Novel role of calpain-3 in the triad-associated protein complex regulating calcium release in skeletal muscle

Abstract

Calpain-3 (CAPN3) is a non-lysosomal cysteine protease that is necessary for normal muscle function, as mutations in CAPN3 result in an autosomal recessive form of limb girdle muscular dystrophy type 2A. To elucidate the biological roles of CAPN3 in skeletal muscle, we performed a search for potential substrates and interacting partners. By yeast-two-hybrid analysis we identified the glycolytic enzyme aldolase A (AldoA) as a binding partner of CAPN3. In co-expression studies CAPN3 degraded AldoA; however, no accumulation of AldoA was observed in total extracts from CAPN3-deficient muscles suggesting that AldoA is not an in vivo substrate of CAPN3. Instead, we found CAPN3 to be necessary for recruitment of AldoA to one specific location, namely the triads, which are structural components of muscle responsible for calcium transport and excitation-contraction coupling. Both aldolase and CAPN3 are present in the triad-enriched fraction and are able to interact with ryanodine receptors (RyR) that form major calcium release channels. Levels of triad-associated AldoA and RyR were decreased in CAPN3-deficient muscles compared with wild-type. Consistent with these observations we found calcium release to be significantly reduced in fibers from CAPN3-deficient muscles. Together, these data suggest that CAPN3 is necessary for the structural integrity of the triad-associated protein complex and that impairment of calcium transport is a phenotypic feature of CAPN3-deficient muscle.

Endogenous Calpain-3 Activation Is Primarily Governed by Small Increases in Resting Cytoplasmic [Ca2+] and Is Not Dependent on Stretch

“…Each individual myofibril in a skeletal muscle fiber is surrounded by the sarcoplasmic reticulum, and the RyRs in the sarcoplasmic reticulum are located at the triad junctions, which are positioned approximately in register with the N2A line of titin in each sarcomere along a myofibril. It has been suggested that some calpain-3 is directly associated with the RyRs (22); the experiments here indicate, however, that any such amount must be at most a very small proportion of the total calpain-3 pool, because disrupting the sarcoplasmic reticulum and all other membranes within the fiber with the detergent Triton X-100 led to rapid diffusional loss of virtually all the RyRs and other sarcoplasmic reticulum proteins within 10 min with very little accompanying washout of calpain-3 (Fig. 3).…”

“…Immunofluorescent confocal microscopy revealed that in adult human muscle, most calpain was localized in two transverse bands per sarcomere, one on each side of the Z-band, in the vicinity of the N2A line on titin (21). Recently, calpain-3 has also been reported to interact with the ryanodine receptor-Ca 2ϩ release channels (RyRs) 2 at the triad junctions (22), which are positioned closely in register with the titin N2A lines, leaving it unclear as to how much of the calpain-3 is associated with titin and how much with the triads. One issue with the study of Kramerova et al (22) is that most of the calpain-3 segregating with the RyRs was autolyzed rather than in its native full-length form, leaving doubt about whether calpain-3 may have altered in position over the time scale involved in the prolonged spinning procedures used in preparation of the triad fractions, perhaps with autolysis altering its diffusibility or binding sites.…”

Triadopathies: An Emerging Class of Skeletal Muscle Diseases

“…Kramerova et al [101] identified CAPN3 localization at the triad (amongst other places). They showed that Capn3 knockout in mice was associated with decreased expression of RyR1 in skeletal muscle, as well as with impaired SR calcium release [101]. Based on these results, the authors proposed that impaired triad function was an important aspect of the disease process in patients with LGMD2A.…”

“…Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A (LGMD2A), a slowly and variably progressive disorder associated with proximal muscle weakness [100]. Kramerova et al [101] identified CAPN3 localization at the triad (amongst other places). They showed that Capn3 knockout in mice was associated with decreased expression of RyR1 in skeletal muscle, as well as with impaired SR calcium release [101].…”

Inherited progressive limb-girdle muscular dystrophy type 2A (calpainopathy): a review of literature

“…Кроме того, американские исследователи во главе с I. Kramerova выявили участие белка кальпаина-3 и в транспорте кальция. Авторы обнаружили существование белково-го комплекса, образованного гликолитическим фер-ментом альдолазой, кальпаином-3 и высвобождающим кальций рианодиновым рецептором (RyR-каналом), и предположили, что кальпаин-3 крайне необходим для поддержания целостности этого комплекса в ске-летных мышцах [37]. Все вышесказанное можно схе-матически суммировать и предположить возможный сценарий патогенеза КПМД2А (рис.…”