Program

PO3-10-39

Salvianolic acid A suppressing mitochondria-associated endoplasmic reticulum membrane (MAM) to limit myocardial ischemia/reperfusion injury in mice

[Speaker] Shou-Bao Wang:1
[Co-author] Dan-Shu Wang:1, Yi-Huang Lin:1, Rong-Rong Wang:1, Lian-Hua Fang:1, Yang Lv:2, Guan-Hua Du:1
1:Beijing Key Laboratory of Drug Target Research and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science & Peking Union Medical College, China, 2:Beijing Key Laboratory of Polymorphic Drugs, Institute of Materia Medica, Chinese Academy of Medical Science & Peking Union Medical College, China

Background: Although the endoplasmic reticulum (ER) and mitochondria play distinct cellular roles, the physical interactions between the two organelles defined as mitochondria-associated ER membranes (MAM), which are essential for cellular homeostasis, such as calcium, lipid and metabolite exchange. Salvianolic acid A ((2R)-3-(3, 4-dihydroxyphenyl)-2-[(E)-3-[2-[(E)-2-(3, 4-dihydroxyphenyl) ethenyl]-3, 4-dihydroxyphenyl] prop-2-enoyl] oxypropanoic acid, SAA) is the main active constituent of Salvia miltiorrhiza. In spite of a large body of evidence showing protective effects of SAA on the myocardium during ischemia/reperfusion (I/R), the underlying mechanisms have not been clearly elucidated. Here we investigated whether SAA exerts the cardioprotective role through modulation of MAM.
Methods: Ischemia/reperfusion injury was established in cultured neonatal rat cardiomyocytes (NRCMs) subjected to hypoxia/reoxygenation and using left coronary artery occlusion and reperfusion in mice. Organelle-targeted fluorescent proteins were used in NRCMs to investigate the alterations in ER-mitochondria juxtaposition. We also employed PLA in mice heart cryosection as an additional strategy in to imply the alterations in MAM. Altered ER-mitochondrial protein expression was measured in total lysate and enriched fractions of MAM using Percoll-based subcellular fractionation of heart tissues. MAM was functionally indicated by mitochondrial calcium overload, compromised mitochondrial oxidative capacity and augmented oxidative stress. We used adenovirus-mediated gene transfer to express a recombinant construct encoding a synthetic linker that increases ER-mitochondria contact sites in the heart of animals to verify the effects of SAA on MAM.
Results: SAA significantly decreased I/R-induced cell death in both in vitro and in vivo models. ER-mitochondrial apposition and MAM function are increased significantly in I/R injured cells and mice. SAA treatment reduced the level of MAM, attenuated the mitochondrial calcium overload and improved mitochondrial oxidative capacity in animals. Downregulation of PACS-2, IP3R1, and VDAC1 in enriched MAM subfraction, proteins important for ER-mitochondria tethering or calcium transport, was observed in SAA treated NRCMs and mice. Experimental induction of ER-mitochondria interactions impaired the protective effects of SAA in the context of mitochondrial calcium overload, oxidative stress, and mitochondrial oxidative capacity in I/R animals.
Coclusions: These findings suggested that SAA suppresses mitochondria-associated endoplasmic reticulum membrane (MAM) to limit myocardial ischemia/reperfusion injury in mice.
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