Tongyang Huoxue decoction (TYHX) ameliorating hypoxia/ reoxygenation-induced disequilibrium of calcium homeostasis via regulating -tubulin in rabbit sinoatrial node cells

Ethnopharmacological relevance: ss-tubulin is a skeletal protein of sinoatrial node cells (SANCs) that maintains the physiological structure of SANCs and inhibits calcium overload. Tongyang Huoxue decoction (TYHX) is widely used to treat sick sinus syndrome (SSS) owing to its effects on calcium channels regulation and SANCs protection. Aim of the study: This study focuses on the mechanism of TYHX in improving the hypoxia/reoxygenation (H/R)induced disequilibrium of calcium homeostasis in SANCs via regulating ss-tubulin. Materials and methods: Real-Time PCR (RT-PCR) and Western Blot were adopted to detect the mRNA and protein expression levels of calcium channel regulatory molecules. Laser confocal method was employed to examine ss-tubulin structure and fluorescence expression levels in SANCs, as well as calcium wave and calcium release levels. Results: It was found that the fluorescence expression level decreased and the ss-tubulin structure of SANCs was damaged after H/R treatment. The mRNA and protein expression levels of SERCA2a/CaV1.3/NCX and ss-tubulin decreased, while the mRNA and protein expression of RyR2 increased. The results of calcium wave and calcium transient experiments showed that the fluorescence expression level of Ca2+ increased and calcium overload occurred in SANCs. After treatment with TYHX, the mRNA and protein expression levels of SERCA2a/CaV1.3/ NCX and ss-tubulin increased, while the mRNA and protein expression levels of RyR2 decreased and the cell structure was restored. Interestingly, the regulation of TYHX on calcium homeostasis was further enhanced after Ad-ss-tubulin treatment and counteracted after siRNA-ss-tubulin treatment. These results suggest that TYHX could maintain calcium homeostasis via regulating ss-tubulin, thus protecting against H/R-induced SANCs injury, which may be a new target for SSS treatment.