Background: Autosomal-recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated cardiomyopathy (CNM5). Loss of SPEG is associated with defective triad formation, abnormal excitation-contraction coupling, calcium mishandling, and disruption of focal adhesion complex in skeletal muscles.
Objectives: To elucidate the underlying molecular pathways, we have utilized multi-omics tools and analysis to obtain a comprehensive view of the complex biological processes and molecular functions. Skeletal muscles from two-month-old SPEG-deficient and wildtype mice were used for RNA sequencing (n = 4 per genotype) and mass spectrometry (n = 4 for wildtype; n = 3 for SPEG-deficient mice) to profile the transcriptomics, interactomics, proteomics, and phospho-proteomics.
Results: We identified that SPEG interacts with myospryn complex proteins CMYA5, FSD2, RyR1 which are critical for triad formation, and SPEG deficiency results in myospryn complex abnormalities (protein levels decreased to 22% ± 3% for CMYA5 (P < 0.05) and 18% ± 3% for FSD2 (P < 0.01)). In addition, transcriptional and protein profiles of SPEG-deficient muscle revealed defective mitochondrial function including aberrant accumulation of enlarged mitochondria (P < 0.0001) on electron microscopy. Furthermore, SPEG regulates RyR1 phosphorylation at S2902 (phosphorylation level decreased to 55% ± 15% at S2902; P < 0.05), and its loss affects JPH2 phosphorylation at multiple sites. On analyzing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix-receptor interaction (P < 1e-15) and peroxisome proliferator-activated receptors signaling (P < 9e-14), which may be due to defective triad and mitochondrial abnormalities.
Conclusions: We have elucidated the critical role of SPEG in triad as it works closely with myospryn complex, phosphorylates JPH2 and RyR1, and demonstrated that its deficiency is associated with mitochondrial abnormalities. This study emphasizes the importance of using multi-omics techniques to comprehensively analyze the molecular anomalies of myopathy and lay a foundation to develop novel and precise therapies for patients carrying SPEG mutations.