RESUMEN
AIM: Activity of early embryonic cardiomyocytes relies on spontaneous Ca(2+) oscillations that are induced by interplay between sarcoplasmic reticulum (SR) - Ca(2+) release and ion currents of the plasma membrane. In a variety of cell types, Ca(2+) -activated K(+) current (IK(Ca) ) serves as a link between Ca(2+) signals and membrane voltage. This study aimed to determine the role of IK (Ca) in developing cardiomyocytes. METHODS: Ion currents and membrane voltage of embryonic (E9-11) mouse cardiomyocytes were measured by patch clamp; [Ca(2+) ]i signals by confocal microscopy. Transcription of specific genes was measured with RT-qPCR and Ca(2+) -dependent transcriptional activity using NFAT-luciferase assay. Myocyte structure was assessed with antibody labelling and confocal microscopy. RESULTS: E9-11 cardiomyocytes express small conductance (SK) channel subunits SK2 and SK3 and have a functional apamin-sensitive K(+) current, which is also sensitive to changes in cytosolic [Ca(2+) ]i . In spontaneously active cardiomyocytes, inhibition of IK (Ca) changed action and resting potentials, reduced SR Ca(2+) load and suppressed the amplitude and the frequency of spontaneously evoked Ca(2+) oscillations. Apamin caused dose-dependent suppression of NFAT-luciferase reporter activity, induced downregulation of a pattern of genes vital for cardiomyocyte development and triggered changes in the myocyte morphology. CONCLUSION: The results show that apamin-sensitive IK (Ca) is required for maintaining excitability and activity of the developing cardiomyocytes as well as having a fundamental role in promoting Ca(2+) - dependent gene expression.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Potenciales de la Membrana/fisiología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Potasio/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Apamina/metabolismo , Femenino , Ratones , Músculo Liso/metabolismo , Embarazo , Transcripción GenéticaRESUMEN
AIM: Spontaneous activity of embryonic cardiomyocytes originates from sarcoplasmic reticulum (SR) Ca(2+) release during early cardiogenesis. However, the regulation of heart rate during embryonic development is still not clear. The aim of this study was to determine how endothelin-1 (ET-1) affects the heart rate of embryonic mice, as well as the pathway through which it exerts its effects. METHODS: The effects of ET-1 and ET-1 receptor inhibition on cardiac contraction were studied using confocal Ca(2+) imaging of isolated mouse embryonic ventricular cardiomyocytes and ultrasonographic examination of embryonic cardiac contractions in utero. In addition, the amount of ET-1 peptide and ET receptor a (ETa) and b (ETb) mRNA levels were measured during different stages of development of the cardiac muscle. RESULTS: High ET-1 concentration and expression of both ETa and ETb receptors was observed in early cardiac tissue. ET-1 was found to increase the frequency of spontaneous Ca(2+) oscillations in E10.5 embryonic cardiomyocytes in vitro. Non-specific inhibition of ET receptors with tezosentan caused arrhythmia and bradycardia in isolated embryonic cardiomyocytes and in whole embryonic hearts both in vitro (E10.5) and in utero (E12.5). ET-1-mediated stimulation of early heart rate was found to occur via ETb receptors and subsequent inositol trisphosphate receptor activation and increased SR Ca(2+) leak. CONCLUSION: Endothelin-1 is required to maintain a sufficient heart rate, as well as to prevent arrhythmia during early development of the mouse heart. This is achieved through ETb receptor, which stimulates Ca(2+) leak through IP3 receptors.