Deficient Cardiolipin Remodelling Alters Muscle Fibre Composition and Neuromuscular Connectivity in Barth Syndrome

Figures

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  FIGURE 1. Cardiolipin species and skeletal muscle phenotype of Taz PM . (a) Mass spectrometry quantification of individual monolysocardiolipin (MLCL) species and total MLCL (sum of individual species) in isolated mitochondria from age‐matched wt and Taz PM gastrocnemius muscles. n = 12 per genotype. (b) Mass spectrometry quantification of individual cardiolipin (CL) species and total CL (sum of individual species). (c) Calculation of total MLCL to total CL ratio. (d) Body weight n = 10 per genotype, (e) maximal weight lifted per mouse and (f) duration of hanging from a high bar. n = 10 per genotype. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 2. Skeletal muscle fibres are smaller and exhibit a shift towards a faster myosin heavy chain in the Taz PM mice. (a) Immunofluorescence for laminin (magenta), type 1 myosin heavy chain (MHC)(blue), type 2A MHC (green) and type 2B MHC (red) was performed on age‐matched wt and Taz PM tibialis anterior muscle cross‐sections. Unstained muscle fibres are inferred to be type 2× MHC (black). (b) Frequency distribution of fibre size separated by MHC expression. (c) Mean fibre type‐specific cross‐sectional area (CSA) fibre size. ND = none detected. (d) The proportion of fibres staining positive for each MHC. (e) Mean area of all fibres. n = 3–4 per genotype. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 3. Motor unit number decreases in Taz PM hindlimb. (a) Single motor unit potential (SMUP), (b) motor unit number estimate (MUNE), (c) compound muscle action potential from onset to peak (CMAP(o‐p)) and (d) compound muscle action potential from peak to trough (CMAP(p‐t)) were measured in vivo from age‐matched wt and Taz PM triceps surae muscles. n = 6–7 per genotype. **p < 0.01.

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  FIGURE 4. Proteins of the neuromuscular junction are dysregulated in muscle of Taz PM . Representative immunofluorescent imaging for synapsin, β tubulin III, BTX and merged images for (a) wt and (b) Taz PM gastrocnemius. (c) Western blot images for synaptosomal‐associated protein‐25 (SNAP25), α tubulin, β tubulin, cholinergic receptor nicotinic alpha 1 subunit (CHRNA1), muscle associated receptor tyrosine kinase (MUSK), dedicator of cytokinesis 7 (DOK7), Rapsyn and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). (d) Quantification of western blots for presynaptic proteins SNAP25, α tubulin and β tubulin. (e) Quantification of the western blots for the postsynaptic proteins CHRNA1, MUSK, DOK7 46‐kDa band, DOK7 53‐kDa band and Rapsyn. n = 3 per genotype.

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  FIGURE 5. Mitochondrial morphology is deranged without consistent changes in mitochondrial protein content in muscle of Taz PM mice. (a) Representative TEM images of cross‐section (top) and longitudinal sections (bottom) of wt (left) and Taz PM (right) gastrocnemius muscle. Note that Taz PM mitochondria are swollen and disorganized, mainly lacking cristae (indicated via red M) or are swollen with only sparsely organized cristae (red M*) compared to smaller wt mitochondria (white M). Skeletal muscle appears as polygons in cross sections (top panels) and in repeating, banded pattern in longitudinal sections (bottom panels). Scale bars: Top = 800 nm, Bottom = 1 μm. (b) Western blots for mitochondrial proteins citrate synthase (CS), voltage dependent anion‐selective channel 1 (VDAC1), succinate dehydrogenase (SDHA), ATP synthase F1 subunit alpha (ATP5a1) and NADH ubiquinone oxidoreductase subunit B8 (NDUFB8), mitochondrial calcium uniporter (MCU), transmembrane protein 65 (TMEM65) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). (c) Quantification of citrate synthase blot. (d) Mitochondrial protein western blot quantifications. n = 3 per genotype.

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  FIGURE 6. The energetic state in gastrocnemius muscle of Taz PM mice is the same as that of wt mice. (a) Adenine nucleotide (ATP, ADP and AMP) analysis by ultra‐performance liquid chromatography (UPLC) of gastrocnemius muscles. (b) Adenine nucleotide calculations of energy charge = (ATP + (0.5*ADP))/(ATP + ADP + AMP), ADP/ATP ratio and AMP/ATP ratio. (c) Calculation for the sum of adenine nucleotides (AdN Sum = ATP + ADP + AMP) and UPLC analysis of uric acid. (d) UPLC analysis of phosphocreatine (PCr). (e) UPLC analysis of NAD+, NADH and NAD+/NADH ratio. n = 3 per genotype.

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  FIGURE 7. Stress signalling is increased in muscle of Taz PM mice. (a) Western blots for phosphoSer51‐eukaryotic translation initiation factor 2A (eIF2A S51), total eukaryotic translation initiation factor 2A (eIF2A), C/EBP homologous protein (CHOP), Protein Death Ligand Enhancer (DELE1), p53, mouse double minute 2 homolog (MDM2), β‐catenin, glycogen synthase kinase 3 alpha (GSK3 α), glycogen synthase kinase 3 beta (GSK3 β) and GAPDH. (b) Quantification of the western blots for ISR proteins eIF2A and CHOP and mitochondrial stress response DELE1 long form (DELE1‐L) and processed form (DELE1‐S). (c) Quantification of western blots for p53, MDM2, β‐catenin, GSK3 α and GSK3 β. n = 3 per genotype.