Autophagy and Inflammasome Activation in Dilated Cardiomyopathy

Figures

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  Figure 1. Fibrosis, hypertrophy, and loss of proteostasis characterize the hearts of dilated cardiomyopathy (DCM) patients. (A) Fibrosis. Microimages of trichrome stained control and diseased hearts, showing interstitial and perivascular fibrosis (blue). Dot plots indicate the volume fraction occupied by fibrosis; an unpaired t test was employed. (B) Hypertrophy. Confocal images of cardiac sections showing α-sarcomeric actin (ASA, red) and desmin (green) stained cardiomyocytes. Cardiomyocyte cross-sectional area and length are shown in dot plots; unpaired t test and Mann–Whitney test, respectively, were employed. (C–E) Protein misfolding. Microimages of polyubiquitinated protein labeled cardiac sections (brown). Normalized integrated optical density (IOD) values, obtained dividing individual IOD values by the mean IOD of controls, of the immunohistochemistry staining are shown in dot plots; an unpaired t test was employed (C). Confocal images of Congo red (CR) stained cardiac sections of control, DCM, and AL amyloidosis (AL) patients. CR was excited with a UV laser, while the fluorescence emission range was set at 596–615 nm employing a monochromator. The integrated fluorescence intensity…

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  Figure 2. Defects of the autophagy lysosomal pathway characterize the hearts of DCM patients. (A,B) Autophagy. Western blots (WBs) of whole protein extracts of 3 control hearts and 3 DCM explanted hearts. Blotted proteins were incubated with antibodies against Beclin 1, Atg5, Atg7, and GAPDH. Densitometric analyses of 6 DCM and 6 controls are shown in dot plots; unpaired t tests (Beclin 1 and Atg 5) and Mann–Whitney tests (Atg 7) were employed (A). Microimages of p62SQSTM1-labeled cardiac sections (brown, (B)). Quantitative analysis of p62SQSTM1 staining is shown in dot plots; a Mann–Whitney test was employed. (C–E) Lysosome dysfunction and biogenesis. Confocal images of LAMP2- (red) and ASA-labeled (white) cardiac sections. Arrows point to enlarged lysosomes. Normalized IFI values of LAMP2 staining are shown in dot plots; a Mann–Whitney test was employed (C). Confocal images of Galectin3- (red) and ASA-labeled (white) cardiac sections. Arrow points to a myocyte with Galectin3 punctae. Dot plots display the fraction of cardiomyocytes showing Galectin3 punctae in their cytoplasm; an unpaired t test was employed (D). Confocal images of TFEB- (red) and ASA-stained (white) cardiac sections.…

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  Figure 3. Evidence of the accumulation of dysfunctional mitochondria, lipoperoxidation products, and activated inflammasomes in explanted hearts. (A,B) Mitochondrial dysfunction. Confocal images of cardiac sections immunostained for mitochondria (green) and Parkin (red). Higher magnifications of the insets are shown, as single channel images of mitochondria and Parkin, in the right portion of each figure (A). Microimages of Parkin immunohistochemistry stainings of cardiac sections (brown). Normalized IOD values of Parkin are shown in dot plots; a Mann–Whitney test was employed (B). (C–E) Oxidative stress and redox signaling. Microimages of 4-hydroxynonenal (4HNE)-labeled cardiac sections (brown). Normalized IOD values of 4HNE are shown in dot plots; an unpaired t test was employed (C). Confocal images of cardiac sections immunostained for 53BP1 (red) and ASA (white). The fraction of cardiomyocytes showing 53BP1 nuclear positivity is shown in dot plots; a Mann–Whitney test was employed (D). APE/REF immunohistochemistry staining of cardiac sections (brown). Results of the quantitative assessment of the nuclear expression of APE/REF are shown in the right panel as normalized IOD; a…

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  Figure 4. DCM is associated with alterations of amino acid metabolism. (A,B) Metabolomic analysis. 1D NMR spectra of 12 control (green lines) and 12 DCM hearts (red lines). The full spectra are shown in the bottom panel, while the top panel shows the 3.75–4.45 ppm region and the peaks assigned to lactic acid and 6-phosphogluconic acid (A). Heatmap summarizing the results of the hierarchical clustering analysis generated employing the top 25 features ranked by t tests. Each colored cell on the map corresponds to a concentration value in the data table, with samples in columns and features/compounds in rows (B). (C) WB and real-time PCR analyses. Representative WB of whole-protein extracts of 3 control hearts and 3 DCM explanted hearts. Blotted proteins were incubated with antibodies against PP2Cm and GAPDH. Results of the densitometric analyses (n = 6 controls and 6 DCM) are reported in the bottom-left panel; a Mann–Whitney test was employed. The bottom-right panel summarizes the results of the RT-PCR analysis of miR-22 expression in control (n = 12) and DCM (n = 37) hearts; a Mann–Whitney test was employed. Fold change was computed using miR-92a as a housekeeping gene.

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  Figure 5. Cardiac pericytes isolated from diseased hearts are senescent and accumulate dysfunctional mitochondria. (A,B) In vitro and in vivo characterizations of CPc. Confocal images of cultured CPc (n = 5 D-CPc and n = 5 EDCM-CPc) stained for NG2 (green, left panel), PDGFRα and PDGFRβ (green and red, central panel), and Tbx18 (red, right panel; (A)). Confocal images showing that mostly PDGFRβ+ cells (green) are localized in and around arterioles and capillaries and coexpress smooth muscle actin (red). ASA is shown in white (B). (C–G) Senescence and ALP dysfunction of EDCM-CPc. Epifluorescence images of CPc isolated from normal (D-CPc; n = 5) and pathologic hearts (EDCM-CPc; n = 4) show positivity to p16INK4A (green, C), γH2A.X (green) and Ki67 (red, D), lipofuscins (green, €), Galectin 3 (red) and LAMP2 (green, (F)), and mitochondria (green, (G)). Nuclei are labeled by DAPI in blue. Dot plots in C–G indicate the results of the quantitative analysis; Mann–Whitney tests were employed. (H) Profiles of mitochondria bioenergetics measurements. Plots in (i) display average (±S.D.) OCR of 4 D- and 4 EDCM-CPc, measured at baseline and after the addition of the stressors oligomycin, FCCP,…

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  Figure 6. Inhibition of miR-22 reverses EDCM-CPc senescence. Results of RT-PCR analysis of EDCM-CPc treated with either anti-miR22 or a negative control (NC; (A)). (B–F) Reversal of EDCM-CPc senescence and ALP dysfunction by miR-22 inhibition. Confocal images of EDCM-CPc treated with either anti-miR22 or NC showing positivity to p16INK4A (green, (B)), γH2A.X (green) and Ki67 (red, (C)), lipofuscins (green) and Galectin 3 (red, D), mitochondria (green, (E)), and TFEB (yellow, (F)). Nuclei are labeled by DAPI in blue. (G) Effects of miR-22 inhibition on mTOR targets, PP2Cm, and autophagy. Representative WB of whole protein extracts of 6–7 EDCM-CPc treated with either anti-miR22 or NC (G). Blotted proteins were incubated with antibodies against 4EBP1, p-4EBP1Thr37/46, PP2Cm, LC3A/B or p62SQSTM1. Results of the quantitative analyses are reported in the right panels. (H) Effects of miR-22 inhibition on IL1β secretion. ELISA assay was used for the detection of IL1β released in the culture supernatant of 7 EDCM-CPc treated with either anti-miR22 or NC (H). Dot plots indicate the results of quantitative analyses; paired t tests (p16INK4A, Ki67, lipofuscin, mitochondria, TFEB, 4EBP1,…

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  Figure 7. Autophagy and inflammation in DCM. Illustration summarizing the main findings described in human DCM samples. Blue ellipses indicate the key actors of this process. Dashed arrows indicate correlative or literature findings. Arrows with blunt ends indicate inhibition, while pointed arrows indicate activation.