Cardiovascular Protective Effects of NP-6A4, a Drug with the FDA Designation for Pediatric Cardiomyopathy, in Female Rats with Obesity and Pre-Diabetes

Figures

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  Figure 1. Obese female ZDF (ZDF-F) rats have reduced HDL but increased alkaline phosphatase in plasma and increased NT-pro-BNP (the heart failure marker) and microvascular damage in myocardium compared to healthy female ZL (ZL-F) rats. Five-month-old ZDF-F rats (n = 5) exhibit a reduction in plasma HDL (A) and an increase in alkaline phosphatase (B) and cardiac pro-BNP protein levels (C) compared to ZL-F rats (n = 6) * p < 0.05. (D,E) Representative images of cardiac sections of ZL-F and ZDF-F rats showing NT-pro-BNP expression using horseradish peroxidase staining. Red arrows mark brown clusters corresponding to NT-pro-BNP staining. (F,G) Representative images from Masson’s Trichrome staining of cardiac-tissue sections from ZDF-F rats. All ZDF-F rats had one or more vessels in the heart with pink-stained surrounding regions indicative of a vascular leak and edema and cell debris (blood vessel marked by yellow arrow) (G). None of the ZL-F rat hearts showed such damage (F). Scale bar: 100 µm. (H) shows the quantification of the damaged microvascular area as a percentage of the total whole heart section area for ZL-F and ZDF-F rats using Apeiro ImageScope annotation tools. **** p < 0.0001.

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  Figure 2. Effects of NP-6A4 and co-treatment with PD123319 on cardiac microvascular density and blood vessel damage in ZDF-F rats. Representative images of the heart sections of ZDF-F rats treated with saline and NP-6A4 and stained with endothelial marker Isolectin B4 (IB4) or Masson’s Trichrome are shown. NP-6A4-treated heart sections exhibit a doubling of IB4 signal indicating microvascular density increased by 200% in response to NP-6A4 compared to saline treatment (A,B). Co-treatment with PD123319 suppressed this effect (A,B) Scale bar: 50 µm. Trichrome-stained heart sections of ZDF-F rats treated with saline exhibit one or more regions of vascular damage indicated by pink-stained areas and marked by yellow arrows caused by cardiomyocyte loss and/or edema (C). This type of damage was not detected in five out of six ZDF-F rats treated with NP-6A4; however, ZDF-F rats treated with NP-6A4 + PD123319 showed similar vascular damage surrounding at least one blood vessel (C) Scale bar: 100 µm. (D) shows the quantification of damaged microvascular area as a percentage of the total whole heart slice area for ZDF-F rats treated with saline, or NP-6A4 or NP-6A4 + PD123319 (NP + PD) using Apeiro…

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  Figure 3. Effects of NP-6A4 and co-treatment with PD123319 on cardiomyocyte hypertrophy, cardiac fibrosis, and AT2R protein expression in ZDF-F rats. Representative images of heart sections of ZDF-F rats treated with wheat germ agglutinin (WGA) staining and picrosirius staining are shown. WGA staining (A,B) show that rats treated with NP-6A4 have a 61.7% reduction in cardiomyocyte surface area compared to rats treated with saline or NP-6A4 + PD123319 (NP+PD). Scale bar: 50 µm. Quantification of picrosirius red (PSR) staining of collagen in the heart sections of ZDF-F rats showed that there was no difference in the PSR staining between saline and NP-6A4 treated rats, but NP6A4 + PD123319 (NP+PD) treatment that inhibited all AT2R signaling increased PSR positivity by 27% (C,D). Scale bar: 100 µm. Immunoblotting of heart tissue lysates from ZDF-F rats with anti-AT2R antibody (E) showed that AT2R expression was increased by 240% in NP-6A4- compared to saline- or NP-6A4 + PD123319-treated rats (F). ** p < 0.01, *** p < 0.001.

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  Figure 4. IPA-predicted activation of the canonical pathway Superpathway of Inositol Compounds in the heart tissues of ZDF-F ats treated with NP-6A4IPA-predicted Z score: 2.234. The orange color indicates the positive Z score and activation of the pathway.

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  Figure 5. Effect of treatment with NP-6A4 or co-treatment with PD123319 on the cardiac autophagy status of ZDF-F rats. (A) New autophagy signaling pathway consisting of 8 proteins activated in response to NP-6A4 in ZDF-F rat heart as predicted by Ingenuity Pathway Analysis of the differentially expressed proteins between saline and NP-6A4 treated rat hearts (orange color indicates activation: see Prediction legend) in the Diseases and Functions category. The fold change of each protein in NP-6A4-treated rat hearts compared to those treated with saline (top number below each protein) and p value (bottom number below each protein) are shown. Z score = 1.664; p value = 0.005. (B) Representative images of immunoblotting analysis of cardiac lysates from ZDF-F rats treated with saline, NP-6A4, or NP-6A4 + PD123319 and probed with anti-LC3, anti-Rubicon, anti-p62, and anti-Actin antibodies are shown. An increase in LC3-I and LC3-II levels and a reduction in Rubicon and p62 levels is seen in ZDF-F rat heart lysates treated with NP-6A4 compared to those treated with saline or NP-6A4 + PD123319. Graphs show quantification of LC3-II (C), Rubicon (D), and p62 (E) in ZDF-F rat heart lysates after…

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  Figure 6. IPA-predicted activation of Reelin Signaling in the heart tissues of ZDF-F rats co-treated with AT2R antagonist PD123319. IPA-predicted Z score: 2.0. Orange color indicates activation of the pathway.

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  Figure 7. Effects of co-treatment with AT2R antagonist PD123319 on cardiac signaling of young ZDF-F rats. IPA analysis of differentially expressed proteins between NP-6A4 and NP-6A4+ PD123319-treated rat hearts 6 predicted that co-treatment with PD123319 resulted in activating a signaling network that (A) increases cell death of muscle cells (orange indicates activation: Z score: 1.943; p value = 0.0015) and another signaling network that (B) suppresses ATP concentration (blue indicates suppression: Z score: −1.982; p value =0.008) in the Diseases and Functions Category. Fold changes of each protein (top number below each protein) in heart lysates of ZDF rats treated with NP-6A4 + PD123319 versus those treated with NP-6A4 alone and corresponding p values (bottom number below each protein) are shown in (A,B).