Growth-Associated Protein-43 Loss Promotes Ca2+ and ROS Imbalance in Cardiomyocytes

Figures

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  Figure 1. Key factors involved in cardiac hypertrophy. (A) Western blots of GATA4, Nkx-2.5, MyH7, cMyH, and α-actinin proteins derived from WT or GAP-43−/− cardiomyocytes, and the corresponding densitometric analyses. (B) Western blotting of p-CaMKII and Met281/282 oxidized CaMKII (ox-CaMKII) proteins from WT or GAP-43−/− cardiomyocytes and the corresponding densitometric analyses. The densitometric analyses in (A,B) are plotted as the ratio between the optical density (OD) × mm2 of each band and the OD × mm2 of the corresponding loading control (GAPDH, β-tubulin, Vinculin, and CaMKII). (C) Representative images of WT and GAP-43−/− cardiomyocytes stained for α-actinin and quantitative analyses of cell area (Bar = 20 µm). Data are expressed as means ± S.E.M. from N = three biological samples in panels (A–C) with n = 143 WT and 147 GAP-43−/− cardiomyocytes in panel (C). * p < 0.05; *** p < 0.001 (Student’s t-test).

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  Figure 2. Ca2+ transient abnormalities. (A) Representative Ca2+ signal abnormality patterns detected by AnomalyExplorer software (ver. 1.0) [15] in WT (left image) and GAP-43−/− (right image) cardiomyocytes loaded with Fluo-4. Each color-coded represents both normal Ca2+ signals (in gray) and different abnormalities such as low peaks, double peaks, medium peaks, oscillations, and irregular phases. (B) Quantitative analysis of the different Ca2+ transient abnormalities. The values are means ± S.E.M. from N = three biological samples; a total number of 251 WT and 318 GAP-43−/− cardiomyocytes were examined * p < 0.05, ** p < 0.01, *** p < 0.001 (Students’ t-tests). (C) Pie charts indicate the cell percentages showing different Ca2+ transient signals.

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  Figure 3. CaM inhibitor restored Ca2+ dyshomeostasis in GAP-43−/− cardiomyocytes. (A) Representative Ca2+ signal abnormality patterns detected by AnomalyExplorer software [15] in WT (left image) and GAP-43−/− (right image) cardiomyocytes loaded with Fluo-4 and treated with 20 μM W7, a CaM inhibitor. Each color-coded chart represents both normal Ca2+ signals (in gray) and different abnormalities such as low peaks, double peaks, medium peaks, oscillations, and irregular phases. (B) Quantitative analysis of the different Ca2+ transient abnormalities. The values are means ± S.E.M. from N = three biological samples; a total of 1589 WT+W7 and 1547 GAP-43−/−+W7 cardiomyocytes were examined (the data related to WT and GAP-43−/− cardiomyocytes are those shown in Figure 2). * p < 0.05, ** p < 0.01, **** p < 0.0001 (ANOVA with post-hoc Tukey’s multiple comparison test). (C) Pie charts indicate the cell percentage showing different Ca2+ transient signals.

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  Figure 4. Impact of the antioxidant NAC on spontaneous Ca2+ oscillations in GAP-43−/− cardiomyocytes. (A) Representative Ca2+ signal abnormality patterns detected by AnomalyExplorer software [15] in WT (left image) and GAP-43−/− (right image) cardiomyocytes loaded with Fluo-4 and treated with 1 mM NAC, a pharmacological antioxidant. Each color-coded chart represents both normal Ca2+ signals (in gray) and different abnormalities such as low peaks, double peaks, medium peaks, oscillations, and irregular phases. (B) Quantitative analysis of the different Ca2+ transient abnormalities. The values are means ± S.E.M. from N = three biological samples, a total number of 739 WT+NAC and 681 GAP-43−/− + NAC cardiomyocytes were examined (the data related to WT and GAP-43−/− cardiomyocytes are those shown in Figure 2). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (ANOVA with post-hoc Tukey’s multiple comparison test).

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  Figure 5. Expression levels of Ca2+ handling proteins. (A) Immunoblots of Cav 1.2, RyR2, pRyR2, SERCA2, pPLB, and calcineurin from WT or GAP-43−/− cardiomyocytes. (B) Corresponding densitometric analyses of Western blotting in panel A. The densitometric analyses are plotted as the ratio between the optical density (OD) × mm2 of each band and the OD × mm2 of the corresponding loading control (vinculin, β-actin or GAPDH); Western blotting of pPLB was stripped and reprobed for GAPDH, and Western blotting of calcineurin was stripped and reprobed for GAPDH. Data are expressed as means ± S.E.M. from N= three biological samples. * p < 0.05, ** p < 0.01 (Student’s t-test).

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  Figure 6. ROS levels in GAP-43−/− cardiomyocytes. (A) Representative confocal images of WT or GAP-43−/− cardiomyocytes, without or with 1 mM NAC or 20 µM W7, loaded with H2DCFDA. (B) Quantitative analysis of ROS levels in cardiomyocytes, expressed as a ratio between cell mean fluorescence intensity (Arbitrary Unity, A.U.) and cell area (µm2). The values are means ± S.E.M. from N = three biological samples; a total number of 375 WT, 329 WT + NAC, 440 WT + W7, 300 GAP-43−/−, 280 GAP-43−/− + NAC, 429 GAP-43−/− + W7 cardiomyocytes were examined. ** p < 0.01, **** p < 0.0001 (ANOVA with post-hoc Tukey’s multiple comparison test).

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  Figure 7. Mitochondria morphology and metabolism changes in GAP-43−/− cardiomyocytes. (A) Representative high-magnification confocal images (corresponding to insert dotted-line boxes) of mitochondria in WT and GAP-43−/− cardiomyocytes immunostained with anti-TOM20 (bar = 20 µm; the insert solid-line boxes show the low magnification acquired field). The white arrows indicate the different mitochondria morphology in WT and in GAP-43−/− cardiomyocytes. (B) Representative confocal images of JC-1-stained mitochondria in WT and GAP-43−/− cardiomyocytes (bar = 20 µm). (C) Mitochondrial membrane potential in WT and GAP-43−/− cardiomyocytes loaded with JC-1, plotted as the ratio of red and green fluorescence of JC-1. Data are means ± S.E.M. from N = three biological samples; a total number of 523 WT and 681 GAP-43−/− cardiomyocytes were examined. (D) Lactate released in cardiomyocyte cell medium. (E) Glucose levels in cardiomyocyte cell medium. Data in panels (D,E) are means ± S.E.M. from N = six biological samples of WT cardiomyocyte cultures and from five biological samples of GAP-43−/− cardiomyocyte cultures. * p < 0.05; ** p < 0.01 (Student’s t-tests).

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  Figure 8. Increased mitochondrial Ca2+ levels in GAP-43−/− cardiomyocytes. (A) Representative confocal images of WT and GAP-43−/− cardiomyocytes monitored in the absence (top images) or presence (bottom images) of Ru360, a specific mitochondrial Ca2+ uptake inhibitor. Cells were loaded with Fluo-4 (green fluorescence) and Mitotracker Deep Red (red fluorescence) in order to measure mitochondrial Ca2+. (B) Quantitative analysis of mitochondrial Ca2+ levels calculated in cell regions in which Fluo-4 and Mito-Tracker Deep Red signals co-localized. For each cell, the mitochondrial Fluo-4 mean fluorescence intensity value (expressed as arbitrary unit, A.U.), and area were calculated; the data were expressed as mean fluorescence per area unit (A.U./µm2). The values are means ± S.E.M. from N = three biological samples; a total number of 739 WT, 415 GAP-43−/−, 551 WT+Ru360, 546 GAP-43−/−+Ru360 cardiomyocytes were examined. * p < 0.05, **** p < 0.0001 (ANOVA with post-hoc Tukey’s multiple comparison test).

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  Figure 9. Increased superoxide anion levels in GAP-43−/− cardiomyocytes. (A) Representative confocal images of WT cardiomyocytes and GAP-43−/− cardiomyocytes without (top images) or with W7 or Ru360 (bottom left and bottom right images, respectively) loaded with MitoSOX, a mitochondrial superoxide anion indicator. (B) Quantitative analysis of O2●− levels expressed as a ratio between cell mean fluorescence intensity (O2●−) and area unit (µm2). Data are means ± S.E.M. derived from N = three biological samples; a total number of 577 WT, 947 GAP-43−/−, 908 GAP-43−/− + W7, 808 GAP-43−/−+Ru360 cardiomyocytes were examined. **** p < 0.0001 (ANOVA with post-hoc Tukey’s multiple comparison test).