PINK1 Phosphorylates Drp1S616 to Improve Mitochondrial Fission and Inhibit the Progression of Hypertension-Induced HFpEF

Figures

• 

  Figure 1. Hypertension-induced HFpEF model. (A–D) Heart weight (A), ratio of HW to BW (B), LV weight (C), and ratio of LV weight to BW (D); n = 4 per group. (E–K) Representative images of M-mode ultrasound (E), interventricular septal end systolic thickness (IVSs), interventricular septal end diastolic thickness (IVSd), LV end systolic internal diameter (LVIDs), LV end diastolic internal diameter (LVIDd) (F), end-systolic volume (ESV), end-diastolic volume (EDV) (G), E/A (H), E/E′ (I), ejection fraction (EF) (J), and fraction of shortened (FS) (K); n = 4 per group. (L,M) Representative images of LV H&E staining (L) and quantitative analysis of myocardial cell area (4 samples per group, 3–5 fields of view per sample, Scale bar: 20 μm) (M). (N,O) Representative images of Picro Sirius Red staining (N) and quantitative analysis of collagen fiber area fraction (4 samples per group, 3–5 fields analyzed per sample, Scale bar: 50 μm) (O). The data are mean ± SD, * Compared with the C group, p < 0.05.

• 

  Figure 2. Mitochondrial dysfunctions in hypertension-induced HFpEF. (A) mRNA expression of mitochondrial function-related genes CS, SDH, PDH, ATPase, SIRT3, PGC-1α, NRF-1 in the LV; n = 4 per group. (B) Relative ATP content in the LV (normalized using protein concentration); n = 4 per group. (C–F) Representative images of mitochondrial transmission electron microscopy in the LV (Scale bar: 2 μm, 500 nm) (C), quantitative analysis of the number of mitochondria per unit field of view (analysis of more than 100 mitochondria per sample, 4 samples per group) (D), quantitative analysis of mitochondrial area (analysis of more than 100 mitochondria per sample, 4 samples per group) (E), and quantitative analysis of mitochondrial fission index (ratio of mitochondrial number to mitochondrial area, n = 4 per group) (F). (G) mRNA expression of Drp1, a key gene for mitochondrial fission in the left ventricle of rats, and Mfn2, a key gene for mitochondrial fusion (n = 4 in each group). The data are mean ± SD, * Compared with the C group, p < 0.05.

• 

  Figure 3. pDrp1S616-mediated mitochondrial fission. (A) WB analysis of total protein levels of Drp1 and phosphorylation of Drp1S616 in the LV; n = 4 per group. (B) Drp1 protein level on mitochondria, Drp1 protein level in cytoplasm, and relative ratio of mitochondrial Drp1 to cytoplasmic Drp1 after the separation of LV mitochondria and cytoplasmic proteins; n = 4 per group. (C) Representative images of immunofluorescence of the LV (red: mitochondrial outer membrane marker, Tom20, green: Drp1, blue: DAPI, Scale bar: 20 μm), and (D) Manders co-localization coefficient (MCC) quantitative analysis of Tom20 and Drp1 (5 fields analyzed per sample, 4 samples per group). The data are mean ± SD, * Compared with the C group, p < 0.05.

• 

  Figure 4. PINK1 knockout in H9C2 cardiomyocytes impairs mitochondrial fission and function. (A,B) PINK1 protein expression (A) and mRNA expression (B) in the LV; n = 4 per group. (C,D) PINK1 mRNA expression (C) and PINK1 protein expression, Drp1 total protein level and Drp1S616 phosphorylation level (D) in H9C2 cardiomyocytes after PINK1 knockout; n = 3 per group. (E,F) Drp1 protein level on mitochondria (E) and Drp1 protein level in cytoplasm and relative ratio of mitochondrial Drp1 to cytoplasmic Drp1 (F) after separation of mitochondrial and cytoplasmic proteins of H9C2 cardiomyocytes; n = 3 per group. (G,H) Representative images of immunofluorescence of H9C2 cardiomyocytes (red: mitochondrial outer membrane marker, Tom20, green: Drp1, blue: DAPI, Scale bar: 2 μm) (G) and Manders co-localization coefficient (MCC) quantitative analysis of Tom20 and Drp1 (more than 30 cells were analyzed per group) (H). (I,J) Representative images of MitoTracker Red fluorescent staining of mitochondrial morphology (Scale bar: 2 μm) (I) and quantitative analysis of mitochondrial aspect ratio (more than 30 cells analyzed per group) (J). (K) mRNA expression of mitochondrial function-related genes CS, SDH,…

• 

  Figure 5. Overexpression of Drp1WT, rather than Drp1S616A, improve mitochondrial fission and function. (A) PINK1 protein expression, Drp1 total protein level and Drp1S616 phosphorylation level after PINK1KO cardiomyocytes overexpressed Drp1WT or Drp1S616A; n = 3 per group. (B,C) Drp1 protein levels on mitochondria and in cytoplasm after separation of mitochondrial and cytoplasmic proteins of H9C2 cardiomyocytes; n = 3 per group. (D,E) Representative images of immunofluorescence of H9C2 cardiomyocytes (red: mitochondrial outer membrane marker, Tom20, green: Drp1, blue: DAPI, Scale bar: 2 μm) (D) and Manders co-localization coefficient (MCC) quantitative analysis of Tom20 and Drp1 (more than 30 cells analyzed per group) (E). (F,G) mRNA expression of mitochondrial function-related gene PGC-1α (F) and CS, SDH, PDH, ATPase (G) in H9C2 cardiomyocytes, n = 3 per group. (H,I) Representative images of TMRE mitochondrial membrane potential fluorescence staining (Scale bar: 5 μm) (H) and quantitative analysis of mitochondrial membrane potential fluorescence intensity (more than 30 cells were analyzed per group) (I). (J) Relative ATP content of H9C2 cardiomyocytes (normalized using protein…

• 

  Figure 6. PINK1 overexpression improves hypertension-induced HFpEF phenotypes. (A–D) Heart weight (A), ratio of heart weight to body weight (B), left ventricle weight (C), and ratio of left ventricle weight to body weight (D); n = 4 per group. (E–K) Representative images of M-mode ultrasound (E), IVSs, IVSd, LVIDs, LVIDd (F), ESV, EDV (G), E/A (H), E/E′ (I), EF (J), and FS (K); n = 4 per group. (L,M) Representative images of H&E staining of rat left ventricle (L) and quantitative analysis of myocardial cell area (4 samples per group, 3–5 fields of view per sample, Scale bar: 20 μm) (M). (N,O) Representative images of Picro Sirius Red staining (N) and quantitative analysis of collagen fiber area fraction (4 samples per group, 3–5 fields analyzed per sample, Scale bar: 50 μm) (O). The data are mean ± SD, * p < 0.05 compared to the other group.

• 

  Figure 7. PINK1 overexpression stimulates pDrp1S616-mediated fission to improve mitochondrial function. (A) Drp1 total protein expression, Drp1S616 phosphorylation level, and PINK1 protein expression in the LV; n = 4 per group. (B) PINK1 mRNA expression in the LV; n = 4 per group. (C,D) Drp1 protein level on mitochondria, Drp1 protein level in cytoplasm (C) and the relative ratio of mitochondrial Drp1 to cytoplasmic Drp1 (D) after the separation of LV mitochondria and cytoplasmic proteins; n = 4 per set. (E,F) Representative images of immunofluorescence of the LV (red: mitochondrial outer membrane marker, Tom20, green: Drp1, blue: DAPI, Scale bar: 20 μm) (E) and Manders co-localization coefficient (MCC) quantitative analysis of Tom20 and Drp1 (5 fields analyzed per sample, 4 samples per group) (F). (G–J) Representative images of mitochondrial transmission electron microscopy in the LV (Scale bar: 2 μm, 500 nm) (G) and quantitative analysis of the number of mitochondria per unit field of view (analysis of >100 mitochondria per sample, 4 samples per group) (H), quantitative analysis of mitochondrial area (more than 100 mitochondria analyzed per sample, 4 samples per group) (I), and…

• 

  Figure 8. Molecular mechanism of PINK1-regulated hypertension-induced HFpEF. PINK1 increases phosphorylation level of Drp1S616 and mitochondrial localization of Drp1 to stimulate mitochondrial fission, thereby restoring mitochondrial function and ultimately slowing the development of hypertension-induced HFpEF.