Action potential studies were conducted on 4- to 6-day-old monolayer cultures plated directly onto fibronectin-coated 9×22-mm glass coverslips. 18 The cells were preplated to reduce fibroblast proliferation, cultured initially in serum-containing medium, then switched to serum-free medium after 24 hours. A standard trypsin dissociation method was used to prepare newborn ventricle cell cultures. The present study investigated this possibility.Īdult rats were anesthetized with ketamine-xylazine before cardiectomy, and neonatal rats decapitated, in accordance with Institutional Animal Care and Use Committee protocols of Columbia University. Thus, HCN2 activation might differ, for example, when expressed in neonatal versus adult ventricular myocytes. Instead, it seems likely that unidentified accessory proteins or other cellular factors influence the voltage dependence of individual HCN isoforms. 16 17 Thus, the intrinsic voltage dependence of different HCN isoforms is insufficient to explain the functional diversity of native cardiac I f, either regionally or developmentally. However, when HCN2 and HCN4 were expressed in mammalian cell lines, activation voltages differed by <10 mV. This suggests that HCN2 is inherently negatively activating, and its relative abundance determines the activation threshold in different regions of the heart or at different ages. Ventricle contains HCN2 and HCN4, with the HCN2/HCN4 mRNA ratio being greater in adult than newborn. 14 15 Sinus node and Purkinje fibers, in which I f activates at less negative potentials, contain largely HCN1 and HCN4. 11 12 13 Three of these (HCN1, HCN2, and HCN4) are present in heart, and their relative message level varies with region and age. The molecular and cellular bases for the regional variability of activation voltages in the normal adult heart and the regulation of ventricular activation voltage by development remain to be determined, but such understanding is critical to any future therapeutic application of the expressed current in myocardium.įour members of the HCN gene family are currently known. Interestingly, I f activates at less negative voltages in newborn ventricle (−70 mV in rat 8 10 ). However, I f is present in both automatic 1 and nonautomatic 2 3 4 5 6 regions of the heart, and the threshold voltage varies widely among cardiac regions, being least negative in sinus node (−40 mV in rabbit 7 ) and most negative in ventricle (−108 mV or more negative 5 8 9 ). The recent cloning of the HCN gene family, representing the molecular correlate of the pacemaker current I f, raises the possibility of using genetic approaches for the treatment of rhythm disorders. The full text of this article is available at. Thus, ventricular maturational state influences the voltage dependence of expressed HCN2, resulting in distinct physiological impact of expressed channels in neonate and adult myocytes. In adult, where HCN2 activates more negatively, the effect was evident only during anodal excitation, requiring significantly less stimulus energy than control (2149☒66 versus 3140☒79 mV In the neonate, AdHCN2 caused a significant increase in spontaneous rate compared with control (88±5 versus 48±4 bpm). This did not result from developmental differences in basal cAMP, because saturating cAMP in the pipette caused an equivalent positive shift in both preparations. ![]() The expressed current exhibited an 18-mV difference in activation (V 1/2 −95.9☑.9 in adult −77.6☑.6 mV in neonate), comparable to the 22-mV difference between native I f in adult and neonatal cultures (V 1/2 −98.7 versus −77.0 mV). We therefore prepared an adenoviral construct (AdHCN2) of HCN2, the dominant ventricular isoform at either age, and used it to infect neonatal and adult rat ventricular myocytes to investigate the role of maturation on current gating. ![]() ![]() However, heterologously expressed HCN2 and HCN4, the putative molecular correlates of ventricular I f, exhibit only a modest difference in activation voltage. Customer Service and Ordering InformationĪbstract-Ventricular pacemaker current ( I f) shows distinct voltage dependence as a function of age, activating outside the physiological range in normal adult ventricle, but less negatively in neonatal ventricle.Stroke: Vascular and Interventional Neurology.Journal of the American Heart Association (JAHA).Circ: Cardiovascular Quality & Outcomes.Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB).
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