No abstract

The study of human cardiac tissue development is hampered by the lack of a suitable in vitro model. We describe the phenotypic properties of cardiomyocytes derived from human embryonic stem (ES) cells. Human ES cells were cultivated in suspension and plated to form aggregates termed embryoid bodies (EBs). Spontaneously contracting areas appeared in 8.1% of the EBs. Cells from the spontaneously contracting areas within EBs were stained positively with anti-cardiac myosin heavy chain, anti--alpha-actinin, anti-desmin, anti--cardiac troponin I (anti-cTnI), and anti-ANP antibodies. Electron microscopy revealed varying degrees of myofibrillar organization, consistent with early-stage cardiomyocytes. RT-PCR studies demonstrated the expression of several cardiac-specific genes and transcription factors. Extracellular electrograms were characterized by a sharp component lasting 30 +/- 25 milliseconds, followed by a slow component of 347 +/- 120 milliseconds. Intracellular Ca(2+) transients displayed a sharp rise lasting 130 +/- 27 milliseconds and a relaxation component lasting 200--300 milliseconds. Positive and negative chronotropic effects were induced by application of isoproterenol and carbamylcholine, respectively. In conclusion, the human ES cell--derived cardiomyocytes displayed structural and functional properties of early-stage cardiomyocytes. Establishment of this unique differentiation system may have significant impact on the study of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering.

Abstract-A major function of haptoglobin (Hp) is to bind hemoglobin (Hb) to form a stable Hp-Hb complex and thereby prevent Hb-induced oxidative tissue damage. Clearance of the Hp-Hb complex can be mediated by the monocyte/macrophage scavenger receptor CD163. We recently demonstrated that diabetic individuals homozygous for the Hp 2 allele (Hp 2-2) were at 500% greater risk of cardiovascular disease (CVD) compared with diabetic individuals homozygous for the Hp 1 allele (Hp 1-1). No differences in risk by Hp type were seen in individuals without diabetes. To understand the relationship between the Hp polymorphism and diabetic CVD, we sought to identify differences in antioxidant and scavenging functions between the Hp types and to determine how these functions were modified in diabetes. The scavenging function of Hp was assessed using rhodamine-tagged and 125 I-Hp in cell lines stably transfected with CD163 and in macrophages expressing endogenous CD163. We found that the rate of clearance of Hp 1-1-Hb by CD163 is markedly greater than that of Hp 2-2-Hb. Diabetes is associated with an increase in the nonenzymatic glycosylation of serum proteins, including Hb. The antioxidant function of Hp was assessed with glycosylated and nonglycosylated Hb. We identified a severe impairment in the ability of Hp to prevent oxidation mediated by glycosylated Hb. We propose that the specific interaction between diabetes, CVD, and Hp genotype is the result of the heightened urgency of rapidly clearing glycosylated Hb-Hp complexes from the subendothelial space before they can oxidatively modify low-density lipoprotein to atherogenic oxidized low-density lipoprotein.

Since cardiac transplantation is limited by the small availability of donor organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. To determine the compatibility of human embryonic stem cellderived cardiomyocytes (hESC-CMs) (7 to 55 days old) with the myocardium, we investigated their functional properties regarding intracellular Ca ] i transient and contraction, indicating that at this developmental stage, contraction depends on transsarcolemmal Ca 2؉ influx rather than on sarcoplasmic reticulum Ca 2؉ release. Third, in agreement with the notion that a voltage-dependent Ca 2؉ current is present in hESC-CMs and contributes to the mechanical function, verapamil completely blocked contraction. Fourth, whereas hESC-CMs expressed SERCA2 and NCX at levels comparable to those of the adult porcine myocardium, calsequestrin and phospholamban were not expressed. Our study shows for the first time that functional properties related to intracellular Ca 2؉ handling of hESC-CMs differ markedly from the adult myocardium, probably due to immature sarcoplasmic reticulum capacity. STEM CELLS 2006;24:236 -245

Sudden cardiac death caused by ventricular arrhythmias is a disastrous event, especially when it occurs in young individuals. Among the five major arrhythmogenic disorders occurring in the absence of a structural heart disease is catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a highly lethal form of inherited arrhythmias. Our study focuses on the autosomal recessive form of the disease caused by the missense mutation D307H in the cardiac calsequestrin gene, CASQ2. Because CASQ2 is a key player in excitation contraction coupling, the derangements in intracellular Ca2+ handling may cause delayed afterdepolarizations (DADs), which constitute the mechanism underlying CPVT. To investigate catecholamine-induced arrhythmias in the CASQ2 mutated cells, we generated for the first time CPVT-derived induced pluripotent stem cells (iPSCs) by reprogramming fibroblasts from skin biopsies of two patients, and demonstrated that the iPSCs carry the CASQ2 mutation. Next, iPSCs were differentiated to cardiomyocytes (iPSCs-CMs), which expressed the mutant CASQ2 protein. The major findings were that the β-adrenergic agonist isoproterenol caused in CPVT iPSCs-CMs (but not in the control cardiomyocytes) DADs, oscillatory arrhythmic prepotentials, after-contractions and diastolic [Ca2+]i rise. Electron microscopy analysis revealed that compared with control iPSCs-CMs, CPVT iPSCs-CMs displayed a more immature phenotype with less organized myofibrils, enlarged sarcoplasmic reticulum cisternae and reduced number of caveolae. In summary, our results demonstrate that the patient-specific mutated cardiomyocytes can be used to study the electrophysiological mechanisms underlying CPVT.

In view of the therapeutic potential of cardiomyocytes derived from induced pluripotent stem (iPS) cells (iPS-derived cardiomyocytes), in the present study we investigated in iPS-derived cardiomyocytes, the functional properties related to [Ca2+]i handling and contraction, the contribution of the sarcoplasmic reticulum (SR) Ca2+ release to contraction and the b-adrenergic inotropic responsiveness. The two iPS clones investigated here were generated through infection of human foreskin fibroblasts (HFF) with retroviruses containing the four human genes: OCT4, Sox2, Klf4 and C-Myc. Our major findings showed that iPS-derived cardiomyocytes: (i) express cardiac specific RNA and proteins; (ii) exhibit negative force–frequency relations and mild (compared to adult) post-rest potentiation; (iii) respond to ryanodine and caffeine, albeit less than adult cardiomyocytes, and express the SR-Ca2+ handling proteins ryanodine receptor and calsequestrin. Hence, this study demonstrates that in our cardiomyocytes clones differentiated from HFF-derived iPS, the functional properties related to excitation–contraction coupling, resemble in part those of adult cardiomyocytes.

Induced pluripotent stem cells (iPSC) are generated from fully differentiated somatic cells that were reprogrammed into a pluripotent state. Human iPSC which can be obtained from various types of somatic cells such as fibroblasts or keratinocytes can differentiate into cardiomyocytes (iPSC-CM), which exhibit cardiac-like transmembrane action potentials, intracellular Ca2+ transients and contractions. While major features of the excitation-contraction coupling of iPSC-CM have been well-described, very little is known on the ultrastructure of these cardiomyocytes. The ultrastructural features of 31-day-old (post-plating) iPSC-CM generated from human hair follicle keratinocytes (HFKT-iPSC-CM) were analysed by electron microscopy, and compared with those of human embryonic stem-cell-derived cardiomyocytes (hESC-CM). The comparison showed that cardiomyocytes from the two sources share similar proprieties. Specifically, HFKT-iPSC-CM and hESC-CM, displayed ultrastructural features of early and immature phenotype: myofibrils with sarcomeric pattern, large glycogen deposits, lipid droplets, long and slender mitochondria, free ribosomes, rough endoplasmic reticulum, sarcoplasmic reticulum and caveolae. Noteworthy, the SR is less developed in HFKT-iPSC-CM. We also found in both cell types: (1) ‘Ca2+-release units’, which connect the peripheral sarcoplasmic reticulum with plasmalemma; and (2) intercellular junctions, which mimic intercalated disks (desmosomes and fascia adherens). In conclusion, iPSC and hESC differentiate into cardiomyocytes of comparable ultrastructure, thus supporting the notion that iPSC offer a viable option for an autologous cell source for cardiac regenerative therapy.

Background-The sinoatrial node is the main impulse-generating tissue in the heart. Atrioventricular conduction block and arrhythmias caused by sinoatrial node dysfunction are clinically important and generally treated with electronic pacemakers. Although an excellent solution, electronic pacemakers incorporate limitations that have stimulated research on biological pacing. To assess the suitability of potential biological pacemakers, we tested the hypothesis that the spontaneous electric activity of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) exhibit beat rate variability and power-law behavior comparable to those of human sinoatrial node. Methods and Results-We recorded extracellular electrograms from hESC-CMs and iPSC-CMs under stable conditions for up to 15 days. The beat rate time series of the spontaneous activity were examined in terms of their power spectral density and additional methods derived from nonlinear dynamics. The major findings were that the mean beat rate of hESC-CMs and iPSC-CMs was stable throughout the 15-day follow-up period and was similar in both cell types, that hESC-CMs and iPSC-CMs exhibited intrinsic beat rate variability and fractal behavior, and that isoproterenol increased and carbamylcholine decreased the beating rate in both hESC-CMs and iPSC-CMs. Conclusions-This is the first study demonstrating that hESC-CMs and iPSC-CMs exhibit beat rate variability and power-law behavior as in humans, thus supporting the potential capability of these cell sources to serve as biological pacemakers. Our ability to generate sinoatrial-compatible spontaneous cardiomyocytes from the patient's own hair (via keratinocyte-derived iPSCs), thus eliminating the critical need for immunosuppression, renders these myocytes an attractive cell source as biological pacemakers. (Circulation. 2012;125:883-893.)