Cardiovascular diseases (CVDs) are the leading cause of mortality and morbidity globally, representing approximately a third of all deaths every year. The greater part of these cases is represented by myocardial infarction (MI), or heart attack as it is better known, which occurs when declining blood flow to the heart causes injury to cardiac tissue. Mesenchymal stem cells (MSCs) are multipotent stem cells that represent a promising vector for cell therapies that aim to treat MI due to their potent regenerative effects. However, it remains unclear the extent to which MSC-based therapies are able to induce regeneration in the heart and even less clear the degree to which clinical outcomes could be improved. Exosomes, which are small extracellular vesicles (EVs) known to have implications in intracellular communication, derived from MSCs (MSC-Exos), have recently emerged as a novel cell-free vector that is capable of conferring cardio-protection and regeneration in target cardiac cells. In this review, we assess the current state of research of MSC-Exos in the context of MI. In particular, we place emphasis on the mechanisms of action by which MSC-Exos accomplish their therapeutic effects, along with commentary on the current difficulties faced with exosome research and the ongoing clinical applications of stem-cell derived exosomes in different medical contexts.

In an ethnically homogeneous population of women living in Tuscany, Italy, the relationships between age, body weight, bone mineral density and the vitamin D receptor (VDR) gene polymorphism were studied, with the objective of recognizing patients at risk for osteoporosis. In 275 women bone mineral density was measured by Dual Energy X-rays Absorptiometry (DEXA). In 50 of them the individual genetic pattern for VDR was evaluated by DNA extraction followed by PCR amplification of the VDR gene, and digestion with the restriction enzyme BsmI. Age and bone mineral density were inversely related (R2 = 0.298). Body weight was associated with bone mineral density (R2 = 0.059), but not with age. In osteoporotic women, mean (± SD) body weight was 59.9 ± 6.5 Kg, lower than that recorded in non osteoporotic women (64.2 ± 9.4 Kg), even though not significantly different (p = 0.18). No association was found between VDR gene polymorphism, bone density or body weight. The performance of anthropometric and genetic components appear to be poor, and, at least for the time being, bone mineral density measurement by means of MOC-DEXA represents the optimal method to detect women at risk for postmenopausal osteoporosis.

Aims Altered mechanical load in response to injury is a main driver of myocardial interstitial fibrosis. No current in vitro model can precisely modulate mechanical load in a multicellular environment while maintaining physiological behaviour. Living myocardial slices (LMS) are a 300 μm-thick cardiac preparation with preserved physiological structure and function. Here we apply varying degrees of mechanical preload to rat and human LMS to evaluate early cellular, molecular, and functionality changes related to myocardial fibrosis. Methods and resultsLeft ventricular LMS were obtained from Sprague Dawley rat hearts and human cardiac samples from healthy and failing (dilated cardiomyopathy) hearts. LMS were mounted on custom stretchers and two degrees of diastolic load were applied: physiological sarcomere length (SL) (SL = 2.2 μm) and overload (SL = 2.4 μm). LMS were maintained for 48 h under electrical stimulation in circulating, oxygenated media at 37°C. In overloaded conditions, LMS displayed an increase in nucleus translocation of Yes-associated protein (YAP) and an up-regulation of mechanotransduction markers without loss in cell viability. Expression of fibrotic and inflammatory markers, as well as Collagen I deposition were also observed. Functionally, overloaded LMS displayed lower contractility (7.48 ± 3.07 mN mm À2 at 2.2 SL vs. 3.53 ± 1.80 mN mm À2 at 2.4 SL). The addition of the profibrotic protein interleukin-11 (IL-11) showed similar results to the application of overload with enhanced fibrosis (8% more of collagen surface coverage) and reduced LMS contractility at physiological load. Conversely, treatment with the Transforming growth factor β receptor (TGF-βR) blocker SB-431542, showed down-regulation of genes associated with mechanical stress, prevention of fibrotic response and improvement in cardiac function despite overload (from 2.40 ± 0.8 mN mm À2 to 4.60 ± 1.08 mN mm À2 ). Conclusions The LMS have a consistent fibrotic remodelling response to pathological load, which can be modulated by a TGF-βR blocker. The LMS platform allows the study of mechanosensitive molecular mechanisms of myocardial fibrosis and can lead to the development of novel therapeutic strategies.

Cardiac fibroblasts regulate the development of the adult cardiomyocyte phenotype and cardiac remodeling in disease. We investigate the role that cardiac fibroblasts-secreted extracellular vesicles (EVs) have in the modulation of cardiomyocyte Ca2+ cycling–a fundamental mechanism in cardiomyocyte function universally altered during disease. EVs collected from cultured human cardiac ventricular fibroblasts were purified by centrifugation, ultrafiltration and size-exclusion chromatography. The presence of EVs and EV markers were identified by dot blot analysis and electron microscopy. Fibroblast-conditioned media contains liposomal particles with a characteristic EV phenotype. EV markers CD9, CD63 and CD81 were highly expressed in chromatography fractions that elute earlier (Fractions 1–15), with most soluble contaminating proteins in the later fractions collected (Fractions 16–30). Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were treated with fibroblast-secreted EVs and intracellular Ca2+ transients were analyzed. Fibroblast-secreted EVs abbreviate the Ca2+ transient time to peak and time to 50% decay versus serum-free controls. Thus, EVs from human cardiac fibroblasts represent a novel mediator of human fibroblast-cardiomyocyte interaction, increasing the efficiency of hiPSC-CM Ca2+ handling.

Introduction Mechanical overload plays an important role in the progression of chronic heart failure (CHF). Beside the numerous neurohormonal and chemical stimuli that contribute to structural and functional changes in the myocardium, intercellular communication mediated by extracellular vesicles (EVs), also plays a pivotal role in disease progression. Exosomes are a subset of EVs ranging between 30–150 nm; these mediators carry molecular cargoes such as miRNAs, mRNAs, DNA and several proteins. However, it is still unclear how specific pathological stimuli may induce a change in their release from tissues or a difference in their cargo content. Living myocardial slices (LMS) are ultra-thin sections of heart tissue, ranging from 100–400 μm in thickness. They retain the multicellularity, electromechanical physiology, biochemistry and extracellular matrix of the adult heart. Purpose To investigate whether mechanical load influences both the released amount and content of LMS-derived exosomes after 24 hours culture. Methods and results LMS were prepared from the left ventricle of male Sprague Dawley rats using a high precision vibratome. LMS were stretched in the direction of the muscle fibers at a sarcomere length (SL) of 2.2 μm to recapitulate physiological preload, and at 2.4 μm SL to recapitulate a condition of volume overload. LMS were cultured for 24 hours under electromechanical stimulation. Media containing LMS-derived exosomes was harvested after culture and processed for exosome isolation by size exclusion chromatography columns. Particle size and concentration were assessed by nanoparticle tracking analysis and protein quantity by microBCA assay. There was no significant difference in the size of exosomes between overload and physiological conditions, with an average mean size of 113.475±8.35 nm for the physiological and 129.3±16.35 nm for the overload condition (p=0.61; n=4 physiological; n=3 overload). Although there was not significant difference in exosome amount between physiological and overload condition, (7.00E+10±2.53E+09 vs 1.04E+11±5.77E+09 particles/ml) (p=0.57; n=4 physiological; n=3 overload), exosomes released from overloaded LMS showed a significant increase in protein content compared to physiologically loaded LMS (150.57±25.682 vs 66.045±9.855 μg/ml) (p=0.04; n=3 overload; n=2 physiological). Conclusions Mechanical load influences the cargo content of EVs secreted from LMS after 24 hours culture under electromechanical stimulation. Understanding how mechanical load correlates with specific cargoes in EVs will reveal novel therapeutic targets for the treatment of CHF. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): British Heart Foundation (BHF)

Engagement of the sarcoplasmic reticulum (SR) Ca2+ stores for excitation–contraction (EC)-coupling is a fundamental feature of cardiac muscle cells. Extracellular matrix (ECM) proteins that form the extracellular scaffolding supporting cardiac contractile activity are thought to play an integral role in the modulation of EC-coupling. At baseline, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show poor utilisation of SR Ca2+ stores, leading to inefficient EC-coupling, like developing or human CMs in cardiac diseases such as heart failure. We hypothesised that integrin ligand–receptor interactions between ECM proteins and CMs recruit the SR to Ca2+ cycling during EC-coupling. hiPSC-CM monolayers were cultured on fibronectin-coated glass before 24 h treatment with fibril-forming peptides containing the integrin-binding tripeptide sequence arginine–glycine–aspartic acid (2 mM). Micropipette application of 40 mM caffeine in standard or Na+/Ca2+-free Tyrode’s solutions was used to assess the Ca2+ removal mechanisms. Microelectrode recordings were conducted to analyse action potentials in current-clamp. Confocal images of labelled hiPSC-CMs were analysed to investigate hiPSC-CM morphology and ultrastructural arrangements in Ca2+ release units. This study demonstrates that peptides containing the integrin-binding sequence arginine–glycine–aspartic acid (1) abbreviate hiPSC-CM Ca2+ transient and action potential duration, (2) increase co-localisation between L-type Ca2+ channels and ryanodine receptors involved in EC-coupling, and (3) increase the rate of SR-mediated Ca2+ cycling. We conclude that integrin-binding peptides induce recruitment of the SR for Ca2+ cycling in EC-coupling through functional and structural improvements and demonstrate the importance of the ECM in modulating cardiomyocyte function in physiology.

Background Gene therapy based on viral vectors is a promising new approach and offers great potential for the study and the treatment of cardiac diseases. Nucleic acid-based transfection of human heart tissue could be considered a valid and translational alternative to the use of transgenic animal models in cardiovascular research. Purpose Here we explore the use of Living Myocardial Slices (LMS) as a platform for nucleic acid-based therapies. Rat LMS and Adeno-Associated viruses (AAV) were used to optimise and analyse gene transfer efficiency, viability, tissue functionality, and cell tropism in cardiac tissue. Human cardiac samples from failing (dilated cardiomyopathy) hearts were also used to validate the human model. In addition, we transfected and tested for the first time the regenerative effect of AAV-miR-199a in both healthy and pathologically overloaded LMS. Methods LMS (300 μm thick) were prepared and glued to PTFE-coated holders, mounted on custom stretchers, and cultured at physiological sarcomere length for 72-hours under electrical stimulation. Two recombinant AAV serotypes (AAV-6 and AAV-9) at different multiplicity of infection (MOI) expressing green fluorescent protein (GFP) or the miR-199 transgene were added to the surface of the LMS. Results AAV6 proved to be the most suitable serotype and 20,000 MOI the most efficient dose, as these conditions were proven not to affect LMS contractility and to have the highest transduction and penetrability efficiency. This serotype exhibited preferential cell tropism to cardiomyocytes and stromal cells (40% of transduction in both) in rat LMS, with lower efficiency in endothelial cells, (20% of transduction). In contrast, AAV6 in human failing LMS showed higher transduction efficiency in endothelial cells and myofibroblasts. Treatment with miR-199a significantly increased the active force of pathologically overstretched LMS, downregulated the expression of its target genes, and affected the proliferative capacity of LMS. Conclusion LMS can be used as a model for nucleic acid-based therapies and their transfection protocol can be adapted to samples obtained from failing human hearts. With great translational relevance, this model would accelerate preclinical studies of novel nucleic acid-based therapies for heart failure. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

No abstract