Our previous finding of a reduced energy metabolism in slow- and fast-twitch skeletal muscle fibres from the murine model of Duchenne muscular dystrophy (the mdx mouse) led us to examine the importance of intracellular glucose availability for a normal energy turnover. To this end, basal and KCl-stimulated (20.9 mM total extracellular K+) rates of glucose uptake (GUP) and heat production were measured in isolated, glucose-incubated (5 mM) soleus and extensor digitorum longus muscles from mdx and control C57B1/10 mice, in the presence and in the absence of insulin (1.7 nM). Under all conditions and for both muscle types, glucose uptake values for mdx and control muscles were similar although heat production was lower in mdx muscles. The marked stimulation of GUP by insulin in both mdx and control muscles had only minor effects on heat production. In contrast, glucose deprivation or inhibition of glycolysis with 2-deoxy-D-glucose (5 mM) significantly decreased heat production in control muscles only, which attenuated, although did not suppress, the difference in basal heat production between mdx and control muscles. Stimulation of heat production by a short-chain fatty acid salt (octanoate, 2 mM) was significantly less marked in mdx than in control muscles. Increased cytoplasmic synthesis of CoA by addition of 5 mM pantothenate (vitamin B5) increased the thermogenic response to glucose more in mdx than in control muscles. We conclude that the low energy turnover in mdx-mouse muscle fibres is not due to a decrease of intracellular glucose availability, but rather to a decreased oxidative utilization of glucose and free fatty acids. We suggest that some enzyme complex of the tricarboxylic acid cycle or inefficiency of CoA transport in the mitochondria could be involved.

Recent reports suggest that the nuclear envelope possesses specific ion transport mechanisms that regulate the electrolyte concentrations within the nucleoplasm and perinuclear space. In this work, intact nuclei were isolated from sheep cardiac cells. After chromatin digestion, the nuclear envelopes were sonicated and four nuclear vesicle populations were separated by sucrose step gradients (SF1-SF4). These fractions were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their protein content was analyzed by Western blot, using lamin and SEC 61 antibodies. The lamins, which are associated with the inner nuclear membrane, were present in three fractions, SF2, SF3, and SF4, with a lower amount in SF2. The SEC 61 protein, a marker of the rough endoplasmic reticulum, was detected in small amounts in SF1 and SF2. Upon fusion of vesicles into bilayers, the activities of nuclear ionic channels were recorded in 50 mM trans/250 mM cis KCl or CsCl, pH 7.2. Two types of Cl- selective channels were recorded: a large conducting 150-180-pS channel displaying substates, and a low conducting channel of 30 pS. They were both spontaneously active into bilayers, and their open probability was poorly voltage dependent at negative voltages. Retinoic acid (10(-8) M) increases the po of the large Cl- conducting channel, whereas ATP modifies the kinetics of the low conductance anion selective channel. Our data also suggest that this anionic channel is mainly present in the SF3 and SF4 population. The presence of a 181 +/- 10 pS cation-selective channel was consistently observed in the SF2 population. The behavior of this channel was voltage dependent in the voltage range -80 to +60 mV. Furthermore, we report for the first time the activity of a channel exclusively present in the SF3 and SF4 fractions, shown to contain mainly inner membrane vesicles. This cation selective channel displays a 75-pS conductance in 50 mM trans/250 mM cis K-gluconate. It is concluded that the bilayer reconstitution technique is an attractive approach to studying the electrophysiological properties of the inner and outer membranes of the nuclear envelope.

SUMMARY1. The rate of energy expended for the clearance of sarcoplasmic Ca2+ by sarcoreticular Ca2+ uptake processess, plus the concomitant metabolic reactions, was evaluated from measurements of resting heat production by mouse soleus muscle before and after indirect inhibition of Ca2+ uptake by sarcoplasmic reticulum (SR).2. Direct inhibition of the Ca2+, Mg2+-ATPase of SR membrane in intact muscle preparations exposed to the specific inhibitor 2,5-di(tert-butyl-1 ,4-benzohydroquinone (tBuBHQ) slowly increased the rate of heat production (E). Indirect inhibition of SR Ca2+ uptake was obtained by reducing sarcoplasmic Ca2+ concentration (Ca2+) as a consequence of reducing Ca2+ release from the SR using dantrolene sodium. This promptly decreased E by 12 %. Exposure of the preparations to an Mg2+-enriched environment (high Mg2+) or to the chemical phosphatase 2,3-butanedione monoxime (BDM), two other procedures aimed at decreasing SR Ca2+ release, also acutely decreased E, by 20 and 24 %, respectively.3. Subthreshold-for-contracture depolarization of the sarcolemma achieved by increasing extracellular K+ concentration to 11-8 mm induced a biphasic increase of E: an initial peak to 290 % of basal E, followed by a plateau phase at 140 % of basal E during which resting muscle tension was increased by less than 3 %. Most, if not all, of the plateau-phase metabolic response was quickly suppressed by dantrolene or high Mg2+ or BDM. Another means of increasing SR Ca2+ cycling was to partially remove the calmodulin-dependent control of SR Ca2+ release using the calmodulin inhibitor W-7. The progressive increase in E with 30 pm-W-7 was largely reduced by dantrolene or high Mg2+ or BDM.4. In the presence of either dantrolene or BDM to prevent the effect of W-7 on SR Ca2+ release, exposure of the muscle to W-7 acutely suppressed about 3 % of E. This and the above results confirm that the plasmalemmal, calmodulin-dependent Ca2+-ATPase, although a qualitatively essential part of the Ca2+ homeostatic system of the cell, can only be responsible for a very minor part of the energy expenditure devoted to the homeostasis of Ca 2+. Active Ca2+ uptake by SR which, at least in the submicromolar range of Cai , is expected to be responsible for most of this Ca2+-dependent energy expenditure, might dissipate up to 25-40% of total metabolic energy in the intact mouse soleus under basal and near-basal conditions. MS 9801 22PHY 455

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Muscles from the mdx mouse (X-linked genetic disorder similar to Duchenne muscular dystrophy) lack dystrophin-associated transsarcolemmal proteins and show reduced maintenance metabolic rates. Here, microcalorimetric comparisons of metabolic stimulation by exogenous substrates in isolated muscles revealed substrate-selective limitation of chemical reaction rates through both glycolytic and TCA-cycle pathways, identical in slow- and fast-twitch mdx muscles. This systemic approach, as opposed to comparisons of single-enzyme activities, sheds new light on the function of dystrophin and associated proteins. The in vivo efficiency of metabolic pathways may depend on stabilization of enzyme complexes by dystrophin-associated elements of the cytoskeleton.