Pgam5 aggravates hyperglycemia-induced myocardial dysfunction through disrupting Phb2-dependent mitochondrial dynamics

Abstract

This study aims to elucidate the roles of Phosphoglycerate Mutase Family Member 5 (Pgam5) and Prohibitin 2 (Phb2) in the context of hyperglycemia-induced myocardial dysfunction, a critical aspect of diabetic cardiomyopathy. The research employed primary cardiomyocytes, which were then subjected to hyperglycemia treatment to mimic diabetic conditions. We used siRNA transfection to knock down Pgam5 and overexpressed Phb2 using adenovirus transfection to assess their individual and combined effects on cardiomyocyte health. Mitochondrial function was evaluated through measurements of mitochondrial membrane potential using the JC-1 probe, and levels of mitochondrial reactive oxygen species (ROS) were assessed. Additionally, the study involved qPCR analysis to quantify the transcriptional changes in genes related to mitochondrial fission and mitophagy. Our findings indicate that hyperglycemia significantly reduces cardiomyocyte viability and impairs mitochondrial function, as evidenced by decreased mitochondrial membrane potential and increased ROS levels. Pgam5 knockdown was observed to mitigate these adverse effects, preserving mitochondrial function and cardiomyocyte viability. On the molecular level, Pgam5 was found to regulate genes associated with mitochondrial fission (such as Drp1, Mff, and Fis1) and mitophagy (including Parkin, Bnip3, and Fundc1). Furthermore, overexpression of Phb2 countered the hyperglycemia-induced mitochondrial dysfunction and normalized the levels of key mitochondrial antioxidant enzymes. The combined data suggest a protective role for both Pgam5 knockdown and Phb2 overexpression against hyperglycemia-induced cellular and mitochondrial damage. The study elucidates the critical roles of Pgam5 and Phb2 in regulating mitochondrial dynamics in the setting of hyperglycemia-induced myocardial dysfunction. By modulating mitochondrial fission and mitophagy, Pgam5 and Phb2 emerge as key players in preserving mitochondrial integrity and cardiomyocyte health under diabetic conditions. These findings contribute significantly to our understanding of the molecular mechanisms underlying diabetic cardiomyopathy and suggest potential therapeutic targets for mitigating myocardial dysfunction in diabetes.

Keywords: Pgam5; Phb2; diabetic cardiomyopathy; mitochondrial fission; mitophagy.

Figure 1

Pgam5 knockdown attenuates the hyperglycemia-mediated cardiomyocyte death and dysfunction. A. Cell viability was determined by MTT assay. B-G. Pgam5 siRNA was transfected into cardiomyocytes and then the contractile was measured, including resting length remained, peak shortening (PK), maximal velocity of shortening (dL/dt), and time-to-peak shortening, maximal velocity of relengthening (-dL/dt), and time-to-90% relengthening (TR90). #p<0.05.

Figure 2

Mitochondrial dysfunction was induced by hyperglycemia due to increased Pgam5. A. JC-1 probe was used to stain mitochondrial membrane potential. B. ROS production was measured in cardiomyocytes. C-F. The activities of catalase, glutaredoxin, thioredoxin reductase (TrxR), and peroxiredoxin were determined by ELISA. #p<0.05.

Figure 3

Mitophagy was activated whereas mitochondrial fission was inhibited by Pgam5 deletion in the setting of hyperglycemia. A-C. RNA was isolated from cardiomyocytes and then the transcription of Drp1, Mff, and Fis1 was measured via qPCR. D-F. RNA was isolated from cardiomyocytes and then the transcription of Parkin, Fundc1, and Bnip3 was measured via qPCR. #p<0.05.

Figure 4

Pgam5 controlled mitophagy and mitochondrial fission through Phb2. A-C. RNA was isolated from cardiomyocytes and then the transcription of Drp1, Mff, and Fis1 was measured via qPCR. Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. D-F. RNA was isolated from cardiomyocytes and then the transcription of Parkin, Fundc1, and Bnip3 was measured via qPCR. Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. #p<0.05.

Figure 5

Phb2 overexpression reduced mitochondrial dysfunction upon hyperglycemia stress. A. JC-1 probe was used to stain mitochondrial membrane potential. B. ROS production was measured in cardiomyocytes. Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. C-F. The activities of catalase, glutaredoxin, thioredoxin reductase (TrxR), and peroxiredoxin were determined by ELISA. Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. #p<0.05.

Figure 6

Phb2 overexpression maintained cardiomyocyte viability and function upon hyperglycemia stress. A. Cell viability was determined by MTT assay. Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. B-G. Pgam5 siRNA was transfected into cardiomyocytes and then the contractile was measured, including resting length remained, peak shortening (PK), maximal velocity of shortening (dL/dt), and time-to-peak shortening, maximal velocity of relengthening (-dL/dt), and time-to-90% relengthening (TR90). Phb2 adenovirus were transfected into cardiomyocytes before hyperglycemia treatment. #p<0.05.