In animal models, the cardiotropic hormone relaxin has been shown to protect the heart against ischemia and reperfusion‐induced damage, acting by multiple mechanisms that primarily involve the coronary vessels. This in vit ro study evaluates whether relaxin also has a direct protective action on cardiac muscle cells. H9c2 rat cardiomyoblasts and primary mouse cardiomyocytes were subjected to hypoxia and reoxygenation. In some experiments, relaxin was added preventatively before hypoxia; in others, at reoxygenation. To elucidate its mechanisms of action, we focused on Notch‐1, which is involved in heart pre‐ and postconditioning to ischemia. Inactivated RLX was used as negative control. Relaxin (17 nmol/L, EC ₅₀ 4.7 nmol/L), added 24 h before hypoxia or at reoxygenation, protected against cardiomyocyte injury. In fact, relaxin significantly increased cell viability (assayed by trypan blue and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide), decreased apoptosis (assayed by TUNEL and bax/bcl‐2 ratio), and reduced nitroxidative damage (assayed by nitrotyrosine expression and 8‐hydroxy‐deoxyguanosine levels). These effects were partly attributable to the ability of relaxin to upregulate Notch‐1 signaling; indeed, blockade of Notch‐1 activation with the specific inhibitor DAPT reduced relaxin‐induced cardioprotection during hypoxia and reoxygenation. This study adds new mechanistic insights on the cardioprotective role of relaxin on ischemic and oxidative damage—Boccalini, G., Sassoli, C., Formigli, L., Bani, D., Nistri, S., Relaxin protects cardiac muscle cells from hypoxia/reoxygenation injury: involvement of the Notch‐1 pathway. FASEB J. 29, 239–249 (2015). www.fasebj.org

Notch signaling is a major intercellular coordination mechanism highly conserved throughout evolution. In vertebrates, Notch signaling is physiologically involved in embryo development, including mesenchymal cell commitment, formation of heart tissues and angiogenesis. In post-natal life, Notch signaling is maintained as a key mechanism of cell-cell communication and its dysregulations have been found in pathological conditions such as ischemic and fibrotic diseases. In the heart, Notch takes part in the protective response to ischemia, being involved in pre- and postconditioning, reduction of reperfusion-induced oxidative stress and myocardial damage, and cardiomyogenesis. Conceivably, the cardioprotective effects of Notch may depend on neo-angiogenesis, thus blunting lethal myocardial ischemia, as well as on direct stimulation of cardiac cells to increase their resistance to injury. Another post-developmental adaptation of Notch signaling is fibrosis: being involved in the orientation of mesenchymal cell fate, Notch can modulate the differentiation of pro-fibrotic myofibroblasts, e.g. by reducing the effects of the profibrotic cytokine TGF-β. In conclusion, Notch can regulate the interactions between heart muscle and stromal cells and switch cardiac repair from a pro-fibrotic default pathway to a pro-cardiogenic one. These features make Notch signaling a suitable target for new cardiotropic therapies.

Smoking is regarded as a major risk factor for the development of cardiovascular diseases (CVD). This study investigates whether serelaxin (RLX, recombinant human relaxin‐2) endowed with promising therapeutic properties in CVD, can be credited of a protective effect against cigarette smoke (CS)‐induced vascular damage and dysfunction. Guinea pigs exposed daily to CS for 8 weeks were treated with vehicle or RLX, delivered by osmotic pumps at daily doses of 1 or 10 μg. Controls were non‐smoking animals. Other studies were performed on primary guinea pig aortic endothelial (GPAE) cells, challenged with CS extracts (CSE) in the absence and presence of 100 ng/ml (17 nmol/l) RLX. In aortic specimens from CS‐exposed guinea pigs, both the contractile and the relaxant responses to phenylephrine and acetylcholine, respectively, were significantly reduced in amplitude and delayed, in keeping with the observed adverse remodelling of the aortic wall, endothelial injury and endothelial nitric oxide synthase (eNOS) down‐regulation. RLX at both doses maintained the aortic contractile and relaxant responses to a control‐like pattern and counteracted aortic wall remodelling and endothelial derangement. The experiments with GPAE cells showed that CSE significantly decreased cell viability and eNOS expression and promoted apoptosis by sparkling oxygen free radical‐related cytotoxicity, while RLX counterbalanced the adverse effects of CSE. These findings demonstrate that RLX is capable of counteracting CS‐mediated vascular damage and dysfunction by reducing oxidative stress, thus adding a tile to the growing mosaic of the beneficial effects of RLX in CVD.

Cigarette smoke (CS) is the major etiologic factor of chronic obstructive pulmonary disease (COPD), characterized by airway remodeling, lung inflammation and fibrosis, emphysema and respiratory failure. The current therapies can improve COPD management but cannot arrest its progression and reduce mortality. Hence, there is a major interest in identifying molecules susceptible of development into new drugs to prevent or reduce CS-induced lung injury. Serelaxin (RLX), or recombinant human relaxin-2, is a promising candidate because of its anti-inflammatory and antifibrotic properties highlighted in lung disease models. Here we used a guinea pig model of CS-induced lung inflammation and remodeling reproducing some of the hallmarks of COPD. Animals exposed chronically to CS (8 weeks) were treated with vehicle or RLX, delivered by osmotic pumps (1 or 10 μg/day) or aerosol (10 μg/ml/day) during CS treatment. Controls were non-smoking animals. RLX maintained airway compliance to a control-like pattern, likely because of its capability to counteract lung inflammation and bronchial remodeling. In fact, treatment of CS-exposed animals with RLX reduced the inflammatory recruitment of leukocytes, accompanied by a significant reduction of the release of pro-inflammatory cytokines (TNFα, IL-1β). Moreover, RLX was able to counteract the adverse bronchial remodeling and emphysema induced by CS exposure by reducing goblet cell hyperplasia, smooth muscle thickening and fibrosis. Of note, RLX delivered by aerosol has shown a comparable efficacy to systemic administration in reducing CS-induced lung dysfunction and damage. In conclusion, RLX emerges as a new molecule to counteract CS-induced inflammatory lung diseases.