Sarcolemmal Alterations in Unloaded Rat Heart after Heterotopic Transplantation

Following heterotopic transplantation, the rat heart undergoes atrophy and exhibits delayed cardiac relaxation without any changes in contraction and systolic Ca2+ transients. Furthermore, the sarcoplasmic reticular Ca2+ uptake and release activities were reduced and Ca2+ influx through L-type Ca2+ channels was increased in the atrophied heart. Since Ca2+ movements at sarcolemma are intimately involved in the regulation of intracellular Ca2+ concentration, the present study was undertaken to test if sarcolemma plays any role to maintain cardiac function in the atrophied heart.The characteristics of sarcolemmal Ca2+ pump and Na+–Ca2+ exchange activities were examined in 8 weeks heterotopically isotransplanted rat hearts which did not support hemodynamic load and underwent atrophy. Sarcolemmal ATP (adenosine triphosphate)-dependent Ca2+ uptake and Ca2+-stimulated ATPase (adenosine triphosphatase) activities were increased without any changes in Na+–K+ ATPase activities in the transplanted hearts. Although no alterations in the Na+-dependent Ca2+ uptake were evident, Na+-induced Ca2+ release was increased in the transplanted heart sarcolemmal vesicles. The increase in Na+-induced Ca2+ release was observed at different times of incubation as well as at 5, 20, and 40 mM Na+. The sarcolemma from transplanted hearts also showed higher contents of phosphatidic acid, sphingomyelin, and cholesterol.These results indicate that increases in the sarcolemmal, Ca2+ transport activities in unloaded heart may provide an insight into adaptive mechanism to maintain normal contractile behavior of the atrophic heart.

Insulin sensitivity is independent of lipid binding protein trafficking at the plasma membrane in human skeletal muscle: effect of a 3-day, high-fat diet

The aim of the present study was to investigate lipid-induced regulation of lipid binding proteins in human skeletal muscle and the impact hereof on insulin sensitivity. Eleven healthy male subjects underwent a 3-day hypercaloric and high-fat diet regime. Muscle biopsies were taken before and after the diet intervention, and giant sarcolemmal vesicles were prepared. The high-fat diet induced decreased insulin sensitivity, but this was not associated with a relocation of FAT/CD36 or FABPpm protein to the sarcolemma. However, FAT/CD36 and FABPpm mRNA, but not the proteins, were upregulated by increased fatty acid availability. This suggests a time dependency in the upregulation of FAT/CD36 and FABPpm protein during high availability of plasma fatty acids. Furthermore, we did not detect FATP1 and FATP4 protein in giant sarcolemmal vesicles obtained from human skeletal muscle. In conclusion, this study shows that a short-term lipid-load increases mRNA content of key lipid handling proteins in human muscle. However, decreased insulin sensitivity after a high-fat diet is not accompanied with relocation of FAT/CD36 or FABPpm protein to the sarcolemma. Finally, FATP1 and FATP4 protein was located intracellularly but not at the sarcolemma in humans.

T3 increases lactate transport and the expression of MCT4, but not MCT1, in rat skeletal muscle

Triiodothyronine (T3) regulates the expression of genes involved in muscle metabolism. Therefore, we examined the effects of a 7-day T3 treatment on the monocarboxylate transporters (MCT)1 and MCT4 in heart and in red (RG) and white gastrocnemius muscle (WG). We also examined rates of lactate transport into giant sarcolemmal vesicles and the plasmalemmal MCT1 and MCT4 in these vesicles. Ingestion of T3 markedly increased circulating serum T3 ( P < 0.05) and reduced weight gain ( P < 0.05). T3 upregulated MCT1 mRNA (RG +77, WG +49, heart +114%, P < 0.05) and MCT4 mRNA (RG +300, WG +40%). However, only MCT4 protein expression was increased (RG +43, WG +49%), not MCT1 protein expression. No changes in MCT1 protein were observed in any tissue. T3 treatment doubled the rate of lactate transport when vesicles were exposed to 1 mM lactate ( P < 0.05). However, plasmalemmal MCT4 was only modestly increased (+13%, P < 0.05). We conclude that T3 1) regulates MCT4, but not MCT1, protein expression and 2) increases lactate transport rates. This latter effect is difficult to explain by the modest changes in plasmalemmal MCT4. We speculate that either the activity of sarcolemmal MCTs has been altered or else other MCTs in muscle may have been upregulated.

Muscle contraction increases lactate transport while reducing sarcolemmal MCT4, but not MCT1

Rates of lactate uptake into giant sarcolemmal vesicles were determined in vesicles collected from rat muscles at rest and immediately after 10 min of intense muscle contraction. This contraction period reduced muscle glycogen rapidly by 37–82% in all muscles examined ( P < 0.05) except the soleus muscle (no change P > 0.05). At an external lactate concentration of 1 mM lactate, uptake into giant sarcolemmal vesicles was not altered ( P > 0.05), whereas at an external lactate concentration of 20 mM, the rate of lactate uptake was increased by 64% ( P < 0.05). Concomitantly, the plasma membrane content of monocarboxylate transporter (MCT)1 was reduced slightly (−10%, P < 0.05), and the plasma membrane content of MCT4 was reduced further (−25%, P < 0.05). In additional studies, the 10-min contraction period increased the plasma membrane GLUT4 ( P < 0.05) while again reducing MCT4 (−20%, P < 0.05) but not MCT1 ( P > 0.05). These studies have shown that intense muscle contraction can increase the initial rates of lactate uptake, but only when the external lactate concentrations are high (20 mM). We speculate that muscle contraction increases the intrinsic activity of the plasma membrane MCTs, because the increase in lactate uptake occurred while plasma membrane MCT4 was decreased and plasma membrane MCT1 was reduced only minimally, or not at all.

Impaired sarcolemmal vesicle lactate uptake and skeletal muscle MCT1 and MCT4 expression in obese Zucker rats

The present experiments were undertaken to characterize 1) the hindlimb muscle mass lactate uptake and 2) the expression of monocarboxylate transporter isoforms MCT1 and MCT4, as well as lactate dehydrogenase (LDH) isozyme distribution, in various skeletal muscles of Zucker fa/fa rats taken as a model of insulin resistance-related obesity. Initial lactate uptake at six different concentrations was measured in sarcolemmal vesicles (SV) by use ofl-[U-14C]lactate. Compared with controls, the maximal rate of lactate uptake and affinity were decreased in SV of Zucker rats (∼30%) in which MCT4 content was significantly decreased ( P < 0.05). MCT4 expression was decreased in soleus, extensor digitorum longus, and red tibialis anterior (RTA; P < 0.05), but not in white tibialis anterior, whereas MCT1 expression was decreased only in RTA of Zucker rats ( P < 0.05). Obesity led to a shift toward type M-LDH isozyme in mixed muscles. We conclude that obesity leads to changes in muscular MCT1 and MCT4 expression, which, when associated with LDH isozyme redistribution, may contribute to the hyperlactatemia noted in insulin resistance.

Localisation of intracellular calcium stores in the striated muscles of the jellyfishPolyorchis penicillatus: possible involvement in excitation–contraction coupling

SUMMARYWhen jellyfish striated muscles were stimulated directly, the amplitude of contractile tension increased as the stimulation frequency increased. Application of 10 mmol l–1 caffeine reduced the amplitude of contractile tension and abolished this facilitatory relationship, indicating that calcium stores participate in excitation–contraction coupling. Calcium stores were identified ultrastructurally using enzymatic histochemistry to localize CaATPases, and potassium dichromate to precipitate calcium. Electron energy-loss spectroscopy was used to verify the presence of calcium in precipitates. Both CaATPase and calcium were localised in membrane-bound vesicles beneath the sarcolemma. We concluded that sub-sarcolemmal vesicles could act as calcium stores and participate in excitation–contraction coupling.

Altered cardiac Na+/H+ exchange in phospholipase D-treated sarcolemmal vesicles

Cardiac sarcolemmal Na+/H+ exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na+/H+ exchange. The present study was carried out to determine the effects on Na+/H+ exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 ± 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 ± 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 ± 2% inhibition of Na+/H+ exchange. Na+/H+exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na+. All of these parameters of Na+/H+ exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na+ was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na+/H+ exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na+/H+ exchange.