Postnatal development of Ca²⁺‐sequestration by the sarcoplasmic reticulum of fast and slow muscles in normal and dystrophic mice

Abstract

Ca2'-uptake activities of the sarcoplasmic reticulum (SR) were determined with a Ca2 +-sensitive electrode in homogenates from fast-and slow-twitch muscles from both normal and dystrophic mice (C57BL/6J strain) of different ages. Immunochemical quantification of tissue Ca2 +-ATPase content allowed determination of the specific Ca2+-transport activity of the enzyme. In 3-week-old mice of the dystrophic strain specific Ca2+ transport was already significantly lower than in the normal strain. It progressively decreased with maturation and reached only 40 -50% and 30 -50% of the normal values in fast-and slow-twitch muscles of adult dystrophic animals, respectively. Tissue contents of calsequestrin were reduced in both types of muscle leading to an increased Ca2 + -ATPase to calsequestrin protein ratio. Equal amounts of the Ca2 + -ATPase protein (detected by Coomassie blue staining of polyacrylamide gels) were present in SR vesicles isolated by Ca2 +-oxalate loading from adult normal and dystrophic fast-twitch muscles. However, the specific ATP-hydrolysing activity of the enzyme was approximately 50% lower in dystrophic than in normal SR. The reduced ATP-hydrolysing activity was correlated with decreased Ca2 '-transport activity, phosphoprotein formation and fluorescein isothiocyanate labeling as determined in total microsomal and heavy SR fractions. Although the Ca2+ and ATP affinities of the enzyme were unaltered, its ATPase activity was reduced at all levels of ATP in the dystrophic SR. Taken together, these findings point to a markedly impaired function of the SR and an increase in the population of inactive SR Ca2 ' -ATPase molecules in murine muscular dystrophy.An increase of free Ca2+ concentration in the cytosol has been connected to the pathomechanism of muscular dystrophy (for reviews see [l-31). Increased Ca2+ concentrations in the sarcoplasm [4 -61 could be due to an increased Ca2+ influx caused by an elevated permeability of the sarcolemma (7 -lo], or from a reduced capacity of Ca2+ uptake by the sarcoplasmic reticulum (SR) [l 1 -151. An imbalance in the Ca'+-regulating system could also be related to a reduced cytosolic Ca2 +-binding capacity of the diseased muscle fiber. A decrease in parvalbumin, the major cytosolic Ca2+-binding protein, was previously observed in fast-twitch muscles of adult dystrophic mice of the C57BL/6J(d~~'/dy~~) strain [16].In this context, we were interested in studying other Ca2+-sequestering proteins in dystrophic muscles, especially those of the sarcoplasmic reticulum (SR). In this study we investigated Ca2+ transport into the SR, tissue contents and catalytic properties of the SR Ca2+-ATPase, as well as the tissue concentration of calsequestrin, the putative Ca2 +-storage protein localised in luminal spaces of the terminal cisternae of the SR [17]. Murine muscular dystrophy is not clinically manifest at birth, but develops 3 -4 weeks post partum. Therefore, we followed the time courses of these parameters in hindlimb fast-and slow-twitch muscles from birth to 36 weeks of a...

Immunochemical quantification of sarcoplasmic reticulum Ca2+-ATPase and calsequestrin in muscle biopsies from patients with myotonia congenita and paramyotonia congenita Eulenburg

“…This large variability of the Ca 2 + -ATPase concentrations is consistent with previous findings that the Ca 2 +-ATPase content varies with fibre type Maier et al, 1986) and muscle maturation Leberer and H/irtner, 1988) in rabbit, rat and mouse muscles. The lowest concentration of Ca 2 + -ATPase was detected in an extensor digitorum longus muscle of a 14-year-old boy (control specimen 5, Table 1), whereas the highest Ca2+-ATPase content was 51.1 • 28.1 mg/g muscle protein (mean + SD, n--12) in all control muscles.…”

Fiber Types in Mammalian Skeletal Muscles

“…Both isoforms can be found in slow fibers, whereas only CASQ1 is expressed in fast fibers (177,680). Calsequestrin content is greater in fast than in slow fibers (466) and a quantitative analysis on single murine fibers (554) points to a concentration of 36 M in fast fibers (only CASQ1) and 11 M in slow fibers (CASQ1 and CASQ2). CASQ plays two important roles: 1) calcium buffer since, due to the large number of acidic residues, each CASQ1 molecule binds up 80 calcium ions and each CSAQ2 up to 60 calcium ions, and 2) modulator of calcium release due to its interaction with RyR (64).…”

“…As mentioned above, CASQ is the most important calcium binding protein inside SR. Due to the larger SR volume (202) and the greater abundance of CASQ (466,554), the SR of fast fibers is only filled with calcium for 35% of its capacity at resting concentrations of cytosolic free calcium: thus any increase of cytosolic free calcium will be followed by a fast and effective resequestration into the SR with little increase of intrareticular free calcium concentration (128,242). In contrast, in slow fibers, the SR is completely saturated with calcium at resting concentrations of cytosolic free calcium (242); thus during the reuptake phase of a cytosolic calcium transient the saturation of SR will slow down the removal of calcium from cytosol by backinhibition of the Ca 2ϩ pump (367).…”

Drastic reduction of calsequestrin-like proteins and impaired calcium binding in dystrophicmdxmuscle

“…Although earlier studies (18,39,53,62,71) have analyzed the dystrophic chicken and merosin-deficient dy mouse, no comprehensive study has addressed the status of key Ca 2ϩ regulatory membrane proteins in an established animal model of x-linked Duchenne muscular dystrophy such as the mdx mouse. Leg and torso mdx skeletal muscle fibers do not exhibit all of the observed pathobiochemical changes as seen in muscle specimens from patients afflicted with Duchenne muscular dystrophy (17).…”