THR-123, a novel BMP-7 mimetic that activates Akt phosphorylation and inhibits cardiomyocyte apoptosis and inflammation, protects the heart from myocardial injury in a Rat model

Figures

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  Figure 1. Animal study design, compound treatment and blood collection schedule in the animal model of myocardial injury following ischemia (LAD model).

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  Figure 2. Cross sectional slice of heart. Unaffected, perfused region stained Evans blue dye prior to sacrifice.

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  Figure 3. Preventing and repairing myocardial ischemic injury by THR-123.

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  Figure 4. THR-123 treated animals showed marked decline in myocardial infarction size compared to PBS treated animals. Animals underwent necropsy on post-surgical day 7. Each occlusion was confirmed by ECG analysis during the surgery and ultimately after Evans blue injection on Day 7 at necropsy. All but one rat demonstrated successful occlusion by Evans Blue at necropsy. Animals PBS-treated, BMP-7 treated or THR-123 treated were evaluated at necropsy. Areas of infarct were clearly identified in all heart samples and morphometry was performed on all the harvested slices. Treatment-related differences in gross pathology and morphometry were observed. Results are expressed as Ratio of Area of Infarct (AI) to Area of Risk (AR) (Mean ± SD) see Table 1. *Vehicle (PBS) vs. BMP mimetic (THR-123), P = 0.008. △ Vehicle (PBS) vs. BMP-7, P = 0.748. Cohen's d values & interpretation for the group comparisons are shown in Table 2.

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  Figure 5. THR-123 (BMP mimetic) reduced the left ventricular area weighted ratio AN/AR [the area occupied by necrosis (AN) to area at risk (AR)] by 84% compared to vehicle (PBS), in the LAD occlusion model of myocardial infarction. Animals PBS-treated, BMP-7 treated or BMP mimetic (THR-123) -treated were evaluated at necropsy. Areas of infarct were clearly identified in all heart samples and morphometry was performed on all the harvested slices. Results are expressed as Ratio of Area of Necrosis (AN) to Area of Risk (AR) (Mean ± SD). *Vehicle (PBS) vs. BMP mimetic (THR-123), P = 0.006. △ Vehicle (PBS) vs. BMP-7, P = 0.48. Cohen’s d values & interpretation for the group comparisons are shown in Table 2.

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  Figure 6. THR-123 (BMP mimetic) reduced VN/VR ratio [volume of necrosis (VN) to volume at risk (VR)] in the LAD occlusion model of myocardial infarction. Animals PBS-treated, BMP-7 treated or BMP mimetic (THR-123) -treated were evaluated at necropsy. Areas of Infarct were clearly identified in all heart samples and morphometry was performed on all the harvested slices. Results are expressed as ratio of Volume of Necrosis (VN) to Volume at Risk (VR) (Mean ± SD). *Vehicle (PBS) vs. BMP mimetic (THR-123), P = 0.008. △ Vehicle (PBS) vs. BMP-7, P = 0.748. Cohen’s d values & interpretation for the group comparisons are shown in Table 2.

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  Figure 7. THR-123 (BMP mimetic) reduced the degree of cardiac inflammation in the LAD occlusion model of myocardial infarction. Animals PBS-treated, BMP-7 treated or BMP mimetic (THR-123) -treated were evaluated for inflammation. * Vehicle (PBS) vs. BMP mimetic (THR-123), P = 0.0029. △ Vehicle (PBS) vs. BMP-7, P = 0.0114. Cohen’s d values & interpretation for the group comparisons are shown in Table 2.

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  Figure 8. THR-123 (BMP mimetic) significantly increased cellular Akt phosphorylation compared to doxorubicin (Adriamycin) alone. Cell lysates were prepared & analyzed for AKT phosphorylation by ELISA. *Doxorubicin alone vs. Doxorubicin + BMP mimetic (THR-123, 100 µM), P < 0.001. △ Doxorubicin alone vs. Doxorubicin + BMP mimetic (THR-123, 500 µM), P < 0.001.

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  Figure 9. THR-123 (BMP mimetic) effectively inhibited doxorubicin (Adriamycin) induced caspase-3 activity (apoptosis marker) in cardiomyocytes. Cell lysates were prepared & analyzed for Caspase-3 by ELISA. ★Cardiomyocytes were cultured in medium without serum vs. medium containing Doxorubicin, P = 0.0014. △ Doxorubicin vs. BMP-7, P= 0.001. ⋆ Doxorubicin vs. BMP mimetic (THR-123 100 µM), P=0.004. ▴ Doxorubicin vs. BMP mimetic (THR-123 500 µM), P= 0.001.

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  Figure 10. THR-123 (BMP mimetic) effectively inhibited LPS (lipopolysaccharide) induced inflammation (pro- inflammatory cytokine IL-6 release) in cardiomyocytes. Cardiomyocytes were treated with lipopoly-saccharide (LPS, 100 ng/mL) for 12 h, followed by treatment with BMP-7 (143 nM) or BMP mimetic (4 to 500 µM) for 24 h., as indicated in ‘Methods’. Culture supernatants were assayed for pro-inflammatory cytokine release (IL-6) using ELISA. ★ LPS vs. LPS + BMP-7, P < 0.001. △ LPS vs. LPS + BMP-7 mimetic (THR-123, 4 µM), P < 0.001. ⋆ LPS vs. LPS + BMP-7 mimetic (THR-123, 20 µM), P < 0.001. ▴LPS vs. LPS + BMP-7 mimetic (THR-123, 100 µM), P< 0.001. ◻ LPS vs. LPS + BMP-7 mimetic (THR-123, 500 µM), P < 0.001.

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  Figure 11. THR-123 (BMP mimetic) effectively inhibited LPS (lipopolysaccharide) induced caspase-3 activity (apoptosis marker) in cardiomyocytes. Cell lysates were prepared & analyzed for Caspase-3 by ELISA. ★ Medium alone vs. LPS, P < 0.001. △ LPS vs. LPS + BMP-7, P = 0.825. ⋆ LPS vs. LPS + BMP-7 mimetic (THR-123, 4 µM, P = 0.327. ▴ LPS vs. LPS + BMP-7 mimetic (THR-123, 20 µM, P < 0.001. ◻ ◻°LPS vs. LPS + BMP-7 mimetic (THR-123, 100 µM, P < 0.001. ◼ LPS vs. LPS + BMP-7 mimetic (THR-123, 500 µM, P < 0.001.

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  Figure 12. THR-123 significantly increased Akt Ser473 phosphorylation (p < 0.001) in cardiomyocytes subjected to ischemic injury. Cell lysates were prepared & analyzed for AKT phosphorylation by ELISA. ★ Serum-free medium vs. Medium + PBS, P = 0.157. △ Serum-free Medium vs. Medium + BMP mimetic (THR-123, 100 µM), P < 0.001.