PKM2 interacts with and phosphorylates PHB2 to sustain mitochondrial quality control against septic cerebral-cardiac injury

Abstract

Sepsis induces profound disruptions in cellular homeostasis, particularly impacting mitochondrial function in cardiovascular and cerebrovascular systems. This study elucidates the regulatory role of the Pyruvate Kinase M2 (PKM2)- Prohibitin 2 (PHB2) axis in mitochondrial quality control during septic challenges and its protective effects against myocardial and cerebral injuries. Employing LPS-induced mouse models, we demonstrate a significant downregulation of PKM2 and PHB2 in both heart and brain tissues post-sepsis, with corresponding impairments in mitochondrial dynamics, including fission, fusion, and mitophagy. Overexpression of PKM2 and PHB2 not only restores mitochondrial function, as evidenced by normalized ATP production and membrane potential but also confers resistance to oxidative stress by mitigating reactive oxygen species generation. These cellular mechanisms translate into substantial in vivo benefits, with transgenic mice overexpressing PKM2 or PHB2 displaying remarkable resistance to sepsis-induced cardiomyocyte and neuronal apoptosis, and organ dysfunction. Our findings highlight the PKM2-PHB2 interaction as a novel therapeutic target for sepsis, providing a foundation for future research into mitochondrial-based interventions to treat this condition. The study's insights into the molecular underpinnings of sepsis-induced organ failure pave the way for potential clinical applications in the management of sepsis and related pathologies.

Keywords: PHB2; PKM2; mitochondrial quality control; septic cerebrovascular damage.

Figure 1

Suppression of PKM2 and PHB2 in Cardiac and Cerebral Tissues Post-Sepsis. A-B. RNA was isolated from heart tissues and then the transcriptions of PKM2 or PHB2 were measured via qPCR. C-D. RNA was isolated from brain tissues and then the transcription of PKM2 or PHB2 was measured via qPCR. E-F. Proteins were isolated from heart tissues and then the expression of PKM2 and PHB2 were determined by western blots. G-H. Proteins were isolated from brain tissues and then the expression of PKM2 and PHB2 were determined by western blots. I-J. Immunofluorescence of PKM2 or PHB2 in HL-1 cells. DAPI was used to stain nucleus. K-L. Immunofluorescence of PKM2 or PHB2 in HL-1 cells. DAPI was used to stain nucleus. *p<0.05 vs. PBS group.

Figure 2

PKM2-PHB2 Interaction Preserves Mitochondrial Function during Inflammatory Stress in HL-1 cells. A. Co-IP assay was used to measure the interaction between PKM2 and PHB2 in HL-1 cells. B. ELISA kit was used to measure the ATP production in HL-1 cells. C. Immunofluorescence of mitochondrial membrane potential in HL-1 cells upon LPS exposure. D. Immunofluorescence of mROS in HL-1 cells upon LPS exposure. *p<0.05 vs. PBS group, #p<0.05 vs. LPS+Ad-Ctrl group.

Figure 3

PKM2-PHB2 Interaction Preserves Mitochondrial Function during Inflammatory Stress in N2a cells. A. Co-IP assay was used to measure the interaction between PKM2 and PHB2 in N2a cells. B. ELISA kit was used to measure the ATP production in N2a cells. C. Immunofluorescence of mitochondrial membrane potential in N2a cells upon LPS exposure. D. Immunofluorescence of mROS in N2a cells upon LPS exposure. *p<0.05 vs. PBS group, #p<0.05 vs. LPS+Ad-Ctrl group.

Figure 4

Association Between Reduced PKM2 and PHB2 Expression and Mitochondrial Quality Control Dysfunction in LPS-treated HL-1 cells. A-D. RNA was isolated from heart tissues and then the transcriptions of Drp1, Fis1, Mfn2 and Opa1 were measured via qPCR in LPS-treated HL-1 cells. E. The mitophagy activity was determined by mito-Kemia assay in LPS-treated HL-1 cells. F-G. RNA was isolated from heart tissues and then the transcriptions of Sirt3 and PGC1α were measured via qPCR in LPS-treated HL-1 cells. *p<0.05 vs. PBS group, #p<0.05 vs. LPS+Ad-Ctrl group.

Figure 5

Association Between Reduced PKM2 and PHB2 Expression and Mitochondrial Quality Control Dysfunction in LPS-treated N2a cells. A-D. RNA was isolated from heart tissues and then the transcriptions of Drp1, Fis1, Mfn2 and Opa1 were measured via qPCR in LPS-treated N2a cells. E. The mitophagy activity was determined by mito-Kemia assay in LPS-treated N2a cells. F-G. RNA was isolated from heart tissues and then the transcriptions of Sirt3 and PGC1α were measured via qPCR in LPS-treated N2a cells. *p<0.05 vs. PBS group, #p<0.05 vs. LPS+Ad-Ctrl group.

Figure 6

Overexpression of PKM2 and PHB2 Mitigates Sepsis-Induced Cardiomyocyte and Neuronal Death. A-B. Cell viability was determined by MTT assay in LPS-treated HL-1 cells or N2a cells. C-F. TUNEL staining of apoptotic cells in LPS-treated HL-1 cells or N2a cells. G-H. ELISA kit was used to detect the activity of caspase-3 in LPS-treated HL-1 cells or N2a cells. *p<0.05 vs. PBS group, #p<0.05 vs. LPS+Ad-Ctrl group.

Figure 7

PKM2 Overexpression Ameliorates Sepsis-Induced Cardiovascular and Cerebral Dysfunction In Vivo. A-C. Heart function was measured via echocardiography. *p<0.05 vs. WT group, #p<0.05 vs. LPS+WT group.

Figure 8

PHB2 Overexpression Confers Resilience Against Sepsis-Induced Cardiovascular and Cerebral Dysfunction. A-C. Heart function was measured via echocardiography. *p<0.05 vs. WT group, #p<0.05 vs. LPS+WT group.