Metformin attenuates ER stress-induced mitochondrial dysfunction

Abstract

Endoplasmic reticulum (ER) stress, a disturbance of the ER function, contributes to cardiac injury. ER and mitochondria are closely connected organelles within cells. ER stress contributes to mitochondrial dysfunction, which is a key factor to increase cardiac injury. Metformin, a traditional anti-diabetic drug, decreases cardiac injury during ischemia-reperfusion. Metformin also inhibits ER stress in cultured cells. We hypothesized that metformin can attenuate the ER stress-induced mitochondrial dysfunction and subsequent cardiac injury. Thapsigargin (THAP, 3 mg/kg) was used to induce ER stress in C57BL/6 mice. Cell injury and mitochondrial function were evaluated in the mouse heart 48 hours after 1-time THAP treatment. Metformin was dissolved in drinking water (0.5 g/250 ml) and fed to mice for 7 days before THAP injection. Metformin feeding continued after THAP treatment. THAP treatment increased apoptosis in mouse myocardium compared to control. THAP also led to decreased oxidative phosphorylation in heart mitochondria-oxidizing complex I substrates. THAP decreased the calcium retention capacity, indicating that ER stress sensitizes mitochondria to mitochondrial permeability transition pore opening. The cytosolic C/EBP homologous protein (CHOP) content was markedly increased in THAP-treated hearts compared to control, particularly in the nucleus. Metformin prevented the THAP-induced mitochondrial dysfunction and reduced CHOP content in cytosol and nucleus. Thus, metformin reduces cardiac injury during ER stress through the protection of cardiac mitochondria and attenuation of CHOP expression.

Copyright © 2017 Elsevier Inc. All rights reserved.

Figures

Figure 1. Administration of metformin decreases cardiac injury in THAP-treated mouse hearts

THAP (3 mg/kg, I.P.) was used to induce the ER stress for 48 hours in C57BL/6 mice. In metformin-treated hearts, mice first received metformin (dissolved in the drinking water) for one week. Then, THAP was administered to mice once and mice followed for 48 hours (Panel A). Apoptotic cell death was assessed using TUNEL staining (green color, Panel B). Total nuclei were quantified using DAPI staining (blue color, Panel B). Arrowhead indicated a typical TUNEL positive nucleus. THAP significantly increased apoptotic cell death compared to vehicle (Panel B&C). Metformin treatment markedly decreased apoptotic cell death in the THAP-treated hearts with no effect in untreated hearts. Mean ± SEM. *p

Figure 2. Administration of metformin activates AMPK and decreases CHOP expression in cytosol and nucleus in THAP-treated mouse hearts

THAP treatment increased the content of CHOP in cytosol (Panel A) and nucleus (Panel B) compared to vehicle. Metformin treatment dramatically decreased the expression of CHOP in cytosol and nucleus in THAP-treated hearts (Panel A&B). In metformin treated hearts, phosphorylated AMPK content was increased compared to non-metformin-treated hearts (Panel C); supporting that metformin feeding activates the AMPK as expected. The potential mechanisms of metformin protection during the ER stress was summarized in the Panel D. Mean ± SEM. *p

Figure 3. Administration of metformin improves mitochondrial function in THAP-treated mouse hearts

Compared to vehicle, THAP treatment decreased the rate of oxidative phosphorylation when glutamate + malate was used as complex I substrate (Panel A). Metformin feeding protected oxidative phosphorylation rates in the THAP-treated hearts oxidizing complex I substrates. Metformin feeding alone did not affect the oxidative phosphorylation with complex I substrates (Panel A). There were no differences in the oxidation of succinate (+rotenone) between groups (Panel B). Mean ± SEM; * p <0.05 vs. other groups. N=6 in each group.

Figure 4. Administration of metformin decreases MPTP opening and ROS generation in THAP-treated mouse hearts

Calcium retention capacity (CRC) was used to measure the sensitivity to MPTP opening in isolated mitochondria (Panel A). The CRC was decreased in the THAP-treated heart mitochondria compared to vehicle, suggesting that the ER stress sensitizes to MPTP opening in cardiac mitochondria (Panel A&B). Metformin treatment improved the CRC in the THAP-treated mitochondria (Panel A&B), indicating that metformin decreases MPTP opening during the ER stress. THAP treatment also increased the ROS generation in isolated mitochondria oxidizing complex II substrates (Panel D) but not complex I substrates (Panel C) compared to vehicle. Again, metformin decreased the ROS generation in the TAHP-treated mitochondria (Panel D). Mean ± SEM. *p <0.05 vs. other groups. n=6 in each group.