Background: Myocardial injury induced by ischemia-reperfusion (I/R) remains a major barrier to improved clinical outcomes and continues to compromise human health. Adenosine diphosphate-ribosylation factor GTPase-activating protein (ArfGAP) with homology 3 (SH3) domain, ankyrin repeat, and pleckstrin homology (PH) domain-containing protein 3 (ASAP3) is a key regulator involved in cytoskeletal remodeling, membrane dynamics, and signal transduction. Its potential role in the pathogenesis of myocardial I/R injury, however, remains unclear. This study aims to elucidate the function of ASAP3 in myocardial I/R injury.

Methods: A hypoxia-reperfusion (H/R) injury model was established in H9c2 cells. The expression of ASAP3 and ras-related C3 botulinum toxin substrate 1 (RAC1) was modulated by transfection. Cell viability and apoptosis were subsequently assessed. Additionally, intracellular Ca²⁺ accumulation, mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential, and adenosine triphosphate (ATP) levels were evaluated. Endoplasmic reticulum (ER) stress and mitochondria-ER crosstalk were also investigated. Co-immunoprecipitation (Co-IP) experiments were conducted to verify the ASAP3-RAC1 interaction.

Results: ASAP3 expression was significantly downregulated following H/R injury (p < 0.01), concomitant with reduced cell viability and increased apoptosis in H9c2 cells (p < 0.01). Overexpression of ASAP3 significantly improved cell viability and decreased apoptosis under H/R conditions (p < 0.01). Furthermore, ASAP3 overexpression reduced intracellular Ca²⁺ accumulation (p < 0.01), preserved mitochondrial and ER function as evidenced by increased JC-1 and ATP levels, and reduced ROS production and ER stress markers (p < 0.01). Co-IP experiments confirmed a physical interaction between ASAP3 and RAC1, suggesting a possible direct regulatory mechanism. Notably, RAC1 overexpression abolished the protective effects of ASAP3 (p < 0.01), highlighting an antagonistic interplay between these proteins.

Conclusions: Overexpression of ASAP3 in cells subjected to H/R injury inhibits RAC1 activation and facilitates mitochondria-endoplasmic reticulum crosstalk, thereby alleviating cardiomyocyte injury.