RESUMEN
Mutations in lysosomal-associated membrane protein 2 (LAMP-2) gene are associated with Danon disease, which often leads to cardiomyopathy/heart failure through poorly defined mechanisms. Here, we identify the LAMP-2 isoform B (LAMP-2B) as required for autophagosome-lysosome fusion in human cardiomyocytes (CMs). Remarkably, LAMP-2B functions independently of syntaxin 17 (STX17), a protein that is essential for autophagosome-lysosome fusion in non-CMs. Instead, LAMP-2B interacts with autophagy related 14 (ATG14) and vesicle-associated membrane protein 8 (VAMP8) through its C-terminal coiled coil domain (CCD) to promote autophagic fusion. CMs derived from induced pluripotent stem cells (hiPSC-CMs) from Danon patients exhibit decreased colocalization between ATG14 and VAMP8, profound defects in autophagic fusion, as well as mitochondrial and contractile abnormalities. This phenotype was recapitulated by LAMP-2B knockout in non-Danon hiPSC-CMs. Finally, gene correction of LAMP-2 mutation rescues the Danon phenotype. These findings reveal a STX17-independent autophagic fusion mechanism in human CMs, providing an explanation for cardiomyopathy in Danon patients and a foundation for targeting defective LAMP-2B-mediated autophagy to treat this patient population.
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Autofagosomas/metabolismo , Enfermedad por Depósito de Glucógeno de Tipo IIb/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/metabolismo , Fusión de Membrana , Miocitos Cardíacos/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Autofagosomas/patología , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Técnicas de Inactivación de Genes , Enfermedad por Depósito de Glucógeno de Tipo IIb/genética , Enfermedad por Depósito de Glucógeno de Tipo IIb/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Lisosomas/genética , Lisosomas/patología , Miocitos Cardíacos/patología , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismoRESUMEN
OBJECTIVE: Cardiac troponin I (cTnI) is an essential physiological and pathological regulator of cardiac relaxation. Significant to this regulation, the post-translational modification of cTnI through phosphorylation functions as a key mechanism to accelerate myofibril relaxation. Similar to phosphorylation, post-translational modification by acetylation alters amino acid charge and protein function. Recent studies have demonstrated that the acetylation of cardiac myofibril proteins accelerates relaxation and that cTnI is acetylated in the heart. These findings highlight the potential significance of myofilament acetylation; however, it is not known if site-specific acetylation of cTnI can lead to changes in myofilament, myofibril, and/or cellular mechanics. The objective of this study was to determine the effects of mimicking acetylation at a single site of cTnI (lysine-132; K132) on myofilament, myofibril, and cellular mechanics and elucidate its influence on molecular function. METHODS: To determine if pseudo-acetylation of cTnI at 132 modulates thin filament regulation of the acto-myosin interaction, we reconstituted thin filaments containing WT or K132Q (to mimic acetylation) cTnI and assessed in vitro motility. To test if mimicking acetylation at K132 alters cellular relaxation, adult rat ventricular cardiomyocytes were infected with adenoviral constructs expressing either cTnI K132Q or K132 replaced with arginine (K132R; to prevent acetylation) and cell shortening and isolated myofibril mechanics were measured. Finally, to confirm that changes in cell shortening and myofibril mechanics were directly due to pseudo-acetylation of cTnI at K132, we exchanged troponin containing WT or K132Q cTnI into isolated myofibrils and measured myofibril mechanical properties. RESULTS: Reconstituted thin filaments containing K132Q cTnI exhibited decreased calcium sensitivity compared to thin filaments reconstituted with WT cTnI. Cardiomyocytes expressing K132Q cTnI had faster relengthening and myofibrils isolated from these cells had faster relaxation along with decreased calcium sensitivity compared to cardiomyocytes expressing WT or K132R cTnI. Myofibrils exchanged with K132Q cTnI ex vivo demonstrated faster relaxation and decreased calcium sensitivity. CONCLUSIONS: Our results indicate for the first time that mimicking acetylation of a specific cTnI lysine accelerates myofilament, myofibril, and myocyte relaxation. This work underscores the importance of understanding how acetylation of specific sarcomeric proteins affects cardiac homeostasis and disease and suggests that modulation of myofilament lysine acetylation may represent a novel therapeutic target to alter cardiac relaxation.
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Calcio/metabolismo , Miocardio/metabolismo , Miofibrillas/metabolismo , Troponina I/metabolismo , Acetilación , Animales , Femenino , Ventrículos Cardíacos/citología , Lisina/metabolismo , Miocitos Cardíacos/metabolismo , Ratas Endogámicas Dahl , Ratas Sprague-DawleyRESUMEN
Succinylation is a post-translational modification of protein lysine residues with succinyl groups derived from succinyl CoA. Succinylation is considered a significant post-translational modification with the potential to impact protein function which is highly conserved across numerous species. The role of succinylation in the heart, especially in heart failure and myofibril mechanics, remains largely unexplored. Mechanical parameters were measured in myofibrils isolated from failing hearts of ischemic cardiomyopathy patients and non-failing donor controls. We employed mass spectrometry to quantify differential protein expression in myofibrils from failing ischemic cardiomyopathy hearts compared to non-failing hearts. In addition, we combined peptide enrichment by immunoprecipitation with liquid chromatography tandem mass spectrometry to quantitatively analyze succinylated lysine residues in these myofibrils. Several key parameters of sarcomeric mechanical interactions were altered in myofibrils isolated from failing ischemic cardiomyopathy hearts, including lower resting tension and a faster rate of activation. Of the 100 differentially expressed proteins, 46 showed increased expression in ischemic heart failure, while 54 demonstrated decreased expression in ischemic heart failure. Our quantitative succinylome analysis identified a total of 572 unique succinylated lysine sites located on 181 proteins, with 307 significantly changed succinylation events. We found that 297 succinyl-Lys demonstrated decreased succinylation on 104 proteins, while 10 residues demonstrated increased succinylation on 4 proteins. Investigating succinyl CoA generation, enzyme activity assays demonstrated that α-ketoglutarate dehydrogenase and succinate dehydrogenase activities were significantly decreased in ischemic heart failure. An activity assay for succinyl CoA synthetase demonstrated a significant increase in ischemic heart failure. Taken together, our findings support the hypothesis that succinyl CoA production is decreased and succinyl CoA turnover is increased in ischemic heart failure, potentially resulting in an overall decrease in the mitochondrial succinyl CoA pool, which may contribute to decreased myofibril protein succinylation in heart failure.
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Cardiomiopatías/metabolismo , Insuficiencia Cardíaca/metabolismo , Proteínas Mitocondriales/metabolismo , Isquemia Miocárdica/metabolismo , Miocardio/metabolismo , Miofibrillas/metabolismo , Ácido Succínico/metabolismo , Acilación , Cardiomiopatías/complicaciones , Humanos , Lisina/metabolismo , Metilación , Persona de Mediana Edad , Isquemia Miocárdica/complicaciones , Proteómica , Reproducibilidad de los Resultados , Donantes de TejidosRESUMEN
Heart failure is a morbid disorder characterized by progressive cardiomyocyte (CM) dysfunction and death. Interest in cell-based therapies is growing, but sustainability of injected CMs remains a challenge. To mitigate this, we developed an injectable biomimetic Reverse Thermal Gel (RTG) specifically engineered to support long-term CM survival. This RTG biopolymer provided a solution-based delivery vehicle of CMs, which transitioned to a gel-based matrix shortly after reaching body temperature. In this study we tested the suitability of this biopolymer to sustain CM viability. The RTG was biomolecule-functionalized with poly-l-lysine or laminin. Neonatal rat ventricular myocytes (NRVM) and adult rat ventricular myocytes (ARVM) were cultured in plain-RTG and biomolecule-functionalized-RTG both under 3-dimensional (3D) conditions. Traditional 2D biomolecule-coated dishes were used as controls. We found that the RTG-lysine stimulated NRVM to spread and form heart-like functional syncytia. Regarding cell contraction, in both RTG and RTG-lysine, beating cells were recorded after 21 days. Additionally, more than 50% (p value < 0.05; n = 5) viable ARVMs, characterized by a well-defined cardiac phenotype represented by sarcomeric cross-striations, were found in the RTG-laminin after 8 days. These results exhibit the tremendous potential of a minimally invasive CM transplantation through our designed RTG-cell therapy platform.
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Biomimética/métodos , Células Madre Embrionarias/citología , Laminina/química , Lisina/química , Miocitos Cardíacos/citología , Polímeros/química , Ingeniería de Tejidos/métodos , Animales , Animales Recién Nacidos , Células Cultivadas , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Cardiac hypertrophy is a strong predictor of morbidity and mortality in patients with heart failure. Small molecule histone deacetylase (HDAC) inhibitors have been shown to suppress cardiac hypertrophy through mechanisms that remain poorly understood. We report that class I HDACs function as signal-dependent repressors of cardiac hypertrophy via inhibition of the gene encoding dual-specificity phosphatase 5 (DUSP5) DUSP5, a nuclear phosphatase that negatively regulates prohypertrophic signaling by ERK1/2. Inhibition of DUSP5 by class I HDACs requires activity of the ERK kinase, mitogen-activated protein kinase kinase (MEK), revealing a self-reinforcing mechanism for promotion of cardiac ERK signaling. In cardiac myocytes treated with highly selective class I HDAC inhibitors, nuclear ERK1/2 signaling is suppressed in a manner that is absolutely dependent on DUSP5. In contrast, cytosolic ERK1/2 activation is maintained under these same conditions. Ectopic expression of DUSP5 in cardiomyocytes results in potent inhibition of agonist-dependent hypertrophy through a mechanism involving suppression of the gene program for hypertrophic growth. These findings define unique roles for class I HDACs and DUSP5 as integral components of a regulatory signaling circuit that controls cardiac hypertrophy.
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Cardiomegalia/metabolismo , Fosfatasas de Especificidad Dual/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Histona Desacetilasas/metabolismo , Animales , Animales Recién Nacidos , Benzamidas/farmacología , Cardiomegalia/genética , Núcleo Celular/enzimología , Células Cultivadas , Fosfatasas de Especificidad Dual/genética , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Immunoblotting , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Pirimidinas/farmacología , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Little is known about the function of the cytoplasmic histone deacetylase HDAC6 in striated muscle. Here, we addressed the role of HDAC6 in cardiac and skeletal muscle remodeling induced by the peptide hormone angiotensin II (ANG II), which plays a central role in blood pressure control, heart failure, and associated skeletal muscle wasting. Comparable with wild-type (WT) mice, HDAC6 null mice developed cardiac hypertrophy and fibrosis in response to ANG II. However, whereas WT mice developed systolic dysfunction upon treatment with ANG II, cardiac function was maintained in HDAC6 null mice treated with ANG II for up to 8 wk. The cardioprotective effect of HDAC6 deletion was mimicked in WT mice treated with the small molecule HDAC6 inhibitor tubastatin A. HDAC6 null mice also exhibited improved left ventricular function in the setting of pressure overload mediated by transverse aortic constriction. HDAC6 inhibition appeared to preserve systolic function, in part, by enhancing cooperativity of myofibrillar force generation. Finally, we show that HDAC6 null mice are resistant to skeletal muscle wasting mediated by chronic ANG-II signaling. These findings define novel roles for HDAC6 in striated muscle and suggest potential for HDAC6-selective inhibitors for the treatment of cardiac dysfunction and muscle wasting in patients with heart failure.
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Angiotensina II , Cardiomegalia/enzimología , Insuficiencia Cardíaca/enzimología , Histona Desacetilasas/metabolismo , Músculo Esquelético/enzimología , Atrofia Muscular/enzimología , Miocardio/enzimología , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Cardiomegalia/prevención & control , Modelos Animales de Enfermedad , Fibrosis , Insuficiencia Cardíaca/inducido químicamente , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/prevención & control , Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/deficiencia , Histona Desacetilasas/genética , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Atrofia Muscular/inducido químicamente , Atrofia Muscular/patología , Atrofia Muscular/prevención & control , Miocardio/patología , Transducción de Señal , Volumen Sistólico , Sístole , Factores de Tiempo , Función Ventricular Izquierda , Remodelación VentricularRESUMEN
Introduction Transthyretin amyloid cardiomyopathy (ATTR-CM) is a progressive, infiltrative form of heart failure (HF). Nevertheless, ATTR-CM is a largely underrecognized and misdiagnosed condition. This study's objective was to develop an efficient model to assess the chance of ATTR-CM in patients with HF. Methods This was an observational study of patients with HF who had a confirmed diagnosis of ATTR-CM and those with HF but without known ATTR-CM between January 1, 2019, and July 1, 2021. Patient characteristics were extracted from administrative and claims electronic databases and compared between the groups. A propensity score for having ATTR-CM was modeled. Samples of 50 control patients with the highest and lowest propensity scores were adjudicated to assess whether further workup to evaluate for ATTR-CM was warranted for each patient. The sensitivity and specificity of the model were calculated. Results Thirty-one patients with confirmed ATTR-CM and 7620 patients without known ATTR-CM were included in the study. Patients with ATTR-CM were more likely to be Black and to have atrial flutter/fibrillation, cardiomegaly, HF with preserved ejection fraction, pericardial effusion, carpal tunnel syndrome, joint disorders, and lumbar spinal stenosis and to use a diuretic (all p < 0.05). A propensity model with 16 inputs was developed (c-statistic = 0.875). The model's sensitivity and specificity were 71.9% and 95.2%, respectively. Conclusion The propensity model developed in this study provided an efficient means for identifying patients with HF who are more likely to have ATTR-CM and may warrant further workup.
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Neuropatías Amiloides Familiares , Fibrilación Atrial , Cardiomiopatías , Insuficiencia Cardíaca , Humanos , Prealbúmina , Cardiomiopatías/complicaciones , Neuropatías Amiloides Familiares/complicaciones , Neuropatías Amiloides Familiares/diagnóstico , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/epidemiologíaRESUMEN
Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.
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Miofibrillas , Sarcómeros , Animales , Conectina/química , Conectina/genética , Conectina/metabolismo , Histona Desacetilasa 6/genética , Histona Desacetilasa 6/metabolismo , Humanos , Ratones , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Miofibrillas/metabolismo , Ratas , Sarcómeros/metabolismoRESUMEN
Small molecule histone deacetylase (HDAC) inhibitors block adverse cardiac remodeling in animal models of heart failure. The efficacious compounds target class I, class IIb and, to a lesser extent, class IIa HDACs. It is hypothesized that a selective inhibitor of a specific HDAC class (or an isoform within that class) will provide a favorable therapeutic window for the treatment of heart failure, although the optimal selectivity profile for such a compound remains unknown. Genetic studies have suggested that class I HDACs promote pathological cardiac remodeling, while class IIa HDACs are protective. In contrast, nothing is known about the function or regulation of class IIb HDACs in the heart. We developed assays to quantify catalytic activity of distinct HDAC classes in left and right ventricular cardiac tissue from animal models of hypertensive heart disease. Class I and IIa HDAC activity was elevated in some but not all diseased tissues. In contrast, catalytic activity of the class IIb HDAC, HDAC6, was consistently increased in stressed myocardium, but not in a model of physiologic hypertrophy. HDAC6 catalytic activity was also induced by diverse extracellular stimuli in cultured cardiac myocytes and fibroblasts. These findings suggest an unforeseen role for HDAC6 in the heart, and highlight the need for pre-clinical evaluation of HDAC6-selective inhibitors to determine whether this HDAC isoform is pathological or protective in the setting of cardiovascular disease.
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Histona Desacetilasas/metabolismo , Hipertensión/enzimología , Miocardio/enzimología , Adenoviridae/genética , Animales , Enfermedades Cardiovasculares , Células Cultivadas , Ventrículos Cardíacos/enzimología , Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/biosíntesis , Histona Desacetilasas/genética , Hipertensión/patología , Masculino , Ratones , Miocitos Cardíacos/enzimología , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Remodelación VentricularRESUMEN
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for biomedical research. However, they are immature, which is a barrier to modeling adult-onset cardiovascular disease. Here, we sought to develop a simple method that could drive cultured hiPSC-CMs toward maturity across a number of phenotypes, with the aim of utilizing mature hiPSC-CMs to model human cardiovascular disease. hiPSC-CMs were cultured in fatty acid-based medium and plated on micropatterned surfaces. These cells display many characteristics of adult human cardiomyocytes, including elongated cell morphology, sarcomeric maturity, and increased myofibril contractile force. In addition, mature hiPSC-CMs develop pathological hypertrophy, with associated myofibril relaxation defects, in response to either a pro-hypertrophic agent or genetic mutations. The more mature hiPSC-CMs produced by these methods could serve as a useful in vitro platform for characterizing cardiovascular disease.
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Cardiomiopatía Hipertrófica/fisiopatología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/fisiología , Línea Celular , Células Cultivadas , Medios de Cultivo/química , Ácidos Grasos/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Modelos Biológicos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miofibrillas/fisiología , Fenilefrina/farmacología , Sarcómeros/fisiología , Análisis de Secuencia de ARN , Transducción de SeñalRESUMEN
Using neonatal rat ventricular myocytes, we previously reported that the expression of a dominant negative form of the c-Fos proto-oncogene (AFos) inhibited activator protein 1 activity and blocked the induction of the pathological gene profile stimulated by phenylephrine (PE) while leaving growth unaffected. We now extend these observations to the adult rat ventricular myocyte (ARVM) to understand the relationship between gene expression, growth, and function. Ventricular myocytes were isolated from adult rats and infected with adenovirus expressing beta-galactosidase (control) or AFos. The cells were subsequently treated with PE, and protein synthesis, gene program, calcium transients, and contractility were evaluated. As seen with the neonatal rat ventricular myocytes, in control cells PE stimulated an increase in protein synthesis, induced the pathological gene profile, and exhibited both depressed contractility and calcium transients. Although ARVMs expressing AFos still had PE-induced growth, pathological gene expression as well as contractility and calcium handling abnormalities were inhibited. To determine a possible mechanism of the preserved myocyte function in AFos-expressing cells, we examined phospholamban (PLB) and sarco(endo)plasmic reticulum calcium-ATPase proteins. Although there was no change in total PLB or sarco(endo)plasmic reticulum calcium-ATPase expression in response to PE treatment, PE decreased the phosphorylation of PLB at serine-16, an observation that was prevented in AFos-expressing cells. In conclusion, although PE-induced growth was unaffected in AFos-expressing ARVMs, the expression of the pathological gene profile was inhibited and both contractile function and calcium cycling were preserved. The inhibition of functional deterioration was, in part, due to the preservation of PLB phosphorylation.
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Proteínas de Unión al Calcio/fisiología , Genes fos/fisiología , Ventrículos Cardíacos/citología , Contracción Miocárdica/fisiología , Miocitos Cardíacos/fisiología , Adenoviridae/genética , Animales , Calcio/metabolismo , Cardiotónicos/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Genes fos/genética , Hipertrofia , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Fenilefrina/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/fisiología , Ratas , Ratas Sprague-Dawley , Factor de Transcripción AP-1/fisiología , beta-Galactosidasa/genética , beta-Galactosidasa/fisiologíaRESUMEN
Heart failure with preserved ejection fraction (HFpEF) is a major health problem without effective therapies. This study assessed the effects of histone deacetylase (HDAC) inhibition on cardiopulmonary structure, function, and metabolism in a large mammalian model of pressure overload recapitulating features of diastolic dysfunction common to human HFpEF. Male domestic short-hair felines (n = 31, aged 2 months) underwent a sham procedure (n = 10) or loose aortic banding (n = 21), resulting in slow-progressive pressure overload. Two months after banding, animals were treated daily with suberoylanilide hydroxamic acid (b + SAHA, 10 mg/kg, n = 8), a Food and Drug Administration-approved pan-HDAC inhibitor, or vehicle (b + veh, n = 8) for 2 months. Echocardiography at 4 months after banding revealed that b + SAHA animals had significantly reduced left ventricular hypertrophy (LVH) (P < 0.0001) and left atrium size (P < 0.0001) versus b + veh animals. Left ventricular (LV) end-diastolic pressure and mean pulmonary arterial pressure were significantly reduced in b + SAHA (P < 0.01) versus b + veh. SAHA increased myofibril relaxation ex vivo, which correlated with in vivo improvements of LV relaxation. Furthermore, SAHA treatment preserved lung structure, compliance, blood oxygenation, and reduced perivascular fluid cuffs around extra-alveolar vessels, suggesting attenuated alveolar capillary stress failure. Acetylation proteomics revealed that SAHA altered lysine acetylation of mitochondrial metabolic enzymes. These results suggest that acetylation defects in hypertrophic stress can be reversed by HDAC inhibitors, with implications for improving cardiac structure and function in patients.
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Diástole , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/fisiopatología , Inhibidores de Histona Desacetilasas/uso terapéutico , Animales , Presión Sanguínea/efectos de los fármacos , Gatos , Diástole/efectos de los fármacos , Modelos Animales de Enfermedad , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/fisiopatología , Inhibidores de Histona Desacetilasas/farmacología , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/fisiopatología , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Miofibrillas/efectos de los fármacos , Miofibrillas/metabolismo , Fenotipo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Volumen Sistólico/efectos de los fármacos , Vorinostat/farmacología , Vorinostat/uso terapéuticoRESUMEN
Myofibril based mechanical studies allow evaluation of sarcomeric protein function. We describe a novel method of obtaining myofibrils from primary cardiomyocyte culture. Adult rat ventricular myocytes (ARVMs) were obtained by enzymatic digestion and maintained in serum free condition. ARVMs were homogenized in relaxing solution (pCa 9.0) with 20% sucrose, and myofibril suspension was made. Myofibrils were Ca2+-activated and relaxed at 15°C. Results from ARVM myofibrils were compared to myofibrils obtained from ventricular tissue skinned with Triton X-100. At maximal Ca2+-activation (pCa 4.5) myofibril mechanical parameters from ARVMs were 6.8 ± 0.9 mN/mm2 (resting tension), 146.8 ± 13.8 mN/mm2 (maximal active tension, P0), 5.4 ± 0.22 s-1 (rate of force activation), 53.4 ± 4.4 ms (linear relaxation duration), 0.69 ± 0.36 s-1 (linear relaxation rate), and 10.8 ± 1.3 s-1 (exponential relaxation rate). Force-pCa curves were constructed from Triton skinned tissue, ARVM culture day 1, and ARVM culture day 3 myofibrils, and pCa50 were 5.79 ± 0.01, 5.69 ± 0.01, and 5.71 ± 0.01, respectively. Mechanical parameters from myofibrils isolated from ARVMs treated with phenylephrine were compared to myofibrils isolated from time-matched non-treated ARVMs. Phenylephrine treatment did not change the kinetics of activation or relaxation but decreased the pCa50 to 5.56 ± 0.03 (vehicle treated control: 5.67 ± 0.03). For determination of protein expression and post-translational modifications, myofibril slurry was re-suspended and resolved for immunoblotting and protein staining. Troponin I phosphorylation was significantly increased at serine 23/24 in phenylephrine treated group. Myofibrils obtained from ARVMs are a viable method to study myofibril mechanics. Phenylephrine treatment led to significant decrease in Ca2+-sensitivity that is due to increased phosphorylation of TnI at serine 23/24. This culture based approach to obtaining myofibrils will allow pharmacological and genetic manipulation of the cardiomyocytes to correlate biochemical and biophysical properties.
RESUMEN
There are no approved drugs for the treatment of heart failure with preserved ejection fraction (HFpEF), which is characterized by left ventricular (LV) diastolic dysfunction. We demonstrate that ITF2357 (givinostat), a clinical-stage inhibitor of histone deacetylase (HDAC) catalytic activity, is efficacious in two distinct murine models of diastolic dysfunction with preserved EF. ITF2357 blocked LV diastolic dysfunction due to hypertension in Dahl salt-sensitive (DSS) rats and suppressed aging-induced diastolic dysfunction in normotensive mice. HDAC inhibitor-mediated efficacy was not due to lowering blood pressure or inhibiting cellular and molecular events commonly associated with diastolic dysfunction, including cardiac fibrosis, cardiac hypertrophy, or changes in cardiac titin and myosin isoform expression. Instead, ex vivo studies revealed impairment of cardiac myofibril relaxation as a previously unrecognized, myocyte-autonomous mechanism for diastolic dysfunction, which can be ameliorated by HDAC inhibition. Translating these findings to humans, cardiac myofibrils from patients with diastolic dysfunction and preserved EF also exhibited compromised relaxation. These data suggest that agents such as HDAC inhibitors, which potentiate cardiac myofibril relaxation, hold promise for the treatment of HFpEF in humans.
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Presión Sanguínea/fisiología , Histona Desacetilasas/metabolismo , Animales , Presión Sanguínea/genética , Conectina/metabolismo , Femenino , Insuficiencia Cardíaca , Hemodinámica/fisiología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Ácidos Hidroxámicos/uso terapéutico , Ratones , Miocardio/metabolismo , Miocitos Cardíacos , Miosinas/metabolismo , Ratas , Ratas Sprague-Dawley , Disfunción Ventricular Izquierda/metabolismoRESUMEN
BACKGROUND: Although cardiovascular disease is the primary killer of women in the United States, women and female animals have traditionally been omitted from research studies. In reports that do include both sexes, significant sexual dimorphisms have been demonstrated in development, presentation, and outcome of cardiovascular disease. However, there is little understanding of the mechanisms underlying these observations. A more thorough understanding of sex-specific cardiovascular differences both at baseline and in disease is required to effectively consider and treat all patients with cardiovascular disease. METHODS AND RESULTS: We analyzed contractility in the whole rat heart, adult rat ventricular myocytes (ARVMs), and myofibrils from both sexes of rats and observed functional sex differences at all levels. Hearts and ARVMs from female rats displayed greater fractional shortening than males, and female ARVMs and myofibrils took longer to relax. To define factors underlying these functional differences, we performed an RNA sequencing experiment on ARVMs from male and female rats and identified ≈600 genes were expressed in a sexually dimorphic manner. Further analysis revealed sex-specific enrichment of signaling pathways and key regulators. At the protein level, female ARVMs exhibited higher protein kinase A activity, consistent with pathway enrichment identified through RNA sequencing. In addition, activating the protein kinase A pathway diminished the contractile sexual dimorphisms previously observed. CONCLUSIONS: These data support the notion that sex-specific gene expression differences at baseline influence cardiac function, particularly through the protein kinase A pathway, and could potentially be responsible for differences in cardiovascular disease presentation and outcomes.
Asunto(s)
Miocitos Cardíacos/metabolismo , Transcriptoma , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ecocardiografía , Femenino , Regulación de la Expresión Génica , Masculino , Contracción Miocárdica , Miofibrillas/genética , Miofibrillas/metabolismo , ARN/química , ARN/aislamiento & purificación , ARN/metabolismo , Ratas , Ratas Sprague-Dawley , Análisis de Secuencia de ARN , Caracteres Sexuales , Transducción de Señal/genéticaRESUMEN
The ability of the adult heart to regenerate cardiomyocytes (CMs) lost after injury is limited, generating interest in developing efficient cell-based transplantation therapies. Rigid carbon nanotubes (CNTs) scaffolds have been used to improve CMs viability, proliferation, and maturation, but they require undesirable invasive surgeries for implantation. To overcome this limitation, we developed an injectable reverse thermal gel (RTG) functionalized with CNTs (RTG-CNT) that transitions from a solution at room temperature to a three-dimensional (3D) gel-based matrix shortly after reaching body temperature. Here we show experimental evidence that this 3D RTG-CNT system supports long-term CMs survival, promotes CMs alignment and proliferation, and improves CMs function when compared with traditional two-dimensional gelatin controls and 3D plain RTG system without CNTs. Therefore, our injectable RTG-CNT system could potentially be used as a minimally invasive tool for cardiac tissue engineering efforts.
Asunto(s)
Nanotubos de Carbono , Animales , Gelatina , Miocitos Cardíacos , Ratas , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
BACKGROUND: Although induction of activator protein-1 (AP-1) transcription factor activity has been observed in cardiac hypertrophy, a direct role for AP-1 in myocardial growth and gene expression remains obscure. METHODS AND RESULTS: Hypertrophy was induced in cultured neonatal rat cardiomyocytes with phenylephrine or overexpression of a constitutively active MAP3K, MKK6. In both treatment groups, induction of the pathological gene profile was observed, ie, expression of beta-myosin heavy chain (betaMHC), atrial/brain natriuretic peptides (ANP/BNP), and skeletal alpha-actin (sACT) was increased, whereas expression for alpha-myosin heavy chain (alphaMHC) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA) genes was repressed. The role of AP-1 in the hypertrophic phenotype was evaluated with the use of an adenoviral construct expressing a dominant negative mutant of the c-Fos proto-oncogene (AdAFos). Although AFos did not change the myocyte growth response, it abrogated the gene profile to both agonists, including the upregulation of both alphaMHC and SERCA expression. CONCLUSIONS: Although c-Fos/AP-1 is necessary for induction of the pathological/fetal gene program, it does not appear to be critical for cardiomyocyte hypertrophy.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Genes fos/fisiología , Hipertrofia/genética , Miocitos Cardíacos/patología , Factor de Transcripción AP-1/fisiología , Animales , Células Cultivadas , Perfilación de la Expresión Génica , Genes fos/genética , Hipertrofia/etiología , Mutación , Proteínas Proto-Oncogénicas c-fos/administración & dosificación , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/farmacología , Ratas , Transducción GenéticaRESUMEN
Alterations in TR [thyroid hormone (TH) receptor]1 isoform expression have been reported in models of both physiologic and pathologic cardiac hypertrophy as well as in patients with heart failure. In this report, we demonstrate that TH induces hypertrophy as a direct result of binding to the TRalpha1 isoform and, moreover, that overexpression of TRalpha1 alone is also associated with a hypertrophic phenotype, even in the absence of ligand. The mechanism of TH and TRalpha1-specific hypertrophy is novel for a nuclear hormone receptor and involves the transforming growth factor beta-activated kinase (TAK1) and p38. Mitigating TRalpha1 effects, both TRalpha2 and TRbeta1 attenuate TRalpha1-induced myocardial growth and gene expression by diminishing TAK1 and p38 activities, respectively. These findings refine our previous observations on TR expression in the hypertrophied and failing heart and suggest that manipulation of thyroid hormone signaling in an isoform-specific manner may be a relevant therapeutic target for altering the pathologic myocardial program.
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Quinasas Quinasa Quinasa PAM/metabolismo , Miocitos Cardíacos/citología , Receptores alfa de Hormona Tiroidea/metabolismo , Hormonas Tiroideas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adenoviridae/metabolismo , Animales , Western Blotting , Núcleo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Citosol/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Hipertrofia , Imidazoles/farmacología , Inmunoprecipitación , Concentración 50 Inhibidora , Ligandos , Microscopía Fluorescente , Modelos Biológicos , Células Musculares/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Unión Proteica , Isoformas de Proteínas , Piridinas/farmacología , Conejos , Ratas , Receptores de Hormona Tiroidea/metabolismo , Ribonucleasas/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Tension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G). These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations.
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Acoplamiento Excitación-Contracción/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/fisiología , Miofibrillas/fisiología , Fenómenos Biomecánicos , Miosinas Cardíacas/genética , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Diferenciación Celular , Línea Celular , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Cinética , Mutación , Miocitos Cardíacos/citología , Miofibrillas/ultraestructura , Cadenas Pesadas de Miosina/genética , Nanoestructuras/química , Cultivo Primario de CélulasRESUMEN
Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. However, current approaches are inefficient. Here we demonstrate that pro-fibrotic signalling potently antagonizes cardiac reprogramming. Remarkably, inhibition of pro-fibrotic signalling using small molecules that target the transforming growth factor-ß or Rho-associated kinase pathways converts embryonic fibroblasts into functional cardiomyocyte-like cells, with the efficiency up to 60%. Conversely, overactivation of these pro-fibrotic signalling networks attenuates cardiac reprogramming. Furthermore, inhibition of pro-fibrotic signalling dramatically enhances the kinetics of cardiac reprogramming, with spontaneously contracting cardiomyocytes emerging in less than 2 weeks, as opposed to 4 weeks with GHMT alone. These findings provide new insights into the molecular mechanisms underlying cardiac conversion of fibroblasts and would enhance efforts to generate cardiomyocytes for clinical applications.