Chronic lactate exposure promotes cardiomyocyte cytoskeleton remodelling

Figures

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  Fig. 1. Effect of carbon source on cardiomyocyte growth, survival, and redox state. A) Growth curves of AC16 cardiomyocyte cell line in medium supplemented with glucose 5.5 mM (red, GG) or with lactate 8 mM (blue, LG). B) Lactate Dehydrogenase (LDH) levels in GG (red) or LG (blue) after 24, 48 and 72 h of growth. C) Cytofluorimetric analysis of the cell cycle distribution phases in AC16 cardiomyocyte growth for 72 h with glucose 5.5 mM (GG) or lactate 8 mM (LG). M = mitotic phase cells, S = synthesis phase cells, G0/G1 = cells in G0 or G1 phases. D) Measurement of ROS levels in AC16 cardiomyocyte cell growth for 72 h with glucose 5.5 mM (red, GG) or lactate 8 mM (blue, LG) by using the 2′-7′-Dichlorodihydrofluorescein diacetate assay (DCFH-DA) (*p < 0.05; ***p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 2. LC-MS analysis of 72-h growth cells in presence of glucose (GG) or lactate (LG) as carbon source; A) STRING representation of 14 statistically relevant proteins. B) Dysregulation of 14 statistically relevant proteins LG (A-blue bar) versus GG (B-red bar). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 3. Effect of lactate on cardiomyocyte morphology and cytoskeleton. A) Representative cell morphology images, by optical microscopy, of AC16 cardiomyocyte growth in glucose 5.5 mM (GG) or lactate 8 mM (LG) for 72 h. B) Representative confocal microscopy images of microfilament pattern by a phalloidin/DAPI staining in AC16 cardiomyocyte in GG and LG cells at 72 h. C) Quantification of relative phalloidin fluorescence performed by ImageJ software. Histogram reports the mean values and standard deviations of four image from three independent biological experiments (*p < 0.05).

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  Fig. 4. Lactate exposure affects cardiomyocytes metabolism. A) OCR measurement in AC16 cardiomyocyte growth with glucose 5.5 mM (red bar, GG) or lactate 8 mM (blue bar, LG) for 72 h. B) Relative abundance of phosphoenolpyruvate by metabolomic GC-MS analysis reported as fold decrease in LG (blue bar) respect to GG (red bar) cells. C) Relative abundance of alpha-ketoglutarate and succinate. D) Representative immunoblot image and histogram of PDH-1Eα; normalization of immunoblot was performed on Coomassie-stained PVDF membrane (original image of Western blot is reported in Fig. S2). E) Glucose uptake in AC16 cardiomyocyte growth in glucose 5.5 mM (GG) or lactate 8 mM (LG) for 72 h. All histograms display the results as mean values and SD of three independent biological experiments (*p < 0.05; **p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 5. Relative abundance of amino acids by metabolomic GC-MS analysis reported as fold increase (or decrease) in cells growth with lactate 8 mM (blue bar, LG) respect to those treated with glucose 5.5 mM (red bar, GG). A) asparagine; B) aspartic acid; C) histidine; D) leucine; E) phenylalanine; F) glycine; G) methionine; H) serine; I) glutamic acid; L) isoleucine; M) tyrosine; N) valine. All histograms display the results as mean values and SD of three independent biological experiments (*p < 0.05; **p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 6. Schematization of endogenous lactate effects on cardiomyocyte cell. Created with BioRender.com.

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