Sotagliflozin ameliorates doxorubicin-induced cardiomyopathy by regulating inflammation and mitochondrial dysfunction through the AMPK/mTOR pathway

Figures

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  Figure 1. SOTA ameliorates DOX-induced cardiac dysfunction in mice. (A) Echocardiographic changes in cardiac function in motion-mode: LVIDd, LVIDs, LV Vol d, LV Vol s, LVFS, LVEF and E/A of mice 10 days after treatment with DOX combined with SOTA. (B) Representative echocardiogram images of mice. All data are expressed as the mean ± SD (n=6). **P<0.01 compared with the Control untreated groups and ##P<0.01 compared with the DOX-alone treated group. ns, not significant; SOTA, sotagliflozin; DOX, doxorubicin; LVIDd, left ventricular internal dimensions in diastole; LVIDs, left ventricular internal dimensions in systole; LV Vol d, left ventricular volume in diastole; LV Vol s, left ventricular volume in systole; LVFS, left ventricular fractional shortening rate; LVEF, left ventricular ejection fraction; E/A, early/atrial; HR, heart rate; BPM, beats per minute; Resp, respiration rate; T, temperature; ECG, electrocardiogram.

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  Figure 2. SOTA ameliorates DOX-induced myocardial fibrosis and morphological alteration in mice. (A) Representative images of H&E staining of heart tissues in mice. Scale bar, 20 µm. (B) Representative images of Sirius red staining of heart tissues in mice. Scale bar, 20 µm. (C) Western blotting bands and (D) semi-quantitative results of collagen I, collagen III and MMP-9 proteins of heart tissue in mice. (E) Changes in serum LDH, CK-MB, cTn-I and BNP levels were detected using commercial ELISA kits. All data are expressed as the mean ± SD (n=6). **P<0.01 compared with the Control untreated group; #P<0.05, ##P<0.01 compared with the DOX alone group. ns, not significant; SOTA, sotagliflozin; DOX, doxorubicin; LDH, lactate dehydrogenase; CK-MB, creatine kinase isoenzyme; cTn-I, cardiac troponin I; BNP, brain natriuretic peptide; H&E, hematoxylin and eosin; MMP-9, matrix metallopeptidase 9.

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  Figure 3. SOTA attenuates DOX-induced cardiac apoptosis, inflammation and mitochondrial dysfunction in vivo. (A) Representative images of TUNEL staining and (B) the quantitative results of heart tissue in mice. Scale bar, 20 µm. (C) Representative images of western blotting and (D) statistical results of the C cas-3, Bax and Bcl-2 proteins of heart tissue in mice. (E) The levels of IL-6, IL-1β and TNF-α in mice heart tissue as determined by commercial ELISA kits. (F) Changes in ROS, ATP, H2O2 and Ca2+ levels in mice heart tissue were detected by commercial ELISA kits. All data are expressed as the mean ± SD (n=6). **P<0.01 compared with Control untreated group, ##P<0.01 compared with DOX alone group. ns, not significant; SOTA, sotagliflozin; DOX, doxorubicin; C cas-3, cleaved caspase-3; ROS, reactive oxygen species; H2O2, hydrogen peroxide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4',6-diamidino-2-phenylindole; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; IL-6, interleukin-6; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; ATP, adenosine triphosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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  Figure 4. SOTA alleviates DOX-induced cardiotoxicity and cardiomyocyte apoptosis in vitro. (A) Cell viability of H9c2 cells treated with DOX (0, 5, 10, 20 and 30 µM) at 12 h. (B) Cell viability of H9c2 cells treated with DOX (10 µM) combined with SOTA (0, 5, 10, 20 and 40 µM) at 12 h. (C) LDH levels in H9c2 cells were detected by a commercial ELISA kit. (D) Representative TUNEL staining images and (E) the quantitative results of H9c2 cells. Scale bar, 20 µm. (F) Statistical results of C cas-3, Bax and Bcl-2 proteins of H9c2 cells and (G) representative images of western blotting. All data are expressed as the mean ± SD (n=3). *P<0.05, **P<0.01 compared with Control untreated groups, #P<0.05 and ##P<0.01 compared with DOX alone group. ns, not significant; SOTA, sotagliflozin; DOX, doxorubicin; C cas-3, cleaved capsase-3; LDH, lactate dehydrogenase.

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  Figure 5. SOTA attenuates DOX-induced cardiac inflammation and ROS in vitro. (A) IL-6, IL-1β and TNF-α levels in H9c2 cells as determined by commercial ELISA kits. (B) Representative images of DCFH-DA staining and (C) the quantitative results of ROS levels in H9c2 cells. All data are expressed as the mean ± SD (n=3). Scale bar, 25 µm. **P<0.01 compared with Control untreated group; ##P<0.01 compared with DOX alone group. SOTA, sotagliflozin; DOX, doxorubicin; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; IL-6, interleukin-6; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; DAPI, 4',6-diamidino-2-phenylindole.

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  Figure 6. SOTA attenuates DOX-induced cardiac mitochondrial dysfunction in vitro. (A) Representative images of Rhod2-AM staining and (B) mitochondrial Ca2+ quantitative results in H9c2 cells. Scale bar, 25 µm. (C) H2O2 levels in H9c2 cells as determined by commercial ELISA kits. (D) Representative images of JC-1 staining and (E) statistical results of JC-1 aggregates to monomers in H9c2 cells. Scale bar, 25 µm. (F) ATP levels in H9c2 cells as determined by ELISA kits. All data are expressed as the mean ± SD (n=3). **P<0.01 compared with Control untreated group, #P<0.05 and ##P<0.01 compared with DOX alone group. SOTA, sotagliflozin; DOX, doxorubicin; H2O2, hydrogen peroxide; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; DAPI, 4',6-diamidino-2-phenylindole; ATP, adenosine triphosphate.

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  Figure 7. SOTA attenuates DOX-induced cardiomyocyte apoptosis by activating the AMPK/mTOR pathway in vitro. H9c2 cells were treated concomitantly with DOX and SOTA and with 10 µM of the AMPK/mTOR pathway inhibitor compound C. (A) Representative images of western blotting and (B) statistical results of p-AMPK/AMPK and p-mTOR/mTOR proteins in H9c2 cells treated with different concentrations of DOX. (C) Representative images of western blotting and (D) statistical results of p-AMPK/AMPK and p-mTOR/mTOR proteins in the presence of compound C. (E) Statistical results of C cas-3 and (F) Bcl-2/Bax proteins. (G) Quantitative results of H9c2 cells and (H) representative images of TUNEL staining. Scale bar, 20 µm. All data are expressed as the mean ± SD (n=3). *P<0.05, **P<0.01 compared with DOX group, #P<0.05 and ##P<0.01 compared with SOTA + DOX group. SOTA, sotagliflozin; DOX, doxorubicin; p-, phosphorylated; AMPK, 5' AMP-activated protein kinase; C cas-3, cleaved caspase 3; mTOR, mechanistic target of rapamycin; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4',6-diamidino-2-phenylindole; GAPDH,…

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  Figure 8. SOTA attenuates DOX-induced cardiac mitochondrial dysfunction by activating the AMPK/mTOR pathway in vitro. (A) Representative images of DCFH-DA staining and (B) representative images of JC-1 staining in H9c2 cells. (C) Quantitative results of ROS levels. (D) Quantitative mitochondrial Ca2+ levels in H9c2 cells. (E) Statistical results of the ratio of JC-1 aggregates to monomers. All data are expressed as the mean ± SD (n=3). **P<0.01 compared with DOX group, #P<0.05 and ##P<0.01 compared with SOTA + DOX group. SOTA, sotagliflozin; mTOR, mechanistic target of rapamycin; DOX, doxorubicin; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; DAPI, 4',6-diamidino-2-phenylindole.

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  Figure 9. Schematic diagram showing protective signaling of SOTA on doxorubicin-induced cardiomyopathy. SOTA, sotagliflozin; AMPK, 5' AMP-activated protein kinase; ROS, reactive oxygen species; mTOR, mechanistic target of rapamycin.