sAC-Dependent Mitochondrial Dynamics and Endothelial Barrier Protection: A Therapeutic Target for Cardiovascular Dysfunction

Background: Accumulating evidence suggests that cyclic AMP (cAMP) signaling plays a key role in promoting mitochondrial function, with mitochondrial cAMP levels regulated by type 10 soluble adenylyl cyclase (sAC). Our recent work showed that knockdown of sAC in cardiac cells leads to mitochondrial dysfunction, which may contribute to cardiac pathophysiology. Mitochondrial dynamics, including fission and fusion, are essential for maintaining mitochondrial health and are disrupted in cardiac diseases such as heart failure. In this study, we investigated the effects of sAC overexpression on mitochondrial dynamics in endothelial cells (ECs), aiming to explore its potential therapeutic role in restoring mitochondrial function and improving cardiac outcomes.

Methods: The study is performed in human microvascular endothelial cell line (HMEC-1) and primary human umbilical vein endothelial cells (HUVEC). Mitochondrial dynamics are studied by live-cell imaging using fluorescent chemical probes. Human sAC is over-expressed using lentiviral-based system. Substrate metabolism was assessed by OmniLog system (Biolog Inc.) using MitoPlates™.

Results: Overexpression of sAC in both HMEC-1 cells and HUVECs resulted in increased cellular cAMP levels, which were accompanied by the appearance of small-sized mitochondria and the disappearance of long, networked mitochondria. This shift in mitochondrial morphology is likely due to enhanced mitochondrial fission and/or reduced fusion. In addition, the mitochondrial DNA-to-nuclear DNA (mitDNA/nDNA) ratio was elevated in sAC-overexpressing cells, suggesting an increase in mitochondrial biogenesis. These effects were reversed upon treatment with LRE1, a pharmacological inhibitor of sAC.
Further investigation revealed that the changes in mitochondrial morphology induced by sAC overexpression were reversed by PKI (1 μM), a specific inhibitor of PKA, but not by ESI-09 (1 μM), an inhibitor of Epac, indicating that the effect is mediated through the sAC/cAMP/PKA pathway.
Metabolic analysis using MitoPlate™ showed that sAC overexpression in ECs increased pyruvate and other oxidative phosphorylation (OXPHOS) substrate metabolism, suggesting that these mitochondria are metabolically active. Additionally, sAC-overexpressing ECs exhibited higher levels of ROS production and enhanced AMPK phosphorylation. Importantly, the hyperpermeability induced by thrombin and TNF-α in ECs was ameliorated in sAC-overexpressing cells, indicating a protective effect on the endothelial barrier.

Conclusion: Overexpression of sAC enhances mitochondrial biogenesis and metabolic activity in endothelial cells, mediated through the cAMP/PKA pathway. Additionally, sAC overexpression improves endothelial barrier function, protecting against hyperpermeability induced by thrombin and TNF-α. These findings suggest that sAC could be a potential therapeutic target for improving endothelial function and mitigating inflammation-related vascular dysfunction.