Hee Cheol Cho

Regenerative Potential of Cardiosphere-Derived Cells Expanded From Percutaneous Endomyocardial Biopsy Specimens

Background— Ex vivo expansion of resident cardiac stem cells, followed by delivery to the heart, may favor regeneration and functional improvement. Methods and Results— Percutaneous endomyocardial biopsy specimens grown in primary culture developed multicellular clusters known as cardiospheres, which were plated to yield cardiosphere-derived cells (CDCs). CDCs from human biopsy specimens and from comparable porcine samples were examined in vitro for biophysical and cytochemical evidence of cardiogenic differentiation. In addition, human CDCs were injected into the border zone of acute myocardial infarcts in immunodeficient mice. Biopsy specimens from 69 of 70 patients yielded cardiosphere-forming cells. Cardiospheres and CDCs expressed antigenic characteristics of stem cells at each stage of processing, as well as proteins vital for cardiac contractile and electrical function. Human and porcine CDCs cocultured with neonatal rat ventricular myocytes exhibited biophysical signatures characteristic of myocytes, including calcium transients synchronous with those of neighboring myocytes. Human CDCs injected into the border zone of myocardial infarcts engrafted and migrated into the infarct zone. After 20 days, the percentage of viable myocardium within the infarct zone was greater in the CDC-treated group than in the fibroblast-treated control group; likewise, left ventricular ejection fraction was higher in the CDC-treated group. Conclusions— A method is presented for the isolation of adult human stem cells from endomyocardial biopsy specimens. CDCs are cardiogenic in vitro; they promote cardiac regeneration and improve heart function in a mouse infarct model, which provides motivation for further development for therapeutic applications in patients.

Functional Integration of Electrically Active Cardiac Derivatives From Genetically Engineered Human Embryonic Stem Cells With Quiescent Recipient Ventricular Cardiomyocytes Insights Into the Development of Cell-Based Pacemakers

Background—Human embryonic stem cells (hESCs) derived from blastocysts can propagate indefinitely in culture while maintaining pluripotency, including the ability to differentiate into cardiomyocytes (CMs); therefore, hESCs may provide an unlimited source of human CMs for cell-based therapies. Although CMs can be derived from hESCs ex vivo, it remains uncertain whether a functional syncytium can be formed between donor and recipient cells after engraftment. Methods and Results—Using a combination of electrophysiological and imaging techniques, here we demonstrate that electrically active, donor CMs derived from hESCs that had been stably genetically engineered by a recombinant lentivirus can functionally integrate with otherwise-quiescent, recipient, ventricular CMs to induce rhythmic electrical and contractile activities in vitro. The integrated syncytium was responsive to the &bgr;-adrenergic agonist isoproterenol as well as to other pharmacological agents such as lidocaine and ZD7288. Similarly, a functional hESC-derived pacemaker could be implanted in the left ventricle in vivo. Detailed optical mapping of the epicardial surface of guinea pig hearts transplanted with hESC-derived CMs confirmed the successful spread of membrane depolarization from the site of injection to the surrounding myocardium. Conclusions—We conclude that electrically active, hESC-derived CMs are capable of actively pacing quiescent, recipient, ventricular CMs in vitro and ventricular myocardium in vivo. Our results may lead to an alternative or a supplemental method for correcting defects in cardiac impulse generation, such as cell-based pacemakers.

Gene Therapy to Inhibit the Calcium Channel β Subunit Physiological Consequences and Pathophysiological Effects in Models of Cardiac Hypertrophy

Calcium cycling figures prominently in excitation-contraction coupling and in various signaling cascades involved in the development of left ventricular hypertrophy. We hypothesized that genetic suppression of the L-type calcium channel accessory &bgr;-subunit would modulate calcium current and suppress cardiac hypertrophy. A short hairpin RNA template sequence capable of mediating the knockdown of the L-type calcium channel accessory &bgr;-subunit gene was incorporated into a lentiviral vector (PPT.CG.H1.&bgr;2). Transduction of ventricular myocytes in vivo with the active short hairpin RNA partially inhibited the L-type calcium current. In neonatal rat cardiomyocytes, L-type calcium channel accessory &bgr;-subunit gene knockdown reduced calcium transient amplitude. Similarly, [3H]leucine incorporation was attenuated in PPT.CG.H1.&bgr;2-transduced neonatal rat cardiomyocytes compared with nonsilencing controls in a phenylephrine-induced hypertrophy model. In vivo gene transfer attenuated the hypertrophic response in an aortic-banded rat model of left ventricular hypertrophy, with reduced left ventricular wall thickness and heart weight/body weight ratios in PPT.CG.H1.&bgr;2-injected rats at four weeks post transduction. Fractional shortening was preserved in rats treated with PPT.CG.H1.&bgr;2. These findings indicate that knockdown of L-type calcium channel accessory &bgr;-subunit is capable of attenuating the hypertrophic response both in vitro and in vivo without compromising systolic performance. Suppression of the calcium channel &bgr; subunit may represent a novel and useful therapeutic strategy for left ventricular hypertrophy.

Transcriptional Suppression of Connexin43 by TBX18 Undermines Cell-Cell Electrical Coupling in Postnatal Cardiomyocytes

T-box transcription factors figure prominently in embryonic cardiac cell lineage specifications. Mesenchymal precursor cells expressing Tbx18 give rise to the hearts pacemaker, the sinoatrial node (SAN). We sought to identify targets of TBX18 transcriptional regulation in the heart by forced adenoviral overexpression in postnatal cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) transduced with GFP showed sarcolemmal, punctate Cx43 expression. In contrast, TBX18-transduced NRCMs exhibited sparse Cx43 expression. Both the transcript and protein levels of Cx43 were greatly down-regulated within 2 days of TBX18 transduction. Direct injection of TBX18 in the guinea pig heart in vivo inhibited Cx43 expression. The repressor activity of TBX18 on Cx43 was highly specific; protein levels of Cx45 and Cx40, which comprise the main gap junctions in the SAN and conduction system, were unchanged by TBX18. A reporter-based promoter assay demonstrated that TBX18 directly represses the Cx43 promoter. Phenotypically, TBX18-NRCMs exhibited slowed intercellular calcein dye transfer kinetics (421 ± 54 versus control 127 ± 43 ms). Intracellular Ca2+ oscillations in control NRCM monolayers were highly synchronized. In contrast, TBX18 overexpression led to asynchronous Ca2+ oscillations, demonstrating reduced cell-cell coupling. Decreased coupling led to slow electrical propagation; conduction velocity in TBX18 NRCMs slowed by more than 50% relative to control (2.9 ± 0.5 versus 14.3 ± 0.9 cm/s). Taken together, TBX18 specifically and directly represses Cx43 transcript and protein levels. Cx43 suppression leads to significant electrical uncoupling, but the preservation of other gap junction proteins supports slow action potential propagation, recapitulating a key phenotypic hallmark of the SAN.

Biological pacemakers as a therapy for cardiac arrhythmias

Purpose of review Cardiac rhythm disorders are caused by malfunctions of impulse generation or conduction. Malfunctions of impulse generation, that is, defects in pacemaking, are often life-threatening. Present therapies span a wide array of approaches, but remain largely palliative. Recent progress in understanding of the underlying biology of pacemaking opens up new prospects for better alternatives to the present routine. Specifically, development and use of biological pacemakers could prove to be advantageous to the conventional approaches. Recent findings We review the current state of the art in gene and cell-based approaches to correct cardiac rhythm disturbances. These include genetic suppression of an ionic current, embryonic as well as adult stem cell therapies, novel synthetic pacemaker channels, and adult somatic cell-fusion approach. Summary Biological pacemaking can be achieved by modulating ionic currents by gene transfer or by delivering engineered pacemaker cells into normally quiescent myocardium. The present state of development is proof-of-concept; we are now working on reducing to practice a stable, reliable biological product as an alternative to electronic pacemakers.

Taking the Cells Out of Cell Therapy

Regenerative therapy for myocardial infarction (MI) seeks to regrow healthy heart muscle that, once lost, has traditionally been assumed to be gone forever. After a decade of stuttering progress ([1][1]), we now have reason to believe that therapeutic regeneration is indeed possible in human beings

22. Creation of a Biological Pacemaker by Cell Fusion

As an alternative to electronic pacemakers, we explored the feasibility of converting ventricular myocytes into pacemakers by somatic cell fusion. The idea is to create chemically induced fusion between myocytes and syngeneic fibroblasts engineered to express HCN1 pacemaker channels (HCN1-fibroblasts). HCN1-fibroblasts were fused with freshly isolated guinea pig ventricular myocytes using polyethylene-glycol 1500. In vivo fused myocyte-HCN1-fibroblast cells exhibited spontaneously oscillating action potentials; the firing frequency increased with beta-adrenergic stimulation. The heterokaryons created ectopic ventricular pacemaker activity in vivo at the site of cell injection. Coculture of nonfused HCN1-fibroblasts and myocytes without polyethylene-glycol 1500 revealed no evidence of dye transfer, demonstrating that the I(f)-mediated pacemaker activity arises from heterokaryons rather than electrotonic coupling. This nonviral, non-stem cell approach enables autologous, adult somatic cell therapy to create biopacemakers.