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(Circulation Research. 1999;85:e7-e16.)
© 1999 American Heart Association, Inc.

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Ca²⁺ Influx Through Ca²⁺ Channels in Rabbit Ventricular Myocytes During Action Potential Clamp

Influence of Temperature

José L. Puglisi, Weilong Yuan, José W. M. Bassani, Donald M. Bers

From the Department of Physiology (J.L.P., W.Y., D.M.B.), Loyola University Chicago, Maywood, Ill; Departamento de Engenharia Biomédica (J.L.P., J.W.M.B.), Universidade Estadual de Campinas, UNICAMP, Brazil.

Correspondence to Donald M. Bers, PhD, Department of Physiology, Loyola University Medical School, 2160 South First Ave, Maywood, IL 60153. E-mail dbers{at}luc.edu

Abstract—Ca²⁺ influx via Ca²⁺ current (I_Ca) during the action potential (AP) was determined at 25°C and 35°C in isolated rabbit ventricular myocytes using AP clamp. Contaminating currents through Na⁺ and K⁺ channels were eliminated by using Na⁺- and K⁺-free solutions, respectively. DIDS (0.2 mmol/L) was used to block Ca²⁺-activated chloride current (I_Cl(Ca)). When the sarcoplasmic reticulum (SR) was depleted of Ca²⁺ by preexposure to 10 mmol/L caffeine, total Ca²⁺ entry via I_Ca during the AP was 12 µmol/L cytosol (at both 25°C and 35°C). Similar Ca²⁺ influx at 35°C and 25°C resulted from a combination of higher and faster peak I_Ca, offset by more rapid I_Ca inactivation at 35°C. During repeated AP clamps, the SR gradually fills with Ca²⁺, and consequent SR Ca²⁺ release accelerates I_Ca inactivation during the AP. During APs and contractions in steady state, total Ca²⁺ influx via I_Ca was reduced by 50% but was again unaltered by temperature (5.6±0.2 µmol/L cytosol at 25°C, 6.0±0.2 µmol/L cytosol at 35°C). Thus, SR Ca²⁺ release is responsible for sufficient I_Ca inactivation to cut total Ca²⁺ influx in half. However, because of the kinetic differences in I_Ca, the amount of Ca²⁺ influx during the first 10 ms, which presumably triggers SR Ca²⁺ release, is much greater at 35°C. I_Ca during a first pulse, given just after the SR was emptied with caffeine, was subtracted from I_Ca during each of 9 subsequent pulses, which loaded the SR. These difference currents reflect I_Ca inactivation due to SR Ca²⁺ release and thus indicate the time course of local [Ca²⁺] in the subsarcolemmal space near Ca²⁺ channels produced by SR Ca²⁺ release (eg, maximal at 20 ms after the AP activation at 35°C). Furthermore, the rate of change of this difference current may reflect the rate of SR Ca²⁺ release as sensed by L-type Ca²⁺ channels. These results suggest that peak SR Ca²⁺ release occurs within 2.5 or 5 ms of AP upstroke at 35°C and 25°C, respectively. I_Cl(Ca) might also indicate local [Ca²⁺], and at 35°C in the absence of DIDS (when I_Cl(Ca) is prominent), peak I_Cl(Ca) also occurred at a time comparable to the peak I_Ca difference current. We conclude that SR Ca²⁺ release decreases the Ca²⁺ influx during the AP by 50% (at both 25°C and 35°C) and that changes in I_Ca (and I_Cl(Ca)), which depend on SR Ca²⁺ release, provide information about local subsarcolemmal [Ca²⁺]. The full text of this article is available at http://www.circresaha.org.

Key Words: Ca²⁺ current • cardiac muscle • excitation-contraction coupling • sarcoplasmic reticulum Ca²⁺ release

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