Activation and inactivation properties of normal and denervated mammalian skeletal muscle

The effect of denervation on the activation and inactivation of excitationcontraction coupling in fastand slow-twitch rat skeletal muscle was assessed using potassium (K-) contractures and the two-microelectrode point voltage clamp technique with visual endpoint determination of contraction threshold. Solutions containing sulphate as the impenneant anion were used to obtain better control of depolarization in K-contracture and voltage clamp experiments. Denervation shifted the voltage dependence of the half maximal activation of K-contractures by -12 mV in fast-twitch extensor digitorum longus (EDL)/compared to only a -1.3 mV in slow-twitch soleus fibres. The overall effect of denervation was to make the activation curves similar for the two types of muscle. The point of initial tension generation in activation curves has been used to predict contraction thresholds for the muscle fibres. Following denervation, changes in the °l<~pe of the activation curves^gges&S^tfiS ffie’Sntract’ thresholds for soleus and EDL became similar. To test/this observation, the twomicroelectrode point voltage clamp technique was used to accurately determine contraction thresholds for a variety of test pulse durations (5-500 msec) in normal and denervated soleus and EDL muscle fibres. Results demonstrated that contraction thresholds were significantly more negative in soleus compared to EDL (by about 13 mV). A shift in contraction thresholds at all pulse durations was observed following denervation. Contraction thresholds for soleus became more positive (4 mV) while those for EDL became more negative (6 mV), thereby making contraction thresholds in the two muscles similar. The effect of conditioning depolarizations on the peak height for test contractures induced by exposure to solutions containing 200 mM K+ (200K) was examined in normal and denervated preparations. Only small negative shifts (1-2 mV) in the voltages for half maximal inactivation were observed between normal and denervated muscle (both soleus and EDL), and the slope factors were hardly altered. An extensive study was carried out to examine the membrane potential, Vt, at which threshold contractions were elicited by brief test pulses in polarized and depolarized EDL, soleus and stemomastoid muscle fibres. The effects of test pulse duration, inactivation of E-C coupling and denervation were studied. The Vtholding potential curve for test pulses with durations of 10 msec to 50 msec demonstrated (a) a shallow negative slope component with a minimum Vt at a holding potential of -40 mV in soleus fibres, or -35 mV in EDL and stemomastoid fibres and (b) a steep positive slope component at more positive holding potentials. Characteristics of both components of the V^-holding potential curve were examined. It was concluded that the effectsof depolarization on threshold contractions elicited by brief test pulses were very different from, and could not be predicted by, the effects of depolarization on maximum 200K contracture tension. The full time course of maximal contractures in 200 mM K+ was examined in normal and denervated EDL and soleus. Of particular interest was the decay of contracture tension brought about by the inactivation of E-C coupling. It was found that 200K contractures in normal soleus and EDL decayed more rapidly if preceded by a 5, 10 or 30 minute conditioning depolarization in 30K. The decay of contractures in denervated muscle were slower than normal and five minute conditioning depolarizations had a lesser effect on the decay of 200K contracture tension in denervated muscle. The results are described by a five state model for activation of the voltage sensor for E-C coupling which includes two inactivated states. Denervation of EDL resulted in a distinctive biphasic decay of 200K contracture tension which was interpreted in terms of the unmasking of a second inactive state. The results show that denervation has a strong effect on the time course and voltage dependence of contraction. Most of these effects can be explained by an effect of denervation on the voltage sensor for E-C coupling. In the above experiments, the osmolarity and ionic strength of the high potassium solutions were elevated and the same as that of the 200K solution. To examine the effect the high ionic strength had on K-contractures, sulphate was replaced by methanesulphonate in some experiments. Results indicated a -11 mV shift in the voltage for half maximal activation in methanesulphonate compared to results obtained with sulphate containing solutions. A hyperpolarization of the membrane potential in most elevated potassium solutions was also observed. The results did not show whether sulphate or methanesulphonate produced anomalous effects on the voltage activation of contraction. Time did not permit a further investigation of this point. Finally, a series of voltage clamp experiments were carried out to examine the effects of the perchlorate anion on the contraction threshold for soleus, for a variety of test pulse durations (5-500 msec). Results demonstrated a progressive and parallel negative shift in the contraction threshold upon exposure to solutions containing 0 mM, 2 mM and 10 mM perchlorate. TABLE OF CONTENTS iii Acknowledgements Abstract i Table of contents iii Chapter 1 GENERAL INTRODUCTION 1.1 Structure of skeletal muscle 1 1.2 The contractile mechanism 1 1.3 Skeletal muscle sarcolemma 2 1.4 The transverse (T-) tubule 3 1.5 Distribution of ion channels in the sarcolemma and T-system 3 1.6 The skeletal muscle action potential 4 1.7 The triad 6 1.8 Protein structure of the triad 6 1.8. 1 The dihydropyridine (DHP) receptor of the T-tubule 6 1.8. 2 The ryanodine receptor of the terminal cistemae 8 1.9 Excitation-contraction coupling 10 1.9. 1 Voltage dependence of activation 10 1.9. 2 Asymmetric charge movement 10 1.9. 3 Components of asymmetric charge movement 11 1.9. 4 Charge movement and the DHP-sensitive Ca^+ channel 12 1.9. 5 The role of the DHP receptor in E-C coupling 12 1.10 Models for excitation contraction coupling 13 1.11 Historical perspectives on activation and inactivation of excitation-contraction coupling 16 1. 11.1 Contractile inactivation 16 1.11.2 Force membrane potential relationship 17 1.11.3 Effects of external calcium on force production and inactivation 17 1.11.4 Caffeine and contractile inactivation 18 1.11.5 Contractile inactivation under voltage clamp conditions 18 1.11.6 Effects of temperature on inactivation and repriming 19 1.11.7 Further effects of low [Ca^ + ]0 and other divalent cations on activation and inactivation of E-C coupling 19 1.11.8 Characterization of E-C coupling processes by modification of the response of the voltage sensor 21 1.12.1 Activation and inactivation of asymmetric charge movement 24 1.12.2 Effects of low external calcium and calcium channel blockers on charge movement 25 1.12.3 Effects of surgical and drug treatment on charge movement 26 1.13 Effects of denervation on properties of skeletal muscle 27 1.14 Aims of the experiments 28 Chapter 2 GENERAL METHODS 2.1 Biological preparations 29 2.2 Macrodissection 29 2.3 Microdissection 30 2.4 Denervation surgery 31 2.5 Solutions 31 2.6 Potassium contracture technique 33 2.7. 1 Two-microelectrode voltage clamp 34 2.7. 2 Capacitance neutralization 35 2.7. 3 Contraction threshold measurement 35 28 S+afis-bicoJl -tesHskj procedures Chapter 3 ACTIVATION AND INACTIVATION OF CONTRACTION IN NORMAL AND DENERVATED RAT SOLEUS AND EDL 3.