The Role of Lactate in Mitochondrial Metabolism of DOX-Induced Senescent AC16 Cells

Abstract

Senescent cells accumulate with age in organ and tissue causing the decline of functionality and various pathological conditions including cardiovascular disease. Regular exercise induces continuous exposure to lactate that contribute to adaptive process through mitochondrial biogenesis and improve of metabolic process. Lactate accumulation during exercise also appears to be associated with exercise-induced mitochondrial adaptation. Improvement of mitochondria function through lactate exposure could be a tool to prevent cardiomyocytes senescence and cardiac aging. The aim of the following article is to investigate the role of lactate in Doxorubicin-induced senescent AC16 human cardiomyocytes cell mitochondrial metabolism. We assessed the metabolic behaviour in senescent cardiomyocytes after chronic lactate exposure and provided a discussion of the effect of this metabolite in regulating mitochondrial physiology during cardiac aging.

Keywords: DOX‐induced senescent AC16 cells; metabolomics; mitochondrial dysfunction; proteomics.

Figure 1

A Representative Immunoblot image and histogram of p21. Senescent cells (S) were grown in glucose 5.5 mM SG; glucose 5.5 mM + lactate 8 mM SGL and lactate 8 mM SL; no‐senescent cells (C) were grown in glucose 5.5 mM CG; glucose 5.5 mM + lactate 8 mM CGL and lactate 8 mM CL. Normalization of immunoblot was performed on Coomassie‐stained PVDF membrane. Histogram displays the results as mean values and SD of three independent biological experiments (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (B) Optical microscope images; all panels are the same magnification, scale bars for all images: 200 µm. (C) Nucleus area measurement performed using Image J. (D) Actin cytoskeleton by a phalloidin (red)/DAPI (blue) staining and representative immunoblot image and histogram of Actin. Histogram displays the results as mean values and SD of three independent biological experiments (****p < 0.0001). Scale bars for all images: 37 µm.

Figure 2

Cellular growth and viability assay. (A) Number of cells 48 h after the growth in different carbon sources. (B) Representative image of flow cytometry analysis of annexin V/propidium iodide‐stained. (C) Histograms that represent the percentage of live, apoptotic, and necrotic cells obtained by flow cytometric analysis. Results are reported as mean of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 3

Mitochondrial function. (A) Representative image of flow cytometry analysis of TMRE stained, on the X‐axis the relative fluorescence, on the Y‐axis the number of events. Senescent cells histograms show two peaks: one with low fluorescence intensity (M1) and another with high intensity (M2). (B) Graph that represent the Oxygen consume rate at basal level, after the addition of CCCP (maximal) and at the end of its effect (post). Results are reported as mean of three independent experiments (*p < 0.05).

Figure 4

Mitochondria number and size. (A) Representative confocal images of AC16 mitochondria. Organelles were stained with Mitotracker Red (red signal) and Nuclei with Hoechst 33342 (blue signal). The respective reproductions of mitochondrial skeleton were reported in the bottom panel (black and grey). Scale bars for control images are 20 µm, scale bars for senescent 52 µm. Mitochondria skeletons were reported in the bottom panel and the quantification of their (B) intensity, (C) number and (D) size calculated by ImageJ were reported in the histogram graphs as mean values and SEM of three independent experiments (**p < 0.01; ***p < 0.001). (E) Representative immunoblot image and histogram of PGC1α. Normalization of immunoblot was performed on Coomassie‐stained PVDF membrane (*p < 0.05).

Figure 5

The oxidative phosphorylation system (OXPHOS). Representative immunoblot image of all complex of OXPHOS. Senescent (S) and no‐senescent (C) cells separately grown on media with three different carbon sources (glucose 5.5 mM (G); glucose 5.5 mM + lactate 8 mM (GL) and lactate 8 mM (L)). Normalization of immunoblot was performed on Coomassie‐stained PVDF membrane. Histograms display the statistically significant differences identified in CI, CII, and CV. Results are reported as mean values and SD of three independent biological experiments (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 6

ATP levels and ROS measurement. (A) Measurement of ATP level. (B) Measurement of ROS levels by using the 2′‐7′‐Dichlorodihydrofluorescein diacetate assay (DCFH‐DA). Results are reported as mean values and SD of three independent biological experiments (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 7

Graphic summary of the pathways involving the studied enzymes.

Figure 8

Representative immunoblot image and histogram of (A) Pyruvate dehydrogenase (PDH); (B) Citrate synthase; (C) Succinate dehydrogenase subunit A (SDHA); (D) Lactate dehydrogenase A (LDHA); (E) Lactate dehydrogenase B (LDHB). SG and CG: glucose 5.5 mM; SGL and CGL: glucose 5.5 mM + lactate 8 mM; SL and CL: lactate 8 mM. Normalization of immunoblot was performed on Coomassie‐stained PVDF membrane. Histograms display the results as mean values and SD of three independent biological experiments (*p < 0.05; **p < 0.01).

Figure 9

(A) Levels of extracellular lactate normalized for the quantity of proteins; (B) Levels of intracellular lactate normalized for the quantity of proteins. (C) Representative immunoblot image and histogram of monocarboxylate transporter 4 (MCT4): SG and CG: glucose 5.5 mM; SGL and CGL: glucose 5.5 mM + lactate 8 mM; SL and CL: lactate 8 mM. Normalization of immunoblot was performed on Coomassie‐stained PVDF membrane Histograms display the results as mean values and SD of three independent biological experiments (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).