Mechanisms of force inhibition by halothane and isoflurane in intact rat cardiac muscle

1. We investigated the mechanisms underlying the negative inotropic effect of the volatile anaesthetics halothane and isoflurane using twenty-two intact, right ventricular trabeculae of rat. [Ca2+](i) was measured qualitatively using either fluo-3 or fura-2, loaded into the cytosol via the acetoxymethyl (AM) ester form. Diastolic sarcomere length was adjusted to 2.1-2.2 mu m and experiments were performed at 21-23 degrees C. 2. Halothane (0.25-3 %) and isoflurane (0.48-4 %) produced dose-dependent decreases in the amplitudes of the intracellular Ca2+ transients and twitch force. When the fluorescent Ca2+ indicator signals were corrected for changes in autofluorescence, neither volatile anaesthetic significantly changed diastolic [Ca2+](i). 3. The ability of halothane and isoflurane to induce Ca2+ release from the sarcoplasmic reticulum of quiescent trabeculae was examined. When the superfusate was Ca2+ and Na+ free (thereby preventing Na+-Ca2+ exchange and Ca2+ influx), 2% halothane, but not 4% isoflurane, evoked a transient increase in [Ca2+](i). 4. Halothane and isoflurane produced reversible, dose-dependent changes in cellular autofluorescence, the pattern of which was consistent with an increase in concentration of the reduced forms of nicotinamide adenine nucleotides and flavoproteins. This observation supports the putative inhibitory action of volatile anaesthetics at the site of Complex I of the mitochondrial electron transport chain. 5. Addition of the fatty acid hexanoate, a substrate that can be metabolized in the face of Complex I inhibition, did not appreciably attenuate the anaesthetic-induced negative inotropy; however, it greatly diminished autofluorescence changes. 6. To determine whether direct actions of the volatile anaesthetics on the contractile system contributed to the negative inotropy, external [Ca2+] was varied to modulate the amplitude of the Ca2+ transient. In the presence of 2% halothane or 4% isoflurane, restoration of the peak Ca2+ transient to control levels did not restore peak force. Moreover, halothane (1%) and isoflurane (1.6%) each reduced maximal Ca2+-activated force (attained using ryanodine tetani and a high external [Ca2+]) by around 15%. 7. We conclude that the negative inotropic actions of halothane and isoflurane on intact cardiac muscle reflect both reduced availability of Ca2+ and decreased responsiveness of the contractile system to Ca2+. The inhibitory action of the volatile anaesthetics on mitochondrial function does not contribute significantly to the negative inotropy but may lead to changes in cellular autofluorescence and misinterpretation of fluorescent Ca2+ indicator signals.