CTRP5-Overexpression Attenuated Ischemia-Reperfusion Associated Heart Injuries and Improved Infarction Induced Heart Failure

Figures

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  FIGURE 1. C1q/TNF-related protein 5 (CTRP5) expression in mouse heart and eonatal rat cardiomyocytes (NRCMSs) under pathophysiological condition (A) plasma CTRP5 level after myocardial infarction (MI) or sham surgery at different time points, which was quantified by Elisa analysis (N = 7–10); (B) mRNA expression of CTRP5 in mouse heart after MI or sham surgery at different time points (N = 6); (C,D) protein expression of CTRP5 in mouse heart after MI or sham surgery different time points (N = 6); (E,F) protein expression of CTRP5 in NRCMSs after hypoxia for 2, 4 or 6 h firstly and then reoxygenation for 6 and 12 h. Relative protein level of CTRP5 was calculated by ImageJ. Data are shown as mean ± SEM. *p < 0.05 represented the difference between the two groups connected by line was statistically significant.

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  FIGURE 2. Adeno-associated virus serum type 9 (AAV9) mediated CTRP5 overexpression attenuated ischemia/reperfusion (I/R) associated injuries in mouse heart. (A) CTRP5 overexpression reduced I/R induced infarction size. C57B6/L mice were systemically administrated with an AAV vector to express GFP (AAV-GFP, 4.0 × 107 PFU) or human CTRP5 (AAV-CTRP5, 4.0 × 107 PFU) for 3 weeks and then were subjected to I/R surgery. Heart sections were stained with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) after I/R. (B) Quantitative analysis of infarct area (IA, white)/left ventricle area (IA/LV), area at risk (AAR, red)/LV and (IA+AAR)/LV ratios (N = 7–10). (C,D) Representative pictures of TUNEL staining and quantitative analysis for apoptotic nuclei at 24 h after I/R (N = 7–10). (E) Caspase 3 activity analysis (N = 6). (F–H) Blots and relative quantitative analysis of Bax and Bcl2 expression (N = 6, normalized to β-actin). Data are shown as mean ± SEM. *p < 0.05 represented the difference between the two groups connected by line was statistically significant.

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  FIGURE 3. Adeno-associated virus serum type 9 (AAV9) mediated CTRP5 overexpression improved MI-induced cardiac dysfunction and heart failure. (A,B) Representative pictures of PSR staining and quantitative analysis of infarction area showed that CTRP5 overexpression reduced infarction area after 21 days of MI surgery (N = 6). (C) Representative pictures of M-mode echocardiography for assessing cardiac function after at 21st day after MI surgery (n = 7–10). Echocardiographic analysis presented that CTRP5 overexpression decreased (D) LVIDd and (E) LVIDs and increased (F) LVEF and (G) FS. Data are shown as mean ± SEM. *p < 0.05 represented the difference between the two groups connected by line was statistically significant.

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  FIGURE 4. C1q/TNF-related protein 5 (CTRP5) overexpression enhanced AMPK signaling in mouse heart after I/R Mice were administrated with AAV-GFP or AAV-CTRP5 for 3 weeks and then were subjected to sham or I/R surgery. Hearts were harvested for determining the protein levels of (A) CTRP5, phosphorylated AMP-activated protein kinase alpha 2 (p-AMPKα2), AMPKα2, phosphorylated acetyl-CoA carboxylase (p-ACC), ACC, p-mTORC1, mTORC1, and PGC1α. Relative quantification of (B) CTRP3, (C) p-AMPKα2, (D) p-ACC, (E) p-mTORC1, and (F) PGC1α. The relative expression of CTRP3 and PGC1α (normalized to β-actin). The relative expression of p-AMPKα2, p-ACC, and p-mTORC1 was normalized to its corresponding total protein and β-actin respectively. Data are shown as mean ± SEM. *p < 0.05 represented the difference between the two groups connected by line was statistically significant.

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  FIGURE 5. C1q/TNF-related protein 5 (CTRP5) overexpression protected against NRCMs apoptosis Isolated NRCMs were transfected with Ad-GFP or Ad-CTRP5 for 24 h, followed by hypoxia for 24 h firstly and then reoxygenation for 12 h. (A) Western-blot was performed for determining protein expression of CTRP5, p-AMPKα2, AMPKα2, p-ACC, ACC, and β-actin. Relative quantification of (B) CTRP5, (C) p-AMPKα2, and (D) p-ACC. (E–G) Blots and relative quantification of the apoptosis associated proteins of BCL2 and BAX. (H) Caspase 3 activity analysis kit was used to determine cleaved caspase 3 activity. (I,J) Lactate dehydrogenase (LDH) Assay Kit was used to detect the LDH leakage, and compound C (Comp C, 10 mm) was used to inhibit AMPK activity. The relative expression of CTRP5, BCL2, and BAX were normalized to β-actin. The relative expression of p-AMPKα2 and p-ACC were normalized to its corresponding total protein and β-actin respectively. Data are shown as mean ± SEM. *p < 0.05 represented the difference between the two groups connected by line was statistically significant.

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  FIGURE 6. Adeno-associated virus serum type 9 (AAV9) mediated CTRP5 overexpression could not attenuate ischemia/reperfusion associated injuries in AMPKα2-knock out (AMPKα2-KO) mice. (A) AMPKα2-KO mice were administrated with an AAV vector to express GFP (AAV-GFP, 4.0 × 107 PFU) or human CTRP5 (AAV-CTRP5, 4.0 × 107 PFU) for 3 weeks and then were subjected to I/R surgery. (A) Western-blot was performed for determining protein expression levels of CTRP5, p-AMPKα2, AMPKα2, p-ACC, ACC, and β-actin. Relative quantification of (B) CTRP5, (C) p-AMPKα2, and (D) p-ACC in AMPKα2-KO mouse heart tissue. (E) Heart sections were stained with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) after I/R and the results indicated that CTRP5 overexpression could not attenuate I/R associated mouse heart injury. (F) Quantitative analysis of the infarct area (IA, white)/left ventricle area (IA/LV), area at risk (AAR, red)/LV (AAR/LV) and (IA+AAR)/LV (N = 10). (G,H) Representative pictures of TUNEL staining and quantitative analysis for apoptotic nuclei (N = 10). The relative expression of CTRP5 was normalized to β-actin. The relative expression of p-AMPKα2 and p-ACC were normalized to its corresponding…

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  FIGURE 7. Adeno-associated virus serum type 9 (AAV9) mediated CTRP5 overexpression could not attenuate MI-induced cardiac dysfunction in AMPKα2-KO mice. (A) Representative pictures of M-mode echocardiography for assessing cardiac function at the 21st day after MI surgery (n = 7–10). Echocardiographic analysis presented that CTRP5 overexpression showed no protective effects on cardiac function, as evidenced by (B) LVEDs, (C) LVIDd, (D) EF, and (E) FS values. Data are shown as mean ± SEM. NS indicated no significant difference *p < 0.05 represented the difference between the two groups connected by line was statistically significant.