Analysis of excitation‐contraction‐coupling components in chronically stimulated canine skeletal muscle

Abstract

The chronic stirnulation of predominantly fast-twitch mammalian skeleldl muscle causes a transformation to physiological characteristics of slow-twitch skeletal muscle. Here, we report the effects of chronic stimulation on the protein components of the sarcoplasmic reticulum and transverse tubular membranes which are directly involved in excitation-contraction coupling. Comparison of protein composition of microsomal fractions from control and chronically stimulated muscle was performed by immunoblot analysis and also by staining with Coomassie blue or the cationic carbocyanine dye Stains-all. Consistent with previous experiments, a greatly reduced density was observed for the fast-twitch isozyme of Ca2+-ATPase, while the expression of the slow-twitch Ca2+-ATPase was found to be greatly enhanced. Components of the sarcolemma (Na'/K+-ATPase, dystrophin-glycoprotein complex) and the free sarcoplasmic reticulum (Ca2+-binding protein sarcalumenin and a 53-kDa glycoprotein) were not affected by chronic stimulation. The relative abundance of calsequestrin was slightly reduced in transformed skeletal muscle. However, the expression of the ryanodine receptor/Ca2+ -release channel from junctional sarcoplasmic reticulum and the transverse tubular dihydropyridine-sensitive Ca2 + channel, as well as two junctional sarcoplasmic reticulum proteins of 90 kDa and 94 kDa, was greatly suppressed in transformed muscle. Thus, the expression of the major protein components of the triad junction involved in excitation-contraction coupling is suppressed, while the expression of other muscle membrane proteins is not affected in chronically stimulated muscle.In excitation-contraction (EC) coupling of skeletal muscle, Ca2 + release from the sarcoplasmic reticulum (SR) is initiated and coupled to the depolarization of the transverse tubular membrane system [I, 21. The ryanodine receptor (RyR) of rabbit skeletal muscle has been purified and shown to be identical to the Ca2+-release channel of the SR [3-51. The 'foot' structure at the junction between the SR and the transverse tubules is composed of the Ca2 '-release channel and has been proposed to interact with the transverse tubule tetrad [6]. The receptor for the 1,4-dihydropyridine class of Ca2+ blockers (DHPR) functions in skeletal muscle, not only as a Ca2+ channel, but also as the voltage sensor in EC coupling [7,8], and is probably the primary component of the transverse tubule tetrad. The subunit composition of the DHPR from rabbit skeletal muscle has been analyzed [9, 101 and each of the subunits have been cloned [8,11-131. Other components found in the triad fraction of skeletal muscle are thc junctional sarcoplasmic reticulum (JSR) proteins of apparent 90 kDa and 94 kDa [14], which could play a regulatory role in EC

Measurement of Sarcoplasmic Reticulum Ca2+-ATPase Activity and E-Type Mg2+-ATPase Activity in Rat Heart Homogenates

“…Following the procedure described by Ohlendieck et al (14), a microsomal fraction was isolated from rat heart, containing vesicles derived from SR, transverse tubules, and sarcolemma; the protein yield was 1.3 Ϯ 0.2 mg protein ⅐ g Ϫ1 muscle wet weight. When this cardiac mixed membrane fraction was employed to measure the SR Ca 2ϩ -ATPase activity, according to common procedures for skeletal muscle (17), an apparent negative value was obtained (Ϫ0.23 Ϯ 0.03 mol ⅐ min Ϫ1 ⅐ mg Ϫ1 ) since the basal activity, detected before the addition of 0.2 mM CaCl 2 and 4 M ionophore A23187, was higher than the total activity subsequently determined in the same cuvette after the addition of these compounds.…”

Adaptations in human muscle sarcoplasmic reticulum to prolonged submaximal training

“…Provided that CLFS is of sufficient daily volume and duration, RyR protein content decreases dramatically (9,14,26) without changes in RyR1 to RyR2, the isoform that predominates in heart muscles (8). Although not measured, the downregulation in RyR would be expected to be accompanied by a pronounced decrease in Ca 2ϩ -release kinetics (8).…”

“…The CLFS model can also induce large reductions in SERCA content. However, unlike RyR, a shift from SERCA1, the predominant isoform observed in type II muscle fibers, to SERCA2a, the major isoform that exits in slow-twitch (type I) fibers, occurs (3,14,26,27). Because the alteration in SERCA isoform content with CLFS might be expected to change, the Ca 2ϩ affinity as measured by the Hill coefficient and Ca 2ϩ concentration at half-maximal activity (pCa 50 ), it is surprising that no study has apparently addressed this issue (29).…”

Salbutamol changes the molecular and mechanical properties of canine skeletal muscle.

“…Membrane fractions mainly containing vesicles of sarcoplasmic reticulum (SR) were isolated as described by Ohlendieck et al (1991) and stored at -70 'C. This membrane fraction was used to measure amounts of SR proteins.…”