Redox State and Mitochondrial Respiratory Chain Function in Skeletal Muscle of LGMD2A Patients

<div><p>Background</p><p>Calpain-3 deficiency causes oxidative and nitrosative stress-induced damage in skeletal muscle of LGMD2A patients, but mitochondrial respiratory chain function and anti-oxidant levels have not been systematically assessed in this clinical population previously.</p><p>Methods</p><p>We identified 14 patients with phenotypes consistent with LGMD2A and performed <i>CAPN3</i> gene sequencing, CAPN3 expression/autolysis measurements, and <i>in</i><i>silico</i> predictions of pathogenicity. Oxidative damage, anti-oxidant capacity, and mitochondrial enzyme activities were determined in a subset of muscle biopsies.</p><p>Results</p><p>Twenty-one disease-causing variants were detected along the entire <i>CAPN3</i> gene, five of which were novel (c.338 T>C, c.500 T>C, c.1525-1 G>T, c.2115+4 T>G, c.2366 T>A). Protein- and mRNA-based tests confirmed <i>in</i><i>silico</i> predictions and the clinical diagnosis in 75% of patients. Reductions in antioxidant defense mechanisms (SOD-1 and NRF-2, but not SOD-2), coupled with increased lipid peroxidation and protein ubiquitination, were observed in calpain-3 deficient muscle, indicating a redox imbalance primarily affecting non-mitochondrial compartments. Although ATP synthase levels were significantly lower in LGMD2A patients, citrate synthase, cytochrome <i>c</i> oxidase, and complex I+III activities were not different from controls.</p><p>Conclusions</p><p>Despite significant oxidative damage and redox imbalance in cytosolic/myofibrillar compartments, mitochondrial respiratory chain function is largely maintained in skeletal muscle of LGMD2A patients.</p></div

Western blot analyses of OXPHOS expression (complex I–V) and ETC enzyme activities (CS, COX, and complex I+III) in LGMD2A patients.

<p>*Significantly lower ATP synthase expression in calpain-3 deficient muscle (P≤0.05). All Western blot data were normalized to total protein levels (Ponceau S stain) and represent averages of age/gender-matched controls (N = 7 for OXPHOS and enzyme assays; N = 5 for VDAC) and LGMD2A patients (N = 5 for OXPHOS and VDAC; N = 2–3 for enzyme assays). Representative images of OXPHOS blots and Ponceau S stain (42 kDa) are shown.</p

LGMD2A phenotypes, CAPN3 sequence variants, and <i>in</i><i>silico</i> predictions.

A<p>Current age/age at biopsy (or genetic confirmation)/first symptoms noted, ^† = deceased, w = wheelchair, c = childhood;</p>B<p>Phenotypic classification into Erb (scapulohumeral), early pelvifemoral (≤12 y), classical pelvifemoral (Leyden-Möbius;13–29 y), late onset pelvifemoral (≥30 y), or asymptomatic phenotype (hyperCKemia);</p>C<p><i>CAPN3</i> exon/intron;</p>D<p>CAPN3 domains;</p>E<p>Zygosity;</p>F<p>Novelty;</p>G<p><i>In silico</i> predictions of pathogenicity of missense mutations with SIFT (Uni-Prot TrEMBL 2009 Mar) and PolyPhen-2 (v2.2.2r398). Y = pathological; N = benign. Conservation of exchanged/deleted amino acids with UCSC genome browser (Human Feb. 2009 [GRCh37/hg19] Assembly) across 46 species. Effects of intronic mutations on pre-mRNA splicing with Human Splicing Finder program (version 2.4.1).</p>$<p>Mutation in 5′ end of intron 19 creates a cryptic splice site that is used instead of the regular site.</p><p>#Mutation in 5′ end of intron 4 destroys donor splice site.</p>‡<p>Mutation in 3′ end of intron 11 destroys the acceptor splice site.</p><p>*Selected for further biochemical testing.</p

Western blot analyses of anti-oxidant capacity (SOD-1, SOD-2, NRF-2/Keap-1), oxidative damage (lipid peroxidation; 4-HNE), and ubiquitination (Ub) in LGMD2A patients.

<p>*P≤0.05 vs. control. **P≤0.01 vs. control. All data were normalized to total protein levels (Ponceau S stain) and represent averages of age/gender-matched controls (N = 3–4; white bars) and LGMD2A patients (N = 4; black bars). Representative images of blots and Ponceau S stain (40–50 kDa) are shown.</p

Expression and autolysis of CAPN3 in quadriceps muscle of LGMD2A patients.

<p>A) CAPN3 mRNA expression (RT-PCR); B) CAPN3 protein (Western blot); C) Ca²⁺-induced autolytic activity of CAPN3 (Western blot). Calpain-3 autolytic activity is presented as a ratio between autolyzed fragments and total CAPN3 (representing <i>relative autolytic capacity</i> of protein present), but the results should also be interpreted in the context of total protein expression from regular Western blots. Logically, a reduction in total CAPN3, obtained from Ca²⁺-free homogenates, equates to a decrease in <i>total autolytic capacity</i>. All CAPN3 data were normalized to a suitable housekeeping gene (β2-microglobulin), protein (actin), or total proteins levels (Ponceau S stain) and graphed as % age/gender-matched healthy controls. Representative images of CAPN3 blots (56–60 kDa, 94 kDa), Ponceau S stain (40–50 kDa), or actin (42 kDa) are shown in B–C. For total protein expression and autolytic activity, N = 7 (LGMD2A) and N = 8 (control), while N = 4 (LGMD2A) and N = 6 (control) for mRNA. Bars to the far left represent the average of all LGMD2A patients (AVG. LGMD2A). INDT: Indeterminate. *Significantly lower compared to controls (P≤0.05).</p