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. 2020 Apr 21;9(8):e015748.
doi: 10.1161/JAHA.119.015748. Epub 2020 Apr 16.

B7-33, a Functionally Selective Relaxin Receptor 1 Agonist, Attenuates Myocardial Infarction-Related Adverse Cardiac Remodeling in Mice

Teja Devarakonda  1 , Adolfo G Mauro  1 , Geronimo Guzman  1 , Sahak Hovsepian  1 , Chad Cain  1 , Anindita Das  1 , Praveen Praveen  2 , Mohammed Akhter Hossain  2 , Fadi N Salloum  1
Affiliations

B7-33, a Functionally Selective Relaxin Receptor 1 Agonist, Attenuates Myocardial Infarction-Related Adverse Cardiac Remodeling in Mice

Teja Devarakonda et al. J Am Heart Assoc. .

Abstract

Background Human relaxin-2 is a peptide hormone capable of pleiotropic effects in several organ systems. Its recombinant formulation (serelaxin) has been demonstrated to reduce infarct size and prevent excessive scar formation in animal models of cardiac ischemia-reperfusion injury. B7-33, a synthetically designed peptide analogous to B-chain of relaxin-2, invokes signaling at relaxin family peptide receptor 1 (cognate receptor for relaxin-2) by preferentially phosphorylating the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2. We sought to investigate the effects of B7-33 treatment post ischemia-reperfusion injury in mice. Methods and Results Adult male CD1 mice were subjected to ischemia-reperfusion via ligation of left anterior descending artery for 30 minutes, followed by 24 hours or 7 days of reperfusion. Echocardiography was performed to assess cardiac function, and cardiac tissue was stained to determine infarct size at 24 hours. B7-33 significantly reduced infarct size (21.99% versus 45.32%; P=0.02) and preserved fractional shortening (29% versus 23%; P=0.02) compared with vehicle. The difference in fractional shortening further increased at 7 days post myocardial infarction (29% versus 20% for B7-33 and vehicle groups, respectively). In vitro, primary cardiomyocytes were isolated from adult hearts and subjected to simulated ischemia-reperfusion injury (simulated ischemia reoxygenation). B7-33 (50 and 100 nmol/L) improved cell survival and reduced the expression of GRP78 (glucose regulated protein), an endoplasmic reticulum stress marker. Subsequently, B7-33 (100 nmol/L) reduced tunicamycin (2.5 μg/mL) induced upregulation of GRP78 in an extracellular signal-regulated kinase 1/2-dependent manner. Conclusions B7-33 confers acute cardioprotection and limits myocardial infarction-related adverse remodeling in mice by attenuating cardiomyocyte death and endoplasmic reticulum stress as well as preserving cardiac function.

Keywords: adverse cardiac remodeling; echocardiography; endoplasmic reticulum stress; myocardial infarction; relaxin.

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Figures

Figure 1
Figure 1. Schematic illustrating the experimental protocol for in vivo studies.
Left anterior descending coronary (LAD) artery in mice was occluded for 30 minutes, followed by either 24 hours or 7 days of reperfusion. Mice were treated at the onset of reperfusion with either B7‐33 or saline. In the longer‐term group of mice (7 days), mice received regular (twice a day) administration of B7‐33 or saline until time of euthanasia. qPCR indicates quantitative polymerase chain reaction; and TTC, triphenyltetrazolium chloride.
Figure 2
Figure 2. B7‐33 improves cardiac function 24 hours post MI.
A, Fractional shortening (FS) is preserved in mice treated with B7‐33 (0.25 mg/kg) 24 hours post myocardial infarction (29.57±1.74% vs 23.34±1.84%), compared to vehicle. B, Left ventricular internal diameter (LVID) is significantly elevated in vehicle‐treated mice (3.17±0.13 mm) vs B7‐33–treated mice (2.83±0.11 mm). C, Representative sketches of M‐mode acquisitions and subsequent measurements (bar=1 mm). D, No significant differences in LVID at diastole among the experimental groups (for A‐D, n=9 for sham, and n=14–18 for vehicle and B7‐33 groups). E, Infarct size, expressed as a percentage of risk area, is significantly lower in B7‐33 group (21.99±7.17%; n=7) vs vehicle group (45.32±5.28%; n=8) (bar=3.5 mm). For A, B, and D, 1‐way ANOVA was used to determine significance, and if P<0.05, Holm‐Sidak test was used for post hoc analysis. For E, unpaired T test was used. *P<0.05, ***P<0.001.
Figure 3
Figure 3. Analysis of long‐axis echocardiography views 24 hours post MI.
A, Representative tracings from experimental groups demonstrating radial velocity vs time, with individual colors representing the 6 long‐axis left ventricular segments to calculate radial strain dyssynchrony. B, Representative tracings from experimental groups depicting dV/dT as a function of time, with the local maxima corresponding to peak relaxation velocity, estimated via speckle tracking analysis. C, Preserved Max dV/dT in B7‐33–treated group (1.11±0.09 mL/s for B7‐33 vs 0.79±0.08 mL/s for vehicle) 24 hours post myocardial infarction (MI). D, Reduced left ventricular radial strain dyssynchrony in B7‐33–treated group 24 hours post MI (17.51±2.17% vs 28.77±4.13% for vehicle; P<0.05). Dyssynchrony was calculated from SD of left ventricular segmental radial strains, and expressed as a percentage of average radial strain. For C and D, n=9 for sham and n=15 to 21 for vehicle and B7‐33 groups. For C and D, 1‐way ANOVA was used to determine significance, and if P<0.05, Holm‐Sidak test was used for post hoc analysis. *P<0.05.
Figure 4
Figure 4. Real‐time polymerase chain reaction analysis at 24 hours, and assessment of cardiac function at 3 & 7 days post MI.
A through C, Treatment with B7‐33 significantly lowers Chop (CCAAT/enhancer‐binding protein‐homologous protein), Asc (apoptosis‐associated speck‐like protein containing a caspase recruitment domain), and toll‐like receptor 4 (Tlr4) mRNA in extracts from cardiac tissue, 24 hours post myocardial infarction (MI) (for A through C, n=9–10). D, Preserved fractional shortening (FS) 3 and 7 days post MI in B7‐33–treated group (30.73±2.20% and 28.29±1.63% vs 21.66±3.05% and 20.25±2.92% for B7‐33– and vehicle [VEH]‐treated group, respectively). E, Left ventricular internal diameter at systole (LVID‐S) at 3 and 7 days (2.66±0.18 and 2.72±0.2 mm vs 3.31±0.21 and 3.70±0.19 mm for B7‐33 and VEH, respectively). F, LVID at diastole (LVID‐D) for 3 and 7 days (3.90±0.15 and 3.96±0.22 mm vs 4.32±0.12 and 4.58±0.12 mm for B7‐33 and VEH groups, respectively) was significantly lower in B7‐33–treated groups (for D through F, n=4–7 per group). G, LV fibrosis is significantly lower in B7‐33–treated animals (n=7; 2.86% [2.52%‐5.71%]) than in VEH‐treated animals (n=7; 8.27% [7.27%‐9.95%]; bar=2.33 mm). H and I, Treatment with B7‐33 significantly lowers tissue inhibitor of metalloproteases (Timp) 1 and Timp2 mRNA expression in cardiac tissue 7 days post ischemia‐reperfusion (IR) surgery (n=4 for per group). Data expressed as relative normalized expression with respect to (RNE W.R.T) B‐actin. For A through C, H, and I, 1‐way ANOVA was used to determine significance, and if P<0.05, Holm‐Sidak test was used for post hoc analysis. For D through F, unpaired T test was used. For G, Mann‐Whitney U test was used to account for the nonnormal distribution. *P<0.05, **P<0.01.
Figure 5
Figure 5. Simulated ischemia reoxygenation (SIRO) of primary cardiomyocytes and fibroblasts.
A, Primary cardiomyocytes treated with either 50 or 100 nmol/L of B7‐33 in myocyte media following simulated ischemia have a lower trypan blue positive percentage of cells (control [51.33±4.53%] vs 50 nmol/L [34.72±5.20%] or 100 nmol/L [35.03±3.43%] of B7‐33) (n=4–6 experiments/group). Pretreating cardiomyocytes with extracellular signal‐regulated kinase (Erk) 1/2 inhibitor PD98059 followed by coincubation with B7‐33 abolishes the protective effects at both 50 nmol/L (64.28±9.73%) and 100 nmol/L (54.35±2.23%) concentrations of B7‐33 (bar=100 μm). B, Fibroblast viability is higher, compared with control, when treated with 10, 50, or 100 nmol/L of fibroblast media (n=3–5 experiments/group) post SIRO. For A and B, 1‐way ANOVA was used to determine significance, and if P<0.05, Holm‐Sidak test was used for post hoc analysis. *P<0.05, **P<0.01.
Figure 6
Figure 6. Western blot analysis of primary cardiomyocytes treated with B7‐33.
In primary cardiomyocytes, increased phosphorylation of extracellular signal‐regulated kinase (Erk) 1/2 (p‐Erk 1/2) in cells treated with B7‐33, reaching significance at 100 nmol/L (A); reduced expression of ASC (apoptosis‐associated speck‐like protein containing a caspase recruitment domain) in cells 6 hours post simulated ischemia reoxygenation (SIRO), on treatment with B7‐33 (B); and reduced expression of GRP78 in cells 24 hours post SIRO, on treatment with B7‐33 (C) (for A through C, n=3 experiments per group). Western blots are quantified and expressed as arbitrary densitometric units (A.D.U). For A through C, 1‐way ANOVA was used to determine significance, and if P<0.05, Holm‐Sidak test was used for post hoc analysis. *P<0.05. C indicates control.
Figure 7
Figure 7. Western blot analysis of tunicamycin‐induced endoplasmic reticulum (ER) stress in protein lysates from primary cardiomyocytes.
Exposure of primary cardiomyocytes to tunicamycin (TUN) for 24 hours significantly reduces phosphorylation of extracellular signal‐regulated kinase (Erk) 1/2 (p‐Erk 1/2) at both 2.5 and 7.5 μg/mL concentrations of TUN (A); and significantly increases expression of GRP78 at both doses of TUN (B). B7‐33 (100 nmol/L) significantly lowers GRP78, compared with control (C), on coincubation with 2.5 μg/mL TUN (for A and B, n=4–6 experiments/group). Western blots are quantified and expressed as arbitrary densitometric units with respect to (A.D.U WRT) the expression of GAPDH. For A and B, 1‐way ANOVA was used for significance testing, and if P<0.05, Holm‐Sidak test was used for post hoc analysis. *P<0.05, **P<0.01, ****P<0.0001.
Figure 8
Figure 8. Treatment of primary cardiomyocytes with tunicamycin (TUN) with/without PD98059 and/or B7‐33.
A, Representative Western blots of primary cardiomyocytes treated with tunicamycin (TUN) with/without PD98059 and/or B7‐33. B, Exposure to PD98059 (50 μmol/L) significantly lowers phosphorylation of extracellular signal‐regulated kinase (Erk) 1/2 (p‐Erk 1/2) 24 hours post incubation in primary cardiomyocytes treated with TUN with/without B7‐33. No significant difference between p‐Erk 1/2 levels was found between TUN and TUN+B7‐33 groups. C, Coincubating TUN with PD98059 abrogates the reduction in GRP78 observed when treated with B7‐33 (for B and C, n=3 experiments/group). Western blots are quantified and expressed as arbitrary densitometric units with respect to (A.D.U WRT) the expression of GAPDH. For B and C, 1‐way ANOVA was used for significance testing, and if P<0.05, Holm‐Sidak test was used for post hoc analysis. *P<0.05.
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