Systemic Blockade of ACVR2B Ligands Protects Myocardium from Acute Ischemia-Reperfusion Injury

Figures

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  Figure 1. ACVR2B-Fc Reduces Ischemia-Reperfusion Injury and Restores Cardiac Function Mice were treated with vehicle (white columns) or with a soluble decoy receptor ACVR2B-Fc at reperfusion (ACVR2B-Fc at reperfusion, gray columns) or 24 h prior to/at reperfusion (ACVR2B-Fc pretreatment, black columns). IR was achieved by transient ligation of left anterior descending (LAD) coronary artery for 30 min, followed by reperfusion for 24 h. (A) ACVR2B-Fc reduced infarcted area as determined with triphenyltetrazolium chloride (TTC stain). n = 7, 8. (B) ACVR2B-Fc reduced cardiac troponin I release into plasma (p = 0.09). n = 8, 9. (C) As determined with echocardiography, ACVR2B-Fc preserved cardiac function after IR observed as preserved ejection fraction. ACVR2B-Fc also reduced left ventricular diameter in diastole (LVID;d) and increased LV posterior wall thickness (LVPW;d) in IR mice with slightly increased total LV mass. Sham values are shown as a dotted line. n = 19, 9, 18. Full echo data in Table S2. (D) ACVR2B-Fc increased cardiomyocyte cross-sectional area. Masson trichrome stained heart section cut horizontally (above) and representative pictures of single cross-sectional cardiomyocytes…

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  Figure 2. ACVR2B-Fc Reduces Activation of SMAD2, Apoptotic Stress-Induced Signaling Pathways, and Autophagy Mice were treated with vehicle (white columns), with soluble decoy receptor ACVR2B-Fc only at reperfusion (gray columns), or with pretreatment of ACVR2B-Fc 24 h prior to/at reperfusion (black columns). IR was achieved by transient ligation of left anterior descending (LAD) coronary artery for 30 min, followed by reperfusion for 6 h. (A) ACVR2B-Fc reduced apoptosis. n = 5, 6, 6. (B) ACVR2B-Fc reduced expression of Bcl-2 family pro-apoptotic protein Bim. n = 5, 6, 6. (C) ACVR2B-Fc inhibited IR-induced activation of SMAD2 protein in infarcted and peri-infarcted zone. ACVR2B-Fc also reduced IR-induced phosphorylation of JNK but had no effect on activation of p38, ERK1/2, or Akt. n = 5, 6, 6. (D) ACVR2B-Fc reduced autophagosomal lipidated LC3II form and decreased autophagosomal/cytoplasmic/LC3II/LC3I ratio in LV as analyzed 24 h after reperfusion. n = 7, 8, 8. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 3. ACVR2B-Fc Protects Cardiomyocytes in Hypoxic Conditions and Inhibits Myostatin-Activated SMAD2/3 Pathway in Cardiomyocytes (A) ACVR2B-Fc protects adult rat ventricular cardiomyocytes (ARVM) from hypoxia-induced cell death as measured with adenylate kinase release. n = 6. (B) ACVR2B ligand myostatin reduces cell survival in hypoxia. Cell toxicity was assessed with adenylate kinase release, and cell viability was determined with resazurin assay measuring cell metabolism. The hypoxia value is shown as a dotted line. n = 6. (C) Neonatal rat ventricular cardiomyocytes (NRVM) were transfected with CAGA-luc SMAD2/3 reporter, and subsequently luciferase reporter activity was analyzed after treatment with ACVR2B ligands, TGF-β, or the GFRAL receptor ligand GDF15. n = 12. (D) Hypoxia induced SMAD2/3-dependent promoter activity in NRVM while SMAD1/5/8 signaling was not activated. ACVR2B-Fc reduced hypoxia-induced SMAD2/3-dependent promoter activity in NRVM. n = 6. (E) Expression of ACVR2B ligands myostatin (Mstn), activin A (Inhba), and activin B (Inhbb) in infarct and peri-infarct zone in LV 6 and 24 h after IR, analyzed by qPCR. The sham value is shown as a dotted line. n = 5–6 (6 h IR);…

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  Figure 4. ACVR2B-Fc Optimizes Metabolism to Hypoxic Conditions in IR Expression of genes were analyzed with qPCR 24 h after IR from the peri-infarct zone. (A) ACVR2B-Fc upregulated expression of peroxisome proliferator-activated receptor gamma coactivator 1 isoforms PGC1α1 and PGC1α4 and did not affect the gene expression of oxidative phosphorylation enzyme cytochrome C (Cycs), but it increased expression of glycolytic phosphofructokinase enzyme Pfkm and insulin-regulated glucose uptake transporter Glut4. Vehicle-treated IR mouse values are shown as dotted line. n = 9, 8. (B) ACVR2B-Fc upregulated expression of MHC-α (Myh6) fast twitch myosin fibers and Cited4, a transcription factor involved in physiological hypertrophy. Vehicle-treated IR mouse values are shown as a dotted line. n = 9, 8. Data are presented as mean ± SD. *p < 0.05, **p < 0.01. (C) ACVR2B-Fc reduced metabolism in adult mouse cardiomyocytes after 4 h hypoxia followed by 1 h reperfusion as determined by oxygen consumption rate (OCR) with bioenergetic mito stress assay. (D) The values for basal and maximal respiration and spare respiratory capacity were calculated from OCR graphs. n = 13, 11 (normoxia); n = 6, 5 (hypoxia).…

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  Figure 5. ACVR2B-Fc Improves LV Function under Prolonged Cardiac Stress (A) Mice were treated with anthracycline doxorubicin for 2-week period, which induces cardiotoxicity. Mice were treated simultaneously with vehicle or ACVR2B-Fc for 2 weeks and LV tissue was analyzed with high-resolution respirometer. ACVR2B-Fc improved cardiac respiration in doxorubin-stressed hearts. Right panel represents respiration capacity for the whole heart. CI, complex I; CII, complex II; ETS, electron transfer system (maximal uncoupled respiration). n = 7. (B) ACVR2B-Fc also slightly improved citrate synthase activity. n = 7. (C) As analyzed with echocardiography, ACVR2B-Fc preserved cardiac systolic function in cumulative doxorubicin-induced toxicity at 4 weeks. n = 9, 6, 9. Full echo data in Table S4. (D) As analyzed with qPCR from LV at 4 weeks, ACVR2B-Fc reduced doxorubicin-induced upregulation of natriuretic peptides ANP (Nppa) and BNP (Nppb). n = 9, 7, 8. Data are presented as mean ± SD. *p < 0.05, **p < 0.01.