GDF15 attenuates myocardial hypoxic injury by inhibiting mitochondrial damage through BNIP3 pathway.

BACKGROUND

Growth differentiation factor 15 (GDF15), a hypoxia-responsive mediator in myocardial injury, is established as a mitochondrial stress biomarker in skeletal muscle. However, its mechanistic role in regulating mitophagy during cardiac hypoxia remains unknown.

METHODS

In this study, human myocardial cells (AC16) were treated with cobalt chloride (CoCl2) to induce hypoxia. GDF15 expression was analyzed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Cells were transfected with GDF15-knockdown lentivirus (LV-shGDF15) or overexpression lentivirus (LV-GDF15), and proliferation was monitored in real-time under normoxia or hypoxia using the iCELLigence RTCA DP and SP system. Mitochondrial function was assessed through lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, malonaldehyde (MDA) concentration, cytosolic reactive oxygen species (ROS), mitochondrial transmembrane potential (ΔΨm), and oxygen consumption rate (OCR) analysis. To validate sequencing results, BCL2/Adenovirus E1B Interacting Protein 3(BNIP3) was knocked down using siRNA in LV-shGDF15 cells and overexpressed via plasmid in the LV-GDF15 group before evaluating ΔΨm and ROS. The effects on mitochondrial homeostasis were further assessed through RNA sequencing and mitochondrial functional analyses.

RESULTS

The proliferative ability of GDF15 knockdown cells was significantly decreased, accompanied by an increase in lactate dehydrogenase release. Overexpression of GDF15 significantly inhibited the accumulation of ROS and prevented ΔΨm loss. Administering GDF15 (50 ng/ml) to GDF15 knockdown cells effectively reduced MDA and ROS expression and mitigated ΔΨm loss. GDF15 protected AC16 cells from CoCl2-induced hypoxic injury. LV-shGDF15 cells with BNIP3 knockdown and CoCl2 treatment exhibited decreased levels of BNIP3 and LC3B-II, increased ΔΨm, and reduced ROS expression. Conversely, GDF15 overexpression significantly reduces BNIP3 levels, diminishes BNIP3-driven aberrant autophagosome biogenesis, and concurrently enhances lysosomal activity.

CONCLUSION

This study unveils a novel cardioprotective mechanism of GDF15 through BNIP3 suppression and characterizes its temporal expression dynamics under hypoxia. GDF15 mitigates hypoxic injury by downregulating BNIP3, thereby curbing pathological mitophagy, activating lysosomes function, and offering a therapeutic target for ischemic cardiomyopathy.