Excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation-contraction (EC) coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With Ca(2+) handling linked, directly or indirectly, to almost all other properties of cardiomyocytes, a solid understanding of this process will be crucial to fully realizing the potential of this technology. Here, we discuss the implications of differences in EC coupling when considering the potential applications of human iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells

Many cells make life work-multicellularity in stem cell-based cardiac disease modelling

Cardiac disease causes 33% of deaths worldwide but our knowledge of disease progression is still very limited. In vitro models utilising and combining multiple, differentiated cell types have been used to recapitulate the range of myocardial microenvironments in an effort to delineate the mechanical, humoral, and electrical interactions that modulate the cardiac contractile function in health and the pathogenesis of human disease. However, due to limitations in isolating these cell types and changes in their structure and function in vitro, the field is now focused on the development and use of stem cell-derived cell types, most notably, human-induced pluripotent stem cell-derived CMs (hiPSC-CMs), in modelling the CM function in health and patient-specific diseases, allowing us to build on the findings from studies using animal and adult human CMs. It is becoming increasingly appreciated that communications between cardiomyocytes (CMs), the contractile cell of the heart, and the non-myocyte components of the heart not only regulate cardiac development and maintenance of health and adult CM functions, including the contractile state, but they also regulate remodelling in diseases, which may cause the chronic impairment of the contractile function of the myocardium, ultimately leading to heart failure. Within the myocardium, each CM is surrounded by an intricate network of cell types including endothelial cells, fibroblasts, vascular smooth muscle cells, sympathetic neurons, and resident macrophages, and the extracellular matrix (ECM), forming complex interactions, and models utilizing hiPSC-derived cell types offer a great opportunity to investigate these interactions further. In this review, we outline the historical and current state of disease modelling, focusing on the major milestones in the development of stem cell-derived cell types, and how this technology has contributed to our knowledge about the interactions between CMs and key non-myocyte components of the heart in health and disease, in particular, heart failure. Understanding where we stand in the field will be critical for stem cell-based applications, including the modelling of diseases that have complex multicellular dysfunctions

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents 10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1–2% of total c-kitpos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC’s myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs

Criteria for chamber-specific categorisation of human cardiac myocytes derived from pluripotent stem cells

Human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) have great potential application in almost all areas of cardiovascular research. A current major goal of the field is to build on the past success of differentiation strategies to produce CMs with the properties of those originating from the different chambers of the adult human heart. With no anatomical origin or developmental pathway to draw on, the question of how to judge the success of such approaches and assess the chamber specificity of PSC-CMs has become increasingly important; commonly used methods have substantial limitations and are based on limited evidence to form such an assessment. In this article, we discuss the need for chamber-specific PSC-CMs in a number of areas as well as current approaches used to assess these cells on their likeness to those from different chambers of the heart. Furthermore, describing in detail the structural and functional features that distinguish the different chamber-specific human adult cardiac myocytes, we propose an evidence-based tool to aid investigators in the phenotypic characterization of differentiated PSC-CMs. Stem Cells 201

Recovery of the failing heart: emerging approaches and mechanisms in excitation-contraction coupling

Heart failure (HF) is a growing cause of morbidity and mortality globally. All clinical therapies that reduce mortality have been shown to induce reverse remodeling. In this article, we discuss a conceptual approach to the evolving treatment of HF using emerging treatment modalities for the drug-refractory patient. This approach is based on the combinatorial, integrated application of therapies shown to influence reverse remodeling in the laboratory

Role and possible mechanisms of clenbuterol in enhancing reverse remodelling during mechanical unloading in murine heart failure

Aims Combined left ventricular assist device (LVAD) and pharmacological therapy has been proposed to favour myocardial recovery in patients with end-stage heart failure (HF). Clenbuterol (Clen), a β2-adrenoceptor (β2-AR) agonist, has been used as a part of this strategy. In this study, we investigated the direct effects of clenbuterol on unloaded myocardium in HF. Methods and results Left coronary artery ligation or sham operation was performed in male Lewis rats. After 4–6 weeks, heterotopic abdominal transplantation of the failing hearts into normal recipients was performed to induce LV unloading (UN). Recipient rats were treated with saline (Sal) or clenbuterol (2 mg/kg/day) via osmotic minipumps (HF + UN + Sal or HF + UN + Clen) for 7 days. Non-transplanted HF animals were treated with Sal (Sham + Sal, HF + Sal) or clenbuterol (HF + Clen). LV myocytes were isolated and studied using optical, fluorescence, and electrophysiological techniques. Clenbuterol treatment improved in vivo LV function measured with echocardiography (LVEF (%): HF 35.9 ± 2 [16], HF + Clen 52.1 ± 1.4 [16]; P < 0.001; mean ± SEM [n]). In combination with unloading, clenbuterol increased sarcomere shortening (amplitude (µm): HF + UN + Clen 0.1 ± 0.01 [50], HF + UN + Sal 0.07 ± 0.01 [38]; P < 0.001) by normalizing the depressed myofilament sensitivity to Ca2+ (slope of the linear relationship between Ca2+ transient and sarcomere shortening hysteresis loop during relaxation (μm/ratio unit): HF + UN + Clen 2.13 ± 0.2 [52], HF + UN + Sal 1.42 ± 0.13 [38]; P < 0.05). Conclusion Clenbuterol treatment of failing rat hearts, alone or in combination with mechanical unloading, improves LV function at the whole-heart and cellular levels by affecting cell morphology, excitation–contraction coupling, and myofilament sensitivity to calcium. This study supports the use of this drug in the strategy to enhance recovery in HF patients treated with LVADs and also begins to elucidate some of the possible cellular mechanisms responsible for the improvement in LV function

Toll-like receptor 9 protects non-immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2

Toll-like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro-inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium-transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca(2+) handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non-immune cells--including cardiomyocytes--using the same ligand-receptor system