Localization of the Ca²⁺‐dependent proteinases and their inhibitor in normal, fasted, and denervated rat skeletal muscle

Abstract

Immunofluorescence and immunogold localization studies show that the two Ca(2+)-dependent proteinases (mu-calpain for the micromolar Ca(2+)-requiring proteinase and m-calpain for the millimolar Ca(2+)-requiring proteinase) and their protein inhibitor (calpastatin) are located exclusively intracellularly in normal rat soleus muscle. Quantitative immunogold studies indicate that binding of antibodies to both calpains and to calpastatin is approximately two times greater at the Z-disk of myofibrils than it is at the I-band or A-band regions. Mitochondria and nuclei in muscle cells contain both calpains and calpastatin at concentrations approximately one-tenth and one-fifth, respectively, of the concentration at the Z-disk, as estimated by antibody binding. Very little calpain or calpastatin was seen in the cytoplasmic intermyofibrillar spaces, and most of the calpain and calpastatin in muscle cells is associated with intracellular structures. Immunofluorescence results suggest that concentration of m-calpain but not mu-calpain or calpastatin is, in some instances, slightly higher near the intracellular surface of the plasma membrane than elsewhere in the muscle cell. Most m-calpain, however, is distributed throughout the interior of mature rat skeletal muscle cells. Denervation, or fasting and refeeding increases the concentration of the calpains and calpastatin in the muscle cell but does not change their distribution. Some mu- and m-calpain and calpastatin is found extracellularly in denervated soleus muscle or soleus muscle from fasting rats, but the extracellular calpains and calpastatin seem to originate from "leakage" of these proteins out of the cell because serum creatine kinase levels are much higher than normal in denervated or fasting rats.

Changes in the Immunogold Electron-Microscopic Localization of Calpain in Bovine Skeletal Muscle Induced by Conditioning and High-Pressure Treatment

“…The colloidal gold particles were scattered singly or in small aggregates with patches on the myofibrils, the relative distribution of the colloidal particles (Table 1) being about 65% in the I-band region (inclusive of the Z-disk), as compared with 35% in the A-band region. This result almost agrees with those of quantitative studies 9,[19][20][21] which indicated that calpain was primarily located on the Z-disk, with a smaller amount in the I-band and very little in the A-band area.…”

“…7,8) Many studies have shown the calpain localization in muscle by using immunoelectron microscopy. [9][10][11] These have indicated that both the -and m-calpains were located exclusively inside (intracellularly) skeletal muscle cells, and that they tended to be associated with such subcellular structures as mitochondria, nuclei or myofibrils, rather than free in the cell cytoplasm. In normal skeletal muscle, they are located in all regions of the myofibril and the throughout the muscle cell.…”

Calpain activation impairs neuromuscular transmission in a mouse model of the slow-channel myasthenic syndrome

“…In muscle, calpain is immunologically localized to endomysial collagen fibrils, basal lamina, sarcolemma, Z bands, and NMJs, as well as the extracellular space (29,31). Calpains exist in multiple subtypes, differing in calcium sensitivity and function, and are involved in several cellular signaling pathways, myoblast fusion, cytoskeletal protein turnover, neurite outgrowth, and growth cone turning in development (26,(32)(33)(34)(35).…”

Enzymes in Tenderization of Meat – The System of Calpains and Other Systems

“…However, the calpain II is in 52% situated on the Z-line and also in the I-band (27%), and A-band (21%) [Koohmaraie, 1994;Kumamoto et al, 1992]. Whereas, calpain 3 in the largest degree is situated in a sarcomere near the Z-and M-line [Ilian et al, 2004c].…”