Figure 3. Evaluation of Mitochondrial Content in Muscle Specimens With Spinal Muscular Atrophy (SMA) and Controls.

Mitochondrial DNA (mtDNA) copy number was determined by quantitative real-time polymerase chain reaction (PCR) in skeletal muscle samples obtained from 7 patients with SMA-I, 5 with SMA-II, and 4 with SMA-III and from 4 age-matched (12–36 months) healthy control samples. All determinations were performed in quadruplicate using 25 ng of total DNA as a template. The mtDNA levels were normalized to nuclear DNA and expressed as relative quantification (RQ) values; reference (RQ = 1.0), the amount of mtDNA detected in skeletal muscle from age-matched controls. A, Mean (SD) levels of mtDNA in each group after normalization to nuclear DNA content. B, Western blot analysis of protein extracts obtained from 4 SMA-I and 4 control biopsy samples using primary antibodies against porin and β-actin for normalization. C, Ratio of porin to β-actin signals expressed in arbitrary units for control and SMA-I samples. Horizontal bars indicate mean values. D and E, Quantitative reverse transcription–PCR studies to evaluate the levels of the indicated transcripts in skeletal muscle samples obtained from 4 patients with SMA-I, 3 with SMA-II, and 4 with SMA-III and 4 age-matched controls. 18S was used as the control housekeeping gene. All determinations were performed in replicates (n = 4). Results are presented as mean (SD) levels in each group. F, Western blot analysis of protein extracts obtained from 4SMA-I and 4 control biopsy specimens using primary antibodies against respiratory chain subunits encoded by mtDNA (MT-CO1 and MT-CO2) and nuclear DNA (COX4 and SDHA) compared with β-actin levels. G, Results of densitometry analysis of respiratory chain subunits after normalization to β-actin expressed in arbitrary units for controls and SMA samples. Horizontal bars indicate mean values. All P values were calculated using the unpaired 2-tailed t test. COX4I1 indicates cytochrome-c oxidase (COX) 4 isoform 1; MT-ATP6, mitochondrially encoded adenosine triphosphate 6;MT-CO1 and -CO2, mitochondrially encoded COX1 and COX2, respectively; MT-ND1, mitochondrially encoded nicotinamide adenine dinucleotide dehydrogenase; NRF, nuclear respiratory factor; PGC-1α, peroxisome proliferator–activated receptor coactivator 1α; PTEN, phosphatase and tensin homolog; SDHA, succinate dehydrogenase complex subunit A; and TFAM, mitochondrial transcription factor A.

^a P < .01 compared with SMA-I.

^b P < .05 compared with SMA-II.

^c P < .05 compared with control.

^d P < .01 compared with control.