CCK1R2R^-/- ameliorates myocardial damage caused by unpredictable stress via altering fatty acid metabolism

Figures & data

Figure 1. Unpredictable stress-induced heart dysfunction.

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(A) The distance of mice movement in the open field test after stress. (B) Representative electrocardiogram of mice after stress. (C&D) LVSP, LVEDP, +dp/dtmax and -dp/dtmax changes under stress in mice. (E) ELISA was used to measure the levels of EPI and NA in mice serum. (F) The cTnI, CK-MB and LDH content in mice serum was detected by the relevant assay kit. Data is displayed as mean ± SD (n = 6). *p < 0.05, **p < 0.01. LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; +dp/dtmax, maximal left ventricular pressure rising rate; -dp/dtmax, maximum left ventricular pressure drop rate; PCR, polymerase chain reaction; CCK1R, Cholecystokinin A Receptor; ELISA, enzyme-linked immune sorbent assay; EPI, epinephrine; NA, noradrenaline; cTnI, cardiac troponin-I; CK-MB, creatine kinase-MB; LDH, lactate dehydrogenase; TNF-α, Tumor Necrosis Factor-Alpha; IL-6, Interleukin-6; TUNEL, TdT-mediated dUTP nick end labeling; DAPI, 4',6-diamidino-2-phenylindole; SD, Standard deviation.

Figure 2. Unpredictable stress causes inflammation and apoptosis.

(A) The levels of TNF-α, IL-6 and IL-10 in the left ventricle of mice were measured by ELISA. (B) Representative confocal images of TUNEL (red) and DAPI (blue) in heart samples from WT and WT + ST mice (scale bar, 50 µm). Results are displayed as mean ± SD (n = 6). **p < 0.01. TNF-α, Tumor Necrosis Factor-Alpha; IL-6, Interleukin-6; ELISA, enzyme-linked immune sorbent assay; TUNEL, TdT-mediated dUTP nick end labeling; DAPI, 4',6-diamidino-2-phenylindole; WT, wild type; WT + ST mice, wild type mice stimulated by unpredictable stress; SD, Standard deviation.

Figure 3. CCK and CCKR were upregulated after unpredictable stress.

(A) Real-time PCR and western blot were used to detect the expression of CCK1R and CCK2R. (B) The CCK content in left ventricle of WT or WT + ST mice. Results are showed as mean ± SD (n = 6). **p < 0.01. CCK, cholecystokinin; CCKR, cholecystokinin receptor; PCR, polymerase chain reaction; CCK1R, cholecystokinin A receptor; CCK2R, cholecystokinin B receptor; WT, wild type; WT + ST mice, wild type mice stimulated by unpredictable stress; SD, Standard deviation.

Figure 4. Cardiac function and myocardial injury were ameliorated in CCK1R2R^-/- mice.

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(A) The distance of mice movement in the open field test. (B) Representative electrocardiogram of mice. (C) LVSP and LVEDP changes under stress in WT or CCK1R2R-/- mice. (D) +dp/dtmax and -dp/dtmax changes after stress in mice. (E) Corresponding assay kit was used to determine the cTnI, CK-MB and LDH content. (F) Immunofluorescence staining of TUNEL-positive cells in WT, WT + ST, CCK1R2R-/- and CCK1R2R-/-+ST (scale bar, 50 µm). Data is shown as mean ± SD (n = 6). *p < 0.05, **p < 0.01. CCK1R2R-/-, cholecystokinin A receptor and cholecystokinin B receptor knockout; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end diastolic pressure; WT, wild type; +dp/dtmax, maximal left ventricular pressure rising rate; -dp/dtmax, maximum left ventricular pressure drop rate; cTnI, cardiac troponin-I; CK-MB, creatine kinase-MB; LDH, lactate dehydrogenase; TUNEL, TdT-mediated dUTP nick end labeling; WT + ST mice, wild type mice stimulated by unpredictable stress; CCK1R2R-/-+ST mice, CCK1R2R-/- mice stimulated by unpredictable stress; SD, Standard deviation.

Figure 5. Oxidative stress and inflammation were decreased in CCK1R2R^-/- mice.

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(A) The ROS production of mice cardiac muscle tissues was detected by a ROS assay kit (scale bar, 50 µm). (B) Western blot analysis was used to measure the expression of Nrf2, HO-1, Nrf2 (nucleus) and iNOS in the mice cardiac muscle tissues. (C) The mRNA expression of TNF-α, IL-6, and IL-10 was analyzed by real-time PCR. (D) ELISA was used to determine the levels of TNF-α, IL-6, and IL-10 in the mice cardiac muscle tissues. Results are presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01. CCK1R2R-/-, cholecystokinin A receptor and cholecystokinin B receptor knockout; ROS, reactive oxygen species; Nrf2, nuclear factor-erythroid 2-related factor 2; HO-1, Heme Oxygenase 1; iNOS, Inducible Nitric Oxide Synthase; TNF-α, Tumor Necrosis Factor-Alpha; IL-6, Interleukin-6; PCR, polymerase chain reaction; ELISA, enzyme linked immune sorbent assay; SD, Standard deviation.

Figure 6. Overall identification of differential metabolites.

(A) A heat map was used to show the overall metabolic profile differences (red, up-regulated; blue, down-regulated). Each row was a metabolite, and each column was a sample. (B) The number of common and unique differential metabolites in different comparison groups was counted by Venn diagram. (C) Statistics of different expressed metabolites. The red column represented upregulation, and the blue column represented downregulation.

Figure 7. Analysis of differential metabolites between different groups.

(A) Heatmap of differentially expressed metabolites in the left ventricular tissue in WT + ST mice compared to the WT mice, in CCK1R2R-/- mice compared to the WT mice, in CCK1R2R-/-+ST mice compared to the CCK1R2R-/- mice, in CCK1R2R-/-+ST mice compared to the WT + ST mice (red, up-regulated; blue, down-regulated). Each row was a metabolite, and each column was a sample. (B) The difference between groups was shown in volcano plot. WT + ST mice, wild type mice stimulated by unpredictable stress; WT, wild type; CCK1R2R-/-, cholecystokinin A receptor and cholecystokinin B receptor knockout; CCK1R2R-/-+ST mice, CCK1R2R-/- mice stimulated by unpredictable stress.

Figure 8. The relative amounts of metabolites (WT vs WT + ST; WT vs CCK1R2R^-/-) is shown in Z-score plot.

WT, wild type; WT + ST mice, wild type mice stimulated by unpredictable stress; CCK1R2R-/-, cholecystokinin A receptor and cholecystokinin B receptor knockout.

Figure 9. The relative amounts of metabolites (CCK1R2R^-/- vs CCK1R2R^-/-+ST; WT + ST vs CCK1R2R^-/-+ST) is shown in Z-score plot.

CCK1R2R-/-, cholecystokinin A receptor and cholecystokinin B receptor knockout; CCK1R2R-/-+ST mice, CCK1R2R-/- mice stimulated by unpredictable stress; WT + ST mice, wild type mice stimulated by unpredictable stress.

Figure 10. Differential metabolite pathway analysis (WT vs WT + ST; WT vs CCK1R2R^-/-).

(A) Histogram. (B) Network planning. WT, wild type; WT + ST mice, wild type mice stimulated by unpredictable stress; CCK1R2R-/-, cholecystokinin A receptor and cholecystokinin B receptor knockout.

Figure 11. Differential metabolite pathway analysis (CCK1R2R^-/- vs CCK1R2R^-/-+ST; WT + ST vs CCK1R2R^-/-+ST).

(A) Histogram. (B) Network planning. CCK1R2R-/-, cholecystokinin A receptor and cholecystokinin B receptor knockout; CCK1R2R-/-+ST mice, CCK1R2R-/- mice stimulated by unpredictable stress; WT + ST mice, wild type mice stimulated by unpredictable stress.