Phosphocreatine Improves Cardiac Dysfunction by Normalizing Mitochondrial Respiratory Function through JAK2/STAT3 Signaling Pathway In Vivo and In Vitro

Figures

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  Figure 1. Mitochondrial respiration in heart tissue homogenate and isolated heart mitochondria of rats. (a) Healthy heart rat tissue homogenate and the mitochondrial respiration were detected using Oxygraph-2k. Red lines indicate the oxygen consumption rate in response to sequential loading of mitochondrial effectors (indicated by arrows above the graphs). (b) STZ group. (c) The treatment group with PCr (50 mM). (d) Quantitative examinations of oxygen utilization rate in light of effectors. Correlations were performed utilizing data lab programming. (e) Isolated heart mitochondria from healthy rats. (f) STZ group. (g) The treatment group with PCr (50 mM). (h) Quantitative examinations of oxygen utilization rate in light of effectors. The following are added: P: pyruvate (5 mM); G: glutamate (10 mM); M: malate (2 mM); ADP: adenosine diphosphate (5 mM); Cyt C: cytochrome c (10 μM); S: succinate (10 mM); FCCP (0.5 μM steps); rotenone (0.5 μM); antimycin A (2.5 μM). Data are displayed as the mean ± SE (n = 3 per group), and comparisons were performed using DatLab software.

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  Figure 2. PCr decreased pathological changes in diabetic hearts. (a) Effects of PCr on heart size were reduced significantly in treated groups than the diabetic group. (b) Hematoxylin and eosin (H&E) staining of the heart tissue showed that PCr improved the striation of myocardial fibers arranged regularly in treated groups. (c) Effects of PCr on blood glucose levels were reduced remarkably in the treated groups than the STZ group. (d) Effects of PCr on myocardial and antioxidative enzyme activities: MDA level was decreased significantly in the PCr group than in the diabetic group; (e, f) SOD and GSH were increased in treated groups compared with diabetic groups indicating the antioxidant capacity agent was endangered in the diabetic myocardium. (g, h, i) Western blot investigation of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Data are introduced as the mean ± SD (n = 3). ###P < 0.01vs. control; ∗∗P < 0.01 and ∗∗∗P < 0.01vs. the STZ group.

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  Figure 3. PCr modulation on the JAK2/STAT3 signaling pathway. (a) Effects of PCr on p-STAT3 translocation in ordinary and STZ conditions (original magnification 200). (b, c, d) Effects of PCr on the declaration of p-JAK2, JAK2, p-STAT3, and STAT3 in STZ and treatment as well as healthy rats (n = 3). Qualities are expressed as the mean ± SEM. ##P < 0.001 and ###P < 0.005vs. the control group. ∗∗P < 0.01 and ∗∗∗P < 0.001vs. the STZ group.

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  Figure 4. Effects of PCr on STZ-instigated apoptosis in DCM. (a) The TUNEL and DAPI recoloring on the myocardium. The nuclei of TUNEL-positive cells are shown utilizing green fluorescence. (b) Quantification of TUNEL-positive cells. (c) The protein dimensions of Bcl-2, Bax, caspase 3, caspase 9, cleaved caspase 3, cleaved caspase 9, and cytochrome c (mitochondria) were recognized by western blot in tissue homogenate. (d, e, f, g, h, i) Quantifications of western blot. Data are exhibited as the mean ± SD (n = 3). ##P < 0.05 and ###P < 0.01vs. the control group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.01vs. the STZ group.

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  Figure 5. Protective impacts of PCr against MGO-actuated damage and apoptosis in H9C2 cells. (a) Chemical structure of phosphocreatine. (b) Effect of pretreatment with PCr (5~20 mM) for 2 h, on the cell morphology and structure of H9C2 cells by bright image (100x magnification) examination. (c) The apoptosis occurrences of H9C2 were stained by DAPI observed by fluorescence images for (200x final magnification). (d) MGO-induced toxicity (0.2~1.2 mM) on H9C2 cell. (e) Effect of PCr (5~20 mM) on induced MGO H9C2. (f) Cytotoxicity of PCr on H9C2 cells. The effects of PCr on the loss of cell practicality, initiated by MGO. Data are displayed as the mean ± SD (n = 3). ###P < 0.05vs. the control group. ∗∗P < 0.05 and ∗∗∗P < 0.01vs. the MGO group.

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  Figure 6. PCr inhibits MGO-induced apoptosis by modulating Bax, Bcl-2, caspase 3, caspase 9, cleaved caspase 3, cleaved caspase 9, and cytochrome c (mitochondria) in H9C2 cells. (a) Cells were treated with PCr (5~20 mM) for 2 h preceding being presented to 1 mM MGO for 24 h. Cell apoptosis was estimated by flow cytometry. (b) Apoptotic cells represent the percentage of Annexin V single positive and Annexin V/PI twofold positive cells. (c) The protein dimensions of Bcl-2, Bax, caspase 3, caspase 9, cleaved caspase 3, cleaved caspase 9, and cytochrome c (mitochondria) were recognized by western blot in cells. (d, e, f, g, h, i, j, k) Quantifications of western blot. Data are exhibited as the mean ± SD (n = 3). ###P < 0.05, ##P < 0.01, and #P < 0.05vs. the control group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.05vs. the MGO group.

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  Figure 7. PCr enhances mitochondrial respiration. (a) Effects of PCr on mitochondrial respiration in intact H9C2 cells. Agent respiratory explore different avenues regarding control H9C2 cells. (b) MGO-incited H9C2 cells. (c) PCr (20 mM) pretreated with MGO (1 mM) invigorated cells. (d) Quantification for all groups in intact H9C2 cells. The respirometry convention for intact cells included the ROUTINE state which was estimated when a steady oxygen transition had been gotten following expansion of cells into the test chamber. Uncoupled respiration in the LEAK state was estimated when a consistent oxygen motion had been accomplished after the expansion of oligomycin (2.5 μM), FCCP (0.5 μM steps), rotenone (0.5 μM), and antimycin A (2.5 μM). (e) Effect of PCr on permeabilized H9C2 cells in control. (f) MGO-induced H9C2 cells. (g) PCr (20 mM) pretreated with MGO-stimulated cells. (h) Quantification for all the groups in permeabilized H9C2 cells. The following are added: digitonin (8.1 μM, 10 μg/106 cells); P: pyruvate (5 mM); G: glutamate (10 mM); M: malate (2 mM); ADP (5 mM); S: succinate (10 mM); FCCP (0.5 μM steps); rotenone (0.5 μM); antimycin A (2.5 μM). Data are presented as the mean ±…

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  Figure 8. Inhibitory impacts of PCr on MGO-actuated H9C2 ROS overproduction. (a) Cells were treated with convergences of (5~20 mM) PCr or 2 mM NAC for 2 h before being animated with (1 mM) MGO for 24 h. (b) Quantification ROS age. (c) Protective impacts of PCr against MGO-initiated mitochondrial brokenness in H9C2 cells. The impact of PCr on mitochondrial membrane potential. The Δψm in each group was computed as the proportion of red to green fluorescence. (d) Data are presented as the mean ± SD (n = 3). ###P < 0.05vs. the control group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.01vs. the MGO group.

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  Figure 9. Effects of PCr on the expression level of pSTAT signaling pathway. (a) Immunofluorescence recoloring examination of pSTAT confinement. The H9C2 cells were named with pSTAT (red), and the nuclei were recolored with DAPI (blue). (b, c, d) Western blot examination of pSTAT in the cytoplasm and nucleus. Data are introduced as the mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01 significantly different from the control group.

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  Figure 10. Schematic representation influence of PCr against DCM on Bcl-2 family members and other mitochondrial apoptotic and metabolic proteins in cardiomyocytes under various pathological conditions.