Activation of volume-sensitive Cl- channel mediates autophagy-related cell death in myocardial ischaemia/reperfusion injury

Abstract

Excessive reactive oxygen species (ROS) plays an important role in myocardial ischemia/reperfusion (I/R) injury, which triggers not only myocardial cellular apoptosis but also autophagy-related cell death, in which volume-sensitive outwardly rectifying (VSOR) Cl- channel-activated by ROS contributes to cell apoptotic volume decrease, playing an incipient incident of cellular apoptosis. However, whether VSOR Cl- channel concurrently participates in autophagy-related cell death regulation remains unclear. To illuminate the issue, studies underwent in myocardial vitro and vivo I/R model. Rats were performed to ischemia 30 minutes and subsequent reperfusion 24-96 hours, ROS scavenger (NAC), VSOR Cl- channel blocker (DCPIB) and autophagy inhibitor (3MA) were administered respectively. Results showed that oxidative stress, LC3-II stain and inflammation in myocardial tissue were markedly increased, lysosome associated membrane protein-2 (LAMP2) were significantly reduced with I/R group as compared with sham group, reperfusion significantly led to damage in myocardial tissue and heart function, whereas the disorder could be rescued through these agents. Moreover, primary neonatal rat cardiomyocytes hypoxia/reoxygenation model were administered, results showed that VSOR Cl- channel-activated by reoxygenation could cause both cell volume decrease and intracellular acidification, which further increased LC3 and depleted of LAMP2, resulting in autophagy-related cell death. Interestingly, VSOR Cl- channel-blocked by DCPIB could stably maintain the cell volume, intracellular pH, abundant LAMP2 and autophagic intensity regardless of ROS intension derived from reoxygenation injury or adding H2O2. These results first demonstrate that VSOR Cl- channel-activated is a pivotal event to trigger autophagy-related death, which reveals a novel therapeutic target to decrease myocardial I/R injury.

Keywords: Pathology Section; VSOR Cl- channel; autophagy; cell death; ischemia/reperfusion injury; reactive oxygen species.

Figure 1. Inhibition of VSOR Cl⁻ channel, ROS and autophagy restore cardiac function and rescue myocardial injury after I/R

The LV function and myocardial infarct were determined after 24 hours of reperfusion. A. Myocardial infarct size images; B. Representative echocardiographic images; C.-H. The infarct area, myocardial enzyme and echocardiographic analyses. *P < 0.05 compared with sham group; ^#P < 0.05 compared with I/R group.

Figure 2. Inhibition of VSOR Cl⁻ channel, ROS and autophagy reduce rat serum inflammation and suppress myocardial oxidative stress after I/R

*P < 0.05 compared with sham group; ^#P < 0.05 compared with I/R group.

Figure 3. Inhibition of VSOR Cl⁻ channel, ROS and autophagy with NAC, DCPIB and 3MA respectively markedly restrain LC3 level, and stanchly maintain LAMP-2 abundance

A. Immunohistochemical images (×400); B., C. Western blot analysis. *P < 0.05 compared with sham group; ^#P < 0.05 compared with I/R group. These data demonstrate those inhibitors, especially both DCPIB and NAC distinctly inhibit rapid decline in LAMP2 abundance and immoderate soaring LC3 derived from reperfusion injury.

Figure 4. Blockate of VSOR Cl⁻ channel and ROS stably interdict deplete of LAMP2 abundance contribute from I/R at each time

A. Immunohistochemical images (×400); B., C. Western blot analysis. *P < 0.05 compared with sham group; ^#P < 0.05 compared with I/R group. These data demonstrate a decline in LAMP2 abundance distinctly derived from reperfusion injury following to time, and being reversed by DCPIB and NAC.

Figure 5. Blockate of VSOR Cl⁻ channel, ROS and autophagy with NAC, DCPIB and 3MA respectively markedly restrain generated autophagosome, and the first two stanchly maintain lysosome abundance

A. Transmission electron microscopy (TEM) images(×12600); B. TEM analysis. *P < 0.05 compared with sham group; ^#P < 0.05 compared with I/R group.

Figure 6. Activation of VSOR Cl⁻ channel by I/R is associated with excessive reactive oxygen species (ROS) and autophagy

Reoxygenation-induced ROS is mainly composed of H₂O₂. Increased VSOR Cl⁻ currents in H₂O₂ exposed cardiomyocytes present a classic simulate I/R model. H₂O₂-induced VSOR Cl⁻ currents were inhibited by adding DCPIB (10 μM); NAC (15 mM) and 3MA (15 μM), n = 5 for each group. Negligible background Cl⁻ currents recorded under isosmotic solution (Ctrl). H₂O₂-induced Cl⁻ currents exhibiting representative properties of VSOR Cl⁻ currents. Background Cl⁻ currents and H₂O₂-induced VSOR Cl⁻ currents were similer in each group. So we primarily appraise the relationship for the mean current densities of each group after intervention. These data reveal activation of VSOR Cl⁻ channel by H₂O₂, is related to ROS, and being reversed by DCPIB, NAC and 3MA. *P < 0.05 compared with sham group; ^#P < 0.05 compared with I/R group.

Figure 7. Blockate of VSOR Cl⁻ channel and ROS stably maintain LAMP2 abundance and restrain excess autophagy contribute from reoxygenation

A. Representative images of immunofluorescent staining; n = 5 for each group. (×100); B.,C. staining cell analysis. *P < 0.05 compared with sham group; ^#P < 0.05 compared with I/R group.

Figure 8. Blockate of VSOR Cl⁻ channel may restrain excess ROS and stabilize intracellular pH contribute from reoxygenation

A. Immunofluorescent staining images; n = 5 for each group (×400); B. staining cell analysis; C. Cell viability analysis by the MTT-assay; D. intracellular pH analysis. *P < 0.05 compared with sham group; ^#P < 0.05 compared with I/R group.

Figure 9. VSOR Cl⁻ channel blockers causes partial Beclin-1 inhibition in myocardial hypoxia-reoxygenation injury

A., C. Western blot analysis; B. Cell viability was measured by the MTT-assay; D. intracellular H₂O₂ analysis. *P < 0.05 compared with sham group; ^#P < 0.05 compared with I/R group.

Figure 10. The time courses of changes in the mean cell volume

A.,B. TUNEL staining images and analysis (×400); C.,D. Cell volume and cardiomyocyte viability analysis; E. intracellular pH analysis; F. intracellular H₂O₂ analysis. *P < 0.05 compared with sham group; ^#P < 0.05 compared with I/R group.

Figure 11. VSOR Cl⁻ channel-activated directly triggers cell volume decrease, when lockate of VSOR Cl⁻ channel, even adding ROS, which failed to cause cell volume decrease in reoxygenation

A.,D. Cell volume and cardiomyocyte viability analysis; C. intracellular pH analysis; B. intracellular H₂O₂ analysis. *P < 0.05 compared with sham group; ^#P < 0.05 compared with I/R group.