SCN5A 下調介導的心臟纖維化與功能障礙：纖維化訊號傳導、代謝調控及 microRNA-452 之影響解析

SCN5A encodes the Nav1.5 sodium channel, essential for cardiac electrophysiology, and its downregulation is associated with arrhythmia and heart failure (HF). Recent evidence indicates that SCN5A dysfunction contributes to ventricular fibrosis through myocardial remodeling in clinical and experimental models of SCN5A loss-of-function mutations. Additionally, cardiac fibroblast activation and excessive extracellular matrix deposition lead to metabolic alterations that further drive fibrotic progression. These fibroblasts aggravated calcium dynamics impairment in adjacent myocytes through paracrine signaling. However, its direct impact on cardiac fibrosis and metabolism remains unclear. This thesis investigates the fibrotic and metabolic consequences of SCN5A knockdown (Skd) in human cardiac fibroblasts (HCF) and also explores the potential effect of Skd on HCF cytokines on calcium cycling in myocytes through conditioned medium.

Immunoblot analysis demonstrated that Skd increased collagen, α-SMA, and fibronectin expression, indicating enhanced fibrosis in Skd HCF compared to control. MicroRNA deep sequencing and qRT-PCR analysis showed a significant decrease of miR-452-5p, while a bioinformatic study identified maladaptive activation of TGF-β/SMAD4 signaling. The direct targeting of SMAD4 by miR-452-5p was confirmed through luciferase reporter assays. Moreover, metabolomic and lipidomic profiling of Skd HCF showed significant metabolic shifts, with upregulated nitrogen, arginine, and glucose metabolism, but reduced fatty acid oxidation (FAO) and tricarboxylic acid (TCA) cycle activity. These changes coincided with impaired cardiac energy output, exacerbating cellular metabolic stress. Additionally, conditioned medium from SCN5A-knockdown HCF impairs calcium cycling in HL-1 cardiomyocytes, indicating that fibroblast-derived factors can adversely affect myocyte calcium handling.

In contrast, miR-452-5p agomir treatment (10 nM, transfection for 8 hr, then cultured in full medium up to 48 hr) attenuated fibrosis and mitochondrial function, improved adenosine triphosphate (ATP) turnover, and partially reduced ROS accumulation. Furthermore, pathway enrichment analysis revealed that miR-452-5p modulated dysregulated metabolic pathways, particularly FAO, pyruvate metabolism, and the tricarboxylic acid cycle. Lipidomic analysis also identified significant alterations in ceramides, fatty acids, glycerides, and sphingolipids in Skd HCF, which were attenuated by miR-452-5p treatment. In addition, calcium dysregulation in HL-1 cells is independent of miR-452-5p, potentially predisposing to calcium dysregulation and arrhythmogenesis. Furthermore, two weeks after systemic delivery of AAV9-miR-452-5p in HF rats (induced through subcutaneous isoproterenol administration, 100 mg/kg body weight), improved cardiac function, reduced myocardial fibrosis, and metabolic reprogramming were observed, thereby demonstrating its therapeutic potential.

In conclusion, SCN5A downregulation induces metabolic reprogramming and fibrosis, while miR-452-5p restoration alleviates these effects by elevating metabolism and reducing fibrosis, highlighting its therapeutic potential. This study reveals the metabolic-fibrotic role of SCN5A in cardiac fibroblasts and its impact beyond electrophysiological dysfunction.