RESUMEN
Mutations in SCO2 are among the most common causes of COX deficiency, resulting in reduced mitochondrial oxidative ATP production capacity, often leading to hypertrophic cardiomyopathy (HCM). To date, none of the recent pertaining reports provide deep understanding of the SCO2 disease pathophysiology. To investigate the cardiac pathology of the disease, we were the first to generate induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) from SCO2-mutated patients. For iPSC generation, we reprogrammed skin fibroblasts from two SCO2 patients and healthy controls. The first patient was a compound heterozygote to the common E140K mutation, and the second was homozygote for the less common G193S mutation. iPSC were differentiated into cardiomyocytes through embryoid body (EB) formation. To test the hypothesis that the SCO2 mutation is associated with mitochondrial abnormalities, and intracellular Ca2+ -overload resulting in functional derangements and arrhythmias, we investigated in SCO2-mutated iPSC-CMs (compared to control cardiomyocytes): (i) the ultrastructural changes; (ii) the inotropic responsiveness to ß-adrenergic stimulation, increased [Ca2+ ]o and angiotensin-II (AT-II); and (iii) the Beat Rate Variability (BRV) characteristics. In support of the hypothesis, we found in the mutated iPSC-CMs major ultrastructural abnormalities and markedly attenuated response to the inotropic interventions and caffeine, as well as delayed afterdepolarizations (DADs) and increased BRV, suggesting impaired SR Ca2+ handling due to attenuated SERCA activity caused by ATP shortage. Our novel results show that iPSC-CMs are useful for investigating the pathophysiological mechanisms underlying the SCO2 mutation syndrome.
Asunto(s)
Cardiomiopatía Hipertrófica/patología , Proteínas Portadoras/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Mitocondriales/metabolismo , Miocitos Cardíacos/metabolismo , Potenciales de Acción/efectos de los fármacos , Adulto , Arritmias Cardíacas/patología , Cafeína/farmacología , Cardiomiopatía Hipertrófica/fisiopatología , Proteínas Portadoras/genética , Diferenciación Celular , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/ultraestructura , Isoproterenol/farmacología , Masculino , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteínas Mitocondriales/genética , Modelos Biológicos , Chaperonas Moleculares , Mutación/genética , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/ultraestructuraRESUMEN
Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the "voltage-clock," where the hyperpolarization-activated funny current If causes diastolic depolarization that triggers action potential cycling; and (ii) the "Ca(2+) clock," where cyclical release of Ca(2+) from Ca(2+) stores depolarizes the membrane during diastole via activation of the Na(+)-Ca(2+) exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current- and voltage-clamp recordings from the same cell showed the so-called "voltage and Ca(2+) clock" pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the "voltage or Ca(2+) clock" produced a similar depolarization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca(2+)-activated intermediate K(+) conductance (IK(Ca), KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IK(Ca) inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IK(Ca) appears to play a crucial role in human embryonic cardiac automaticity.
Asunto(s)
Células Madre Embrionarias/citología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Nodo Sinoatrial/citología , Nodo Sinoatrial/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Línea Celular , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Humanos , Modelos Cardiovasculares , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Nodo Sinoatrial/efectos de los fármacos , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
In view of the therapeutic potential of cardiomyocytes derived from induced pluripotent stem (iPS) cells (iPS-derived cardiomyocytes), in the present study we investigated in iPS-derived cardiomyocytes, the functional properties related to [Ca(2+) ](i) handling and contraction, the contribution of the sarcoplasmic reticulum (SR) Ca(2+) release to contraction and the b-adrenergic inotropic responsiveness. The two iPS clones investigated here were generated through infection of human foreskin fibroblasts (HFF) with retroviruses containing the four human genes: OCT4, Sox2, Klf4 and C-Myc. Our major findings showed that iPS-derived cardiomyocytes: (i) express cardiac specific RNA and proteins; (ii) exhibit negative force-frequency relations and mild (compared to adult) post-rest potentiation; (iii) respond to ryanodine and caffeine, albeit less than adult cardiomyocytes, and express the SR-Ca(2+) handling proteins ryanodine receptor and calsequestrin. Hence, this study demonstrates that in our cardiomyocytes clones differentiated from HFF-derived iPS, the functional properties related to excitation-contraction coupling, resemble in part those of adult cardiomyocytes.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/metabolismo , Animales , Cafeína/farmacología , Calcio/metabolismo , Calsecuestrina/genética , Calsecuestrina/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Prepucio/citología , Expresión Génica , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones SCID , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/metabolismo , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Factores de Transcripción SOXB1/genética , Retículo Sarcoplasmático/metabolismo , Teratoma/metabolismo , Teratoma/patologíaRESUMEN
Our recent studies demonstrated that propargylamine derivatives such as rasagiline (Azilect, Food and Drug Administration-approved anti-Parkinson drug) and its S-isomer TVP1022 protect cardiac and neuronal cell cultures against apoptotic-inducing stimuli. Studies on structure-activity relationship revealed that their neuroprotective effect is associated with the propargylamine moiety, which protects mitochondrial viability and prevents apoptosis by activating Bcl-2 and protein kinase C-epsilon and by down-regulating the proapoptotic protein Bax. Based on the established cytoprotective and neuroprotective efficacies of propargylamine derivatives, as well as on our recent study showing that TVP1022 attenuates serum starvation-induced and doxorubicin-induced apoptosis in neonatal rat ventricular myocytes (NRVMs), we tested the hypothesis that TVP1022 will also provide protection against doxorubicin-induced NRVM functional derangements. The present study demonstrates that pretreatment of NRVMs with TVP1022 (1 microM, 24 h) prevented doxorubicin (0.5 microM, 24 h)-induced elevation of diastolic [Ca(2+)](i), the slowing of [Ca(2+)](i) relaxation kinetics, and the decrease in the rates of myocyte contraction and relaxation. Furthermore, pretreatment with TVP1022 attenuated the doxorubicin-induced reduction in the protein expression of sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase, Na(+)/Ca(2+) exchanger 1, and total connexin 43. Finally, TVP1022 diminished the inhibitory effect of doxorubicin on gap junctional intercellular coupling (measured by means of Lucifer yellow transfer) and on conduction velocity, the amplitude of the activation phase, and the maximal rate of activation (dv/dt(max)) measured by the Micro-Electrode-Array system. In summary, our results indicate that TVP1022 acts as a novel cardioprotective agent against anthracycline cardiotoxicity, and therefore potentially can be coadmhence, theinistered with doxorubicin in the treatment of malignancies in humans.
Asunto(s)
Cardiotónicos/farmacología , Doxorrubicina/antagonistas & inhibidores , Indanos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Animales , Animales Recién Nacidos , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Cardiotoxinas/farmacología , Células Cultivadas , Conexina 43/metabolismo , Doxorrubicina/efectos adversos , Uniones Intercelulares/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Hepatitis C virus (HCV) is a major public health concern, with over 70 million people infected worldwide, who are at risk for developing life-threatening liver disease. No vaccine is available, and immunity against the virus is not well-understood. Following the acute stage, HCV usually causes chronic infections. However, ~30% of infected individuals spontaneously clear the virus. Therefore, using HCV as a model for comparing immune responses between spontaneous clearer (SC) and chronically infected (CI) individuals may empower the identification of mechanisms governing viral infection outcomes. Here, we provide the first in-depth analysis of adaptive immune receptor repertoires in individuals with current or past HCV infection. We demonstrate that SC individuals, in contrast to CI patients, develop clusters of antibodies with distinct properties. These antibodies' characteristics were used in a machine learning framework to accurately predict infection outcome. Using combinatorial antibody phage display library technology, we identified HCV-specific antibody sequences. By integrating these data with the repertoire analysis, we constructed two antibodies characterized by high neutralization breadth, which are associated with clearance. This study provides insight into the nature of effective immune response against HCV and demonstrates an innovative approach for constructing antibodies correlating with successful infection clearance. It may have clinical implications for prognosis of the future status of infection, and the design of effective immunotherapies and a vaccine for HCV.