Integrins Increase Sarcoplasmic Reticulum Activity for Excitation-Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes.

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.

The science behind soft skills: Do's and Don'ts for early career researchers and beyond. A review paper from the EU-CardioRNA COST Action CA17129.

Soft skills are the elementary management, personal, and interpersonal abilities that are vital for an individual to be efficient at workplace or in their personal life. Each work place requires different set of soft skills. Thus, in addition to scientific/technical skills that are easier to access within a short time frame, several key soft skills are essential for the success of a researcher in today's international work environment. In this paper, the trainees and trainers of the EU-CardioRNA COST Action CA17129 training school on soft skills present basic and advanced soft skills for early career researchers. Here, we particularly emphasize on the importance of transferable and presentation skills, ethics, literature reading and reviewing, research protocol and grant writing, networking, and career opportunities for researchers. All these skills are vital but are often overlooked by some scholars. We also provide tips to ace in aforementioned skills that are crucial in a day-to-day life of early and late career researchers in academia and industry.

Extracellular Vesicles from Human Cardiac Fibroblasts Modulate Calcium Cycling in Human Stem Cell-Derived Cardiomyocytes.

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.

Mechanosensitive molecular mechanisms of myocardial fibrosis in living myocardial slices.

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 RESULTS: Left 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-2 at 2.2 SL vs. 3.53 ± 1.80 mN mm-2 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 ß receptor (TGF-ßR) 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-2 to 4.60 ± 1.08 mN mm-2 ). CONCLUSIONS: The LMS have a consistent fibrotic remodelling response to pathological load, which can be modulated by a TGF-ßR blocker. The LMS platform allows the study of mechanosensitive molecular mechanisms of myocardial fibrosis and can lead to the development of novel therapeutic strategies.

Novel Applications of Mesenchymal Stem Cell-derived Exosomes for Myocardial Infarction Therapeutics.

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.