Search

Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the activation of cardiac fibroblasts (cFbs) to myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments, as most of the cellular-based in vitro reductionist models do not take into account the leading role of ECM cues in driving the progression of the pathology. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-β1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (α-SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere assembly, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis., Competing Interests: Declaration of competing interest Luca Bersanini and Malin Becker are employees of Optics11 Life B.V., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The quest for novel methods to mature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for cardiac regeneration, modelling and drug testing has emphasized a need to create microenvironments with physiological features. Many studies have reported on how cardiomyocytes sense substrate stiffness and adapt their morphological and functional properties. However, these observations have raised new biological questions and a shared vision to translate it into a tissue or organ context is still elusive. In this review, we will focus on the relevance of substrates mimicking cardiac extracellular matrix (cECM) rigidity for the understanding of the biomechanical crosstalk between the extracellular and intracellular environment. The ability to opportunely modulate these pathways could be a key to regulate in vitro hiPSC-CM maturation. Therefore, both hiPSC-CM models and substrate stiffness appear as intriguing tools for the investigation of cECM-cell interactions. More understanding of these mechanisms may provide novel insights on how cECM affects cardiac cell function in the context of genetic cardiomyopathies., (Copyright © 2022. Published by Elsevier Ltd.)

Adult human cardiac mesenchymal progenitor cells (hCmPC) are multipotent resident populations involved in cardiac homeostasis and heart repair . Even if the mechanisms have not yet been fully elucidated, the stem cell differentiation is guided by the mitochondrial metabolism; however, mitochondrial approaches to identify hCmPC with enhanced stemness and/or differentiation capability for cellular therapy are not established. Here we demonstrated that hCmPCs sorted for low and high mitochondrial membrane potential (using a lipophilic cationic dye tetramethylrhodamine methyl ester, TMRM), presented differences in energy metabolism from preferential glycolysis to oxidative rates. TMRM-high cells are highly efficient in terms of oxygen consumption rate, basal and maximal respiration, and spare respiratory capacity compared to TMRM-low cells. TMRM-high cells showed characteristics of pre-committed cells and were associated with higher in vitro differentiation capacity through endothelial, cardiac-like, and, to a lesser extent, adipogenic and chondro/osteogenic cell lineage, when compared with TMRM-low cells. Conversely, TMRM-low showed higher self-renewal potential. To conclude, we identified two hCmPC populations with different metabolic profile, stemness maturity, and differentiation potential. Our findings suggest that metabolic sorting can isolate cells with higher regenerative capacity and/or long-term survival. This metabolism-based strategy to select cells may be broadly applicable to therapies.

The ultimate goal of precision disease modeling is to artificially recreate the disease of affected people in a highly controllable and adaptable external environment. This field has rapidly advanced which is evident from the application of patient-specific pluripotent stem-cell-derived precision therapies in numerous clinical trials aimed at a diverse set of diseases such as macular degeneration, heart disease, spinal cord injury, graft-versus-host disease, and muscular dystrophy. Despite the existence of semi-adequate treatments for tempering skeletal muscle degeneration in dystrophic patients, nonischemic cardiomyopathy remains one of the primary causes of death. Therefore, cardiovascular cells derived from muscular dystrophy patients' induced pluripotent stem cells are well suited to mimic dystrophin-associated cardiomyopathy and hold great promise for the development of future fully effective therapies. The purpose of this article is to convey the realities of employing precision disease models of dystrophin-associated cardiomyopathy. This is achieved by discussing, as suggested in the title echoing William Shakespeare's words, the settlements (or "leagues") made by researchers to manage the constraints ("betwixt mine eye and heart") distancing them from achieving a perfect precision disease model.

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by sudden death in young people and featured by fibro-adipose myocardium replacement, malignant arrhythmias, and heart failure. To date, no etiological therapies are available. Mutations in desmosomal genes cause abnormal mechanical coupling, trigger pro-apoptotic signaling pathways, and induce fibro-adipose replacement. Here, we discuss the hypothesis that the ACM causative mechanism involves a defect in the expression and/or activity of the cardiac Ca 2+ handling machinery, focusing on the available data supporting this hypothesis. The Ca 2+ toolkit is heavily remodeled in cardiomyocytes derived from a mouse model of ACM defective of the desmosomal protein plakophilin-2. Furthermore, ACM-related mutations were found in genes encoding for proteins involved in excitation‒contraction coupling, e.g., type 2 ryanodine receptor and phospholamban. As a consequence, the sarcoplasmic reticulum becomes more eager to release Ca 2+ , thereby inducing delayed afterdepolarizations and impairing cardiac contractility. These data are supported by preliminary observations from patient induced pluripotent stem-cell-derived cardiomyocytes. Assessing the involvement of Ca 2+ signaling in the pathogenesis of ACM could be beneficial in the treatment of this life-threatening disease.

Background: Doxorubicin (DOX) is a chemotherapeutic drug limited in its usefulness by an adverse side effect, cardiotoxicity. The mechanisms leading to this detrimental occurrence are not completely clear, and lately many authors focused their attention on the possible role of microRNAs (miRNAs), small regulators of cardiovascular functions, in this phenomenon. Notably, these molecules recently emerged also as potential circulating biomarkers of several cardiac diseases. Thus, the aim of this study was the simultaneous investigation of circulating and cardiac tissue miRNAs expression upon DOX treatment in vivo., Methods: Twenty C57BL/6 female mice were administered with 24 mg/Kg cumulative dose of DOX or saline (CTRL) for 2 weeks. Echocardiography was performed at baseline and at the end of treatment (T1). Plasma and heart samples were collected at T1, separating atria from left (LV) and right (RV) ventricles, and miRNAs expression was tested by RT-qPCR-based arrays. All putatively DOX-regulated candidates were then validated by single assays in vivo and then evaluated also in murine immortalized cardiomyocytes (HL-1) treated with 1 μM DOX for 24 h. In the end, bioinformatics target prediction was performed for all DOX-miRNAs., Results: Cardiotoxicity onset was diagnosed upon impairment of six cardiac functional parameters in DOX-treated mice at T1. Samples collection, followed by screening and validation steps, identified eleven miRNAs dysregulated by the drug in plasma, while seven resulted as altered in separate heart chambers. Interestingly, miR-34a-5p and miR-451a showed a dysregulation in both plasma and tissue samples of DOX-administered animals, whereas five additional miRNAs presented chamber specific modulation. Of note, in vitro experiments showed a very modest overlap with in vivo results. Bioinformatics prediction analysis performed on miR-34a-5p and miR-451a identified several putative targets presenting no significant association with cardiotoxicity. Anyhow, the same analyses, conducted by combining all miRNAs regulated by DOX in each heart chamber, evidenced a possible dysregulation of the adherens junctions gene network, known to be involved in the onset and progression of dilated cardiomyopathy, an established detrimental side effect of the drug., Conclusions: This is the first work investigating miRNAs regulation by DOX both in plasma and heart districts of treated animals. Our results indicate a strong association of miR-34a-5p and miR-451a to DOX-induced cardiotoxicity. In addition, the observed altered expression of diverse miRNAs in separated cardiac chambers hints at a specific response to the drug, implying the existence of different players and pathways leading to dysfunction onset., (Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Patients affected by Duchenne muscular dystrophy (DMD) develop a progressive dilated cardiomyopathy characterized by inflammatory cell infiltration, necrosis, and cardiac fibrosis. Standard treatments consider the use of β-blockers and angiotensin-converting enzyme inhibitors that are symptomatic and unspecific toward DMD disease. Medications that target DMD cardiac fibrosis are in the early stages of development. We found immunoproteasome dysregulation in affected hearts of mdx mice (murine animal model of DMD) and cardiomyocytes derived from induced pluripotent stem cells of patients with DMD. Interestingly, immunoproteasome inhibition ameliorated cardiomyopathy in mdx mice and reduced the development of cardiac fibrosis. Establishing the immunoproteasome inhibition-dependent cardioprotective role suggests the possibility of modulating the immunoproteasome as new and clinically relevant treatment to rescue dilated cardiomyopathy in patients with DMD., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms. Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients' bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients' hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

SERCA2a is the Ca 2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency., Competing Interests: The authors declare no conflict of interest.

Communication between cardiomyocytes depends upon gap junctions (GJ). Previous studies have demonstrated that electrical stimulation induces GJ remodeling and modifies histone acetylase (HAT) and deacetylase (HDAC) activities, although these two results have not been linked. The aim of this work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation-dependent mechanisms. Field stimulation of HL-1 cardiomyocytes at 0.5 Hz for 24 h significantly reduced connexin43 (Cx43) expression and cell-cell communication. HDAC activity was down-regulated whereas HAT activity was not modified resulting in increased acetylation of Cx43. Consistent with a post-translational mechanism, we did not observe a reduction in Cx43 mRNA in electrically stimulated cells, while the proteasomal inhibitor MG132 maintained Cx43 expression. Further, the treatment of paced cells with the HAT inhibitor Anacardic Acid maintained both the levels of Cx43 and cell-cell communication. Finally, we observed increased acetylation of Cx43 in the left ventricles of dogs subjected to chronic tachypacing as a model of abnormal ventricular activation. In conclusion, our findings suggest that altered electrical activity can regulate cardiomyocyte communication by influencing the acetylation status of Cx43., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cardiovascular disease is the leading cause of morbidity and mortality in the developed world. Although ongoing therapeutic strategies ameliorate symptoms and prolong life for patients with cardiovascular diseases, they do not solve the critical issue related to the loss of cardiac tissue. Accordingly, stem/progenitor cell therapy has emerged as a paramount approach for cardiac repair and regeneration. In this regard, c-kit(+) cells have animated much interest and controversy. These cells are self-renewing, clonogenic, and multipotent and display a noteworthy potential to differentiate into all cardiovascular lineages. However, their functional contribution to cardiomyocyte turnover is one of the centrally debated issues concerning their regenerative potential. Regardless, plentiful preclinical and clinical studies have been conducted which provide evidence for the capacity of c-kit(+) cells to improve cardiac function. The purpose of this review is to give a comprehensive, impartial, critical description and evaluation of the literature on c-kit(+) cells from bench to bedside in order to address their true potential, benefits and controversies.

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-α general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients., (© 2014 by the American Diabetes Association.)

Adult human cardiac mesenchymal-like stromal cells (CStC) represent a relatively accessible cell type useful for therapy. In this light, their conversion into cardiovascular precursors represents a potential successful strategy for cardiac repair. The aim of the present work was to reprogram CStC into functionally competent cardiovascular precursors using epigenetically active small molecules. CStC were exposed to low serum (5% FBS) in the presence of 5 µM all-trans Retinoic Acid (ATRA), 5 µM Phenyl Butyrate (PB), and 200 µM diethylenetriamine/nitric oxide (DETA/NO), to create a novel epigenetically active cocktail (EpiC). Upon treatment the expression of markers typical of cardiac resident stem cells such as c-Kit and MDR-1 were up-regulated, together with the expression of a number of cardiovascular-associated genes including KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, and α-MHC. In addition, profiling analysis revealed that a significant number of microRNA involved in cardiomyocyte biology and cell differentiation/proliferation, including miR 133a, 210 and 34a, were up-regulated. Remarkably, almost 45% of EpiC-treated cells exhibited a TTX-sensitive sodium current and, to a lower extent in a few cells, also the pacemaker I(f) current. Mechanistically, the exposure to EpiC treatment introduced global histone modifications, characterized by increased levels of H3K4Me3 and H4K16Ac, as well as reduced H4K20Me3 and H3s10P, a pattern compatible with reduced proliferation and chromatin relaxation. Consistently, ChIP experiments performed with H3K4me3 or H3s10P histone modifications revealed the presence of a specific EpiC-dependent pattern in c-Kit, MDR-1, and Nkx2.5 promoter regions, possibly contributing to their modified expression. Taken together, these data indicate that CStC may be epigenetically reprogrammed to acquire molecular and biological properties associated with competent cardiovascular precursors.

Preclinical studies performed in cell culture and animal systems have shown the outstanding ability of stem cells to repair ischemic heart and lower limbs by promoting the formation of new blood vessels and new myocytes. In contrast, clinical studies of stem cell administration in patients with myocardial ischemia have revealed only modest, although promising, results. Basic investigations have shown the feasibility of adult cells reprogramming into pluripotent cells by defined factors, thus opening the way to the devise of protocols to ex vivo derive virtually unexhausted cellular pools. In contrast, cellular and molecular studies have indicated that risk factors limit adult-derived stem cell survival, proliferation and engraftment in ischemic tissues. The use of fully reprogrammed cells raises safety concerns; therefore, adult cells remain a primary option for clinicians interested in therapeutic cardiovascular repair. Pharmacologic approaches have been devised to restore the cardiovascular repair ability of failing progenitors from patients at risk. In the present contribution, the most advanced pharmacologic approaches to (re)program, boost, and condition endothelial and cardiac progenitor cells to enhance cardiovascular regeneration are discussed., (Copyright © 2010 Elsevier Inc. All rights reserved.)

During cardiac development, the epicardium is the source of multipotent mesenchymal cells, which give rise to endothelial and smooth muscle cells in coronary vessels and also, possibly, to cardiomyocytes. The aim of the present study was to determine whether stem cells are retained in the adult human and murine epicardium and to investigate the regenerative potential of these cells following acute myocardial infarction. We show that c-kit(+) and CD34(+) cells can indeed be detected in human fetal and adult epicardium and that they represent 2 distinct populations. Both subsets of cells were negative for CD45, a cell surface marker that identifies the hematopoietic cell lineage. Immunofluorescence revealed that freshly isolated c-kit(+) and CD34(+) cells expressed early and late cardiac transcription factors and could acquire an endothelial phenotype in vitro. In the murine model of myocardial infarction, there was an increase in the absolute number and proliferation of epicardial c-kit(+) cells 3 days after coronary ligation; at this time point, epicardial c-kit(+) cells were identified in the subepicardial space and expressed GATA4. Furthermore, 1 week after myocardial infarction, cells coexpressing c-kit(+), together with endothelial or smooth muscle cell markers, were identified in the wall of subepicardial blood vessels. In summary, the postnatal epicardium contains a cell population with stem cell characteristics that retains the ability to give rise to myocardial precursors and vascular cells. These cells may play a role in the regenerative response to cardiac damage.

High-mobility group box 1 protein (HMGB1) is a chromatin protein that is released by inflammatory and necrotic cells. Extracellular HMGB1 signals tissue damage, stimulates the secretion of proinflammatory cytokines and chemokines, and modulates stem cell function. The present study examined exogenous HMGB1 effect on mouse left-ventricular function and myocyte regeneration after infarction. Myocardial infarction was induced in C57BL/6 mice by permanent coronary artery ligation. After 4 hours animals were reoperated and 200 ng of purified HMGB1 was administered in the peri-infarcted left ventricle. This intervention resulted in the formation of new myocytes within the infarcted portion of the wall. The regenerative process involved the proliferation and differentiation of endogenous cardiac c-kit+ progenitor cells. Circulating c-kit+ cells did not significantly contribute to HMGB1-mediated cardiac regeneration. Echocardiographic and hemodynamic parameters at 1, 2, and 4 weeks demonstrated a significant recovery of cardiac performance in HMGB1-treated mice. These effects were not observed in infarcted hearts treated either with the unrelated protein glutathione S-transferase or a truncated form of HMGB1. Thus, HMGB1 appears to be a potent inducer of myocardial regeneration following myocardial infarction.

Objective: Controversy about hematopoietic stem cells reprogramming into cardiac myocytes is currently supported by positive and negative findings. In fact, some reports have shown the ability of stem cells from the bone marrow (BM) to differentiate into cardiac myocytes and to contribute to myocardium repair, while others have reported the opposite., Methods: C-kit(+) cells from mouse bone marrow were co-cultured onto neonatal cardiac myocytes. Hematopoietic stem cell-derived cells were analyzed by investigating the expression of cardiac markers and ion channels and by single-cell electrophysiological recordings., Results: Groups of undifferentiated c-kit(+) cells displayed only outward currents. Co-cultured c-kit(+) stem cells on neonatal cardiac myocytes expressed cardiac markers and Na(+) and Ca(2+) voltage-gated ion channels. However, Na(+) and Ca(2+) currents were not detected by electrophysiological patch-clamp recordings even if caffeine and cyclopiazonic acid treatment showed the presence of intracellular calcium stores. This suggests that these channels, although expressed, were not functional and thus do not allow the coupling between excitation and contraction that is typical of cardiac myocytes. Nevertheless, co-cultured cells had a more hyperpolarized resting membrane potential and, at least in a subset of cells, displayed voltage-gated inward rectifier currents and outward currents. Co-cultured c-kit(+)-derived cells were not connected to surrounding cardiac myocytes through gap junctions. To induce a more pronounced differentiation, co-cultured cells were treated with BMP-4 and TGF-beta, two factors that were shown to trigger a cardiac myocyte differentiation pathway in embryonic stem (ES) cells. Even under these conditions, c-kit(+) cells did not differentiate into functionally active cardiac myocytes. However, TGF-beta/BMP-4-treated cells were hyperpolarized and showed and increased inward rectifier current density., Conclusions: Our study shows that mouse BM hematopoietic stem cells exhibit a limited plasticity to transdifferentiate into cardiac myocytes in culture.