Role of TSPO/VDAC1 Upregulation and Matrix Metalloproteinase-2 Localization in the Dysfunctional Myocardium of Hyperglycaemic Rats

Figures

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  Figure 1. Effects of hyperglycaemia on left ventricle (LV)-dysfunction. (A) Representative monodimensional echocardiographic imaging of normal pellet diet (NPD) and NPD + streptozotocin (STZ) groups at the end of the study. (B) Histograms show cardiac dimensions and functional parameters assessed at the end of the study. Hyperglycemia in NPD + STZ rats significantly increased LV end-systolic diameter (LVESd) and LV end-diastolic diameter (LVEDd), impairing fractional shortening (FS) and ejection fraction (EF) as compared with the NPD group. (C,D) In NPD + STZ animals, a significant thinning of interventricular septum in systole (IVSs) and in dyastole (IVSd) and (E,F) the reduction of left ventricular posterior wall in systole (LVPWs) and in diastole (LVPWd) were detected in comparison with the NPD group. (G,H) No differences in end diastolic left ventricular mass (EDLVM) and in end systolic left ventricular mass (ESLVM) were observed between the two groups. The data are presented as mean ± SEM. * p < 0.05 vs. NPD, Mann–Whitney test, n = 6/group.

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  Figure 2. Cardiac tissue strain analysis. (A) Representative parametric displays, area/volume graphs, and segmental synchronicity page related to endocardial strain analysis of parasternal short-axis view (PSLAX) assessed in radial motion at the end of experimental period. (B) Average of time-to-peak (TPk) of the six segments of cardiac wall in endocardial strain analysis. (C) Whole peak capacity, assessed as the peak average (Pk) of the six segments of cardiac wall, in endocardial strain. (D) Maximum wall delay (MWD) of LV during radial motion in endocardial strain analysis. The data are presented as mean ± SEM. * p < 0.05 vs. NPD, Mann–Whitney test, n = 6/group.

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  Figure 3. Effect of hyperglycaemia on NADPH oxidase expression and superoxide dismutase (SOD) activity in myocardial tissue. (A) In the STZ group, up-regulation of NADPH oxidase was associated with higher detection of superoxide anion, compared with the NPD group. On the left, quantification of NADPH expression related to immunohistochemistry analysis and representative confocal images showing MitoSOX™ staining. Red fluorescence indicates the formation of superoxide anion. (B) SOD activity in mitochondrial fraction remained unchanged between the two groups. The data are presented as mean ± SEM. *** p < 0.001 vs. NPD, Mann–Whitney test, n = 6/group.

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  Figure 4. Effect of hyperglycaemia on translocator protein (TSPO) and voltage-dependent anion-selective channel 1 (VDAC1) expression in myocardial tissue. (A) On the left, representative images showing that, in the STZ group, an up-regulation of TSPO expression was observed in comparison with the NPD group. On the right, quantification of TSPO expression related to immunohistochemistry analysis. (B) Densitometric analysis of VDAC1 expression assessed by Western blot assay showing the enhancement of protein level in mitochondrial fraction under hyperglycemia (on the right, representative images of Western blot) The data are presented as mean ± SEM. * p < 0.05; *** p < 0.001 vs. NPD; Mann–Whitney test, n = 6/group (immunohistochemistry analysis); t-test, n = 6/group (Western blot analysis).

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  Figure 5. Bcl-2 and cytochrome C expression in hyperglycaemic and normoglycaemic animals. Western blot analysis showed no changes in Bcl-2 and cytochrome C expression between the two groups. On the left, representative images of Western blot assay; on the right, densitometric analysis. The data are presented as mean ± SEM. n = 6/group (t-test).

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  Figure 6. Effect of hyperglycaemia on MMP-2 activity and localization in myocardial tissue. (A) Zymogram showed a decreased activation of the extracellular MMP-2 (57 kDa) in NPD + STZ rats compared with the NPD group, whereas no difference of full-length FL-MMP-2 and N-terminal truncated isoform of MMP-2 (NT-MMP-2) activities was detected. (B) Representative images of immunofluorescence analysis. Orange staining is due to colocalization of mitochondria (MitoSOX™-induced coloration) with NT-MMP-2 (green). Blue/white staining is due to colocalization of endoplasmic reticulum (ER-Tracker™-induced blue) with NT-MMP-2 (green). * p < 0.05; vs. NPD. The data are presented as mean ± SEM, t-test n = 6/group.

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  Figure 7. Effect of hyperglycaemia on cardiomyocyte. (A) Under physiologic glucose levels, VDAC1 promotes mitochondrial calcium intake and buffering. (B) High glucose levels directly induce an increase of cytosolic calcium. Moreover, chronic hyperglycaemia causes mitochondrial NADPH overexpression and superoxide anion overproduction, thus promoting TSPO and VDAC1 overexpression. This, in turn, results in their probable interaction aimed to prevent mitochondrial calcium accumulation. On the other hand, enhanced cytosolic calcium promotes endoplasmic reticulum (ER) impairment, translocation of NT-MMP-2 from mitochondria-associated membrane (MAM) into mitochondrial matrix, and mitochondrial dysfunction.