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

TazPM mice exhibited markedly disrupted cardiolipin metabolism, with seven-fold elevated total monolysocardiolipin and five-fold reduced mature cardiolipin levels.

• The TazPM model carries a patient-derived D75H point mutation knocked into the Tafazzin locus.
• This mutation preserves protein abundance but abolishes enzymatic transacylase activity.
• The seven-fold elevation in monolysocardiolipin and five-fold reduction in mature CL confirm deficient transacylase activity.
• Cardiolipin remodelling status was quantified using ultra-performance liquid chromatography.

Male TazPM mice exhibited lower skeletal muscle strength and endurance compared to controls.

• Skeletal muscle function was assessed via weightlifting and hanging tests.
• Both strength and endurance were reduced in TazPM mice.
• The model is male, consistent with the X-linked inheritance of Barth syndrome.

TazPM mice had 32% smaller muscle fibres across all fibre types and a shift toward fast-twitch type 2B fibres.

• Muscle fibre composition and size were evaluated using immunofluorescence and western blotting.
• The fibre size reduction of 32% was observed across all fibre types, not limited to a specific subtype.
• There was a compositional shift towards fast-twitch type 2B fibres, which are described as more susceptible to fatigue.
• This fibre type shift may contribute to the increased fatigability characteristic of Barth syndrome.

Electrophysiological analysis revealed a 60% reduction in motor unit number and an increase in average single motor unit potential in TazPM mice, indicating motor neuron remodelling.

• Motor unit number and single motor unit potential were assessed by in vivo electrophysiology.
• Motor unit number was reduced by 60% in TazPM mice.
• The increase in average single motor unit potential alongside reduced motor unit number is consistent with compensatory motor neuron remodelling.
• These findings indicate neuromuscular remodelling beyond the muscle fibre level.

Neuromuscular junction (NMJ) protein analysis showed decreased MUSK and DOK7 and increased CHRNA1 in TazPM mice, suggesting impaired NMJ integrity.

• NMJ protein levels were assessed by western blotting.
• MUSK and DOK7, which are critical for NMJ formation and maintenance, were decreased.
• CHRNA1 (acetylcholine receptor subunit alpha 1) was increased.
• These changes together suggest dysregulation and impaired integrity of the neuromuscular junction.

TazPM mice exhibited mitochondrial structural abnormalities and reduced expression of key mitochondrial proteins, but resting ATP, phosphocreatine and adenine nucleotide ratios were unchanged.

• Mitochondrial morphology was examined by transmission electron microscopy, revealing structural abnormalities.
• Key mitochondrial proteins NDUFB8, MCU, and TMEM65 showed reduced expression.
• Despite these structural and protein-level changes, resting ATP, phosphocreatine and adenine nucleotide ratios were unchanged in both glycolytic and oxidative muscles.
• Bioenergetics were quantified using ultra-performance liquid chromatography.
• These findings indicate no overt energetic failure at rest.

Stress signalling pathways were markedly activated in TazPM mice, including phosphorylation of eIF2α, increased CHOP, DELE1, and p53 expression, and altered Wnt/β-catenin signalling.

• Stress signalling was assessed by western blotting.
• Activated pathways included phosphorylation of eIF2α (integrated stress response), increased CHOP (unfolded protein response/apoptosis marker), increased DELE1 (mitochondrial stress signalling), and increased p53 (cellular stress/apoptosis).
• Wnt/β-catenin signalling components were also altered.
• These stress pathway activations occurred without overt resting energetic failure, suggesting signalling-driven rather than purely ATP-deficiency-driven pathology.

The authors conclude that myopathy in Barth syndrome arises not solely from mitochondrial ATP insufficiency but from cumulative structural and signalling adaptations.

• Despite widespread mitochondrial structural abnormalities and reduced expression of key mitochondrial proteins, resting energy metabolites were preserved.
• Neuromuscular remodelling encompassed fibre size and type shifts, motor unit loss, NMJ dysregulation, and stress pathway activation.
• The findings suggest that cumulative structural and signalling changes, rather than ATP insufficiency alone, drive the myopathy of Barth syndrome.
• This was demonstrated using a knock-in murine model carrying a patient-derived point mutation that abolishes enzymatic activity while preserving protein abundance.