HydroMg attenuates doxorubicin-induced myocardial injury via promoting mitochondrial function.

Figures

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  Figure 1. HydroMg improves cardiomyocyte viability. (A) H9C2 cell death was quantified by live-cell imaging following a 24-hour HydroMg treatment (n ≥ 3 in each group) Treatment with 1000 μg/mL HydroMg induced significant cell death within 24 hours. In contrast, lower concentrations (0.1–100 μg/mL) showed proliferation rates similar to the control. Scale bars: 200 μm. (B) Effect of HydroMg treatment on H9C2 cells proliferation (24 hours). (C–E) Quantification of H9C2 cells proliferation relative to untreated controls following HydroMg treatment at 6 (C), 12 (D), and 24 hours (E) (n ≥ 3 per group). (F–H) H9C2 cells were treated with HydroMg at concentrations of 10, 1, and 0.1 μg/mL for different durations: 6 (F), 12 (G), and 24 hours (H) (n ≥ 3 per group). Cell viability was measured using the cell counting kit-8 assay. All results are presented as mean ± SEM. **P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). Ctrl: Control.

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  Figure 2. HydroMg inhibits DOX-induced cardiomyocyte death and apoptosis. (A) Experimental scheme. (B) H9C2 cells were treated with HydroMg for 6 hours, followed by doxorubicin intervention for 24 hours, with real-time cell death monitoring via live-cell imaging (n ≥ 3 per group). HydroMg resulted in a dose-dependent reduction in doxorubicin-induced cell death, with the 1 μg/mL concentration significantly enhancing cell proliferation compared with the doxorubicin-only group. Scale bars: 200 μm. (C) Dynamic proliferation curve of H9C2 cells during 24-hour DOX intervention following 6-hour HydroMg pretreatment. (D) Relative proliferation rate of H9C2 cells after 6-hour HydroMg pretreatment combined with 24-hour sustained DOX intervention. (E, F) TUNEL staining (green) was performed to detect H9C2 cells apoptosis in each group after HydroMg and DOX treatment (n ≥ 3 per group). Scale bars: 25 μm. HydroMg (1μg/mL) significantly reduced doxorubicin-induced apoptosis. All results are presented as mean ± SEM. *P < 0.05, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). BF: Bright field; Ctrl: control; DAPI: 4’,6-diamidino-2-phenylindole; DOX: doxorubicin; TUNEL: terminal…

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  Figure 3. HydroMg alleviates DOX-induced mitochondrial dysfunction and ROS overproduction in cardiomyocytes. (A) Mitochondrial network structures in H9C2 cells were analyzed using MitoTracker Deep Red staining and fluorescence microscopy (n ≥ 3 per group). Doxorubicin increased mitochondrial fragmentation in H9C2 cells, which was reduced by HydroMg co-treatment. Scale bar: 8 μm. (B) The average mitochondrial length of mitochondria was analyzed in H9C2 cells from Ctrl, DOX, and DOX + HydroMg groups. (C) Mitochondrial membrane potential in live H9C2 cells was assessed using TMRE staining (n ≥ 3 per group). HydroMg attenuated doxorubicin-induced mitochondrial depolarization. Scale bars: 20 μm. (D) TMRE fluorescence intensity was quantified. (E) Mitochondrial ROS production in H9C2 cells was detected using MitoSOX Red staining (n ≥ 3 per group). HydroMg reduced doxorubicin-induced mitochondrial ROS elevation. Scale bars: 75 μm. (F) MitoSOX fluorescence intensity was quantified. (G) DCFH-DA staining showing ROS production in H9C2 cells (n ≥ 3 per group). HydroMg reduced doxorubicin-induced total ROS elevation. Scale bar: 25 μm. (H) Quantification of DCFH-DA fluorescence intensity. All results…

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  Figure 4. HydroMg ameliorates DOX-induced cardiac contractile dysfunction. (A) Experimental schematic. DOX (15 mg/kg) was administered intravenously on day 0 in 6–8-week-old male C57BL/6 mice, preceded by HydroMg pretreatment via intraperitoneal injection 4–5 hours prior. Cardiac functional parameters were serially monitored until day 6 post-treatment. (B) The survival rate (n ≥ 3 per group) using the Kaplan–Meier method. (C) Cardiac function was evaluated by echocardiography. HydroMg (100 mg/kg) improved cardiac function, increasing LVEF by 12.44% and FS by 9.66% versus doxorubicin alone. (D) EF. (E) FS. (F) Representative images of wheat germ agglutinin staining in cardiac tissue sections (n ≥ 3 per group). HydroMg attenuated doxorubicin-induced cardiomyocyte atrophy (WGA staining). Scale bar: 10 μm. (G) Quantification of cardiomyocyte cross-sectional areas. All results are expressed as mean ± SEM. Statistical analysis was performed using one-way analysis of variance followed by Tukey’s post hoc test for multiple comparisons among groups. *P < 0.05, **P < 0.01, ****P < 0.0001. Ctrl: Control; DOX: doxorubicin; EF: ejection fraction; FS: fractional shortening; i.p.: intraperitoneal…

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  Figure 5. HydroMg attenuates cardiomyocyte apoptosis in mice with doxorubicin-induced myocardial injury. (A) TUNEL staining of cardiac tissue sections (n ≥ 3 per group). HydroMg attenuated doxorubicin-induced cardiomyocyte apoptosis. Scale bars: 10 μm. (B) Quantification of TUNEL-positive cardiomyocytes. (C) Western blot analysis of caspase-3 and Bcl2 protein expression levels in mice cardiac tissues. (D, E) Statistical analysis of relative protein expression levels for caspase-3 (D), and Bcl2 (E) (n = 3 per group). All results are expressed as mean ± SEM. Statistical significance was determined by one-way analysis of variance with Tukey’s post hoc test for multiple comparisons. **P < 0.01, ***P < 0.001, ****P < 0.0001. Ctrl: Control; DAPI: 4’,6-diamidino-2-phenylindole; DOX: doxorubicin; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

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  Figure 6. HydroMg mitigates doxorubicin-induced mitochondrial dysfunction and attenuates ferroptosis. (A, B) Western blot analysis of TOM20 protein expression levels in mouse cardiac tissues (A) and statistical analysis (B). (C, D) Western blot analysis of protein expression levels for CI, CII, CIII, CIV, and CV in mouse cardiac tissues (C) and statistical analysis (D). (E, F) Western blot analysis of SLC7A11 protein expression levels in mouse cardiac tissues (E) and statistical analysis (F). (G, H) Western blot analysis of GPX4 protein expression levels in mouse cardiac tissues (G) and statistical analysis (H). All results are presented as mean ± SEM (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). CI: Complex I; CII: Complex II; CIII: Complex III; CIV: Complex IV; Ctrl: control; CV: Complex V; DOX: doxorubicin; GPX4: glutathione peroxidase 4; SLC7A11: solute carrier family 7 member 11; TOM20: translocase of the outer mitochondrial membrane 20.