STX4 Is Indispensable for Mitochondrial Homeostasis in Skeletal Muscle

ABSTRACT Background Mitochondrial homeostasis is vital for optimal skeletal muscle integrity. Mitochondrial quality control (MQC) mechanisms that are essential for maintaining proper functions of mitochondria include mitochondrial biogenesis, dynamics and mitophagy. Previously, Syntaxin 4 (STX4), traditionally considered a cell surface protein known for glucose uptake in skeletal muscle, was also identified at the outer mitochondrial membrane. STX4 enrichment was sufficient to reverse Type 2 diabetes–associated mitochondrial damage in skeletal muscle by inactivation of mitochondrial fission. However, whether STX4 could modulate skeletal muscle mitochondrial homeostasis through MQC mechanisms involving mitochondrial biogenesis or mitophagy remains to be determined. Methods To determine the requirements of STX4 in mitochondrial structure, function and MQC processes of biogenesis and mitophagy, we implemented our in‐house generated inducible skeletal muscle‐specific STX4‐knockout (skmSTX4‐iKO) mice ( Stx4 fl/fl ; Tg (HSA‐rtTA/TRE‐Cre )/B6) and STX4‐depleted immortalized L6.GLUT4myc myotubes via siRNA knockdown (siSTX4). Results We found that non‐obese skmSTX4‐iKO male mice (> 50% reduced STX4 abundance, soleus and gastrocnemius *** p 60% reduction of STX4 in siSTX4 L6.GLUT4myc myotubes (**** p < 0.0001) also decreased ETC abundance (CI ** p < 0.01, CII *** p < 0.001, CIV ** p < 0.01) without changes in mitochondrial glucose metabolism, as shown by U‐ 13 Cglucose isotope tracing. For MQC, both skmSTX4‐iKO male mice (* p < 0.05) and siSTX4 L6.GLUT4myc myotubes (* p < 0.05) showed decreased mitochondrial DNA levels alongside reduced mRNA expression of mitochondrial biogenesis genes Ppargc1a (PGC1‐α, * p < 0.05) and Tfam (* p < 0.05) in skmSTX4‐iKO soleus muscle and PGC1‐α (mRNA ** p < 0.01, protein * p < 0.05), NRF1 (mRNA ** p < 0.01 and protein * p < 0.05) and Tfam (mRNA * p < 0.05) in siSTX4 L6.GLUT4myc myotubes. Furthermore, live cell imaging using the mt‐Keima mitophagy biosensor in siSTX4 L6.GLUT4myc cells revealed significantly impaired mitochondrial turnover by mitophagy (* p < 0.05) and mitochondria–lysosome colocalization (* p < 0.05). STX4 depletion also reduced canonical mitophagy markers, PINK1 and PARKIN in both skmSTX4‐iKO muscle (PARKIN * p < 0.05, PINK1 ** p < 0.01) and siSTX4 L6.GLUT4myc myotubes (PARKIN ** p < 0.01, PINK1 * p < 0.05). Conclusions Our study demonstrated STX4 as a key mitochondrial regulator required for mitochondrial homeostasis in skeletal muscle.