Yiqi Huoxue formula promotes post-myocardial infarction angiogenesis by modulating mitochondria-associated membranes (MAMs) and calcium homeostasis via the cGMP/PKG signaling pathway

Ethnopharmacological relevance: Therapeutic angiogenesis represents a promising strategy for recovery following myocardial infarction (MI). Yiqi Huoxue Formula (YQHX), a well-known traditional Chinese medicinal prescription, is widely utilized in clinical practice to treat myocardial ischemia and enhance cardiac performance in patients with MI. This study aims to elucidate the regulatory role of YQHX on mitochondria-associated membranes (MAMs) via the cGMP/PKG signaling pathway in promoting post-MI angiogenesis and restoring cardiac function.

Materials and methods: An in vivo MI model was established via left anterior descending (LAD) coronary artery ligation in rats, and an in vitro model was developed using hypoxia-induced injury in human umbilical vein endothelial cells (HUVECs). The effects of YQHX on cardiac function and HUVEC behaviors (proliferation, migration, and tube formation) were evaluated. Myocardial histopathology and endothelial damage were assessed using HE/Masson staining and biochemical assays. Angiogenesis in the infarct border zone was visualized by platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) immunofluorescence. Ultrastructural changes in MAMs, the endoplasmic reticulum (ER), and mitochondria were observed via transmission electron microscopy (TEM). Intracellular Reactive Oxygen Species (ROS) and calcium (CA ²⁺) levels were detected using fluorescent probes. Transcriptomic Sequencing was performed to identify key therapeutic pathways, followed by validation of the cGMP/PKG axis and MAMs-associated proteins (including cGMP, PKG, IP3R2, GRP75, FUNDC1, VDAC1, CYPD, and MCU) using ELISA, Western blotting, and immunofluorescence.

Results: Echocardiography and biochemical analysis demonstrated that high-dose YQHX demonstrated comparable efficacy to the first-line clinical drug Perindopril in preserving cardiac function and mitigating myocardial injury. Immunofluorescence revealed that while the infarct border zone in the MI group exhibited disordered and dysfunctional capillary proliferation, YQHX treatment promoted the formation of organized and functional microvessels. TEM revealed significant disruption of MAMs ultrastructure post-MI. Furthermore, the colocalization of IP3R2 and VDAC1 was markedly reduced in MI tissues and hypoxic HUVECs, an effect that was reversed by YQHX. Transcriptomic analysis identified the cGMP/PKG pathway as a pivotal mechanism. Activation of this pathway by YQHX restored MAMs structural integrity and rescued impaired cytosolic CA²⁺ signaling, as confirmed by TEM, Western blotting, triple-labeling immunofluorescence, and calcium assays. Consequently, YQHX-mediated MAMs repair enhanced HUVEC proliferation, migration, and angiogenic capacity.

Conclusion: This study demonstrates that MI/hypoxia impairs endothelial function by disrupting the structural and functional integrity of MAMs. YQHX effectively preserves MAMs architecture and restores intracellular CA²⁺ signaling via the cGMP/PKG signaling axis, thereby promoting post-MI angiogenesis and improving cardiac performance. These findings identify a novel therapeutic target for MI and highlight the unique advantages of traditional Chinese medicine formulas in modulating subcellular organelle interactions. However, given the inherent variability of botanical Materials, these mechanistic findings are based on a single validated batch, and future multi-batch standardizations are warranted.