Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 19 de 19
Filter
Add more filters










Publication year range
1.
Int J Mol Sci ; 21(19)2020 09 29.
Article in English | MEDLINE | ID: mdl-33003641

ABSTRACT

The limited regenerative capacity of the injured myocardium leads to remodeling and often heart failure. Novel therapeutic approaches are essential. Induced pluripotent stem cells (iPSC) differentiated into cardiomyocytes are a potential future therapeutics. We hypothesized that organ-specific reprogramed fibroblasts may serve an advantageous source for future cardiomyocytes. Moreover, exosomes secreted from those cells may have a beneficial effect on cardiac differentiation and/or function. We compared RNA from different sources of human iPSC using chip gene expression. Protein expression was evaluated as well as exosome micro-RNA levels and their impact on embryoid bodies (EBs) differentiation. Statistical analysis identified 51 genes that were altered (p ≤ 0.05), and confirmed in the protein level, cardiac fibroblasts-iPSCs (CF-iPSCs) vs. dermal fibroblasts-iPSCs (DF-iPSCs). Several miRs were altered especially miR22, a key regulator of cardiac hypertrophy and remodeling. Lower expression of miR22 in CF-iPSCs vs. DF-iPSCs was observed. EBs treated with these exosomes exhibited more beating EBs p = 0.05. vs. control. We identify CF-iPSC and its exosomes as a potential source for cardiac recovery induction. The decrease in miR22 level points out that our CF-iPSC-exosomes are naïve of congestive heart cell memory, making them a potential biological source for future therapy for the injured heart.


Subject(s)
Exosomes/genetics , Heart Failure/therapy , Induced Pluripotent Stem Cells/metabolism , Myocardium/metabolism , Cell Differentiation/genetics , Exosomes/metabolism , Fibroblasts/metabolism , Heart/physiopathology , Heart Failure/genetics , Heart Failure/pathology , Humans , MicroRNAs/genetics , Myocardium/pathology , Myocytes, Cardiac/metabolism
2.
Heart Rhythm ; 13(12): 2379-2387, 2016 12.
Article in English | MEDLINE | ID: mdl-27639456

ABSTRACT

BACKGROUND: Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs), only 3 types of action potentials (APs) exist: nodal-, atrial-, and ventricular-like. OBJECTIVES: To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) During culture development a cardiac precursor cell is present that-depending on age-can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of a nodal phenotype, transient appearance of an atrial phenotype, evolution to a ventricular phenotype, and persistence of transitional phenotypes. METHODS: To test these hypotheses, we (1) performed fluorescence-activated cell sorting analysis of nodal, atrial, and ventricular markers; (2) recorded APs from 280 7- to 95-day-old iPSC-CMs; and (3) analyzed AP characteristics. RESULTS: The major findings were as follows: (1) fluorescence-activated cell sorting analysis of 30- and 60-day-old cultures showed that an iPSC-CMs population shifts from the nodal to the atrial/ventricular phenotype while including significant transitional populations; (2) the AP population did not consist of 3 phenotypes; (3) culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57-70 days) appearance of the atrial phenotype; and (4) beat rate variability was more prominent in nodal than in ventricular cardiomyocytes, while pacemaker current density increased in older cultures. CONCLUSION: From the onset of development in culture, the iPSC-CMs population includes nodal, atrial, and ventricular APs and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial, which appears only transiently yet dominates at 57-70 days of evolution.


Subject(s)
Action Potentials/physiology , Atrial Function/physiology , Atrioventricular Node/physiology , Cell Transdifferentiation/physiology , Induced Pluripotent Stem Cells/physiology , Myocytes, Cardiac/physiology , Ventricular Function/physiology , Cell Differentiation/physiology , Cells, Cultured , Electrophysiological Phenomena , Humans
3.
Can J Cardiol ; 30(11): 1279-87, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25442431

ABSTRACT

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the capacity to differentiate into any specialized cell type, including cardiomyocytes. Therefore, hESC-derived and hiPSC-derived cardiomyocytes (hESC-CMs and hiPSC-CMs, respectively) offer great potential for cardiac regenerative medicine. Unlike some organs, the heart has a limited ability to regenerate, and dysfunction resulting from significant cardiomyocyte loss under pathophysiological conditions, such as myocardial infarction (MI), can lead to heart failure. Unfortunately, for patients with end-stage heart failure, heart transplantation remains the main alternative, and it is insufficient, mainly because of the limited availability of donor organs. Although left ventricular assist devices are progressively entering clinical practice as a bridge to transplantation and even as an optional therapy, cell replacement therapy presents a plausible alternative to donor organ transplantation. During the past decade, multiple candidate cells were proposed for cardiac regeneration, and their mechanisms of action in the myocardium have been explored. The purpose of this article is to critically review the comprehensive research involving the use of hESCs and hiPSCs in MI models and to discuss current controversies, unresolved issues, challenges, and future directions.


Subject(s)
Embryonic Stem Cells/transplantation , Heart Diseases/therapy , Induced Pluripotent Stem Cells/transplantation , Myocytes, Cardiac/pathology , Stem Cell Transplantation/methods , Cell Differentiation , Heart Diseases/pathology , Humans
4.
Tissue Eng Part A ; 20(19-20): 2756-67, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25185111

ABSTRACT

Extracellular matrix (ECM) has been utilized as a biological scaffold for tissue engineering applications in a variety of body systems, due to its bioactivity and biocompatibility. In the current study we developed a modified protocol for the efficient and reproducible derivation of mesenchymal progenitor cells (MPCs) from human embryonic stem cells as well as human induced pluripotent stem cells (hiPSCs) originating from hair follicle keratinocytes (HFKTs). ECM was produced from these MPCs and characterized in comparison to adipose mesenchymal stem cell ECM, demonstrating robust ECM generation by the excised HFKT-iPSC-MPCs. Exploiting the advantages of electrospinning we generated two types of electrospun biodegradable nanofiber layers (NFLs), fabricated from polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA), which provide mechanical support for cell seeding and ECM generation. Elucidating the optimized decellularization treatment we were able to generate an available "off-the-shelf" implantable product (NFL-ECM). Using rat subcutaneous transplantation model we demonstrate that this stem-cell-derived construct is biocompatible and biodegradable and holds great potential for tissue regeneration applications.


Subject(s)
Extracellular Matrix/chemistry , Induced Pluripotent Stem Cells/metabolism , Lactic Acid/chemistry , Nanofibers/chemistry , Polyesters/chemistry , Polyglycolic Acid/chemistry , Regenerative Medicine , Animals , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Hair Follicle/cytology , Hair Follicle/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Keratinocytes/cytology , Keratinocytes/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Mice, SCID , Polylactic Acid-Polyglycolic Acid Copolymer , Rats
6.
Pharmacol Ther ; 134(2): 180-8, 2012 May.
Article in English | MEDLINE | ID: mdl-22269465

ABSTRACT

The attrition rates of drugs in development, many of which attributed to unforeseen cardiotoxic side effects of the drugs being tested in humans that were not realized in preclinical animal models, are a significant problem facing the pharmaceutical industry. Recent advances in human stem cell biology have paved the way for incorporating human cell models into the two key aspects of developing new drugs: discovering new effective entities and screening for their safety. Functional cardiomyocytes can now be derived from human pluripotent stem cells (hPSCs), including both embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells. Moreover, recent studies demonstrate the ability of cardiomyocytes derived from patients' iPSCs to recapitulate the phenotype of several known cardiac diseases. In the present review we describe the knowledge recently gained on this promising human cell source in order to fulfill its potential as a useful tool for drug screening.


Subject(s)
Drug Evaluation, Preclinical/methods , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Animals , Humans
7.
Circulation ; 125(1): 87-99, 2012 Jan 03.
Article in English | MEDLINE | ID: mdl-22095829

ABSTRACT

BACKGROUND: Pericytes represent a unique subtype of microvessel-residing perivascular cells with diverse angiogenic functions and multilineage developmental features of mesenchymal stem cells. Although various protocols for derivation of endothelial and/or smooth muscle cells from human pluripotent stem cells (hPSC, either embryonic or induced) have been described, the emergence of pericytes in the course of hPSC maturation has not yet been elucidated. METHODS AND RESULTS: We found that during hPSC development, spontaneously differentiating embryoid bodies give rise to CD105(+)CD90(+)CD73(+)CD31(-) multipotent clonogenic mesodermal precursors, which can be isolated and efficiently expanded. Isolated and propagated cells expressed characteristic pericytic markers, including CD146, NG2, and platelet-derived growth factor receptor ß, but not the smooth muscle cell marker α-smooth muscle actin. Coimplantation of hPSC-derived endothelial cells with pericytes resulted in functional and rapid anastomosis to the murine vasculature. Administration of pericytes into immunodeficient mice with limb ischemia promoted significant vascular and muscle regeneration. At day 21 after transplantation, recruited hPSC pericytes were found incorporated into recovered muscle and vasculature. CONCLUSIONS: Derivation of vasculogenic and multipotent pericytes from hPSC can be used for the development of vasculogenic models using multiple vasculogenic cell types for basic research and drug screening and can contribute to angiogenic regenerative medicine.


Subject(s)
Extremities/blood supply , Ischemia/surgery , Multipotent Stem Cells/transplantation , Pericytes/transplantation , Pluripotent Stem Cells/transplantation , Recovery of Function/physiology , Animals , Endothelial Cells/transplantation , Extremities/surgery , Humans , Ischemia/pathology , Male , Mice , Mice, Inbred NOD , Mice, Nude , Mice, SCID
8.
J Cell Mol Med ; 16(3): 468-82, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22050625

ABSTRACT

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 Ca(2+) 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 ß-adrenergic agonist isoproterenol caused in CPVT iPSCs-CMs (but not in the control cardiomyocytes) DADs, oscillatory arrhythmic prepotentials, after-contractions and diastolic [Ca(2+) ](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.


Subject(s)
Adrenergic beta-Agonists/pharmacology , Calsequestrin/genetics , Isoproterenol/pharmacology , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Tachycardia, Ventricular/pathology , Adult , Calcium/metabolism , Calcium Signaling , Calsequestrin/metabolism , Cell Differentiation , Child , Excitation Contraction Coupling , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Genes, Recessive , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Male , Membrane Potentials , Mutation, Missense , Myocardium/pathology , Myocytes, Cardiac/pathology , Tachycardia, Ventricular/genetics , Tachycardia, Ventricular/metabolism
9.
J Cell Mol Med ; 15(1): 38-51, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20041972

ABSTRACT

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 [Ca(2+) ](i) handling and contraction, the contribution of the sarcoplasmic reticulum (SR) Ca(2+) 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-Ca(2+) 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.


Subject(s)
Induced Pluripotent Stem Cells/cytology , Myocytes, Cardiac/metabolism , Animals , Caffeine/pharmacology , Calcium/metabolism , Calsequestrin/genetics , Calsequestrin/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Fibroblasts/metabolism , Fluorescent Antibody Technique , Foreskin/cytology , Gene Expression , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Male , Mice , Mice, SCID , Myocardial Contraction/drug effects , Myocytes, Cardiac/drug effects , Octamer Transcription Factor-3/genetics , Proto-Oncogene Proteins c-myc/genetics , RNA, Messenger/metabolism , Ryanodine/pharmacology , Ryanodine Receptor Calcium Release Channel/genetics , Ryanodine Receptor Calcium Release Channel/metabolism , SOXB1 Transcription Factors/genetics , Sarcoplasmic Reticulum/metabolism , Teratoma/metabolism , Teratoma/pathology
10.
Cell Reprogram ; 12(6): 665-78, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20964482

ABSTRACT

Induced pluripotent stem cells (iPSCs) represent an ideal cell source for future cell therapy and regenerative medicine. However, most iPSC lines described to date have been isolated from skin fibroblasts or other cell types that require harvesting by surgical intervention. Because it is desirable to avoid such intervention, an alternative cell source that can be readily and noninvasively isolated from patients and efficiently reprogrammed, is required. Here we describe a detailed and reproducible method to derive iPSCs from plucked human hair follicle keratinocytes (HFKTs). HFKTs were isolated from single plucked hair, then expanded and reprogrammed by a single polycistronic excisable lentiviral vector. The reprogrammed HFKTs were found to be very sensitive to human embryonic stem cell (hESC) growth conditions, generating a built-in selection with easily obtainable and very stable iPSCs. All emerging colonies were true iPSCs, with characteristics typical of human embryonic stem cells, differentiated into derivatives of all three germ layers in vitro and in vivo. Spontenaeouly differentiating functional cardiomyocytes (CMs) were successfully derived and characterized from these HFKT-iPSCs. The contracting CMs exhibited well-coordinated intracellular Ca²+ transients and contractions that were readily responsive to ß-adrenergic stimulation with isoproterenol. The introduction of Cre-recombinase to HFKT-iPSC clones was able to successfully excise the integrated vector and generate transgene-free HFKT-iPSC clone that could be better differentiated into contracting CMs, thereby revealing the desired cells for modeling human diseases. Thus, HFKTs are easily obtainable, and highly reprogrammed human cell source for all iPSC applications.


Subject(s)
Cell Differentiation/physiology , Hair Follicle/chemistry , Heart/embryology , Keratinocytes/physiology , Lentivirus/metabolism , Myocardium/cytology , Cell Culture Techniques/methods , Cells, Cultured , Humans , Keratinocytes/cytology , Lentivirus/genetics , Patch-Clamp Techniques , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/physiology
11.
Gen Physiol Biophys ; 29(3): 222-33, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20817946

ABSTRACT

Gap junctions produce low resistance pathways between cardiomyocytes and are major determinants of electrical conduction in the heart. Altered distribution and function of connexin 43 (Cx43), the major gap junctional protein in the ventricles, can slow conduction, and thus contribute to arrhythmogenesis in experimental models such as ischemic rat heart and pacing-induced atrial fibrillation. The mechanisms underlying reduced gap junctional density and conductance during ischemia may involve decreased Cx43 synthesis, increased degradation and/or Cx43 migration into areas which do not contribute to intercellular communication. To test more rigorously the hypothesis that hypoxia resulting from ischemia causes Cx43 internalization, we infected neonatal rat ventricular myocytes (NRVM) with a Cx43-GFP chimera, which enabled us to investigate by means of confocal microscopy the effect of hypoxia (1% O2 for 5 h) on Cx43 distribution in live cardiomyocytes. Importantly, this protocol permitted each culture to serve as its own control. To this end we used life confocal microscopy analysis to determine in the same pair of myocytes the effects of hypoxia on Cx43-GFP distribution at the gap junctional (GJ) and non-GJ areas. In support of this hypothesis, we found that compared to normoxia, 5 h of hypoxia reduced the Cx43-GFP signal at the GJ areas (defined as the border area) and caused a corresponding increase in the Cx43-GFP signal at the non-border areas, thus providing an additional explanation for the intercellular uncoupling during ischemic conditions.


Subject(s)
Cell Hypoxia/physiology , Connexin 43/metabolism , Myocytes, Cardiac/metabolism , Animals , Animals, Newborn , Biological Transport, Active , Cells, Cultured , Connexin 43/genetics , Gap Junctions/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Microscopy, Confocal , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transfection
12.
Crit Rev Eukaryot Gene Expr ; 20(1): 51-9, 2010.
Article in English | MEDLINE | ID: mdl-20528737

ABSTRACT

Cardiovascular diseases are the most frequent cause of death in the industrialized world, with the main contributor being myocardial infarction. Given the high morbidity and mortality rates associated with congestive heart failure, the shortage of donor hearts for transplantation, complications resulting from immunosuppression, and long-term failure of transplanted organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. Because of their remarkable capacity for expansion and unquestioned cardiac potential, pluripotent human embryonic stem cells (hESC) represent an attractive candidate cell source for obtaining cardiomyocytes. Moreover, a number of recent reports have shown that hESC-derived cardiomyocytes (hESC-CM) survive after transplantation into infarcted rodent hearts, form stable cardiac implants, and result in preserved contractile function. Although the latter successes give good reason for optimism, considerable challenges remain in the successful application of hESC-CM to cardiac repair. Because it is desired that the transplanted cells fully integrate within the diseased myocardium, contribute to its contractile performance, and respond appropriately to various physiological stimuli, it is of crucial importance to be familiar with their functional properties. Therefore, this review describes the characteristics of hESC-CM, including their transcriptional profile, structural and electrophysiological properties, ion channel expression, excitation-contraction coupling, and neurohumoral responsiveness.


Subject(s)
Embryonic Stem Cells/transplantation , Myocardial Infarction/surgery , Myocytes, Cardiac/cytology , Myocytes, Cardiac/transplantation , Adult , Calcium Channels, L-Type/genetics , Electrophysiology/methods , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Humans , Myocardial Contraction/physiology , Myocardium/cytology , Myocytes, Cardiac/physiology , Receptors, Adrenergic, beta/genetics , Receptors, Adrenergic, beta/physiology , Receptors, Muscarinic/genetics , Receptors, Muscarinic/physiology , Regeneration , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transcription, Genetic
13.
Ann N Y Acad Sci ; 1188: 68-77, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20201888

ABSTRACT

Because previous findings showed that in human embryonic stem cell-derived cardiomyocytes (hESC-CM) the machinery for Ca2+-induced release of calcium is immature, we tested the hypothesis that hESC-CM contain functional 1,4,5-inositol triphosphate (IP3)-operated intracellular Ca2+ ([Ca2+]i) stores. We investigated the effects of angiotensin II (AT-II) and endothelin 1 (ET-1), which activate the 1,4,5-IP3 pathway, on [Ca2+]i transients and contractions in hESC-CM. Our major findings were that in hESC-CM, both AT-II (10(-9)-10(-7) M) and ET-1 (10(-9)-10(-7) M) exert inotropic and lusitropic effects. The involvement of 1,4,5-IP3-dependent intracellular Ca2+ release in AT-I-induced effects was supported by these findings: the effects of AT-II were blocked by 2-aminoethoxyphenyl borate (2-APB, a 1,4,5-IP3 receptor blocker) and U73122 (a phosopholipase C blocker); and hESC-CM express AT-II type 1 and IP3 type I and II receptors as determined by fluorescence immunostaining. In conclusion, hESC-CM exhibit functional AT-II and ET-1 signaling pathways, as well as 1,4,5-IP3-operated releasable Ca2+ stores.


Subject(s)
Angiotensin II/metabolism , Calcium/metabolism , Cell Differentiation , Embryonic Stem Cells/metabolism , Endothelin-1/metabolism , Myocytes, Cardiac/metabolism , Signal Transduction , Animals , Cell Line , Embryonic Stem Cells/cytology , Humans , Mice , Mice, Inbred ICR , Myocytes, Cardiac/cytology
14.
Stem Cells ; 26(12): 3130-8, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18818435

ABSTRACT

On the basis of previous findings suggesting that in human embryonic stem cell-derived cardiomyocytes (hESC-CM) the sarcoplasmic reticulum Ca(2+)-induced release of calcium machinery is either absent or immature, in the present study we tested the hypothesis that hESC-CM contain fully functional 1,4,5-inositol trisphosphate (1,4,5-IP(3))-operated intracellular Ca(2+) ([Ca(2+)](i)) stores that can be mobilized upon appropriate physiological stimuli. To test this hypothesis we investigated the effects of angiotensin-II (AT-II) and endothelin-1 (ET-1), which activate the 1,4,5-IP(3) pathway, on [Ca(2+)](i) transients and contractions in beating clusters of hESC-CM. Our major findings were that in paced hESC-CM both AT-II and ET-1 (10(-9) to 10(-7) M) increased the contraction amplitude and the maximal rates of contraction and relaxation. In addition, AT-II (10(-9) to 10(-7) M) increased the [Ca(2+)](i) transient amplitude. The involvement of 1,4,5-IP(3)-dependent intracellular Ca(2+) release in the inotropic effect of AT-II was supported by the findings that (a) hESC-CM express AT-II, ET-1, and 1,4,5-IP(3) receptors determined by immunofluorescence staining, and (b) the effects of AT-II were blocked by 2 microM 2-aminoethoxyphenyl borate (a 1,4,5-IP(3) receptor blocker) and U73122 (a phospholipase C blocker). In conclusion, these findings demonstrate for the first time that hESC-CM exhibit functional AT-II and ET-1 signaling pathways, as well as 1,4,5-IP(3)-operated releasable Ca(2+) stores.


Subject(s)
Angiotensin II/metabolism , Calcium/metabolism , Embryonic Stem Cells/cytology , Endothelin-1/metabolism , Myocytes, Cardiac/metabolism , Animals , Cells, Cultured , Enzyme Inhibitors/pharmacology , Humans , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Mice , Models, Biological , Receptors, Adrenergic, beta/metabolism , Signal Transduction
15.
J Electrocardiol ; 40(6 Suppl): S192-6, 2007.
Article in English | MEDLINE | ID: mdl-17993321

ABSTRACT

Cardiovascular diseases are the most frequent cause of death in the industrialized world, with the main contributor being myocardial infarction. Given the high morbidity and mortality rates associated with congestive heart failure, the shortage of donor hearts for transplantation, complications resulting from immunosuppression, and long-term failure of transplanted organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. Because it is desired that the transplanted cells fully integrate within the diseased myocardium, contribute to its contractile performance, and respond appropriately to various physiological stimuli (eg, beta-adrenergic stimulation), our major long-term goal is to investigate the developmental changes in functional properties and hormonal responsiveness of human embryonic stem cells-derived cardiomyocytes (hESC-CM). Furthermore, because one of the key obstacles in advancing cardiac cell therapy is the low differentiation rate of hESC into cardiomyocytes, which reduces the clinical efficacy of cell transplantation, our second major goal is to develop efficient protocols for directing the cardiomyogenic differentiation of hESC in vitro. To accomplish the first goal, we investigated the functional properties of hESC-CM (<90 days old), respecting the contractile function and the underlying intracellular Ca(2+) handling. In addition, we performed Western blot analysis of the key Ca(2+)-handling proteins SERCA2, calsequestrin, phospholamban and the Na(+)/Ca(2+) exchanger. Our major findings were the following: (1) In contrast to the mature myocardium, hESC-CM exhibit negative force-frequency relationships and do not present postrest potentiation. (2) Ryanodine and thapsigargin do not affect the [Ca(2+)](i) transient and contraction, suggesting that, at this developmental stage, the contraction does not depend on sarcoplasmic reticulum Ca(2+) release. (3) In agreement with the finding that a voltage-dependent Ca(2+) current is present in hESC-CM and contributes to the mechanical function, verapamil completely blocks contraction. (4) Although hESC-CM express SERCA2 and Na(+)/Ca(2+) exchanger at levels comparable to those of the adult human myocardium, calsequestrin and phospholamban are not expressed. (4) In agreement with other reports, hESC-CM are responsive to beta-adrenergic stimulation. These findings show that the mechanical function related to intracellular Ca(2+) handling of hESC-CM differs from the adult myocardium, probably because of immature sarcoplasmic reticulum capacity.


Subject(s)
Action Potentials/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Myocardial Contraction/physiology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/physiology , Tissue Engineering/methods , Calcium/metabolism , Cells, Cultured , Humans
16.
Stem Cells ; 24(2): 236-45, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16322641

ABSTRACT

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 cell-derived cardiomyocytes (hESC-CMs) (7 to 55 days old) with the myocardium, we investigated their functional properties regarding intracellular Ca2+ handling and the role of the sarcoplasmic reticulum in the contraction. The functional properties of hESC-CMs were investigated by recording simultaneously [Ca2+]i transients and contractions. Additionally, we performed Western blot analysis of the Ca2+-handling proteins SERCA2, calsequestrin, phospholamban, and Na+/Ca2+ exchanger (NCX). Our major findings are, first, that hESC-CMs displayed temporally related [Ca2+]i transients and contractions, negative force-frequency relations, and lack of post-rest potentiation. Second, ryanodine, thapsigargin, and caffeine did not affect the [Ca2+]i transient and contraction, indicating that at this developmental stage, contraction depends on transsarcolemmal Ca2+ influx rather than on sarcoplasmic reticulum Ca2+ release. Third, in agreement with the notion that a voltage-dependent Ca2+ 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 Ca2+ handling of hESC-CMs differ markedly from the adult myocardium, probably due to immature sarcoplasmic reticulum capacity.


Subject(s)
Calcium/physiology , Embryo, Mammalian/cytology , Myocardial Contraction/physiology , Myocytes, Cardiac/physiology , Sarcoplasmic Reticulum/physiology , Stem Cells/physiology , Animals , Caffeine/pharmacology , Calcium/metabolism , Dose-Response Relationship, Drug , Heart Conduction System/drug effects , Heart Conduction System/physiology , Humans , Mice , Myocardial Contraction/drug effects , Ryanodine/pharmacology , Thapsigargin/pharmacology , Verapamil/pharmacology
17.
Ann N Y Acad Sci ; 1047: 66-75, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16093485

ABSTRACT

Regeneration of the diseased myocardium by cardiac cell transplantation is an attractive therapeutic modality. Yet, because the transplanted cardiomyocytes should functionally integrate within the diseased myocardium, it is preferable that their properties resemble those of the host. To determine the functional adaptability of human embryonic stem cell-derived cardiomyocytes (hESC-CM) to the host myocardium, the authors investigated the excitation-contraction (E-C) coupling and the responsiveness to common physiological stimuli. The main findings are: (1) hESC-CM readily respond to electrical pacing and generate corresponding [Ca(2+)](i) transients (measured by fura-2 fluorescence) and contractions (measured by video edge detector). (2) In contrast to the mature myocardium, hESC-CM display negative force-frequency relations. (3) The hESC-CM contraction is dependent on [Ca(2+)](o) and blocked by verapamil. (4) Surprisingly, ryanodine, the sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin, and caffeine do not affect the [Ca(2+)](i) transient or contraction. Collectively, these results indicate that at the developmental stage of 45 to 60 days, the contraction is largely dependent on [Ca(2+)](o) rather than on sarcoplasmic reticulum (SR) Ca(2+) stores. The results show for the first time that the E-C coupling properties of hESC-CM differ from the adult myocardium, probably due to immature SR function. Based on these findings, genetic manipulation of hESC-CM toward the adult myocardium should be considered.


Subject(s)
Myocardial Contraction/physiology , Myocytes, Cardiac/cytology , Stem Cells/cytology , Animals , Calcium/metabolism , Cell Differentiation , Cell Transplantation , Heart/embryology , Heart/physiology , Heart Conduction System/physiology , Humans , Mice , Myocardial Contraction/drug effects , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/transplantation , Rats , Ryanodine/pharmacology , Thapsigargin/pharmacology
18.
Cardiovasc Res ; 68(1): 75-86, 2005 Oct 01.
Article in English | MEDLINE | ID: mdl-16005448

ABSTRACT

OBJECTIVE: Cardiac hypertrophy is a compensatory response to increased mechanical load. Since Fas receptor activation is an important component in hypertrophy induced by pressure- and volume-overload, deciphering the underlying signaling pathways is of prime importance. Based on our previous work showing that in mice and rats ventricular myocytes the electrophysiological disturbances and diastolic [Ca2+]i-rise caused by 3 h of Fas activation are dependent on the Fas-->phospholipase C (PLC)-->1,4,5-inositol trisphosphate (1,4,5-IP3)-->sarcoplasmic reticulum (SR) [Ca2+]i release pathway, we tested the hypothesis that this pathway is also critical for Fas-mediated hypertrophy. METHODS: The effects of 24 h Fas activation in cultured neonatal rat ventricular myocytes (NRVM) were analyzed by means of RT-PCR, Western blot, immunofluorescence and fura-2 fluorescence. RESULTS: Fas activation increased nuclei surface area, atrial natriuretic peptide and connexin43 (Cx43) mRNA, the protein levels of total Cx43 and non-phosphorylated Cx43, and sarcomeric actin, all indicating hypertrophy. Concomitantly, Fas activation decreased mRNA of SERCA2a, the ryanodine receptor (RyR) and nuclear IP3R3. Further, Fas activation caused NFAT nuclear translocation. The hypertrophy was abolished by U73122, xestospongin C (blockers of the 1,4,5-IP3 pathway), genistein and by the PI3K blocker LY294002. CONCLUSIONS: Fas-mediated hypertrophy is dependent on the 1,4,5-IP3 pathway, which is functionally inter-connected to the PI3K/AKT/GSK3beta pathway. Both pathways act in concert to cause NFAT nuclear translocation and subsequent hypertrophy.


Subject(s)
Cardiomegaly/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Signal Transduction , fas Receptor/metabolism , Animals , Animals, Newborn , Apoptosis , Calcium-Transporting ATPases/metabolism , Cardiomegaly/pathology , Cell Membrane/metabolism , Cells, Cultured , Fluorescent Antibody Technique , Hypertrophy , Immunoblotting , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Sodium-Calcium Exchanger/metabolism
19.
Cardiovasc Res ; 66(1): 64-73, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15769449

ABSTRACT

OBJECTIVES: Altered gap junctional coupling of ventricular myocytes plays an important role in arrhythmogenesis in ischemic heart disease. Since hypoxia is a major component of ischemia, we tested the hypothesis that hypoxia causes gap junctional remodeling accompanied by conduction disturbances. METHODS: Cultured neonatal rat ventricular myocytes were exposed to hypoxia (1% O(2)) for 15 min to 5 h, connexin43 (Cx43) expression was analyzed, and conduction velocity was measured using the Micro-Electrode Array data acquisition system. RESULTS: After 15 min of hypoxia, conduction velocity was unaffected, while total Cx43, including the phosphorylated and nonphosphorylated isoforms, was increased. After 5 h of hypoxia, total Cx43 protein was decreased by 50%, while the nonphosphorylated Cx43 isoform was unchanged. Confocal analyses yielded a 55% decrease in the gap junctional Cx43 fluorescence signal, a 55% decrease in gap junction number, and a 26% decrease in size. The changes in Cx43 were not accompanied by changes in mRNA levels. The reduction in Cx43 protein levels was associated with a approximately 20% decrease in conduction velocity compared to normoxic cultures. CONCLUSIONS: Short-term hypoxia (5 h) decreases Cx43 protein and conduction velocity, thereby contributing to the generation of an arrhythmogenic substrate.


Subject(s)
Arrhythmias, Cardiac/metabolism , Gap Junctions/metabolism , Hypoxia/metabolism , Myocytes, Cardiac/metabolism , Animals , Animals, Newborn , Arrhythmias, Cardiac/physiopathology , Blotting, Western/methods , Connexin 43/analysis , Connexin 43/genetics , Connexin 43/metabolism , Gap Junctions/chemistry , Heart Conduction System , Heart Ventricles , Immunohistochemistry/methods , Microscopy, Confocal , Myocytes, Cardiac/chemistry , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
SELECTION OF CITATIONS
SEARCH DETAIL
...