Zishen Tongyang Huoxue decoction (TYHX) alleviates sinoatrial node cell ischemia/reperfusion injury by directing mitochondrial quality control via the VDAC1--tubulin signaling axis

Ethnopharmacological relevance: Zishen Tongyang Huoxue decoction (TYHX) has been used clinically for nearly 40 years to treat sick sinus syndrome. Previous reports showed that TYHX can inhibit calcium flux by regulating mitochondrial homeostasis via beta-tubulin and increase sinoatrial node cell (SNC) activity. However, the under-lying mechanisms remain unclear.Aim of the study: We aimed to verify the protective effect of TYHX against SNC ischemia by regulating mito-chondrial quality control (MQC) through beta-tubulin and voltage-dependent anion-selective channel 1 (VDAC1) silencing.Materials and methods: We established an in vitro model of SNC ischemia/reperfusion (I/R) injury and performed rescue experiments by silencing beta-tubulin and VDAC1 expression. Cell-Counting Kit 8 assays were performed to detect cell viabilities, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assays (paired with confocal microscopy) were performed to detect fragmentation. Mitochondrial-energy metabolism was detected using the Seahorse assay system. Reverse transcription-quantitative polymerase chain reaction analysis was performed to detect the mRNA-expression levels of MQC-related genes.Results: TYHX inhibited SNC mitochondrial injury. During I/R simulation, TYHX maintained beta-tubulin stability, regulated synergy between mitophagy and the mitochondrial unfolded-protein response (UPRmt), and inhibited mitochondrial oxidative stress and overactive SNC fission. Next-generation sequencing suggested that mitochondrial-membrane injury caused SNC apoptosis. We also found that TYHX regulated beta-tubulin expression through VDAC1 and inhibited dynamin-related protein 1 migration to mitochondria from the nucleus. After preventing excessive mitochondrial fission, the mitophagy-UPRmt pathway, mitochondrial-membrane potential, and mitochondrial energy were restored. VDAC1 silencing affected the regulatory mechanism of MQC in a beta-tubulin-dependent manner via TYHX.Conclusion: TYHX regulated mitochondrial membrane-permeability through VDAC1, which affected MQC through beta-tubulin and inhibited mitochondrial apoptosis. Our findings may help in developing drugs to protect the sinoatrial node.