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 CD 1 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 GRP 78 (glucose regulated protein), an endoplasmic reticulum stress marker. Subsequently, B7‐33 (100 nmol/L) reduced tunicamycin (2.5 μg/mL) induced upregulation of GRP 78 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.

BackgroundDuchenne muscular dystrophy (DMD) is the most frequently inherited human myopathy and represents the most devastating type of muscular dystrophy. This progressive disease is caused by X‐linked recessive defects in the gene encoding for the structural protein, dystrophin. Young boys with DMD exhibit symptoms between 3–5 years of age, primarily involving skeletal muscles. Although the heart is also impacted, limited exercise tolerance masks early symptoms of cardiomyopathy (DMD‐CM). The defects in dystrophin in cardiac tissue lead to loss of membrane integrity, extracellular calcium influx and subsequent cardiomyocyte necrosis, inflammation and increased overall fibrosis. Respiratory failure has long been the leading cause of death in boys with DMD, however, with the advent of respiratory therapy, patients with DMD may now survive longer to unfortunately die in the 2nd or 3rd decade of life from heart failure. Based on its mito‐protective, anti‐inflammatory, anti‐apoptotic and anti‐fibrotic effects, we believe that hydrogen sulfide (H2S) is a promising candidate for therapy against the devastating consequences of DMD‐CM.Methods‘Humanized’ dystrophic G2 mice (shorter telomeres) were generated by inbreeding G1 mdx4cv/mTR mice. The effects of SG1002, an orally active slow releasing H2S prodrug, were tested in the G2 mice (40 mg/kg) either immediately after weaning (~3 weeks of age; Early Treatment) or after cardiac dysfunction (~7 months of age; Late Treatment). Cardiac function was analyzed by echocardiography. Muscle fibrosis was assessed via picrosirius red staining of diaphragm, gastrocnemius, and heart collected at 2, 4 and 9 months. Cardiac mitochondria and protein were isolated to measure oxidative phosphorylation and NLRP3 expression, respectively.ResultsEarly treatment with SG1002 preserved cardiac function (ejection fraction; EF) throughout study duration, whereas untreated mice exhibited significant decline in EF by 4 months of age and continued to worsen over time (Fig. A). Delayed treatment with SG1002 significantly improved EF within 1 month after treatment initiation. SG1002 also significantly attenuated fibrosis in the diaphragm and gastrocnemius, and had a tendency to decrease cardiac fibrosis by 9 months of age (Fig. B). A steady decline in mitochondrial function was observed over time in untreated mice compared to wild type mice (−26%), which was attenuated with SG1002 (−12%, P<0.05 vs. untreated group). Although there was no difference in NLRP3 expression in the heart of G2 mice at 4 months of age compared to wild type, a significant 2‐fold increase in cardiac NLRP3 expression was observed by 9 months (Fig. C).ConclusionIn a ‘humanized’ mouse model of DMD, our results strongly support daily H2S therapy for preservation of cardiac function, attenuation of skeletal muscle fibrosis and cardiac NLRP3 expression. Accordingly, modulation of the inflammasome and scar formation with H2S may represent an important mechanism to mitigate DMD‐CM as well as skeletal muscle injury.Support or Funding InformationR01HL133167 to FNSThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background: B7-33 is a peptide derived from the B-chain of human relaxin, and is shown to elicit biased signaling at relaxin receptor 1 (RXFP1) in human cell lines. The influence of B7-33 on myocardial ischemia-reperfusion (IR) injury and associated cardiac adverse remodeling is unknown. Methods and Results: Primary cardiomyocytes and cardiac fibroblasts were isolated from adult CD1 mice and subjected to simulated ischemia-reoxygenation (SIRO). Myocytes were placed in 1% O 2 chamber for 40 min, followed by 1 h of reoxygenation with control (myocyte media) or B7-33 (10, 50 or 100 nM)-infused media. Trypan blue staining showed a significant decrease in cell death with B7-33 concentrations of 50 nM and 100 nM ( Fig. A ).Treating myocytes with B7-33 for 15 min exhibited a dose-dependent increase in Erk1/2 phosphorylation, which reached statistical significance at 100 nM ( Fig. B ). Fibroblasts subjected to 4 h of hypoxia and 15 h of reoxygenation had increased viability (MTT assay) with B7-33 at all concentrations ( Fig. C ). In vivo , CD1 mice were subjected to IR injury via coronary artery ligation for 30 min, followed by 24 h of reperfusion. Vehicle (saline) or B7-33 (10 μg/kg) was injected i.p. at the onset of reperfusion. After 24 h, B7-33-treated mice demonstrated decreased infarct size (TTC stain) ( Fig. D ) and preserved fractional shortening (M-mode echo, Fig. E ) compared to vehicle-treated mice. Conclusion: Reperfusion therapy with B7-33 reduces infarct size post-MI, preserves cardiac function and protects cardiomyocytes and fibroblasts against SIRO. We propose that B7-33 might be effective against acute MI and a possible alternative to recombinant relaxin for clinical utility.