High-Fat Diet-Induced Diabetic Cardiomyopathy in Female Zebrafish: Cardiac Pathology and Functional Decline Mediated by Type 2 Diabetes

Figures

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  Figure 1. High-fat diet induces obesity and type 2 diabetes in zebrafish. (A) Representative images comparing body morphology between the N and H groups. (B) Body length of zebrafish after 8-week HFD intervention. (n = 18); (C) body weight of zebrafish (n = 18); (D) body mass index of zebrafish (BMI, n = 18); (E) skeletal muscle low-density lipoprotein cholesterol levels (n = 3). (F) Fasting blood glucose levels (n = 3). (G) IPGTT curves (n = 3). (H) AUC of IPGTT. (I) Skeletal muscle insulin content (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 2. T2DM induces pathological cardiac hypertrophy in zebrafish. (A) Representative images of hearts from N and H groups (scale bar: 1 mm). (B) Zebrafish heart weight (n = 15). (C) Hematoxylin and eosin) staining of longitudinal cardiac sections (scale bar: 50 μm). CM, Compact myocardium; SM, Spongy myocardium. (D) Compact myocardial thickness (n = 3). (E) Transmission electron microscopy images of cardiac myofibrils (scale bar: 2 μm; inset shows magnified view of boxed region, scale bar: 1 μm). Red arrows indicate myofibrillar breaks. (F) Sarcomere length quantification (n = 3). (G) Relative mRNA expression levels of nppa, nppb, tnnt2, myh7, mybpc3 and ppara in zebrafish hearts (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 3. T2DM induces cardiac functional impairment in zebrafish. (A) B-mode echocardiography showing end-diastolic and end-systolic cardiac contours. (B) Ejection fraction (EF) (n = 20). (C) Fractional shortening (FS) (n = 20). (D) Stroke volume normalized to body weight (SV/BW) (n = 18). (E) End-diastolic longitudinal diameter (EDDlong) (n = 20). (F) End-diastolic basal diameter (EDDbase) (n = 20). (G) End-systolic longitudinal diameter (ESDlong) (n = 20). (H) End-systolic basal diameter (ESDbase) (n = 20). (I) Representative pulsed-wave Doppler echocardiographic tracings from groups N and H. (J) E-wave peak velocity (E peak) (n = 18). * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 4. T2DM disrupts lipid and glucose metabolism in zebrafish hearts. (A) Oil Red O staining of cardiac tissues after 8-week HFD intervention, with red indicating lipid droplet deposition (scale bar: 50 μm). (B) Quantification of Oil Red O-positive areas (n = 3). (C–F) Western blot analysis showing T2DM-induced activation of lipid metabolism-related pathways (PGC1-α/PPARα/CPT1a) and (G) downregulation of the glucose metabolism regulator GLUT4 (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 5. T2DM triggers cardiac inflammation and fibrosis in zebrafish. (A) Masson staining of cardiac tissues (blue: collagen fibers; red: cardiomyocytes; scale bar: 20 μm). (B) Quantification of the fibrotic area ratio (n = 3). (C–F) Western blot analysis showing T2DM-induced upregulation of pro-inflammatory proteins (IL-1β, IL-18) and the fibrosis marker α-SMA (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 6. T2DM causes mitochondrial structural damage and dysfunction in zebrafish hearts. (A) TEM images of cardiac mitochondria (scale bar: 2 μm; insets show magnified views of boxed regions, scale bar: 1 μm). Red arrows indicate mitochondrial cristae loss, blue arrows denote mitochondrial swelling, and yellow arrows indicate membrane disruption in the H group. (B) Quantification of the mitochondrial density per field. (C) Mitochondrial respiratory assay traces: the blue curve represents the oxygen concentration, and the red curve reflects the oxygen consumption rate. (D) Mitochondrial complex I activity. (E) Maximal electron transport chain capacity. (F) The combined activity of complexes I and II. (G) Mitochondrial complex IV activity. ns, not significant. * p < 0.05. (n = 3).

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  Figure 7. T2DM induces oxidative stress in zebrafish hearts. (A) Dihydroethidium staining of the cardiac tissues (red: ROS-positive areas; scale bar: 50 μm). (B) Quantification of the ROS accumulation (n = 3). (C–F) Western blot analysis showing T2DM-induced upregulation of the ROS-generating enzyme NOX4 and downregulation of the antioxidant proteins (NRF2, SOD1) (n = 6). * p < 0.05, ** p < 0.01.