Voltage Clamp Analysis of Embryonic Heart Cell Aggregates

I N T R O D U C T I O N T h e voltage c lamp technique has been used by n u m e r o u s investigators to describe the voltageand t i me -dependen t conductances in cardiac tissue. T h e main conclusion f rom these analyses is that the ionic cur ren ts that under l ie the action potent ial in the hear t are m o r e n u m e r o u s and complex than in the classic squid axon (for reviews, see Trau twe in , 1973; McAllister et al., 1975; N o m a and Ir isawa, 1976; Beeler and Reuter , 1977; T rau twe in and McDonald, 1978). A major p rob lem in the quanti tat ive in te rpre ta t ion o f voltage c lamp data in hear t tissue arises out o f the multicellular na tu re of the p repara t ion . H e a r t muscle is com pos ed of individual cells connec ted by junc t ions of low but vary ing resistance, into fibers o f complex geome t ry (DeHaan and Fozzard, 1975; Kensler et al., 1977). Substantial voltage gradients may exist within such fibers, and currents crossing the m e m b r a n e o f one cell may d i f fe r f r o m those f lowing in a distant cell. T h e p rob l em of electrical inhomogene i ty of cardiac tissue and the limitations it places on voltage c lamp analyses have been d o c u m e n t e d and discussed at length. I t is general ly ag reed that, because o f the multicellular na ture o f hear t muscle, it has not been possible to control ideally the m e m b r a n e potential o f any p repa ra t ion . Thus , there r ema in substantial uncertaint ies J. GEN. PHYSIOL. 9 The Rockefeller University Press 9 0022-1295[79/02/0175-2451.00 175 Volume 73 February 1979 175-198 on Jauary 6, 2018 jgp.rress.org D ow nladed fom 176 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y 9 V O L U M E 73 9 1 9 7 9 regarding the absolute magnitude, kinetics, and voltage dependence of the various action currents (Johnson and Lieberman, 1971; Fozzard and Beeler, 1975; RamSn et al., 1975; Attwell and Cohen, 1977). Nevertheless, an understanding of the ionic currents in cardiac muscle is beginning to emerge from application of the voltage clamp. The action potential shape recorded from mammalian ventricular tissue has been reconstructed on the basis of two inward and two outward current systems for which (with the exception of activation kinetics for the fast inward current) experimental evidence exists (Beeler and Reuter, 1977; Trautwein and McDonald, 1978); that of the rhythmically active Purkinje fiber has been computed using nine distinguishable currents (McAllister et al., 1975). We have recendy reviewed the relations between the nine conductances of the McAllister-Noble-Tsien model and the spontaneous oscillatory properties of the heart (DeHaan and DeFelice, 1978). All of the above analyses of cardiac membrane currents have been performed on adult heart tissue. Until now, voltage clamp data defining current-voltage relationships in embryonic cardiac preparations have not been available. Evidence concerning the identity and kinetics of ionic currents in such preparations has been indirect, based upon action potential parameters and the sensitivity of electrical events to (adult) current inhibitors. For example, tetrodotoxin (TTX), which specifically blocks the fast-transient inward sodium current (/Na) in nerve, skeletal muscle, and adult cardiac tissue (reviewed by Narahashi, 1974), has no effect on hearts from embryonic chicks, 2-4 d of age, even at concentrations up to 3 x 10 -5 M. However, after 7 d of development, spontaneous activity can be inhibited by TTX concentrations as low as 3 x 10 -s M (Ishima, 1968; Shigenobu and Sperelakis, 1971; McDonald et al., 1972). On the other hand, the organic compounds, verapamil and D600, which suppress the slow inward current (I~) in adult cardiac tissue (Kohlhardt et al., 1972; Kass and Tsien, 1975), can abolish action potentials in hearts from embryos up to 3 d of age, whereas in hearts 7 d or older, these drugs merely reduce the amplitude and duration of the plateau phase; they do not affect action potential generation at these later stages (Shigenobu et al., 1974; McDonald and Sachs, 1975). In this paper we report on a voltage clamp analysis of a heart tissue model system consisting of a spheroidal aggregate of embryonic ventricular cells in culture (Sachs and DeHaan, 1973). We have shown previously that the cells within such an aggregate are dghdy coupled electrically. The entire aggregate membrane appears to be virtually isopotendal during the voltage changes produced by injecting small current pulses through an intracellular micropipette (Clay et al., 1978), although it deviates somewhat from uniformity during the fast rise time of an action potential (DeHaan and Fozzard, 1975). Junctional impedance between cells is <10% of transmembrane impedance and is independent of intercellular voltage gradients in the range of I0-5-I0 -~ V, from DC to 160 Hz (DeFelice and DeHaan, 1977). In the present report, we test further the degree of spatial homogeneity in this preparation under voltage clamp conditions, when substantial currents are applied in fast-rising steps. With the aid of an exploring voltage electrode, we show that deviation from voltage homogeneity during a clamp step is comparable to that achieved in adult cardiac on Jauary 6, 2018 jgp.rress.org D ow nladed fom NATHAN ANn DEHAAN Voltage Clamp Analysis of Embryonic Heart Cell Aggregates 177 prepara t ions . We also demons t r a t e two kinetically and pharmacologica l ly distinct c o m p o n e n t s o f inward cu r ren t and a delayed ou tward cu r r en t with p roper t i es similar to those observed in adul t hear t tissue. T h e advantages o f spherical geome t ry and small size for voltage c lamp analysis have also been exploi ted in a cul tured p r epa ra t i on of neurob las toma cells, 60-110 /.tin in d i ame te r (Moolenaar and Spector , 1978).