PGAM5-Mediated PHB2 Dephosphorylation Contributes to Diabetic Cardiomyopathy by Disrupting Mitochondrial Quality Surveillance

Fig. 1.. Cardiac PGAM5 expression is upregulated by hyperglycemia and contributes to metabolic disorder. In vivo, the cardiomyocyte-specific Pgam5 knockout (Pgam5CKO) and Pgam5f/f mice were injected intraperitoneally with STZ (50 mg/kg dissolved in 0.1 mol/l of citrate buffer) for 5 consecutive days to induce diabetes. Nondiabetic mice were the age- and sex-matched, which were injected with the same volume of PBS. (A) Analysis of relative transcription levels of Pgam5 in cardiac tissues by qPCR. GAPDH, glyceraldehyde phosphate dehydrogenase. (B) Western blot analysis of PGAM5 expression in mice hearts. (C) Time course for changes in fasting blood glucose levels. Blood glucose was measured at baseline (week 0) and periodically over the course of 24 weeks after the first day of STZ or citrate buffer (vehicle control) injection. (D and E) Determination of fasting serum cholesterol (D) and triglyceride (E) levels in mice, 24 weeks after STZ or vehicle injection. (F) Time course of body weight changes. (G) IPGTT results for Pgam5CKO and Pgam5f/f mice. Tests were performed 9 days after STZ/vehicle treatment. (H) Kaplan-Meier survival curves for the various groups (n = 24 mice). Values are…

Fig. 2.. PGAM5 deficiency sustains myocardial function in diabetic mice. In vivo, the Pgam5CKO and Pgam5f/f mice were injected intraperitoneally with STZ (50 mg/kg dissolved in 0.1 mol/l of citrate buffer) for 5 consecutive days to induce diabetes. Nondiabetic mice were the age- and sex-matched, which were injected with the same volume of PBS. In vitro, cardiomyocytes were isolated from STZ-treated Pgam5CKO and Pgam5f/f mice. (A to G) Echocardiography analysis. (A) LVEF, left ventricular ejection fraction; (B) FS, fractional shortening; (C) LVSd, left ventricular systolic dimension; (D) LVDd, left ventricular diastolic dimension; (E) E/A, early to late (atrial) mitral flow velocity ratio; (F) E/e′, ratio of mitral peak velocity of early filling to early diastolic mitral annular velocity; and (G) e′/a′, ratio of diastolic mitral annulus velocities. (H to M) Analysis of contractile parameters in acutely isolated, single cardiomyocytes from Pgam5CKO and Pgam5f/f mice. (H) Resting length in cardiomyocytes; (I) PS, peaking shortening; (J) +dL/dt, maximal velocity of shortening; (K) TPS, time-to-peak shortening; (L) −dL/dt, maximal velocity of relengthening; and (M) TR90, time-to-90%…

Fig. 3.. PGAM5 deletion prevents diabetes-mediated myocardial structural disorder. In vivo, the Pgam5CKO and Pgam5f/f mice were injected intraperitoneally with STZ (50 mg/kg dissolved in 0.1 mol/l of citrate buffer) for 5 consecutive days to induce diabetes. Nondiabetic mice were the age- and sex-matched, which were injected with the same volume of PBS. (A) Representative histopathological images (H&E staining) depicting myocardial disarray in diabetic mice. (B) Representative histopathological images (Masson trichrome staining) revealing myocardial fibrosis in diabetic mice. (C) Quantification of Masson trichrome staining. (D) Representative histopathological images (Sirius Red staining) revealing myocardial fibrosis in diabetic mice. (E) Quantification of Sirius Red staining. (F) Representative electron microscopy images depicting ultrastructural changes in heart tissue from diabetic mice. Yellow arrows indicate disordered myofibrils and swollen mitochondria. (G) Representative images of TUNEL staining in heart tissues from vehicle- and STZ-treated mice and corresponding quantification data. DAPI, 4′,6-diamidino-2-phenylindole. (H) Quantification of TUNEL staining. Values are presented…

Fig. 4.. PGAM5 ablation attenuates hyperglycemia-mediated mitochondrial damage. Primary neonatal cardiomyocytes isolated from Pgam5CKO and Pgam5f/f mice were cultured under high glucose (30 mmol/l) medium for 48 h to induce hyperglycemic stress in vitro. Cardiomyocytes incubated in normal glucose (5.5 mmol/l) medium were used as control. (A) ELISA-based analysis of ATP production in primary cardiomyocytes. (B) Detection of mitochondrial membrane potential (ΔΨm) in cardiomyocytes loaded with the JC-1 probe. (C) The red-to-green fluorescence ratio was used for semiquantitative analysis of changes in ΔΨm. (D) Analysis of mtROS generation in cardiomyocytes loaded with MitoSOX Red. (E) The levels of mtROS were normalized against those of the control group. (F and G) ELISA-based determination of the activity of mitochondrial respiratory complex I (F) and complex V (G). (H) Detection of mPTP opening in cardiomyocytes loaded with TMRE. (I) Quantification of mPTP in cardiomyocytes. Values are presented as mean ± SEM from 4 independent experiments. #P < 0.05.

Fig. 5.. PGAM5 deletion normalizes MQS in cardiomyocytes exposed to hyperglycemia. In vivo, the Pgam5CKO and Pgam5f/f mice were injected intraperitoneally with STZ (50 mg/kg dissolved in 0.1 mol/l of citrate buffer) for 5 consecutive days to induce diabetes. Nondiabetic mice were the age- and sex-matched, which were injected with the same volume of PBS. Primary neonatal cardiomyocytes isolated from Pgam5CKO and Pgam5f/f mice were cultured under high-glucose (30 mmol/l) medium for 48 h to induce hyperglycemic stress in vitro. Cardiomyocytes incubated in normal glucose (5.5 mmol/l) medium were used as control. (A) Tom20 immunofluorescence was applied for analysis of hyperglycemia-induced alterations of mitochondrial morphology in cardiomyocytes. (B) Mean mitochondrial length was analyzed in at least 100 cardiomyocytes. (C to E) qPCR analysis of Parkin (C), Beclin1 (D), and Atg5 (E) expression in cardiac tissue. (F) Analysis of mitophagy in primary cardiomyocytes expressing the mito-Keima probe. (G) Presentative pictures of cardiomyocyte transfected with mito-Keima. (H and I) Relative transcription levels of Nrf2 (H) and Tfam (I) in cardiac tissue, assessed by qPCR. Values are presented as…

Fig. 6.. PGAM5 binds and dephosphorylates PHB2. Mouse HL-1 cardiomyocyte cell line was cultured under high-glucose medium (30 mmol/l of glucose) for 48 h to induce hyperglycemia damage in vitro. HL-1 cells treated with normal glucose medium (5.5 mmol/l of glucose) was used as the control group. (A) Potential PGAM5-PHB2 interaction, as predicted in the inBio Discover platform. (B) Western blot analysis of p-PHB2S91 expression in cardiac tissues. (C) Western blot signals were normalized against total PHB2 expression. (D) Co-IP assay results indicating interaction between PGAM5 and PHB2. Ig-G, immunoglobulin G. (E) Molecular docking of PHB2 and PGAM5. (F) The potential binding sites between PHB2 and PGAM5 are shown in different colors. (G) Double immunofluorescence of PGAM5 and PHB2 in HL-1 cardiomyocytes exposed to hyperglycemia. (H) Mapping of regions in PHB2. Immunoprecipitation (IP) and immunoblotting (IB) were applied to assess interactions between PHB2 mutants and PGAM5. (I) Mapping of regions in PGAM5. Immunoprecipitation and immunoblotting were applied to assess interactions between region-specific PHB2 and PGAM5 mutants upon transfection into HL-1 cells. Values are presented as mean…

Fig. 7.. PGAM5 deficiency-mediated mitochondrial protection is abolished by transfection of a phosphorylation-defective PHB2 mutant. Primary neonatal cardiomyocytes were isolated from Pgam5CKO and Pgam5f/f mice, transfected with HA-PHB2S91A or HA-PHB2S91D mutant constructs, and exposed to PBS or hyperglycemia. (A) Detection of ΔΨm in cardiomyocytes loaded with the JC-1 probe. (B) The red-to-green fluorescence ratio was used for the semiquantitative analysis of changes in ΔΨm. (C) Analysis of mtROS generation in cardiomyocytes loaded with MitoSOX Red. (D) The levels of mtROS were normalized to those of the control group. (E and F) ELISA-based determination of the activity of mitochondrial respiratory complex I (E) and complex V (F). (G) Analysis of mitophagy in primary cardiomyocytes expressing the mito-Keima probe. (H) Presentative pictures of cardiomyocyte transfected with mito-Keima. (I and J) Analysis of Pgc1α (I) and Nrf2 (J) transcription levels in cultured cardiomyocytes. Values are presented as mean ± SEM from 4 independent experiments. #P < 0.05.

Fig. 8.. In vivo expression of a PHB2S91 phosphorylation mutant confers resistance to DCM. WT, heterozygous Phb2S91D/+, and homozygous Phb2S91D/D mice (n = 6 per type) were subjected to STZ-induced diabetes. (A to G) Echocardiography analysis. (A) LVSd, left ventricular systolic dimension; (B) LVEF, left ventricular ejection fraction; (C) FS, fractional shortening; (D) LVDd, left ventricular diastolic dimension; (E) E/A, early to late mitral flow velocities; (F) e′/a′, ratio of diastolic mitral annulus velocities; and (G) E/e′, ratio of mitral peak velocity of early filling to early diastolic mitral annular velocity. (H) Assessment of myocardial structure by H&E staining. (I) Pathological assessment of myocardial fibrosis by Masson trichrome/Sirius Red staining. (J) Quantification of Masson trichrome staining. (K) Quantification of Sirius Red staining. (L and M) Analysis of relative Tgfβ (L) and Mmp9 (M) transcriptional levels in cardiac samples by qPCR. Values are presented as mean ± SEM from 6 mice per group. #P < 0.05.