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A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Published on: July 13, 2018
Phosphoglycerate mutase 5 exacerbates cardiac ischemia-reperfusion injury through disrupting mitochondrial quality
Hang Zhu1, Ying Tan2, Wenjun Du3
1Department of Cardiology, Chinese PLA General Hospital, Beijing, 100853, China.
Abstract:
The death of cardiomyocytes either through apoptosis or necroptosis is the pathological feature of cardiac ischemia-reperfusion (I/R) injury. Phosphoglycerate mutase 5 (PGAM5), a mitochondrially-localized serine/threonine-protein phosphatase, functions as a novel inducer of necroptosis. However, intense debate exists regarding the effect of PGAM5 on I/R-related cardiomyocyte death. Using cardiac-specific PGAM5 knockout (PGAM5CKO) mice, we comprehensively investigated the precise contribution and molecular mechanism of PGAM5 in cardiomyocyte death. Our data showed that both PGAM5 transcription and expression were upregulated in reperfused myocardium. Genetic ablation of PGAM5 suppressed I/R-mediated necroptosis but failed to prevent apoptosis activation, a result that went along with improved heart function and decreased inflammation response. Regardless of PGAM5 status, mitophagy-related cell death was not apparent following I/R. Under physiological conditions, PGAM5 overexpression in primary cardiomyocytes was sufficient to induce cardiomyocyte necroptosis rather than apoptosis. At the sub-cellular levels, PGAM5 deficiency increased mitochondrial DNA copy number and transcript levels, normalized mitochondrial respiration, repressed mitochondrial ROS production, and prevented abnormal mPTP opening upon I/R. Molecular investigation demonstrated that PGAM5 deletion interrupted I/R-mediated DrpS637 dephosphorylation but failed to abolish I/R-induce Drp1S616 phosphorylation, resulting in partial inhibition of mitochondrial fission. In addition, declining Mfn2 and OPA1 levels were restored in PGAM5CKO cardiomyocytes following I/R. Nevertheless, PGAM5 depletion did not rescue suppressed mitophagy upon I/R injury. In conclusion, our results provide an insight into the specific role and working mechanism of PGAM5 in driving cardiomyocyte necroptosis through imposing mitochondrial quality control in cardiac I/R injury.
Insights
Phosphoglycerate mutase 5 (PGAM5) drives necroptosis in cardiac ischemia-reperfusion injury. Deleting PGAM5 protects heart cells from death, improves heart function, and reduces inflammation by regulating mitochondrial quality control.
Area of Science:
- Cardiovascular Biology
- Cell Death Mechanisms
- Mitochondrial Dynamics
Background:
- Cardiac ischemia-reperfusion (I/R) injury involves cardiomyocyte death via apoptosis and necroptosis.
- Phosphoglycerate mutase 5 (PGAM5) is a mitochondrial phosphatase implicated in necroptosis, but its role in I/R injury is debated.
Purpose of the Study:
- To investigate the precise contribution and molecular mechanism of PGAM5 in cardiomyocyte death during cardiac I/R injury.
- To determine the effect of PGAM5 genetic ablation on I/R-induced pathological changes and cardiac function.
Main Methods:
- Utilized cardiac-specific PGAM5 knockout (PGAM5CKO) mice and primary cardiomyocyte cultures.
- Assessed cardiomyocyte death (apoptosis, necroptosis), inflammation, cardiac function, and mitochondrial parameters (DNA copy number, respiration, ROS, mPTP opening, fission/fusion proteins).
- Examined molecular pathways including Drp1 phosphorylation and mitophagy.
Main Results:
- PGAM5 expression increased in reperfused myocardium.
- PGAM5 ablation suppressed I/R-mediated necroptosis but not apoptosis, improving heart function and reducing inflammation.
- PGAM5 deficiency normalized mitochondrial respiration, repressed ROS production, prevented abnormal mPTP opening, and partially inhibited mitochondrial fission.
- PGAM5 deletion affected Drp1 phosphorylation and Mfn2/OPA1 levels but did not rescue mitophagy.
Conclusions:
- PGAM5 is a key inducer of cardiomyocyte necroptosis in cardiac I/R injury.
- PGAM5 regulates mitochondrial quality control, impacting mitochondrial dynamics and function during I/R.
- Targeting PGAM5 may offer a therapeutic strategy for mitigating I/R-induced cardiac damage.
