Fig 1.
Ischemia decreased bcl-2 content in rabbit SSM.
Panel A: Western blotting shows that ischemia markedly decreased bcl-2 content in SSM isolated from rabbit heart compared to non-ischemic hearts. The subunit 4 of cytochrome oxidase was used as protein loading control. Panel B: Ratio of the intensity of bcl-2 over the intensity of subunit 4 of cytochrome oxidase was decreased in ischemia-damaged SSM compared to control (Mean ± SEM; *p<0.05 vs. control).
Fig 2.
Inhibition of bcl-2 decreases the CRC in ischemia-damaged mitochondria.
Panel A: A representative original tracing of the CRC measurement. The pulses of calcium that required for MPTP opening are significantly decreased in ischemia-damaged SSM (subsarcolemmal mitochondria) compared to control. Panel B: Titration with HA14–1 decreases the CRC in both control and ischemic mitochondria in a dose-dependent manner. The concentration of HA14–1 required to reach maximal MPTP opening in ischemia-damaged SSM are lower than that in control SSM, indicating that the sensitivity to HA14–1 inhibition is increased in the ischemic damaged SSM compared to control (Mean ± SEM; *p<0.05 vs. corresponding control).
Fig 3.
Inhibition of bcl-2 increases the release of AK-2 from ischemia-damaged mitochondria.
In control mitochondria, HA14–1 treatment led to compared to no HA14–1 treated mitochondria (Panel A &B). The AK-2 content was markedly decreased in the ischemia-damaged SSM compared to control (Panel B). HA14–1 treatment further decreased the AK-2 content in ischemia-damaged SSM with corresponding increase in supernatant compared to ischemia alone SSM (Panel A & B). HA14–1 inhibition or ischemia did not lead to the release of apoptosis inducing factor (AIF) from mitochondria (Panel C). Voltage dependent anion channel (VDAC) is used as the protein loading control.
Fig 4.
The effect of bcl-2 inhibition on cytochrome c content in control and ischemia-damaged mitochondria.
Panel A: A representative image shows that titration of HA14–1 leads to decreased cytochrome c content in ischemia-damaged SSM with a corresponding increase in supernatant in a dose-dependent manner. Panel B: The ratio of intensity of cytochrome c to the intensity of subunit 4 of cytochrome oxidase. The decreased cytochrome c content in ischemia-damaged mitochondria compared to control in the absence of HA14–1, suggests that ischemia already leads to partial cytochrome c loss before mitochondria are isolated from ischemic hearts. The further decrease in cytochrome c content in HA14–1 treated ischemic mitochondria indicates that inhibition of bcl-2 can lead to further release of cytochrome c into supernatant (Mean±SEM; *p<0.05 vs. corresponding control).
Fig 5.
Overexpression of bcl-2 improves the CRC in mitochondria following ischemia-reperfusion.
Panel A: The LDH content in coronary effluent in bcl-2 overexpressed mice following 40 min global ischemia and 30 min reperfusion was much lower than that in wild type, supporting that overexpression of bcl-2 reduces cell injury during ischemia-reperfusion. Panel B: Overexpression of bcl-2 improves the CRC in mouse SSM following ischemia-reperfusion compared to wild type, indicating that the sensitivity to MPTP opening is decreased in mitochondria from bcl-2 overexpressed mouse heart. (Mean ± SEM; *p<0.05 vs. pre-ischemia; † p<0.05 vs. wild type; n = 6 in each group).