Cellular, mitochondrial and molecular alterations associate with early left ventricular diastolic dysfunction in a porcine model of diabetic metabolic derangement

Figures

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  Figure 1. Ratio of mitral peak velocity during early filling (E) to early diastolic mitral annular velocity (e′, E/e′ ratio, A), peak left ventricle untwist velocity (B), left atrial volume (LA, C), left ventricle end diastolic diameter (LVEDD, D), posterior wall thickness (PWd, E), and relative wall thickness ((2*PWd)/LVEDD, F) in the hearts of DMetD and Control (CON) swine at baseline (BL) and after 5 months. *p < 0.05 for interaction DMetD and time by two-way ANOVA, †p < 0.05 versus corresponding CON by Bonferroni post-hoc test, and ‡p < 0.05 versus CON by unpaired t-test.

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  Figure 2. Left ventricular weight (LV weight, (A)), myocyte cross-sectional area (examples, (B), and data summary (C)), end-diastolic pressure (EDP)–volume (EDV) relation composed from measurements at preload reduction, (inverted triangle), baseline (open circle) and preload increase (open triangle) (D), myocardial collagen deposition (examples, (E) and data summary (F)), stroke work (SW, G), and dP/dtmax (H) and dP/dtmin (I), plotted as a function of end-diastolic volume, measured at sacrifice in CON and DMetD animals.

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  Figure 3. Single cardiomyocyte passive force (A) in response to increasing myocyte stretch and maximal forces (B). CON = controls (13 cardiomyocytes of 5 swine), DMetD, diabetic metabolic derangement (11 cardiomyocytes of 5 swine). Myocardial titin N2BA/N2B ratio of CON (n = 6) and DMetD (n = 5, C), and typical examples of the titin gels for 3 animals in each group . *p < 0.05 versus CON by two-way ANOVA, †p < 0.05 versus CON by unpaired t-test. Full-length gels are shown in Supplementary Fig. S1.

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  Figure 4. Basal (A) and NAPDH-stimulated (B) superoxide production in LV myocardium, myocardial eNOS expression (C), phosphorylation of eNOS (D), the monomer/dimer ratio (E), and nitric oxide (NO) production (F). CON = controls (n = 8), DMetD, diabetic metabolic derangement (n = 9). *p < 0.05 versus corresponding CON by two-way ANOVA and Bonferroni post hoc (panel A and B) or unpaired t-test (panel C–F), †p < 0.05 versus corresponding untreated by two-way ANOVA and Bonferroni post hoc, ‡p < 0.05 versus corresponding basal by two-way ANOVA and Bonferroni post hoc. Full-length gel blots are shown in Supplementary Fig. S2.

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  Figure 5. Examples of cardiomyocyte ultrastructure by electron microscopy in CON (A) and DMetD (B). M = mitochondrion, G = glycogen, V = vacuole. Scale bar represents 2 μm. The inserts highlight the altered glycogen content. Quantified glycogen content (C) and number of vacuoles (D) were significantly higher in DMetD compared to CON. Mitochondrial density (E) or percentage of connected mitochondria (F) were not different between groups. Total myocardial mitochondrial protein content (G) by Western blotting (see Supplementary Fig. 1) was significantly higher in DMetD compared to CON. Electron transport (ET) capacity (H) however, was significantly lower in DMetD compared to CON, due to NADH-linked dysfunction (see Supplementary Fig. 2), suggestive of an intrinsic mitochondrial dysfunction, independent of mitochondrial protein mass. *p < 0.05 vs. CON by unpaired t-test. Full-length gels and blots are shown in Supplementary Fig. S3.

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  Figure 6. Protein levels of the AMPK light (A) and heavy bands (B), pAMPK (C) and enzyme activity of CS (D), LDH (E) and HOAD (F), in CON and DMetD animals. *p < 0.05 versus CON by unpaired t-test. AMPK AMP-activated protein kinase, pAMPK phosphorylated AMPK, CS citrate synthase, LDH lactate dehydrogenase, HOAD 3-hydroxyacyl-CoA dehydrogenase. Full-length gel blots are shown in Supplementary Fig. S5.

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  Figure 7. Myocardial genes and their networks in DMetD are connected to two major pathways associated with glucose and lipid metabolism disorders.