Mark J. Cody

Comparison of Volatile Anesthetic Effects on Actin–Myosin Cross-bridge Cycling in Neonatal versus Adult Cardiac Muscle

Background: The neonatal myocardium is more sensitive to volatile anesthetics compared with adults. The greater myocardial sensitivity of neonates may be attributable to greater anesthetic effect on force regulation at the level of the cross-bridge. In the current study, the authors compared the effects of 1 and 2 minimum alveolar concentration (MAC) halothane and sevoflurane on cardiac muscle from 0- to 3-day-old (neonate) and 84-day-old (adult) rats. Methods: Triton X-100–skinned muscle strips were maximally activated at pCa (negative logarithm of the Ca2+ concentration) of 4.0, and the following were measured in the presence or absence of anesthetic: Rate of force redevelopment after rapid shortening and restretching (ktr) and isometric stiffness at maximal activation and in rigor. The fraction of attached cross-bridges (&agr;fs) and apparent rate constants for cross-bridge attachment (fapp) and detachment (gapp) were calculated assuming a two-state model for cross-bridge cycling. Anesthetic-induced changes in the mean stiffness per cross-bridge were also estimated from values in rigor versus maximum activation in the presence or absence of anesthetic. Results: Neonatal cardiac muscle displayed significantly smaller &agr;fs, slower ktr, and slower fapp compared with adult cardiac muscle; however, gapp was not significantly different. Halothane, and sevoflurane to a significantly lesser extent, decreased &agr;fs, fapp, and the mean force per cross-bridge and increased gapp to a greater extent in neonates. Conclusions: These data indicate that weaker force production in neonatal cardiac muscle involves, at least in part, less efficient cross-bridge cycling kinetics. The authors conclude that the greater myocardial sensitivity of neonates to volatile anesthetics reflects, at least in part, a direct inhibition of cross-bridge cycling, especially the rates of cross-bridge attachment and detachment.

Effects of volatile anesthetics on sarcolemmal calcium transport and sarcoplasmic reticulum calcium content in isolated myocytes.

Background The surface membrane Ca2+–adenosine triphosphatase and Na+–Ca2+ exchanger transport Ca2+ out of the ventricular myocyte, competing for cytosolic Ca2+ with the Ca2+-adenosine triphosphatase located in the sarcoplasmic reticulum. In this study the authors examined the effects of halothane, isoflurane, and sevoflurane on Ca2+ extrusion from the cell and sarcoplasmic reticulum Ca2+ content. Methods Single myocytes from the right ventricular free wall of adult male ferret hearts were isolated, loaded with the acetoxymethyl ester of the fluorescent Ca2+ indicator fluo-3, and electrically stimulated at 0.25 Hz to reach a steady state level of intracellular Ca2+ stores. The effects of halothane, isoflurane, and sevoflurane (1 minimum alveolar concentration) on the peak and rate of decline of the Ca2+ transient induced by 10 mm caffeine were examined. The peak was used as an index of sarcoplasmic reticulum Ca2+ content, and the rate of decline was used to monitor Ca2+ extrusion from the cell. Results During control conditions, halothane reduced the Ca2+ content of the sarcoplasmic reticulum, isoflurane maintained it, and sevoflurane caused it to increase. Halothane did not affect Ca2+ extrusion from the cell, but both isoflurane and sevoflurane inhibited it. When Na+–Ca2+ exchange was inhibited by ionic substitution, isoflurane and sevoflurane still reduced the rate of Ca2+ efflux from the cell. However, when the sarcolemmal Ca2+–adenosine triphosphatase was inhibited by carboxyeosin, isoflurane and sevoflurane had no effect on Ca2+ efflux. Conclusions These results suggest that isoflurane and sevoflurane inhibit Ca2+ transport from the cell via the sarcolemmal Ca2+–adenosine triphosphatase. This effect seems to counteract the decrease in Ca2+ influx through sarcolemmal L-type Ca2+ channels and maintains sarcoplasmic reticulum Ca2+ stores.

Effects of Isoflurane on Intracellular Calcium and Myocardial Crossbridge Kinetics in Tetanized Papillary Muscles

Background Isoflurane depresses the intracellular Ca2+ transient and force development during a twitch, but its effects on crossbridge cycling rates are difficult to predict because of the transient nature of the twitch. Measurements of the effects of isoflurane on crossbridge cycling kinetics during tetanic contractions, which provide a steady state level of activation in intact cardiac muscle, have not been previously reported. Methods Ferret right ventricular papillary muscles were isolated, and superficial cells were microinjected with the bioluminescent photoprotein aequorin to monitor the intracellular Ca2+ concentration. The rate of tension redevelopment (kTR) was measured during steady state isometric activation (tetanic stimulation, frequency 20 Hz, 1 &mgr;m ryanodine, temperature = 30°C) in the absence of isoflurane (2, 6, and 12 mm extracellular [Ca2+]) and in the presence of 0.5, 1.0, and 1.5 minimum alveolar concentration isoflurane (12 mm extracellular [Ca2+]). Results Intracellular [Ca2+], isometric force, and kTR all increased when the extracellular [Ca2+] increased. Isoflurane (0.5, 1.0, and 1.5 minimum alveolar concentration) caused intracellular [Ca2+], isometric force, and kTR to decrease in a dose-dependent manner in the presence of 12 mm extracellular [Ca2+]. In the presence of increasing concentrations of isoflurane, the relation between intracellular [Ca2+] and force remained unchanged, whereas the relation between intracellular [Ca2+] and kTR was shifted toward higher [Ca2+]. Conclusions These results indicate that isoflurane depresses myocardial crossbridge cycling rates. It appears that this effect is partially mediated by a decrease in the intracellular [Ca2+]. However, additional mechanisms must be considered to explain the shift of the relation between intracellular [Ca2+] and kTR toward higher [Ca2+].

Comparison of cross-bridge cycling kinetics in neonatal vs. adult rat ventricular muscle

The developmental shift in contractile protein isoform expression in the rodent heart likely affects actin-myosin cross-bridge interactions. We compared the Ca2+ sensitivity for force generation and cross-bridge cycling kinetics in neonatal (postnatal days 0–3) and adult (day 84) rats. The force-pCa relationship was determined in Triton-X skinned muscle bundles activated at pCa 9.0 to 4.0. In strips maximally activated at pCa 4.0, the following parameters of cross-bridge cycling were measured: (1) rate of force redevelopment following rapid shortening and restretching (ktr); and (2) isometric stiffness at maximal activation and in rigor. The fraction of attached cross-bridges (αfs) and apparent rate constants for cross-bridge attachment (fapp) and detachment (gapp) were derived assuming a two-state model for cross-bridge cycling. Compared to the adult, the force-pCa curve for neonatal cardiac muscle was significantly shifted to the left. Neonatal cardiac muscle also displayed significantly smaller αfs, slower ktr and fapp; however, gapp was not significantly different between age groups. These data indicate that weaker force production in neonatal cardiac muscle involves, at least in part, less efficient cross-bridge cycling kinetics.

Effects of isoflurane and sevoflurane on intracellular calcium and contractility in pressure-overload hypertrophy

Background: Depression of myocardial contractility as a result of isoflurane appears to be greater in myocardial hypertrophy, and the cellular basis for this difference in susceptibility is not clear. In this study we examined the effects of isoflurane and sevoflurane on contractility and intracellular calcium in an animal model of pressure-overload hypertrophy. Methods: Pressure-overload hypertrophy was established in young male ferrets by banding the main pulmonary artery for 1 month and the effects of isoflurane and sevoflurane on contractility and intracellular calcium ([Ca2+]i) were examined in isolated right ventricular papillary muscles, trabeculae, and myocytes. Intracellular calcium was measured with the bioluminescent photoprotein aequorin in isolated papillary muscles, and also with the fluorescent indicator fluo-3 in isolated ventricular myocytes. In addition, Ca2+ sensitivity was assessed in isolated trabeculae after disruption of the surface membrane with a nonionic detergent (skinned fibers). Results: In the presence of isoflurane and sevoflurane, papillary muscles from banded animals exhibited a greater depression of contractility and isolated ventricular myocytes showed a greater decrease in peak [Ca2+]i. Furthermore, baseline calcium sensitivity was decreased and the slope of the relationship between [Ca2+] and force was increased in skinned trabeculae from banded animals. Isoflurane decreased calcium sensitivity in trabeculae from both normal and banded animals. Conclusions: These results suggest that changes in [Ca2+]i and altered calcium sensitivity are both responsible for the exaggerated effects of some volatile anesthetics on contractility in pressure-overload hypertrophy.