ZP2495 Protects against Myocardial Ischemia/Reperfusion Injury in Diabetic Mice through Improvement of Cardiac Metabolism and Mitochondrial Function: The Possible Involvement of AMPK‐FoxO3a Signal Pathway

Figure 1. Effects of ZP2495, glucagon, and ZP131 on cardiac function and hemodynamic and cardiac glucose metabolism in db/db mice after I/R injury. (a) Representative M-mode echocardiography images. (b) Measurement of left ventricular ejection fraction (LVEF). (c) Measurement of left ventricular fractional shortening (LVFS). (d, e) Measurement of maximal velocity of pressure development and decline (±dP/dt). (f) Representative PET/CT scan images from each group. Higher glucose uptake level is evidenced by an increase in the intensity of red color. (g) Quantification of accumulated 18F-fluorodeoxyglucose (18F-FDG) in the heart. Relative accumulation of the radioactivity in particular regions of interest was expressed as standardized uptake value (SUV). (h) Myocardial infarct size was assessed by Evans blue/2,3,5-triphenyl-2H-tetrazolium chloride (TTC) double staining. Evans blue stained areas (black) indicated nonischemic/reperfused area. TTC-stained areas (red staining) indicated ischemic but viable tissue. Evans blue/TTC staining negative areas indicated infarcted myocardium. (i) Summary of infarct area (INF) per area at risk (AAR). (j) Summary of AAR per left ventricle (LV). Sham: BKS +…

Figure 2. ZP2495 exerted antiapoptotic effect on cardiomyocyte of db/db mice. (a) Representative photomicrograph shows TUNEL-positive cardiomyocyte ratio, which was significantly decreased in the ZP2495 group. Apoptotic nuclei were identified as TUNEL positive (green). Myocardium was stained using a monoclonal antibody against troponin I (cTnI) (red) and total nuclei by DAPI counterstaining (blue) (scale bar = 100 μm). (b) Quantitative analysis of apoptotic nuclei. Apoptotic index was termed as the percentage of apoptotic cells. (c) Representative blots of caspase-3 and cleaved caspase-3 in cardiomyocyte of db/db mice subjected to I/R. (d) Semiquantitative analysis of the Western blots. Sham: BKS + sham group; db/db: db/db + sham group; I/R: db/db + I/R group; ZP2495: db/db + I/R + ZP2495 group; glucagon: db/db + I/R + glucagon group; and ZP131: db/db + I/R + ZP131 group. Presented values are mean ± SEM. ∗P < 0.05 versus the sham group; #P < 0.05 versus the db/db group; $P < 0.05 versus the db/db + I/R group; and £P < 0.05 versus the db/db + I/R + ZP2495 group.

Figure 3. ZP2495 exerted antiapoptotic effect on high-glucose-induced NRVMs after SI/R injury. (a) Apoptosis of the NRVMs determined by annexin V/propidium iodide (PI) double staining and flow cytometry. Region Q2 late apoptotic cells, region Q3 vital cells, and region Q4 early apoptotic cells. (b) Representative images of immunostaining for apoptotic (TUNEL) cells (scale bar = 100 μm). (c) Quantification of apoptosis cell by flow cytometry and TUNEL staining. (d) Quantification of apoptosis cell by TUNEL staining. (e) Activities of caspase-3. Con: normal glucose medium group; HG: high-glucose medium group; HG + SI/R: HG + SI/R group; ZP2495: HG + SI/R + ZP2495 group; glucagon: HG + SI/R + glucagon group; and ZP131: HG + SI/R + ZP131 group. Presented values are mean ± SEM. †P < 0.05 versus the control group; §P < 0.05 versus the HG group; ‡P < 0.05 versus the HG + SI/R group; and &P < 0.05 versus the HG + SI/R + ZP2495 group.

Figure 4. ZP2495 regulated Akt, FoxO3a, and apoptosis-related protein expression. (a) Representative blots of p-Akt, Akt, p-Bad, Bad, p-FoxO3a, FoxO3a, BimEL, MnSOD, catalase, Bax, and Bcl-2 in cardiomyocyte of db/db mice subjected to I/R. (b–g) Semiquantitative analysis of the Western blots. Sham: BKS + sham group; db/db: db/db + sham group; I/R: db/db + I/R group; ZP2495: db/db + I/R + ZP2495 group; glucagon: db/db + I/R + glucagon group; and ZP131: db/db + I/R + ZP131 group. Presented values are mean ± SEM. ∗P < 0.05 versus the sham group; #P < 0.05 versus the db/db group; $P < 0.05 versus the db/db + I/R group; and £P < 0.05 versus the db/db + I/R + ZP2495 group.

Figure 5. ZP2495 improved mitochondrial function and biogenesis in the myocardium of db/db mice subjected to I/R. (a) Representative transmission electron micrographs of mitochondria in the LV myocardium. Original magnification ×10,000 (scale bar = 1 μm) (A1) and ×40,000 (scale bar = 250 nm) (A2). (b–d) Respiration in freshly isolated heart mitochondria from each group mice was analyzed with a SeaHorse XF96 analyzer. Mitochondria were assayed under state 4, that is, substrate but no ADP (sometimes also referred to as state 2), and state 3, that is, substrate and ADP present respiration (VO2) with substrates from complex I (5 mmol/L G/M) (b), complex II (5 mmol/L Succ and 1 mmol/L rotenone) (c), and complex IV (0.5 mmol/L N,N,N9,N9-tetramethyl-p-phenylenediamine (TMPD) and 3 mmol/L sodium ascorbate) (d). State 3 was induced with 1 mmol/L ADP in all cases. (e) The RCR was determined as the ratio of state 3 to state 4. The bars plotted correspond to n = 8 replicates from two experiments. (f) Relative ATP level change in different treatments. Sham: BKS + sham group; db/db: db/db + sham group; I/R: db/db + I/R group; ZP2495: db/db + I/R + ZP2495 group; glucagon: db/db + I/R + glucagon group;…

Figure 6. ZP2495 prevented high-glucose and SI/R-induced mitochondrial depolarization and decreased cytochrome c release and reactive oxygen species (ROS) generation from mitochondria in NRVMs. (a) Mitochondrial membrane potential (MMP) was analyzed by JC-1 fluorescence. Upper panels of green fluorescence indicated cellular uptake of JC-1, while lower panels of red fluorescence suggested depict intact mitochondria. HG and SI/R-treated NRVMs showed significantly reduced red fluorescence as compared with the control group, whereas ZP2495 treatment prevented the breakdown of the MMP as indicated by preservation of the red JC-1 fluorescence (scale bar = 100 μm). (b) FACS analyses of three independent experiments with 10,000 cells per treatment condition reveal a decrease of the red JC-1 fluorescence to 41% of control levels after HG and SI/R treatment, which was prevented by ZP2495. (c) Mitotracker red fluorescence (red), cytochrome c immunoreactivity (green), DAPI staining (blue), and merged images (yellow indicates sites of colocalization) in NRVMs. Note the localization of cytochrome c in mitochondria in the NRVM control cells and ZP2495-treated cells and the diffused localization of…

Figure 7. ZP2495 increased mitochondrial biogenesis in hearts of db/db mice subjected to I/R. (a) Representative blots of p-AMPK, AMPK, p-FoxO3a, FoxO3a, Sirt1, Ace-lysine-PGC-1α, PGC-1α, Nrf-1, and Tfam in cardiomyocyte of db/db mice subjected to I/R. (b–e) Semiquantitative analysis of the Western blots. Sham: BKS + sham group; db/db: db/db + sham group; I/R: db/db + I/R group; ZP2495: db/db + I/R + ZP2495 group; glucagon: db/db + I/R + glucagon group; ZP131: db/db + I/R + ZP131 group. Presented values are mean ± SEM. ∗P < 0.05 versus the sham group; #P < 0.05 versus the db/db group; $P < 0.05 versus the db/db + I/R group; and £P < 0.05 versus the db/db + I/R + ZP2495 group. Data shown are representative of six independent experiments.

Figure 8. Proposed scheme for the protective effect of ZP2495 on cardiomyocyte. A summary of our current hypothesis about the mechanisms that the glucagon and GLP-1 dual-agonist (ZP2495) protects against myocardial I/R injury in db/db diabetic mice. The protective effects of ZP2495 may be associated with the activation of Akt/FoxO3 and AMPK/FoxO3a signaling pathways.