Parkin Insufficiency Accentuates High-Fat Diet–Induced Cardiac Remodeling and Contractile Dysfunction Through VDAC1-Mediated Mitochondrial Ca2+ Overload

Figures

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  Figure 1. Enrichment Analysis Result of Transcriptome and Mitochondrial Protein Profiles in the Human Heart (A) Bubble plot showing top enriched Gene Ontology (GO) terms of biological process. Gene set enrichment analysis (GSEA) result of (B) “Ca2+ import into mitochondria” and (C) “Ca2+ ion import into cytosol.” Distribution of genes in the ranked gene list is shown as “hits.” The enrichment score (green line) represents the running enrichment score at any point in the gene set. The bottom portion exhibits the value of ranking metric that measures a gene’s correlation with a phenotype. P values and the normalized enrichment score (NES) are presented in tabular format. (D) Ridge plot showing GSEA result of enriched biological processes (BPs) related to Ca2+ (P < 0.05, |NES| > 1). (E) Mitochondrial or mitophagy proteins (Parkin, LC3BII-I, p62, PGC1α, UCP2, and VDAC1) in lean and obese human hearts. GAPDH, α-tubulin, or COX IV was used as the loading control. Mean ± SEM, n = 6 to 8 samples/group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 between indicated groups.

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  Figure 2. Metabolic Properties (During a 24-Hour Cycle) in WT and Parkin−/− Mice Fed an LFD or HFD for 24 Weeks (A) O2 consumption (Vo2; 24 hours). (B) Pooled Vo2 (light). (C) Pooled Vo2 (dark). (D) Pooled Vo2 (combined). (E) CO2 production (Vco2; 24 hours). (F) Pooled Vco2 (light). (G) Pooled Vco2 (dark). (H) Pooled Vco2 (combined). (I) Respiratory exchange ratio (RER; 24 hours). (J) Pooled RER (light). (K) Pooled RER (dark). (L) Pooled RER (combined). (M) Heat production (24 hours). (N) Pooled heat production. (O) Total physical activity (24 hours). (P) Pooled total physical activity. Mean ± SEM, n = 7 or 8 mice/group. ∗P < 0.05 vs WT-LFD group (A, E, I, M, O); ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 between indicated groups (B to D, F to H, J to L, N, P). HFD = high-fat diet; LFD = low-fat diet; WT = wild-type.

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  Figure 3. HFD Intake–Induced Body Weight Gain, Glucose Intolerance, Plasma Profile, and Cardiac Remodeling in WT and Parkin−/− Mice (A) Body weight gain over the 24-week feeding period. (B) Fasting blood glucose. (C) Serum triglycerides. (D) Plasma insulin. (E) Homeostasis model assessment–estimated insulin resistance (HOMA-IR) index. (F) Heart weight. (G) Heart weight normalized to tibial length. (H) Representative images of lectin. (I) Pooled cardiomyocyte cross-sectional area. (J) Representative images of Oil red O. (K) Pooled quantitative analysis of lipid content. (L) Representative images of Masson trichrome staining. (M) Pooled quantitative analysis of interstitial fibrotic area (as a percentage of entire cardiac region). Mean ± SEM, n = 5 to 7 mice/group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 between indicated groups. Abbreviations as in Figure 2.

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  Figure 4. HFD Intake–Induced Changes in Echocardiographic Indexes in WT and Parkin−/− Mice (A) Representative echocardiographic images from 4 mouse groups. (B) Left ventricular (LV) wall thickness. (C) Septal thickness. (D) LV end-systolic diameter (LVESD). (E) LV end-diastolic diameter (LVEDD). (F) Fractional shortening. (G) Ejection fraction. (H) LV mass. (I) Heart rate. (J) E/A ratio. (K) Deceleration time (DT). (L) Diastolic blood pressure (BP). (M) Systolic BP. (N) Representative images of pulse-wave velocity (PWV). (O) PWV. Mean ± SEM, n = 7 to 9 mice/group. ∗P < 0.05, ∗∗P < 0.01, or ∗∗∗P < 0.001 between indicated groups. Abbreviations as in Figure 2.

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  Figure 5. Effect of HFD Intake on Cardiomyocyte Contractile and Intracellular Ca2+ Properties in WT and Parkin−/− Mice (A) Resting cell length. (B) Peak shortening (percentage of resting cell length). (C) Maximal velocity of shortening (+dL/dt). (D) Maximal velocity of relengthening (−dL/dt). (E) Time to peak shortening (TPS). (F) Time to 90% relengthening (TR90). (G) Baseline Fura-2 fluorescence intensity (FFI). (H) Increase in FFI (ΔFFI) in response to electric stimuli. (I) Intracellular Ca2+ decay rate. Mean ± SEM, n = 56 (A to F) or 40 (G to I) cells from 4 mice per group. ∗P < 0.05, ∗∗P < 0.01, or ∗∗∗P < 0.001 between indicated groups. Abbreviations as in Figure 2.

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  Figure 6. Effect of HFD Intake on Myocardial Ultrastructure, Mitochondrial O2− Production, Mitochondrial Respiration, and Mitochondrial Integrity in WT and Parkin−/− Mice (A) Representative transmission electron microscopic (TEM) images of mitochondrial ultrastructure. (B) Representative dihydroethidium (DHE) fluorescence images. (C) Representative MitoSOX fluorescence images. (D) Mitochondrial area (percentage total area). (E) Mitochondrial area (per mitochondrion). (F) Mitochondrial circularity index (major axis length/minor axis length). (G, H) Pooled data of mitochondrial reactive oxygen species levels using DHE staining or MitoSOX staining. (I) Baseline mitochondrial Ca2+ levels using Rhod-2. (J) NAD+ level depicting mitochondrial permeability transition pore opening. (K) Mitochondrial aconitase activity. (L) Representative immunoblots of UCP2, PGC1α, and TOM20. GAPDH or α-tubulin was used as the loading control. Quantified mitochondrial protein levels of (M) UCP2, (N) PGC1α, and (O) TOM20. Mean ± SEM, n = 6 or 7 mice (A to H, J to O) or 15 cells from 4 mice (I) per group. ∗P < 0.05, ∗∗P < 0.01, or ∗∗∗P < 0.001 between indicated groups. Abbreviations as in Figure 2.

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  Figure 7. Effect of HFD Intake on Autophagy, Mitophagy, and Mitochondria-Mediated Cell Death in Hearts From WT and Parkin−/− Mice (A) Representative images of mt-Keima. (B) Pooled data of mt-Keima fluorescence signal (561 nm/458 nm). Quantified protein levels and representative immunoblots as insets of (C) LC3BI/II, (D) p62, (E) Parkin, (F) IL-6, (G) TNF-α, (H) FasL, (I) caspase-8, (J) Bax, (K) Bcl-2, (L) cytosolic apoptosis-inducing factor (AIF), (M) cytosolic cytochrome c, (N) mitochondrial cytochrome c, (O) caspase-9, and (P) caspase-3. GAPDH, α-tubulin, or COX IV was used as the loading control. Mean ± SEM, n = 10 images (A, B) or 5 to 7 mice (C to P) per group. ∗P < 0.05, ∗∗P < 0.01, or ∗∗∗P < 0.001 between indicated groups. OE = overexpression; PA = palmitic acid; siRNA = small interfering RNA; other abbreviations as in Figure 2.

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  Figure 8. Effect Of Parkin On Mitochondrial Ca2+ Transport Protein Levels (A) Ca2+ channel in the outer mitochondrial membrane VDAC1. (B) VDAC2. (C) VDAC3. (D) Mitochondrial inner membrane Ca2+ uniporter subunit mitochondrial Ca2+ uniporter (MCU). (E) MICU1. (F) MICU2. (G) MCUb. Insets show representative immunoblots depicting protein levels with GAPDH or α-tubulin as the loading control. (H) Scheme depicting protein degradation pathways and inhibitors. (I, J) Adult murine cardiomyocytes were transduced with LacZ or Parkin adenovirus for 48 hours prior to treatment with proteasome inhibitor MG132 (10 μM, 18 hours), autophagy inhibitor 3-MA (5 mM/L, 18 hours), or lysosome inhibitor bafilomycin A1 (50 nM, 4 hours) in the presence or absence of PA (0.5 mM, 8 hours). (I) Representative immunoblots depicting protein levels of VDAC1 and Parkin. α-Tubulin was used as the loading control. (J) Quantified VDAC1 protein level. Mean ± SEM, n = 5 to 7 mice/group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 between indicated groups (A to G) or vs vehicle control (J). ###P < 0.001 vs vehicle-PA-LacZ (J). Abbreviations as in Figures 2 and 7.

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  Figure 9. Effect of Parkin Transfection and Interventions of VDAC1 or Mitochondrial Permeability Transition Pore Opening on PA-Induced Changes in Ca2+ Homeostasis and Cardiomyocyte Contractile Function Adult mouse cardiomyocytes were transfected with Parkin using adenovirus (Parkin OE) overnight prior to incubation with PA (0.5 mM) for another 8 hours in the presence or absence of the VDAC1 inhibitor DIDS (200 μM, 1 hour), mitochondrial permeability transition pore opening activator HA14-1 (2 μM, 24 hours), or cisplatin (20 μM, 24 hours). (A) Representative confocal microscopic images of mitochondrial Ca2+ fluorescence in adult murine cardiomyocytes stained with Rhod-2 (red). (B) Quantified Rhod-2 fluorescent intensity. (C) Resting cell length. (D) Peak shortening (percentage of resting cell length). (E) Maximal velocity of shortening (+dL/dt). (F) Maximal velocity of relengthening (−dL/dt). (G) TPS. (H) TR90. Mean ± SEM, n = 10 cells from 4 mice (A, B) or 20 cells from 4 mice (C to H) per group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 between indicated groups. Abbreviations as in Figures 2, 5, and 7.