Cardiac stem cells: translation to human studies

The discovery of multiple classes of cardiac progenitor cells in the adult mammalian heart has generated hope for their use as a therapeutic in heart failure. However, successful results from animal models have not always yielded similar findings in human studies. Recent Phase I/II trials of c-Kit (SCIPIO) and cardiosphere-based (CADUCEUS) cardiac progenitor cells have demonstrated safety and some therapeutic efficacy. Gaps remain in our understanding of the origins, function and relationships between the different progenitor cell families, many of which are heterogeneous populations with overlapping definitions. Another challenge lies in the limitations of small animal models in replicating the human heart. Cryopreserved human cardiac tissue provides a readily available source of cardiac progenitor cells and may help address these questions. We review important findings and relative unknowns of the main classes of cardiac progenitor cells, highlighting differences between animal and human studies     

人类心脏发育缺陷的分子机理

为了探索心脏发育的缺陷及遗传机制以及在分子水平上先天性心脏病的发生机理,概述了人类心脏发育缺陷的研究进展,包括人类心脏不对称发育、心脏发育缺陷和心脏缺陷的分子机理.     

Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming

Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes, both in vitro and in vivo. Beyond uses in cell replacement therapy, patient-specific cardiomyocytes may find applications in drug testing, drug discovery, and disease modeling. Recently, approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs), adult heart-derived cardiac progenitor cells (CPCs), and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts, highlighting potential applications and future challenges.     

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes

Summary Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14–15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin andα-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycinc centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells my provide a useful experimental system for the study of human cardiac muscle cell biology.     

Zebrafish heart as a model for human cardiac electrophysiology

The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.     

Programming and reprogramming a human heart cell

The latest discoveries and advanced knowledge in the fields of stem cell biology and developmental cardiology hold great promise for cardiac regenerative medicine, enabling researchers to design novel therapeutic tools and approaches to regenerate cardiac muscle for diseased hearts. However, progress in this arena has been hampered by a lack of reproducible and convincing evidence, which at best has yielded modest outcomes and is still far from clinical practice. To address current controversies and move cardiac regenerative therapeutics forward, it is crucial to gain a deeper understanding of the key cellular and molecular programs involved in human cardiogenesis and cardiac regeneration. In this review, we consider the fundamental principles that govern the “programming” and “reprogramming” of a human heart cell and discuss updated therapeutic strategies to regenerate a damaged heart.     

Efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.     

Activation of calcineurin expression in ischemia-reperfused rat heart and in human ischemic myocardium

Calcineurin (CaN) has been reported as a critical mediator for cardiac hypertrophy and cardiac myocyte apoptosis. In the present study, we investigated the activity and expression of CaN and the effect of calpain in rat heart after ischemia and reperfusion. Rat ischemic heart showed significant increase in CaN activity. Western blot analysis of normal rat heart extract with a polyclonal antibody raised against bovine CaN indicated a prominent immunoreactive band of 60 kDa (CaN A). In ischemic-reperfused hearts, the expression of CaN A was significantly low and immunoreactivity was observed in proteolytic bands of 46 kDa. This may be due to the proteolytic degradation of CaN A in ischemic tissues by m-calpain. We also noticed in vitro proteolysis of bovine cardiac CaN A by m-calpain. Immunohistochemical studies showed strong staining of immunoreactivity in rat hearts that had gone under 30 min ischemia followed by 30 min reperfusion similar to that found in human ischemic heart. Ischemia is associated with multiple alterations in the extracellular and intracellular signaling of cardiomyocytes and may act as an inducer of apoptosis. The increase in CaN activity and strong immunostaining observed in ischemic/perfused rat heart may be due to the calpain-mediated proteolysis of this phosphatase.     

Cellular repressor of E1A-stimulated genes attenuates cardiac hypertrophy and fibrosis

Cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein of 220 amino acids. It has been proposed that CREG acts as a ligand that enhances differentiation and/or reduces cell proliferation. CREG has been shown previously to attenuate cardiac hypertrophy in vitro . However, such a role has not been determined in vivo . In the present study, we tested the hypothesis that overexpression of CREG in the murine heart would protect against cardiac hypertrophy and fibrosis in vivo . The effects of constitutive human CREG expression on cardiac hypertrophy were investigated using both in vitro and in vivo models. Cardiac hypertrophy was produced by aortic banding and infusion of angiotensin II in CREG transgenic mice and control animals. The extent of cardiac hypertrophy was quantitated by two-dimensional and M-mode echocardiography as well as by molecular and pathological analyses of heart samples. Constitutive over-expression of human CREG in the murine heart attenuated the hypertrophic response, markedly reduced inflammation. Cardiac function was also preserved in hearts with increased CREG levels in response to hypertrophic stimuli. These beneficial effects were associated with attenuation of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase 1 (MEK-ERK1)/2-dependent signalling cascade. In addition, CREG expression blocked fibrosis and collagen synthesis through blocking MEK-ERK1/2-dependent Smad 2/3 activation in vitro and in vivo . Therefore, the expression of CREG improves cardiac functions and inhibits cardiac hypertrophy, inflammation and fibrosis through blocking MEK-ERK1/2-dependent signalling.     

基于能量融合积分模型的心脏生理物理活动研究

为了更深入地了解目前靠生理实验及临床手段无法洞察的心脏三维空间的电生理运行机制,分析和表现心脏复杂的电生理活动,从而揭示心脏的生理物理特性,本研究通过人类心肌细胞的动作电位传导数学模型,结合基于心脏解剖数据所建立的真实心脏组织结构的三维空间模型,构建出精细的心脏生物物理融合模型,并将心脏在三维空间中的生物物理活动表现出来.实验结果表明,基于心脏动作电位传导的融合模型,不同时刻的动作电位传导在非匀质性组织内的三维空间中的传播位置、空间关系以及生物物理过程被清晰地显示出来,心脏研究人员从而能够以视觉感知的方式认识和深入理解人类心脏电生物物理系统的功能机制,并有助于进一步推测心脏的生理和病理反应.     

Cardiac differentiation of human pluripotent stem cells

Due to the extremely limited proliferative capacity of adult cardiomyocytes, human embryonic (pluripotent) stem cell derived cardiomyocytes (hESC-CMs) are currently almost the only reliable source of human heart cells which are suited to large-scale production. These cells have the potential for wide-scale application in drug discovery, heart disease research and cell-based heart repair. Embryonic atrial-, ventricular- and nodal-like cardiomyocytes can be obtained from differentiated human embryonic stem cells (hESCs). In recent years, several highly efficient cardiac differentiation protocols have been developed. Significant progress has also been made on understanding cardiac subtype specification, which is the key to reducing the heterogeneity of hESC-CMs, a major obstacle to the utilization of these cells in medical research and future cell-based replacement therapies. Herein we review recent progress in cardiac differentiation of hESCs and cardiac subtype specification, and discuss potential applications in drug screening and cell-based heart regeneration.     

Sarcoplasmic reticulum calsequestrins: Structural and functional properties

Calsequestrin is the major Ca²⁺-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its -helical content and the intrinsic fluorescence upon binding of Ca²⁺. Calsequestrin binds Ca²⁺ with high-capacity and with moderate affinity and it functions as a Ca²⁺ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca²⁺ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsquestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.     

Tbx18+心外膜祖细胞参与成年小鼠部分心脏组织形成

转录因子Tbx18在胚胎心脏发育过程中起重要调控作用,是心外膜祖细胞标记之一|故以Tbx18为标记的阳性祖细胞群被称为：Tbx18+心外膜祖细胞(epicardial progenitor cells, EPCs)。小鼠胚胎、新生和成年期心脏组织细胞的特性区别较大,成年小鼠的心脏属于终末分化组织。但是,Tbx18+EPCs对成年小鼠心脏组织的贡献大小尚存争议。本研究拟定量分析Tbx18+EPCs对成年小鼠心脏组织的贡献大小。采用整体和组织切片X-gal染色检测成年心脏组织LacZ的表达|荧光激活细胞分选法(fluorescence activated cell sorting,FACS)分离成年Tbx18Cre/R26EYFP小鼠心脏组织EYFP+细胞。结果显示,在Tbx18+EPCs遗传谱系示踪小鼠,报告基因LacZ和EYFP在成年小鼠心脏的心室、心房、冠状动脉、室间隔等处表达|成年Tbx18Cre/R26EYFP小鼠心脏组织细胞用FACS分离,分选的EYFP+细胞比例平均约为33.94%。由此可见,成年小鼠心脏的心室、心房、冠状动脉、室间隔等心脏组织均可来源于Tbx18+EPCs|约1/3成年小鼠心脏组织细胞来源于Tbx18+EPCs。故Tbx18+EPCs参与成年小鼠心脏组织的部分形成。     

Serdin1/Lrrc10 is dispensable for mouse development

We have previously identified Serdin1/Lrrc10 as a cardiac-specific message that is expressed early in murine heart development and encodes a novel leucine-rich protein. A high degree of evolutionary conservation with respect to protein sequence, cardiac-specific expression, and cis-regulatory elements suggested that LRRC10 has an important and conserved function in cardiac development. Recently, the zebrafish lrrc10 knockdown models were described with a dramatic early defect in heart looping which supported the notion that Serdin1/Lrrc10 is likely to be essential for heart development in all vertebrates. To determine Lrrc10 function in mammalian cardiac development, we have disrupted the Lrrc10 gene in mice. We report here that, in striking contrast to the zebrafish lrrc10 knockdown, Lrrc10-null mice develop normally and exhibit no discernable phenotype.     

ATP-dependent chromatin remodeling complex SWI/SNF in cardiogenesis and cardiac progenitor cell development

The recent identification of cardiac progenitor cells (CPCs) provides a new paradigm for studying and treating heart disease.To realize the full potential of CPCs for therapeutic purposes,it is essenti...     

Oxygen free radicals in volume overload heart failure

It has been suggested that oxygen free radicals (OFR) depress the excitation-contraction coupling in cardiac muscle. It is possible that a decrease in the cardiac contractility in the failing heart may be due to an increased OFR producing activity of polymorphonuclear (PMN) leukocytes. We studied the OFR producing activity (chemiluminescence) of PMN leukocytes from blood in dogs with heart failure due to chronic volume overload. The animals were divided into two groups: I) normal, (n = 10): II) dogs with mitral insufficiency (MI) of 6 to 9 months duration, (n = 10). Hemodynamic studies were done to establish the presence of heart failure. Blood samples were collected to measure PMN leukocyte chemiluminescence. There was a decrease in the cardiac index and index of myocardial contractility (dp/dt/IIP) and an increase in the left ventricular end-diastolic pressure in dogs with MI indicating left ventricular failure. The peak chemiluminescent activity of the PMN leukocytes in blood of dogs with failure was about four folds greater than that in the blood from normal dogs. These results suggest that there may be an increased OFR generation in dogs with volume overload heart failure. The decrease in the myocardial contractility in the failing heart might be due to an increase in the OFR produced by the PMN leukocytes.