Calcium currents in a fast-twitch skeletal muscle of the rat

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially obscured by nonlinear charge movement. Nonetheless, at physiological temperatures, the rate of calcium channel activation in rat skeletal muscle is about five times faster than activation of calcium channels in frog muscle. This pathway may be an important source of calcium entry in mammalian muscle.     

Proteomic responses of skeletal and cardiac muscle to exercise

Regular exercise is effective in the prevention of chronic diseases and confers a lower risk of death in individuals displaying risk factors such as hypertension and dyslipidemia. Thus, knowledge of the molecular responses to exercise provides a valuable contrast for interpreting investigations of disease and can highlight novel therapeutic targets. While exercise is an everyday experience and can be conceptualized in simple terms, it is also a complex physiological phenomenon and investigation of exercise responses requires sophisticated analytical techniques and careful standardization of the exercise stimulus. Proteomic investigation of exercise is in its infancy but the ability to link changes in function with comprehensive changes in protein expression and post-translational modification holds great promise for advancing physiology. This article highlights recent pioneering work investigating the effects of exercise in skeletal and cardiac muscle that has uncovered novel mechanisms underlying the benefits of physical activity.     

Cellular and molecular responses to increased skeletal muscle loading after irradiation

Irradiation of rat skeletal muscles before increased loading has been shown to prevent compensatory hypertrophy for periods of up to 4 wk, possibly by preventing satellite cells from proliferating and providing new myonuclei. Recent work suggested that stem cell populations exist that might allow irradiated muscles to eventually hypertrophy over time. We report that irradiation essentially prevented hypertrophy in rat muscles subjected to 3 mo of functional overload (OL-Ir). The time course and magnitude of changes in cellular and molecular markers of anabolic and myogenic responses were similar in the OL-Ir and the contralateral nonirradiated, overloaded (OL) muscles for the first 3-7 days. These markers then returned to control levels in OL-Ir muscles while remaining elevated in OL muscles. The number of myonuclei and amount of DNA were increased markedly in OL but not OL-Ir muscles. Thus it appears that stem cells were not added to the irradiated muscles in this time period. These data are consistent with the theory that the addition of new myonuclei may be required for compensatory hypertrophy in the rat.     

Quantitative histochemistry of creatine kinase in rat myocardium and skeletal muscle

Summary Creatine kinase (ec 2.7.3.2) activity was demonstrated in rat myocardium using a polyvinyl alcohol-containing incubation medium and auxiliary enzymes. The activity was quantified by microdensitometry using both endpoint measurements and kinetic measurements. Control reactions were performed in the absence of creatine phosphate and ADP.The linear regression lines of the absorbances of reduced Nitro BT at the isobestic wavelength (585 nm) on incubation time were highly significant for both endpoint and kinetic measurements. The activity obtained from endpoint measurements was about 40% lower. This was caused by loss of the formazan reaction product from the tissue sections when the incubation medium was removed at the end of the reaction. The relationship between creatine kinase activity (test minus control reaction) and section thickness was not linear for either myocardium or skeletal muscle; control reactions, however, showed linear relationships with section thickness for both tissues. Limited penetration of auxiliary enzymes into the sections may be responsible for this disporportionality. Therefore, care should be taken in the interpretation of quantitative data obtained with different tissues.In conclusion, multi-step enzyme reactions can be used for quantitative histochemical purposes provided it is taken into account that the reactivity is not proportional to section thickness.     

Fasting-related autophagic response in slow- and fast-twitch skeletal muscle

This study investigated regulation of autophagy in slow-twitch soleus and fast-twitch plantaris muscles in fasting-related atrophy. Male Fischer-344 rats were subjected to fasting for 1, 2, or 3 days. Greater weight loss was observed in plantaris muscle than in soleus muscle in response to fasting. Western blot analysis demonstrated that LC3-II, a marker protein for macroautophagy, was expressed at a notably higher level in plantaris than in soleus muscle, and that the expression level was fasting duration-dependent. To identify factors related to LC3-II enhancement, autophagy-related signals were examined in both types of muscle. Phosphorylated mTOR was reduced in plantaris but not in soleus muscle. FOXO3a and ER stress signals were unchanged in both muscle types during fasting. These findings suggest that preferential atrophy of fast-twitch muscle is associated with induction of autophagy during fasting and that differences in autophagy regulation are attributable to differential signal regulation in soleus and plantaris muscle.     

Chronic low-frequency stimulation upregulates uncoupling protein-3 in transforming rat fast-twitch skeletal muscle

The purpose of this investigation was to examine the temporal changes in uncoupling protein (UCP)-3 expression, as well as related adaptive changes in mitochondrial density and fast-to-slow fiber type transitions during chronically enhanced contractile activity. We examined the effects of 1-42 days of chronic low-frequency electrical stimulation (CLFS), applied to rat tibialis anterior (TA) for 10 h/day, on the expression of UCP-3 and concomitant changes in myosin heavy chain (MHC) protein expression and increases in oxidative capacity. UCP-3 protein content increased from 1 to 12 days, reaching 1.5-fold over control (P < 0.0005); it remained elevated for up to 42 days. In contrast, UCP-3 mRNA decreased in response to CLFS, reaching a level that was threefold lower than control (P < 0.0007). The activities of the mitochondrial reference enzymes citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35), which are known to increase in proportion to mitochondrial density, progressively increased up to an average of 2.3-fold (P < 0.00001). These changes were accompanied by fast-to-slow fiber type transitions, characterized by a shift in the pattern of MHC expression (P <0.0002): MHCI and MHCIIa expression increased by 1.7- and 4-fold, whereas MHCIIb displayed a 2.4-fold reduction. We conclude that absolute increases in UCP-3 protein content in the early adaptive phase were associated with the genesis of mitochondria containing a normal complement of UCP-3. However, during exposure to long-term CLFS, mitochondria were generated with a lower complement of UCP-3 and coincided with the emergence of a growing population of oxidative type IIA fibers.     

Triacylglycerol metabolism in rat skeletal muscle after exercise

The temporal relationships between triacylglycerol (TG) content and TG lipase activity in slow-twitch (STR) and fast-twitch red (FTR) muscles were determined in rats during recovery from a 2-h swim. Immediately after the exercise, plasma free fatty acid (FFA) was elevated and glycogen concentrations were decreased. TG content was decreased 40% in STR muscle and reduced 45% in FTR muscle. The TG concentration of STR muscle increased in a linear fashion throughout recovery so that control levels were reached within the first 24 h after exercise. TG lipase activity of STR muscle was elevated 36% above control immediately after the swim and continued to increase to 84% above control 24 h after the work. In STR muscle there was a net synthesis of TG, while lipase activity was elevated above that measured in muscle of control rats. TG content of FTR muscle remained 45% below control throughout the first 24 h of recovery, and TG lipase activity increased from 26% (P greater than 0.05) greater than control immediately after exercise to threefold above control 24 h after work. All parameters returned to control levels by 48 h of recovery. These data indicated that a net TG synthesis occurs in STR muscle when lipolytic activity is elevated. In FTR muscle, however, a gradual increase in TG lipase activity that occurs during the first 24 h of recovery accompanies a TG concentration well below the control level throughout this same time frame.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical demonstration of ATP: creatine phosphotransferase in rat skeletal muscle

Summary An attempt was made to locate the ATP: creatine phosphotransferase (creatine kinase, CKase) in rat skeletal muscle by a lead precipitation method. The muscle is not stained at all with creatine phosphate (CP), and only weakly with adenosine diphosphate (ADP) as substrate, while it hydrolyzes adenosine triphosphate (ATP) actively. Taking advantage of this fact, it is possible to demonstrate the CKase activity using both ADP and CP as substrate. The CKase activity thus obtained was located in various profiles of sarcoplasmic reticulum as well as in A bands, the staining being comparable to that obtained with ATP as substrate.A weak activity was found only in cisternal dilatations of sarcoplasmic reticulum when sections were incubated with ADP as substrate.     

Properties of slow- and fast-twitch skeletal muscle from mice with an inherited capacity for hypoxic exercise

Muscle fiber type, myosin heavy chain (MHC) isoform composition, capillary density (CD) and citrate synthase (CS) activity were investigated in predominantly slow-twitch (soleus or SOL) and fast-twitch (extensor digitorum longus or EDL) skeletal muscle from mice with inherited differences in hypoxic exercise tolerance. Striking differences in hypoxic exercise tolerance previously have been found in two inbred strains of mice, Balb/cByJ (C) and C57BL/6J (B6), and their F1 hybrid following exposure to hypobaric hypoxia. Mice from the three strains were exposed for 8 weeks to either normobaric normoxia or hypobaric hypoxia (1/2 atm). Hypoxia exposure led to a slightly higher 2b fiber composition and a lower fiber area of types 1 and 2a in SOL of all mice. In the EDL, muscle fiber and MHC isoform composition remained unaffected by chronic hypoxia. Chronic hypoxia did not significantly affect CD in either muscle from any of the three strains. There were relatively larger differences in CS activity among strains and treatment, and in SOL the highest CS activity was found in the F1 mice that had been acclimated to hypoxia. In general, however, neither differences among strains nor treatment in these properties of muscle vary in a way that clearly relates to inherited hypoxic exercise tolerance.     

Imaging caffeine-induced Ca2+ transients in individual fast-twitch and slow-twitch rat skeletal muscle fibers

Fast-twitch and slow-twitch rat skeletal muscles producedissimilar contractures with caffeine. We used digital imagingmicroscopy to monitor Ca²⁺ (withfluo 3-acetoxymethyl ester) and sarcomere motion in intact, unrestrained rat muscle fibers to study this difference. Changes inCa²⁺ in individual fibers weremarkedly different from average responses of a population. All fibersshowed discrete, nonpropagated, local Ca²⁺ transients occurring randomlyin spots about one sarcomere apart. Caffeine increased localCa²⁺ transients and sarcomeremotion initially at 4 mM in soleus and 8 mM in extensor digitorumlongus (EDL; ~23°C). Ca²⁺release subsequently adapted or inactivated; this was surmounted byhigher doses. Motion also adapted but was not surmounted. Prolonged exposure to caffeine evidently suppressed myofilament interaction inboth types of fiber. In EDL fibers, 16 mM caffeine moderately increasedlocal Ca²⁺ transients. In soleusfibers, 16 mM caffeine greatly increased Ca²⁺ release and producedpropagated waves of Ca²⁺(~1.5-2.5 µm/s). Ca²⁺waves in slow-twitch fibers reflect the caffeine-sensitive mechanism ofCa²⁺-inducedCa²⁺ release. Fast-twitch fiberspossibly lack this mechanism, which could account for their lowersensitivity to caffeine.