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Abstract
Ca2+ disturbances are observed when Ca2+-dependent cysteine proteases malfunction, causing muscle weakness and wasting. For example, loss of calpain-3 (CAPN3) activity leads to limb-girdle muscular dystrophy 2A (LGMD2A). In neuronal excitotoxicity, the cleavage of the Na+-Ca2+ exchanger isoform 3 (NCX3) has been associated with an increase in activity and elevation of the Ca2+ content in the endoplasmic reticulum (ER). Since NCX3 is expressed in skeletal muscle, we evaluated the cleavage of different NCX3 splice variants by CAPN1 and CAPN3. Using Fura-2-based cellular Ca2+ imaging, we showed for the first time that CAPN3 increases NCX3 activity and that only NCX3-AC, the variant predominantly expressed in skeletal muscle, is sensitive to calpain. The silencing of the endogenous CAPN1 and the expression of the inactive form of CAPN3 (C129S CAPN3) confirmed the specificity for CAPN1 and CAPN3. Functional studies revealed that cellular Ca2+ uptake through the reverse mode of NCX3 was significantly increased independently of the mode of activation of the exchanger by either a rise in intracellular Ca2+ ([Ca2+]i) or Na+ ([Na+]i). Subsequently, the sensitivity to CAPN1 and CAPN3 could be abrogated by removal of the six residues coded in exon C of NCX3-AC. Additionally, mutation of the Leu-600 and Leu-601 suggested the presence of a cleavage site at Leu-602. The increased Ca2+ uptake of NCX3 might participate in the Ca2+ refilling of the sarcoplasmic reticulum (SR) after the excitation-contraction uncoupling following exercise and therefore be implicated in the impaired reticular Ca2+ storage observed in LGMD2A.
Highlights
The calpain proteases are Ca2+-dependent cysteine proteases expressed throughout the entire body
Using RT-PCR, mRNA levels of the calpain family members were measured in cardiac tissues and skeletal muscles of different fiber types: slow-twitch fibers predominant in the soleus, fast-twitch fibers dominant in the extensor digitorum longus (EDL), or mixed fibers such as those found in the gastrocnemius and the tibialis anterior (TA)
This study revealed the specificity of CAPN1 and CAPN3 for the variant Na+-Ca2+ exchanger isoform 3 (NCX3)-AC, predominant in skeletal muscle, as an increase of the reverse mode of exchange is observed solely for this variant and implicates the presence of the exon C to be responsible for the CAPN effect
Summary
The calpain proteases are Ca2+-dependent cysteine proteases expressed throughout the entire body. The LGMD2A is an autosomal recessive form of muscular dystrophy characterized by muscle weakness of the proximal limb-girdle muscles eventually leading to a loss of ambulation It derives from a single mutation of CAPN3 causing a defect in CAPN3 function. Previous work from our group showed that these variants have different capacities of exchange in physiological situations [30] It is unclear whether the calpain family cleaves both variants and how the cleavage affects their functional properties. The results were confirmed by the use of a siRNA targeting CAPN1 By expressing both CAPN3 and its inactive form (C129S CAPN3), a novel regulation of NCX3 variants by the skeletal muscle CAPN3 was found. The combination of data concerning the reverse mode triggered by [Ca2+]i and by [Na+]i, together with the use of site-directed mutagenesis, enabled a full characterization of the transport capacities of the exchanger upon regulation by calpain and provided a new insight into the molecular determinants responsible for this sensitivity
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