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Aims: Loss-of-function mutations in the hERG gene causes long-QT syndrome type 2 (LQT2), a condition associated with reduced IKr current. Four different mutation classes define the molecular mechanisms impairing hERG. Among them, Class 2 mutations determine hERG trafficking defects. Lumacaftor (LUM) is a drug acting on channel trafficking already successfully tested for cystic fibrosis and its safety profile is well known. We hypothesize that LUM might rescue also hERG trafficking defects in LQT2 and exert anti-arrhythmic effects. Methods and results: From five LQT2 patients, we generated lines of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harbouring Class 1 and 2 mutations. The effects of LUM on corrected field potential durations (cFPD) and calcium-handling irregularities were verified by multi electrode array and by calcium transients imaging, respectively. Molecular analysis was performed to clarify the mechanism of action of LUM on hERG trafficking and calcium handling. Long-QT syndrome type 2 induced pluripotent stem cell-derived cardiomyocytes mimicked the clinical phenotypes and showed both prolonged cFPD (grossly equivalent to the QT interval) and increased arrhythmias. Lumacaftor significantly shortened cFPD in Class 2 iPSC-CMs by correcting the hERG trafficking defect. Furthermore, LUM seemed to act also on calcium handling by reducing RyR2S2808 phosphorylation in both Class 1 and 2 iPSC-CMs. Conclusion: Lumacaftor, a drug already in clinical use, can rescue the pathological phenotype of LQT2 iPSC-CMs, particularly those derived from Class 2 mutated patients. Our results suggest that the use of LUM in LQT2 patients not protected by ß-blockers is feasible and may represent a novel therapeutic option.
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Aminopiridinas/uso terapéutico , Antiarrítmicos/uso terapéutico , Benzodioxoles/uso terapéutico , Síndrome de QT Prolongado/tratamiento farmacológico , Potenciales de Acción/efectos de los fármacos , Adolescente , Adulto , Calcio/metabolismo , Canal de Potasio ERG1/efectos de los fármacos , Femenino , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Síndrome de QT Prolongado/genética , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Resultado del TratamientoRESUMEN
Background: New treatments are needed to reduce myocardial infarct size (MI) and prevent heart failure (HF) following acute myocardial infarction (AMI), which are the leading causes of death and disability worldwide. Studies in rodent AMI models showed that genetic and pharmacological inhibition of mitochondrial fission, induced by acute ischemia and reperfusion, reduced MI size. Whether targeting mitochondrial fission at the onset of reperfusion is also cardioprotective in a clinically-relevant large animal AMI model remains to be determined. Methods: Adult pigs (30-40 kg) were subjected to closed-chest 90-min left anterior descending artery ischemia followed by 72 h of reperfusion and were randomized to receive an intracoronary bolus of either mdivi-1 (1.2 mg/kg, a small molecule inhibitor of the mitochondrial fission protein, Drp1) or vehicle control, 10-min prior to reperfusion. The left ventricular (LV) size and function were both assessed by transthoracic echocardiography prior to AMI and after 72 h of reperfusion. MI size and the area-at-risk (AAR) were determined using dual staining with Tetrazolium and Evans blue. Heart samples were collected for histological determination of fibrosis and for electron microscopic analysis of mitochondrial morphology. Results: A total of 14 pigs underwent the treatment protocols (eight control and six mdivi-1). Administration of mdivi-1 immediately prior to the onset of reperfusion did not reduce MI size (MI size as % of AAR: Control 49.2 ± 8.6 vs. mdivi-1 50.5 ± 11.4; p = 0.815) or preserve LV systolic function (LV ejection fraction %: Control 67.5 ± 0.4 vs. mdivi-1 59.6 ± 0.6; p = 0.420), when compared to vehicle control. Similarly, there were no differences in mitochondrial morphology or myocardial fibrosis between mdivi-1 and vehicle control groups. Conclusion: Our pilot study has shown that treatment with mdivi-1 (1.2 mg/kg) at the onset of reperfusion did not reduce MI size or preserve LV function in the clinically-relevant closed-chest pig AMI model. A larger study, testing different doses of mdivi-1 or using a more specific Drp1 inhibitor are required to confirm these findings.
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Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/metabolismo , Quinazolinonas/uso terapéutico , Animales , Modelos Animales de Enfermedad , Ecocardiografía , Femenino , Dinámicas Mitocondriales/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Proyectos Piloto , Porcinos , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Activation of signal transducer and activator of transcription 3 (STAT3) is imperative for mammalian development, specifically cardiogenesis. STAT3 phosphorylation and acetylation are key post-translational modifications that regulate its transcriptional activity. Significance of such modifications during human cardiogenesis remains elusive. Using human pluripotent stem cells to recapitulate cardiogenesis, two independently modified STAT3α (92 kDa) isoforms (phosphorylated and acetylated), which perform divergent functions were identified during cardiomyocyte (CM) formation. Phosphorylated STAT3α functioned as the canonical transcriptional activator, while acetylated STAT3α underwent caspase-3-mediated cleavage to generate a novel STAT3ζ fragment (â¼45 kDa), which acted as a molecular adaptor integral to the ErbB4-p38γ signaling cascade in driving CM formation. While STAT3α knockdown perturbed cardiogenesis by eliminating both post-translationally modified STAT3α isoforms, caspase-3 knockdown specifically abrogates the function of acetylated STAT3α, resulting in limited STAT3ζ formation thereby preventing nuclear translocation of key cardiac transcription factor Nkx2-5 that disrupted CM formation. Our findings show the coexistence of two post-translationally modified STAT3α isoforms with distinct functions and define a new role for STAT3 as a molecular adaptor that functions independently of its canonical transcriptional activity during human cardiogenesis. Stem Cells 2017;35:2129-2137.
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Caspasa 3/metabolismo , Miocitos Cardíacos/metabolismo , Factor de Transcripción STAT3/metabolismo , Acetilación , Humanos , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Factores de TranscripciónRESUMEN
Activation of ErbB4 receptor signaling is instrumental in heart development, lack of which results in embryonic lethality. However, mechanism governing its intracellular signaling remains elusive. Using human pluripotent stem cells, we show that ErbB4 is critical for cardiogenesis whereby its genetic knockdown results in loss of cardiomyocytes. Phospho-proteome profiling and Western blot studies attribute this loss to inactivation of p38γ MAPK isoform which physically interacts with NKx2.5 and GATA4 transcription factors. Post-cardiomyocyte formation p38γ/NKx2.5 downregulation is followed by p38α/MEF2c upregulation suggesting stage-specific developmental roles of p38 MAPK isoforms. Knockdown of p38γ MAPK similarly disrupts cardiomyocyte formation in spite of the presence of NKx2.5. Cell fractionation and NKx2.5 phosphorylation studies suggest inhibition of ErbB4-p38γ signaling hinders NKx2.5 nuclear translocation during early cardiogenesis. This study reveals a novel pathway that directly links ErbB4 and p38γ to the transcriptional machinery of NKx2.5-GATA4 complex which is critical for cardiomyocyte formation during mammalian heart development.
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Corazón/embriología , Proteínas de Homeodominio/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 12 Activada por Mitógenos/metabolismo , Organogénesis/fisiología , Receptor ErbB-4/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción GATA4/metabolismo , Proteína Homeótica Nkx-2.5 , Células Madre Embrionarias Humanas/citología , Humanos , Isoenzimas/metabolismo , Factores de Transcripción MEF2/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Fosforilación/fisiologíaRESUMEN
Mechanisms determining intrinsic differentiation bias inherent to human pluripotent stem cells (hPSCs) toward cardiogenic fate remain elusive. We evaluated the interplay between ErbB4 and Epidemal growth factor receptor (EGFR or ErbB1) in determining cardiac differentiation in vitro as these receptor tyrosine kinases are key to heart and brain development in vivo. Our results demonstrate that during cardiac differentiation, cell fate biases exist in hPSCs due to cardiac/neuroectoderm divergence post cardiac mesoderm stage. Stage-specific up-regulation of EGFR in concert with persistent Wnt3a signaling post cardiac mesoderm favors commitment toward neural progenitor cells (NPCs). Inhibition of EGFR abrogates these effects with enhanced (>twofold) cardiac differentiation efficiencies by increasing proliferation of Nkx2-5 expressing cardiac progenitors while reducing proliferation of Sox2 expressing NPCs. Forced overexpression of ErbB4 rescued cardiac commitment by augmenting Wnt11 signaling. Convergence between EGFR/ErbB4 and canonical/noncanonical Wnt signaling determines cardiogenic fate in hPSCs. Stem Cells 2016;34:2461-2470.
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Ectodermo/citología , Miocitos Cardíacos/citología , Neuronas/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Receptor ErbB-4/metabolismo , Diferenciación Celular , Línea Celular , Linaje de la Célula , Receptores ErbB/metabolismo , Humanos , Miocitos Cardíacos/metabolismo , Organogénesis , Vía de Señalización WntRESUMEN
We investigate the effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)-derived progenitors and cardiomyocytes into acutely infarcted myocardium in severe combined immune deficiency mice. A total of 2 × 10(5) progenitors, cardiomyocytes or cell-free saline were injected into peri-infarcted anterior free wall. Sham-operated animals received no injection. Myocardial function was assessed at 2-week and 4-week post-infarction by using echocardiography and pressure-volume catheterization. Early myocardial remodelling was observed at 2-week with echocardiography derived stroke volume (SV) in saline (20.45 ± 7.36 µl, P < 0.05) and cardiomyocyte (19.52 ± 3.97 µl, P < 0.05) groups, but not in progenitor group (25.65 ± 3.61 µl), significantly deteriorated as compared to sham control group (28.41 ± 4.41 µl). Consistently, pressure-volume haemodynamic measurements showed worsening chamber dilation in saline (EDV: 23.24 ± 5.01 µl, P < 0.05; ESV: 17.08 ± 5.82 µl, P < 0.05) and cardiomyocyte (EDV: 26.45 ± 5.69 µl, P < 0.05; ESV: 18.03 ± 6.58 µl, P < 0.05) groups by 4-week post-infarction as compared to control (EDV: 15.26 ± 2.96 µl; ESV: 8.41 ± 2.94 µl). In contrast, cardiac progenitors (EDV: 20.09 ± 7.76 µl; ESV: 13.98 ± 6.74 µl) persistently protected chamber geometry against negative cardiac remodelling. Similarly, as compared to sham control (54.64 ± 11.37%), LV ejection fraction was preserved in progenitor group from 2-(38.68 ± 7.34%) to 4-week (39.56 ± 13.26%) while cardiomyocyte (36.52 ± 11.39%, P < 0.05) and saline (35.34 ± 11.86%, P < 0.05) groups deteriorated early at 2-week. Improvements of myocardial function in the progenitor group corresponded to increased vascularization (16.12 ± 1.49/mm(2) to 25.48 ± 2.08/mm(2) myocardial tissue, P < 0.05) and coincided with augmented networking of cardiac telocytes in the interstitial space of infarcted zone.
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Células Madre Pluripotentes Inducidas/fisiología , Infarto del Miocardio/fisiopatología , Neovascularización Fisiológica/fisiología , Células Madre/fisiología , Remodelación Ventricular/fisiología , Animales , Línea Celular , Femenino , Humanos , Ratones , Ratones SCID , Miocardio/patología , Miocitos Cardíacos/fisiologíaRESUMEN
In this meeting report, particularly addressing the topic of protection of the cardiovascular system from ischemia/reperfusion injury, highlights are presented that relate to conditioning strategies of the heart with respect to molecular mechanisms and outcome in patients' cohorts, the influence of co-morbidities and medications, as well as the contribution of innate immune reactions in cardioprotection. Moreover, developmental or systems biology approaches bear great potential in systematically uncovering unexpected components involved in ischemia-reperfusion injury or heart regeneration. Based on the characterization of particular platelet integrins, mitochondrial redox-linked proteins, or lipid-diol compounds in cardiovascular diseases, their targeting by newly developed theranostics and technologies opens new avenues for diagnosis and therapy of myocardial infarction to improve the patients' outcome.
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Cardiología/tendencias , Enfermedades Cardiovasculares , Nanomedicina Teranóstica/tendencias , Animales , Cardiología/métodos , HumanosRESUMEN
The heart is an electrically conducting organ with networked bioelectric currents that transverse a large segment of interstitial space interspersed with the muscular parenchyma. Non-excitable connective cells in the interstitial space contributed importantly to many structural, biochemical, and physiological activities of cardiac homeostasis. However, contribution of interstitial cells in the cardiac niche has long been neglected. Telocyte is recently recognized as a distinct class of interstitial cell that resides in a wide array of tissues including in the epicardium, myocardium, and endocardium of the heart. They are increasingly described to conduct ionic currents that may have significant implications in bioelectric signaling. In this review, we highlight the significance of telocytes in such connectivity and conductivity within the interstitial bioelectric network in tissue homeostasis.
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Sistema de Conducción Cardíaco/fisiología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Miocardio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Telocitos/fisiología , Antígenos CD34/genética , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Endocardio/citología , Endocardio/fisiología , Expresión Génica , Sistema de Conducción Cardíaco/citología , Homeostasis/fisiología , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Miocardio/citología , Técnicas de Placa-Clamp , Pericardio/citología , Pericardio/fisiología , Canales de Potasio de Rectificación Interna/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Telocitos/citologíaRESUMEN
Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) offer immense value in studying cardiovascular regenerative medicine. However, intrinsic biases and differential responsiveness of hPSCs towards cardiac differentiation pose significant technical and logistic hurdles that hamper human cardiomyocyte studies. Tandem modulation of canonical and non-canonical Wnt signaling pathways may play a crucial role in cardiac development that can efficiently generate cardiomyocytes from pluripotent stem cells. Our Wnt signaling expression profiles revealed that phasic modulation of canonical/non-canonical axis enabled orderly recapitulation of cardiac developmental ontogeny. Moreover, evaluation of 8 hPSC lines showed marked commitment towards cardiac-mesoderm during the early phase of differentiation, with elevated levels of canonical Wnts (Wnt3 and 3a) and Mesp1. Whereas continued activation of canonical Wnts was counterproductive, its discrete inhibition during the later phase of cardiac differentiation was accompanied by significant up-regulation of non-canonical Wnt expression (Wnt5a and 11) and enhanced Nkx2.5(+) (up to 98%) populations. These Nkx2.5(+) populations transited to contracting cardiac troponin T-positive CMs with up to 80% efficiency. Our results suggest that timely modulation of Wnt pathways would transcend intrinsic differentiation biases of hPSCs to consistently generate functional CMs that could facilitate their scalable production for meaningful clinical translation towards personalized regenerative medicine.
RESUMEN
Telocytes (TCs) with exceptionally long cellular processes of telopodes have been described in human epicardium to act as structural supporting cells in the heart. We examined myocardial chamber-specific TCs identified in atrial and ventricular fibroblast culture using immunocytochemistry and studied their electrophysiological property by whole-cell patch clamp. Atrial and ventricular TCs with extended telopodes and alternating podoms and podomers that expressed CD34, c-Kit and PDGFR-ß were identified. These cells expressed large conductance Ca²âº-activated K⺠current (BK(Ca)) and inwardly rectifying K⺠current (IK(ir)), but not transient outward K⺠current (I(to)) and ATP-sensitive potassium current (K(ATP)). The active channels were functionally competent with demonstrated modulatory response to H2 S and transforming growth factor (TGF)-ß1 whereby H2S significantly inhibited the stimulatory effect of TGF-ß1 on current density of both BKCa and IK(ir). Furthermore, H2S attenuated TGF-ß1-stimulated KCa1.1/Kv1.1 (encode BK(Ca)) and Kir2.1 (encode IK(ir)) expression in TCs. Our results show that functionally competent K⺠channels are present in human atrial and ventricular TCs and their modulation may have significant implications in myocardial physiopathology.
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Atrios Cardíacos/citología , Ventrículos Cardíacos/citología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Miocardio/citología , Canales de Potasio de Rectificación Interna/metabolismo , Células del Estroma/fisiología , Antígenos CD34/genética , Antígenos CD34/metabolismo , Separación Celular , Células Cultivadas , Fibroblastos/citología , Fibroblastos/fisiología , Expresión Génica , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Humanos , Sulfuro de Hidrógeno/farmacología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Potenciales de la Membrana/efectos de los fármacos , Miocardio/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
We investigated global and regional effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) in infarcted myocardium. Acute myocardial infarction (MI) was induced by ligation of left coronary artery of severe combined immunodeficient mice before 2 × 10(5) iMSCs or cell-free saline were injected into peri-infarcted anterior free wall. Sham-operated animals received no injection. Global and regional myocardial function was assessed serially at 1-week and 8-week by segmental strain analysis by using two dimensional (2D) speckle tracking echocardiography. Early myocardial remodelling was observed at 1-week and persisted to 8-week with global contractility of ejection fraction and fractional area change in saline- (32.96 ± 14.23%; 21.50 ± 10.07%) and iMSC-injected (32.95 ± 10.31%; 21.00 ± 7.11%) groups significantly depressed as compared to sham control (51.17 ± 11.69%, P < 0.05; 34.86 ± 9.82%, P < 0.05). However, myocardial dilatation was observed in saline-injected animals (4.40 ± 0.62 mm, P < 0.05), but not iMSCs (4.29 ± 0.57 mm), when compared to sham control (3.74 ± 0.32 mm). Furthermore, strain analysis showed significant improved basal anterior wall strain (28.86 ± 8.16%, P < 0.05) in the iMSC group, but not saline-injected (15.81 ± 13.92%), when compared to sham control (22.18 ± 4.13%). This was corroborated by multi-segments deterioration of radial strain only in saline-injected (21.50 ± 5.31%, P < 0.05), but not iMSC (25.67 ± 12.53%), when compared to sham control (34.88 ± 5.77%). Improvements of the myocardial strain coincided with the presence of interconnecting telocytes in interstitial space of the infarcted anterior segment of the heart. Our results show that localized injection of iMSCs alleviates ventricular remodelling, sustains global and regional myocardial strain by paracrine-driven effect on neoangiogenesis and myocardial deformation/compliance via parenchymal and interstitial cell interactions in the infarcted myocardium.
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Células Madre Pluripotentes Inducidas/trasplante , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Remodelación Ventricular/fisiología , Animales , Modelos Animales de Enfermedad , Ecocardiografía , Femenino , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Ratones SCID , Infarto del Miocardio/diagnóstico por imagenRESUMEN
AIMS: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disorder associated with sudden cardiac death. Its pathophysiology is still poorly understood. We aimed to produce an in vitro cellular model of ARVC using patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes and determine whether the model could recapitulate key features of the disease phenotype. METHODS AND RESULTS: Dermal fibroblasts were obtained from a 30-year-old man with a clinical diagnosis of ARVC, harbouring a plakophilin 2 (PKP2) gene mutation. Four stable iPSC lines were generated using retroviral reprogramming, and functional cardiomyocytes were derived. Gene expression levels of desmosomal proteins (PKP2 and plakoglobin) in cardiomyocytes from ARVC-iPSCs were significantly lower compared with cardiomyocytes from control iPSCs (P< 0.01); there were no significant differences in the expression of desmoplakin, N-cadherin, and connexin 43 between the two groups. Cardiomyocytes derived from ARVC-iPSCs exhibited markedly reduced immunofluorescence signals when stained for PKP2 and plakoglobin, but similar levels of staining for desmoplakin, N-cadherin, and connexin 43 compared with control cardiomyocytes. Transmission electron microscopy showed that ARVC-iPSC cardiomyocytes were larger and contained darker lipid droplets compared with control cardiomyocytes. After 2 weeks of cell exposure to adiopgenic differentiation medium, ARVC-iPSC cardiomyocytes were found to contain a significantly greater amount of lipid, calculated using Oil Red O staining, compared with controls (734 ± 35.6 vs. 8.1 ± 0.49 a.u., respectively; n = 7, P = 0.001). CONCLUSION: Patient-specific iPSC-derived cardiomyocytes display key features of ARVC, including reduced cell surface localization of desmosomal proteins and a more adipogenic phenotype.
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Displasia Ventricular Derecha Arritmogénica/patología , Células Madre Pluripotentes Inducidas/patología , Miocitos Cardíacos/patología , Adulto , Displasia Ventricular Derecha Arritmogénica/genética , Displasia Ventricular Derecha Arritmogénica/metabolismo , Agonistas de los Canales de Calcio/farmacología , Desmosomas/metabolismo , Fibroblastos/patología , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Masculino , Modelos Cardiovasculares , Mutación , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Fenotipo , Placofilinas/genética , Placofilinas/metabolismo , gamma Catenina/metabolismoRESUMEN
Cardiac fibroblasts are crucial in pathophysiology of the myocardium whereby their aberrant proliferation has significant impact on cardiac function. Hydrogen sulphide (H2S) is a gaseous modulator of potassium channels on cardiomyocytes and has been reported to attenuate cardiac fibrosis. Yet, the mechanism of H2S in modulating proliferation of cardiac fibroblasts remains poorly understood. We hypothesized that H2S inhibits proliferative response of atrial fibroblasts through modulation of potassium channels. Biophysical property of potassium channels in human atrial fibroblasts was examined by whole-cell patch clamp technique and their cellular proliferation in response to H2S was assessed by BrdU assay. Large conductance Ca(2+)-activated K(+) current (BK(Ca)), transient outward K(+) current (I(to)) and inwardly rectifying K(+) current (IK(ir)) were found in human atrial fibroblasts. Current density of BK(Ca) (IC50 = 69.4 µM; n = 6), I(to) (IC50 = 55.1 µM; n = 6) and IK(ir) (IC50 = 78.9 µM; n = 6) was significantly decreased (P < 0.05) by acute exposure to NaHS (a H2S donor) in atrial fibroblasts. Furthermore, NaHS (100-500 µM) inhibited fibroblast proliferation induced by transforming growth factor-ß1 (TGF-ß1; 1 ng/ml), Ang II (100 nM) or 20% FBS. Pre-conditioning of fibroblasts with NaHS decreased basal expression of Kv4.3 (encode I(to)), but not KCa1.1 (encode BK(Ca)) and Kir2.1 (encode IK(ir)). Furthermore, H2S significantly attenuated TGF-ß1-stimulated Kv4.3 and α-smooth muscle actin expression, which coincided with its inhibition of TGF-ß-induced myofibroblast transformation. Our results show that H2S attenuates atrial fibroblast proliferation via suppression of K(+) channel activity and moderates their differentiation towards myofibroblasts.
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Diferenciación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Atrios Cardíacos/efectos de los fármacos , Sulfuro de Hidrógeno/farmacología , Secuencia de Bases , Cartilla de ADN , Atrios Cardíacos/citología , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Hydrogen sulfide (H2S) is an endogenously generated gaseous transmitter known for its cytoprotective effect mediated by the PI3K-Akt signaling pathway. Human induced pluripotent stem cell (hiPSC)-derived mesenchymal stromal cells (MSCs), or hiPSC-MSCs, represent an alternative source of MSCs for autologous cell therapy. The big-conductance Ca(2+)-activated outward K(+) currents (BKCa), known to mediate cell proliferation, have been detected in >80% of hiPSC-MSCs. The present study aimed to explore the effect of H2S on survival and proliferation of hiPSC-MSCs and investigate the mediatory role of BKCa. METHODS: Effects of H2S on proliferation and survival of hiPSC-MSCs were measured by 5-bromo-2-deoxyuridine incorporation, population doubling and cell cycle assays, and by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay and 4'-6-diamidino-2-phenylindole staining, respectively. BKCa was recorded by means of the whole-cell patch-clamp technique. The expressions of KCa 1.1 (encoding BKCa) and apoptosis-related genes were measured by reverse transcriptase-polymerase chain reaction. The phosphorylation of Akt was assessed by Western blot analysis. RESULTS: Exogenously administered NaHS (an H2S donor, 50-300 µmol/L) significantly promoted proliferation of hiPSC-MSCs. NaHS prevented the hypoxia-induced apoptosis and suppressed BKCa currents without altering the expression levels of α- and ß-KCa 1.1. In addition, NaHS increased the phosphorylation of Akt and decreased the expression of Caspase 8 and Bax in hiPSC-MSCs. Paxilline (1 µmol/L), a BKCa blocker, showed similar effects on promoting cell proliferation and phosphorylation of Akt and suppression of apoptotic genes in hiPSC-MSCs. CONCLUSIONS: Our data confirmed that H2S arguments the proliferation and survival of hiPSC-MSCs through activation of the PI3K-Akt pathway and that such effects could be mediated through inhibition of BKCa.
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Sulfuro de Hidrógeno/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Células Madre Mesenquimatosas/efectos de los fármacos , Apoptosis/efectos de los fármacos , Caspasa 8/biosíntesis , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Indoles/farmacología , Canales de Potasio de Gran Conductancia Activados por el Calcio/efectos de los fármacos , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Técnicas de Placa-Clamp , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
Despite optimal medical therapy, cardiovascular disease remains a leading cause of morbidity and mortality worldwide. One emerging therapeutic approach for treatment of cardiomyopathies is stem cell therapy. Use of stem cells for regenerative medicine has quickly evolved over the last decade. On the basis of encouraging pre-clinical results, stem cell therapy has developed rapidly into a potentially promising treatment for ischemic heart disease, myocardial infarction and congestive heart failure. In this review, we summarize the current state-of-the-art of stem cell therapy and compare collective experiences gleaned from trials using intravenous, intra-coronary and intra-myocardial delivery in exacting credible benefits. We discuss implications of clinical outcomes reported in relation to the delivered stem cells as probable destiny therapy for cardiovascular repair.
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Cardiomiopatías/terapia , Regeneración , Trasplante de Células Madre/métodos , Células Madre , Administración Intravenosa , Corazón , Insuficiencia Cardíaca/terapia , Humanos , Infarto del Miocardio/terapia , Medicina RegenerativaRESUMEN
Mitochondrial dysfunction induced by acute cardiac ischemia-reperfusion (IR), may increase susceptibility to arrhythmias by perturbing energetics, oxidative stress production and calcium homeostasis. Although changes in mitochondrial morphology are known to impact on mitochondrial function, their role in cardiac arrhythmogenesis is not known. To assess action potential duration (APD) in cardiomyocytes from the Mitofusins-1/2 (Mfn1/Mfn2)-double-knockout (Mfn-DKO) compared to wild-type (WT) mice, optical-electrophysiology was conducted. To measure conduction velocity (CV) in atrial and ventricular tissue from the Mfn-DKO and WT mice, at both baseline and following simulated acute IR, multi-electrode array (MEA) was employed. Intracellular localization of connexin-43 (Cx43) at baseline was evaluated by immunohistochemistry, while Cx-43 phosphorylation was assessed by Western-blotting. Mfn-DKO cardiomyocytes demonstrated an increased APD. At baseline, CV was significantly lower in the left ventricle of the Mfn-DKO mice. CV decreased with simulated-ischemia and returned to baseline levels during simulated-reperfusion in WT but not in atria of Mfn-DKO mice. Mfn-DKO hearts displayed increased Cx43 lateralization, although phosphorylation of Cx43 at Ser-368 did not differ. In summary, Mfn-DKO mice have increased APD and reduced CV at baseline and impaired alterations in CV following cardiac IR. These findings were associated with increased Cx43 lateralization, suggesting that the mitofusins may impact on post-MI cardiac-arrhythmogenesis.
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Conservadores de la Densidad Ósea , Traumatismos Craneocerebrales , Ratones , Animales , Electrofisiología Cardíaca , IsquemiaRESUMEN
AIMS: Hypertrophic cardiomyopathy (HCM) is characterized by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM. METHODS AND RESULTS: Human cardiomyocytes derived from control-induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy. CONCLUSION: This study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing.
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Cardiomiopatía Hipertrófica/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Peroxidasa/antagonistas & inhibidores , Función Ventricular Izquierda/efectos de los fármacos , Animales , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Cardiomiopatía Hipertrófica/enzimología , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/fisiopatología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Hipertrofia Ventricular Izquierda/enzimología , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/fisiopatología , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/patología , Masculino , Ratones Endogámicos C57BL , Mutación Missense , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Peroxidasa/metabolismo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Dysregulation of matrix synthesis during myocardial fibrosis in post-infarct ventricular remodeling contributes to ventricular dysfunction. Bone marrow stem cell transplantation prevents functional deterioration following myocardial infarction. However, effect of myocardial extracellular matrix (ECM) on stem cell differentiation is poorly understood. We investigate the role of collagen matrices and integrin system in cardiac differentiation and engraftment of stem cells in infarcted myocardium. Sternum-derived bone marrow mesenchymal stem cells (MSCs) were differentiated into cardiomyocyte-like cells (CLCs). They were characterized using RT-PCR, immunofluorescence, flow cytometry and functional integrin neutralization assays. CLCs were injected into peri-infarct borders of injured myocardium of Wistar rats one week following left anterior descending (LAD) artery ligation. Cardiac function was analyzed via pressure-volume relationships. Cardiac differentiated CLCs displayed collagen V specificity, which was absent in undifferentiated MSCs. Collagen V, but not collagen I matrix, promoted attachment, proliferation and cardiac differentiation of CLCs. In contrast to beta(1), alpha(v) integrin contributed minimally in the attachment of CLCs on collagen matrices. However, inhibition of alpha(v)beta(3,) but not alpha(2)beta(1) integrin, selectively attenuated troponin T, sarcomeric alpha-actin and ryanodine 2 receptor gene expression in CLCs. Both MSC and CLC transplantation prevented chamber dilatation and improved contractile function. However, systolic activity in MSC transplanted animals was accompanied by heightened wall stress as demonstrated by elevated myocardial end-diastolic pressure and prolonged tissue relaxation time. Localization of CLCs in the vicinity of collagen V-expressing myofibers promoted their integration into cardiac syncytium. CLCs may facilitate hemodynamic recovery by preserving tissue elasticity in the peri-infarct borders that sustains contractile efficiency for functional recovery in an actively remodeling infarcted myocardium.
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Diferenciación Celular/fisiología , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Células Madre Mesenquimatosas/fisiología , Miocardio/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Células Cultivadas , Colágeno/metabolismo , Femenino , Perfilación de la Expresión Génica , Hemodinámica , Humanos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Miocardio/citología , Ratas , Ratas WistarRESUMEN
Normal cardiac contractile and relaxation functions are critically dependent on a continuous energy supply. Accordingly, metabolic perturbations and impaired mitochondrial bioenergetics with subsequent disruption of ATP production underpin a wide variety of cardiac diseases, including diabetic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, anthracycline cardiomyopathy, peripartum cardiomyopathy, and mitochondrial cardiomyopathies. Crucially, there are no specific treatments for preventing the onset or progression of these cardiomyopathies to heart failure, one of the leading causes of death and disability worldwide. Therefore, new treatments are needed to target the metabolic disturbances and impaired mitochondrial bioenergetics underlying these cardiomyopathies in order to improve health outcomes in these patients. However, investigation of the underlying mechanisms and the identification of novel therapeutic targets have been hampered by the lack of appropriate animal disease models. Furthermore, interspecies variation precludes the use of animal models for studying certain disorders, whereas patient-derived primary cell lines have limited lifespan and availability. Fortunately, the discovery of human-induced pluripotent stem cells has provided a promising tool for modelling cardiomyopathies via human heart tissue in a dish. In this review article, we highlight the use of patient-derived iPSCs for studying the pathogenesis underlying cardiomyopathies associated with metabolic perturbations and impaired mitochondrial bioenergetics, as the ability of iPSCs for self-renewal and differentiation makes them an ideal platform for investigating disease pathogenesis in a controlled in vitro environment. Continuing progress will help elucidate novel mechanistic pathways, and discover novel therapies for preventing the onset and progression of heart failure, thereby advancing a new era of personalized therapeutics for improving health outcomes in patients with cardiomyopathy.
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Cardiomiopatías/metabolismo , Metabolismo Energético , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Antraciclinas/toxicidad , Cardiomiopatías/inducido químicamente , Cardiomiopatías/genética , Cardiomiopatías/patología , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Cardiotoxicidad , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/metabolismo , Cardiomiopatías Diabéticas/patología , Femenino , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/patología , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/patología , Periodo Periparto , Fenotipo , Embarazo , Complicaciones Cardiovasculares del Embarazo/genética , Complicaciones Cardiovasculares del Embarazo/metabolismo , Complicaciones Cardiovasculares del Embarazo/patologíaRESUMEN
Stem cell-based therapy for patients with post-infarct heart failure is a relatively new and revolutionary concept in cardiology. Despite the encouraging results from pre-clinical studies, outcomes from most clinical trials remain moderately positive while the clinical benefits are largely attributed to transplanted cell-associated paracrine effects in stimulating angiogenesis and protecting endogenous cardiomyocytes. This scenario indicates that there may be a considerably protracted iterative process of conceptual and procedural refinement before true clinical benefits can be fully materialized. At present, many pressing questions regarding cell therapy remain unanswered. In addition to the primary interest in determining the ideal type of stem cells with best cardiogenic potential in vitro and in vivo, there are growing concerns on the impact of the host cardiac milieu on the transplanted cells, including their survival, migration, engraftment, and trans-differentiation as well as contribution to left ventricular function. Effective cell delivery and tracking methods are central to the unraveling of these questions. To date, cell-delivery modalities are yet to be optimized and strategies for safe and effective assessment of cells transplanted in the recipients are to be established. In this review, we discuss cell delivery and tracking modalities that are adopted in the current pre-clinical and clinical studies. We further discussed emerging technologies that are poised to impact the success of cell therapy.