This study investigated the effects of the oxidants hydrogen peroxide (H2O2) and 2,2′‐dithiodipyridine (DTDP), and reductants, glutathione (GSH) and dithiothreitol (DTT), on the properties of the contractile apparatus of rat fast‐ and slow‐twitch skeletal muscle fibres, in order to assess how oxidation affects muscle function. Skinned muscle fibres were activated in heavily‐buffered Ca2+ solutions. The force‐[Ca2+] relationship before and after various treatments was fitted by a Hill curve described by the maximum Ca2+‐activated force, pCa50 (‐log10[Ca2+] giving half‐maximum force) and nH (the Hill coefficient). Exposing freshly skinned fibres to strong reducing conditions (i.e. 10 mm DTT or 5 mm GSH) had little if any effect on Ca2+ sensitivity (pCa50 or nH). The effect of oxidants H2O2 and DTDP depended on whether the fibre was relaxed (in pCa > 9) or activated during the exposure. In both fast‐ and slow‐twitch fibres a 5 min exposure to 10 mm H2O2 at pCa > 9 had no effect on pCa50, causing only a reduction in nH. In contrast, when fast‐twitch fibres were activated in the presence of 10 mm H2O2 (or 100 μm DTDP) there was a substantial increase in pCa50 (by ≈0.06 and 0.1, respectively), as well as larger decreases in nH than occurred in relaxed fibres, with all effects being reversed by DTT (10 mm, 10 min). In slow‐twitch soleus fibres, the activation‐dependent effect of DTDP was even greater (pCa50 increased by ≈0.35), and it was found that the rate of reversal in DTT was also increased by activation. A separate important phenomenon was that fast‐twitch fibres that had been oxidised with H2O2 or DTDP (while either relaxed or activated) showed a paradoxical increase in Ca2+ sensitivity (≈0.04 and 0.25 increase in pCa50, respectively) when briefly exposed to the endogenous reductant GSH (5 mm, 2 min). This effect was reversed by DTT or longer (> 20 min) exposure to GSH, did not occur in slow‐twitch soleus fibres, and may contribute to post‐tetanic potentiation in fast‐twitch muscle. Maximum force was not affected by any of the above treatments, whereas exposure to a high concentration of DTDP (1 mm) did greatly reduce force production. These findings reveal a number of novel and probably important effects of oxidation on the contractile apparatus in skeletal muscle fibres.

1. Transverse electrical field stimulation (50 V cm-1, 2 ms duration) of mechanically skinned skeletal muscle fibres of the rat elicited twitch and tetanic force responses (36 +/- 4 and 83 +/- 4 % of maximum Ca2+-activated force, respectively; n = 23) closely resembling those in intact fibres. The responses were steeply dependent on the field strength and were eliminated by inclusion of 10 microM tetrodotoxin (TTX) in the (sealed) transverse tubular (T-) system of the skinned fibres and by chronic depolarisation of the T-system. 2. Spontaneous twitch-like activity occurred sporadically in many fibres, producing near maximal force in some instances (mean time to peak: 190 +/- 40 ms; n = 4). Such responses propagated as a wave of contraction longitudinally along the fibre at a velocity of 13 +/- 3 mm s-1 (n = 7). These spontaneous contractions were also inhibited by inclusion of TTX in the T-system and by chronic depolarisation. 3. We examined whether the T-tubular network was interconnected longitudinally using fibre segments that were skinned for only approximately 2/3 of their length, leaving the remainder of each segment intact with its T-system open to the bathing solution. After such fibres were exposed to TTX (60 microM), the adjacent skinned region (with its T-system not open to the solution) became unresponsive to subsequent electrical stimulation in approximately 50 % of cases (7/15), indicating that TTX was able to diffuse longitudinally inside the fibre via the tubular network over hundreds of sarcomeres. 4. These experiments show that excitation-contraction coupling in mammalian muscle fibres involves action potential propagation both transversally and longitudinally within the tubular system. Longitudinal propagation of action potentials inside skeletal muscle fibres is likely to be an important safety mechanism for reducing conduction failure during fatigue and explains why, in developing skeletal muscle, the T-system first develops as an internal longitudinal network.

This study examined the relationship between the level of Ca2+ loading in the sarcoplasmic reticulum (SR) and the amount of Ca2+ released by an action potential (AP) in fast-twitch skeletal muscle fibres of the rat. Single muscle fibres were mechanically skinned and electric field stimulation was used to induce an AP in the transverse-tubular system and a resulting twitch response. Responses were elicited in the presence of known amounts (0-0.38 mM) of BAPTA, a fast Ca2+ buffer, with the SR Ca2+ pump either functional or blocked by 50 microM 2,5-di-tert-butyl-1,4-hydroquinone (TBQ). When Ca2+ reuptake was blocked, an estimate of the amount of Ca2+ released by an AP could be derived from the size of the force response. In a fibre with the SR loaded with Ca2+ at the endogenous level (approximately 1.2 mM, expressed as total Ca2+ per litre fibre volume; approximately one-third of maximal loading), a single AP triggered the release of approximately 230 microM Ca2+. If a second AP was elicited 10 ms after the first, only a further approximately 60 microM Ca2+ was released, the reduction probably being due to Ca2+ inactivation of Ca2+ release. When Ca2+ reuptake was blocked, APs applied 15 s apart elicited similar amounts of Ca2+ release (approximately 230 microM) on the first two or three occasions and then progressively less Ca2+ was released until the SR was fully depleted after a total of approximately eight APs. When the SR was loaded to near-maximal capacity (approximately 3-4 mM), each AP (or pair of APs 10 ms apart) still only released approximately the same amount of Ca2+ as that released when the fibre was endogenously loaded. Consistent with this, successive APs (15 s apart) elicited similar amounts of Ca2+ release approximately 10-16 times before the amount released declined, and the SR was fully depleted of Ca2+ after a total release calculated to be approximately 3-4 mM. When the SR was loaded maximally, increasing the [BAPTA] above 280 microM resulted in an increase in the amount of Ca2+ released per AP, probably because the greater level of cytoplasmic Ca2+ buffering prevented Ca2+ inactivation from adequately limiting Ca2+ release. These results show that the amount of Ca2+ released by AP stimulation in rat fast-twitch fibres normally stays virtually constant over a wide range of SR Ca2+ content, in spite of the likely large change in the electrochemical gradient for Ca2+. This was also found to be the case in toad twitch fibres. This constancy in Ca2+ release should help ensure precise regulation of force production in fast-twitch muscle in a range of circumstances.

1. World-wide epidemiological and experimental animal studies demonstrate that adversity in fetal life, resulting in intrauterine growth restriction, programmes the offspring for a greater susceptibility to ischaemic heart disease and heart failure in adult life. 2. After cardiogenesis, cardiomyocyte endowment is determined by a range of hormones and signalling pathways that regulate cardiomyocyte proliferation, apoptosis and the timing of multinucleation/terminal differentiation. 3. The small fetus may have reduced cardiomyocyte endowment owing to the impact of a suboptimal intrauterine environment on the signalling pathways that regulate cardiomyocyte proliferation, apoptosis and the timing of terminal differentiation.

1 The eect of the b 2 -adrenoceptor agonist, terbutaline, was investigated on simultaneously measured force and intracellular free calcium ([Ca 2+ ] i ) in intact rat soleus muscle ®bres, and on contractile protein function and Ca 2+ content of the sarcoplasmic reticulum (SR) in skinned ®bres. 2 Terbutaline (10 mM) had no signi®cant eect on either resting force or [Ca 2+ ] i . Exposure to terbutaline increased both the integral of the indo-1 ratio transient and peak twitch force by 37%. 3 At sub-maximal (10 Hz) stimulation frequencies, terbutaline accelerated force relaxation but had highly variable eects on tetanic force amplitude. The corresponding indo-1 ratio transients were signi®cantly larger, and faster to decay than the controls. 4 Terbutaline increased tetanic force at near maximal stimulation frequencies (50 Hz) by increasing tetanic [Ca 2+ ] i . Force relaxation was accelerated at this frequency with no signi®cant change in the indo-1 ratio transient decay rate. 5 All of terbutaline's eects on force and indo-1 ratio transients in intact ®bres were completely blocked and reversed by ICI 118551 (1 mM). IntroductionA long-standing question in b-adrenoceptor pharmacology has been the mechanism of action by which sympathomimetic amines exert distinct eects on the contractility of dierent skeletal muscle types (Bowman, 1980). Twitches and unfused tetanic contractions are potentiated and slowed by b 2 -agonists in fast-twitch muscles (Bowman & Zaimis, 1958;Bowman et al., 1962;Tashiro, 1973;Holmberg & Waldeck, 1980;Cairns & Dulhunty, 1993a), whereas slow-twitch muscles show decreases (Bowman & Zaimis, 1958;Bowman et al., 1962;Holmberg & Waldeck, 1979;Tashiro, 1973), increases (Cairns & Dulhunty, 1993a) or no change (Slack et al., 1997) of contraction amplitude in the presence of a consistently accelerated relaxation.In order to determine the mechanisms underlying such dierences it is necessary to consider the factors in¯uencing the amplitude and time course of skeletal muscle contraction as well as the signal transduction pathway of b 2 -adrenoceptors. Given that SR Ca 2+ handling is a key determinant of mammalian skeletal muscle contraction, and that activation of b 2 -adrenoceptors leads to activation of the cyclic AMP/ cyclic AMP-dependent protein kinase (PKA) pathway, then it is likely that the dierences and variability in response to b 2 -agonists are related to dierences in the phosphorylation of key proteins involved in SR Ca 2+ release and uptake. Two such proteins are the SR Ca 2+ release channel/ryanodine receptor (RR), which is found in all skeletal muscles, and phospholamban (PLB), an inhibitory protein associated with the SR Ca 2+ uptake pump in slow-twitch and cardiac muscles (Kirchberger & Tada, 1976). Phosphorylation of the RR leads to an increase in its open probability (Suko et al., 1993;Hain et al., 1994), whilst phosphorylation of PLB releases its basal inhibitory eect on the SR Ca 2+ uptake pump (Kirchberger & Tada, 1976).Simultaneous measurement of force and the intracell...