Ca2+ influx through Ca2+ channels in rabbit ventricular myocytes during action potential clamp - Influence of temperature

Abstract

Ca2+ influx via Ca2+ current (I-Ca) during the action potential (AP) was determined at 25 degrees C and 35 degrees 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 approximate to 12 mu mol/L cytosol (at both 25 degrees C and 35 degrees C). Similar Ca2+ influx at 35 degrees C and 25 degrees C resulted from a combination of higher and faster peak I-Ca offset by more rapid I-Ca inactivation at 35 degrees 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 approximate to 50% but was again unaltered by temperature (5.6+/-0.2 mu mol/L cytosol at 25 degrees C, 6.0+/-0.2 mu mol/L cytosol at 35 degrees C). Thus, SR Ca2+ release is responsible for sufficient I, 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 degrees C. Ic, during a first pulse, given just after the SR was emptied with caffeine, was subtracted from Ic, during each of 9 subsequent pulses, which loaded the SR. These difference currents reflect lc, 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 degrees 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 degrees C and 25 degrees C, respectively. I-Cl(Ca) might also indicate local [Ca2+], and at 35 degrees 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 approximate to 50% (at both 25 degrees C and 35 degrees 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.