice and measured changes in gene expression by microarray following infarction. Regardless of infarct size, survival was better in females, while mortality occurred 3-10 days following infarction in males. Two days following infarction, males developed significant ventricular dilation, the best predictor of mortality in humans. Three days following infarction, we measured gene expression by microarray, comparing male versus female and sham versus surgery/infarction. In general, our results indicate a higher relative level of gene induction in females versus males and identified programs for angiogenesis, extracellular matrix remodeling, and immune response. This pattern of gene expression was linked to less pathologic remodeling in female hearts, including increased capillary density and decreased fibrosis. In summary, our results suggest an association between improved survival and less pathologic remodeling and the relative induction of gene expression in females following myocardial infarction.
Asunto(s)
Regulación de la Expresión Génica , Infarto del Miocardio/genética , Caracteres Sexuales , Animales , Capilares/patología , Femenino , Fibrosis , Pruebas de Función Cardíaca , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Masculino , Ratones , Infarto del Miocardio/mortalidad , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Análisis de Supervivencia , Remodelación Ventricular/genéticaRESUMEN
BACKGROUND: Induced pluripotent stem cells (iPSCs) can be differentiated into potentially unlimited lineages of cell types for use in autologous cell therapy. However, the efficiency of the differentiation procedure and subsequent function of the iPSC-derived cells may be influenced by epigenetic factors that the iPSCs retain from their tissues of origin; thus, iPSC-derived cells may be more effective for treatment of myocardial injury if the iPSCs were engineered from cardiac-lineage cells, rather than dermal fibroblasts. METHODS AND RESULTS: We show that human cardiac iPSCs (hciPSCs) can be generated from cardiac fibroblasts and subsequently differentiated into exceptionally pure (>92%) sheets of cardiomyocytes (CMs). The hciPSCs passed through all the normal stages of differentiation before assuming a CM identity. When using the fibrin gel-enhanced delivery of hciPSC-CM sheets at the site of injury in infarcted mouse hearts, the engraftment rate was 31.91%±5.75% at Day 28 post transplantation. The hciPSC-CM in the sheet also appeared to develop a more mature, structurally aligned phenotype 28 days after transplantation and was associated with significant improvements in cardiac function, vascularity, and reduction in apoptosis. CONCLUSIONS: These data strongly support the potential of hciPSC-CM sheet transplantation for the treatment of heart with acute myocardial infarction.
Asunto(s)
Células Madre Pluripotentes Inducidas/trasplante , Infarto del Miocardio/terapia , Miocitos Cardíacos/citología , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Fibroblastos/patología , Humanos , Masculino , Ratones , Infarto del Miocardio/patología , Resultado del TratamientoRESUMEN
BACKGROUND: Recent studies indicate that a1-adrenergic receptors (a1-ARs) are cardioprotective by preventing cardiac myocyte death and augmenting contractility in heart failure. Although G-protein-coupled receptors are assumed to localize to and signal at the plasma membrane, we previously demonstrated that endogenous a1-ARs localize to the nuclei in adult cardiac myocytes. However, the functional consequence of this nuclear localization remains unclear. Here, we attempted to reconcile nuclear localization of a1-ARs with their physiologic function by examining a1-AR-induced contractility in adult cardiac myocytes. METHODS AND RESULTS: By measuring shortening in unloaded, cultured adult cardiac myocytes, we found that the a1A-subtype regulated contractility through phosphorylation of cardiac troponin I (cTnI) at the protein kinase C (PKC) site, threonine 144. Reconstitution of an a1A-subtype nuclear localization mutant in cardiac myocytes lacking a1-ARs failed to rescue nuclear a1A-mediated phosphorylation of cTnI and myocyte contractility. Leptomycin B, the nuclear export inhibitor, also blocked a1A-mediated phosphorylation of cTnI. These data indicate that a1-AR signaling originates in the nucleus. Consistent with these observations, we localized the a1A-subtype to the inner nuclear membrane, identified PKCa, d, and e in the nucleus, and found that a1-ARs activate PKCd in nuclei isolated from adult cardiac myocytes. Finally, we found that a PKCd nuclear localization mutant blunted a1-induced phosphorylation of cTnI. CONCLUSIONS: Together, our data identify a novel, "inside-out" nuclear a1A-subtype/PKCd/cTnI-signaling pathway that regulates contractile function in adult cardiac myocytes. Importantly, these data help resolve the discrepancy between nuclear localization of a1-ARs and a1-AR-mediated physiologic function.
Asunto(s)
Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Membrana Nuclear/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Transducción de Señal , Animales , Activación Enzimática , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fosforilación , Proteína Quinasa C-delta/genética , Proteína Quinasa C-delta/metabolismo , Receptores Adrenérgicos alfa 1/deficiencia , Receptores Adrenérgicos alfa 1/genética , Factores de Tiempo , Transfección , Troponina I/metabolismoRESUMEN
Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCR oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.
Asunto(s)
Núcleo Celular/metabolismo , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Transducción de Señal , Animales , Células Cultivadas , Transferencia Resonante de Energía de Fluorescencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Miocitos Cardíacos/citología , Multimerización de Proteína , Receptores Adrenérgicos alfa 1/análisis , Receptores Adrenérgicos alfa 1/genéticaRESUMEN
Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKCepsilon and zeta and troponin T (cTnT) associated with PKC alpha, delta, and epsilon. Based on its association with cTnI, we hypothesized that PKCzeta is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKCzeta: the pseudo-substrate region (A119E), 3'-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKCzeta using PKC phospho-motif antibodies to determine the phosphorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKCzeta T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKCzeta. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKCzeta A119E. Both PKCzeta A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKCzeta exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKCzeta is a novel regulator of myofilament protein phosphorylation.
Asunto(s)
Chaperonas Moleculares/metabolismo , Proteínas Musculares/metabolismo , Miocardio/enzimología , Procesamiento Proteico-Postraduccional/fisiología , Sarcómeros/enzimología , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Sustitución de Aminoácidos/inmunología , Animales , Anticuerpos/inmunología , Isoenzimas/genética , Isoenzimas/inmunología , Isoenzimas/metabolismo , Masculino , Chaperonas Moleculares/genética , Chaperonas Moleculares/inmunología , Proteínas Musculares/genética , Proteínas Musculares/inmunología , Mutación Missense/inmunología , Miocardio/citología , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/inmunología , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Ratas , Sarcómeros/genética , Sarcómeros/inmunología , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/inmunología , Quinasas p21 Activadas/metabolismoRESUMEN
Presence of A+U-rich elements (AREs) within 3'-untranslated regions (3'UTRs) of numerous mRNAs has been associated with rapid mRNA turnover; however, the interaction of specific factors with AREs is also associated with mRNA stabilization. Recently, two ARE binding proteins with putative mRNA destabilizing (AUF1) and stabilizing (HuR) properties have been described. However, no direct comparison ofAUF1 and HuR binding properties has been made. Therefore, we examined the relative affinities of p37AUF1 and HuR for a diverse set ofARE-containing mRNAs encoding beta-adrenergic receptors, a proto-oncogene, and a cytokine. We find that high-affinity AUF1 binding appears to require elements beyond primary nucleotide sequence. In contrast, binding of HuR appears considerably less constrained. As a functional correlate, we determined the ability of these specific mRNA sequences to affect the stability of chimeric beta-globin mRNA constructs. Although the relative affinity ofAUF1 and HuR are generally predictive of mRNA stability, we find that certain mRNA sequences do not conform to these generalizations.