M. Roselle Abraham

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.

Antiarrhythmic Engineering of Skeletal Myoblasts for Cardiac Transplantation

Skeletal myoblasts are an attractive cell type for transplantation because they are autologous and resistant to ischemia. However, clinical trials of myoblast transplantation in heart failure have been plagued by ventricular tachyarrhythmias and sudden cardiac death. The pathogenesis of these arrhythmias is poorly understood, but may be related to the fact that skeletal muscle cells, unlike heart cells, are electrically isolated by the absence of gap junctions. Using a novel in vitro model of myoblast transplantation in cardiomyocyte monolayers, we investigated the mechanisms of transplant-associated arrhythmias. Cocultures of human skeletal myoblasts and rat cardiomyocytes resulted in reentrant arrhythmias (spiral waves) that reproduce the features of ventricular tachycardia seen in patients receiving myoblast transplants. These arrhythmias could be terminated by nitrendipine, an l-type calcium channel blocker, but not by the Na channel blocker lidocaine. Genetic modification of myoblasts to express the gap junction protein connexin43 decreased arrhythmogenicity in cocultures, suggesting a specific means for increasing the safety (and perhaps the efficacy) of myoblast transplantation in patients.

Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (KATP) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with KATP channel-dependent membrane excitability remains elusive. Here, we identify that the response of KATP channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the KATP channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and KATP channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with KATP channels and associated functions.

Magnetic Resonance Imaging Overestimates Ferumoxide-Labeled Stem Cell Survival After Transplantation in the Heart

Background— Stem cell labeling with iron oxide (ferumoxide) particles allows labeled cells to be detected by magnetic resonance imaging (MRI) and is commonly used to track stem cell engraftment. However, the validity of MRI for distinguishing surviving ferumoxide-labeled cells from other sources of MRI signal, for example, macrophages containing ferumoxides released from nonsurviving cells, has not been thoroughly investigated. We sought to determine the relationship between the persistence of iron-dependent MRI signals and cell survival 3 weeks after injection of syngeneic or xenogeneic ferumoxides-labeled stem cells (cardiac-derived stem cells) in rats. Methods and Results— We studied nonimmunoprivileged human and rat cardiac-derived stem cells and human mesenchymal stem cells doubly labeled with ferumoxides and β-galactosidase and injected intramyocardially into immunocompetent Wistar-Kyoto rats. Animals were imaged at 2 days and 3 weeks after stem cell injection in a clinical 3-T MRI scanner. At 2 days, injection sites of xenogeneic and syngeneic cells (cardiac-derived stem cells and mesenchymal stem cells) were identified by MRI as large intramyocardial signal voids that persisted at 3 weeks (50% to 90% of initial signal). Histology (at 3 weeks) revealed the presence of iron-containing macrophages at the injection site, identified by CD68 staining, but very few or no β-galactosidase–positive stem cells in the animals transplanted with syngeneic or xenogeneic cells, respectively. Conclusions— The persistence of significant iron-dependent MRI signal derived from ferumoxide-containing macrophages despite few or no viable stem cells 3 weeks after transplantation indicates that MRI of ferumoxide-labeled cells does not reliably report long-term stem cell engraftment in the heart.

Noninvasive quantification and optimization of acute cell retention by in vivo positron emission tomography after intramyocardial cardiac-derived stem cell delivery.

OBJECTIVES The aim of this study was to quantify acute myocardial retention of cardiac-derived stem cells (CDCs) and evaluate different delivery methods with positron emission tomography (PET). BACKGROUND Success of stem cell transplantation for cardiac regeneration is partially limited by low retention/engraftment of the delivered cells. A clinically applicable method for accurate quantification of cell retention would enable optimization of cell delivery. METHODS The CDCs were derived from syngeneic, male Wistar Kyoto (WK) rats labeled with [(18)F]-fluoro-deoxy-glucose ((18)FDG) and injected intramyocardially into the ischemic region of female WK rats after permanent left coronary artery ligation. The effects of fibrin glue (FG), bradycardia (adenosine), and cardiac arrest were examined. Imaging with (18)FDG PET was performed for quantification of cell retention. Quantitative polymerase chain reaction (PCR) for the male-specific SRY gene was performed to validate the PET results. RESULTS Myocardial retention of cells suspended in phosphate-buffered saline 1 h after delivery was 17.6 +/- 11.5% by PCR and 17.8 +/- 7.3% by PET. When CDCs were injected immediately after induction of cardiac arrest, retention was increased to 75.6 +/- 18.6%. Adenosine slowed the ventricular rate and doubled CDC retention (35.4 +/- 5.3%). A similar increase in CDC retention was observed after epicardial application of FG at the injection site (37.5 +/- 8.2%). The PCR revealed a significant increase in 3-week cell engraftment in the FG animals (22.1 +/- 18.6% and 5.3 +/- 3.1%, for FG and phosphate-buffered saline, respectively). CONCLUSIONS In vivo PET permits accurate measurement of CDC retention early after intramyocardial delivery. Sealing injection sites with FG or lowering ventricular rate by adenosine might be clinically translatable methods for improving stem cell engraftment in a beating heart.

Proarrhythmic Potential of Mesenchymal Stem Cell Transplantation Revealed in an In Vitro Coculture Model

Background— Mesenchymal stem cells (MSCs) are bone marrow stromal cells that are in phase 1 clinical studies of cellular cardiomyoplasty. However, the electrophysiological effects of MSC transplantation have not been studied. Although improvement of ventricular function would represent a positive outcome of MSC transplantation, focal application of stem cells has the potential downside of creating inhomogeneities that may predispose the heart to reentrant arrhythmias. In the present study we use an MSC and neonatal rat ventricular myocyte (NRVM) coculture system to investigate potential proarrhythmic consequences of MSC transplantation into the heart. Methods and Results— Human MSCs were cocultured with NRVMs in ratios of 1:99, 1:9, and 1:4 and optically mapped. We found that conduction velocity was decreased in cocultures compared with controls, but action potential duration (APD80) was not affected. Reentrant arrhythmias were induced in 86% of cocultures containing 10% and 20% MSCs (n=36) but not in controls (n=7) or cocultures containing only 1% MSCs (n=4). Immunostaining, Western blot, and dye transfer revealed the presence of functional gap junctions involving MSCs. Conclusions— Our results suggest that mixtures of MSCs and NRVMs can produce an arrhythmogenic substrate. The mechanism of reentry is probably increased tissue heterogeneity resulting from electric coupling of inexcitable MSCs with myocytes.

Magnetic Resonance–Based Anatomical Analysis of Scar-Related Ventricular Tachycardia. Implications for Catheter Ablation

In catheter ablation of scar-related monomorphic ventricular tachycardia (VT), substrate voltage mapping is used to electrically define the scar during sinus rhythm. However, the electrically defined scar may not accurately reflect the anatomical scar. Magnetic resonance–based visualization of the scar may elucidate the 3D anatomical correlation between the fine structural details of the scar and scar-related VT circuits. We registered VT activation sequence with the 3D scar anatomy derived from high-resolution contrast-enhanced MRI in a swine model of chronic myocardial infarction using epicardial sock electrodes (n=6, epicardial group), which have direct contact with the myocardium where the electrical signal is recorded. In a separate group of animals (n=5, endocardial group), we also assessed the incidence of endocardial reentry in this model using endocardial basket catheters. Ten to 12 weeks after myocardial infarction, sustained monomorphic VT was reproducibly induced in all animals (n=11). In the epicardial group, 21 VT morphologies were induced, of which 4 (19.0%) showed epicardial reentry. The reentry isthmus was characterized by a relatively small volume of viable myocardium bound by the scar tissue at the infarct border zone or over the infarct. In the endocardial group (n=5), 6 VT morphologies were induced, of which 4 (66.7%) showed endocardial reentry. In conclusion, MRI revealed a scar with spatially complex structures, particularly at the isthmus, with substrate for multiple VT morphologies after a single ischemic episode. Magnetic resonance–based visualization of scar morphology would potentially contribute to preprocedural planning for catheter ablation of scar-related, unmappable VT.

Dedifferentiation and Proliferation of Mammalian Cardiomyocytes

Background It has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle. Methodology/Principal Findings Here we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1) cardiomyocyte purification from rat hearts, and 2) genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs), while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP) continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit+. Conclusions/Significance Contradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness, including the expression of c-kit and the capacity for multipotency.

ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex

ATP‐sensitive K+ (KATP) channels are unique metabolic sensors formed by association of Kir6.2, an inwardly rectifying K+ channel, and the sulfonylurea receptor SUR, an ATP binding cassette protein. We identified an ATPase activity in immunoprecipitates of cardiac KATP channels and in purified fusion proteins containing nucleotide binding domains NBD1 and NBD2 of the cardiac SUR2A isoform. NBD2 hydrolyzed ATP with a twofold higher rate compared to NBD1. The ATPase required Mg2+ and was insensitive to ouabain, oligomycin, thapsigargin, or levamisole. K1348A and D1469N mutations in NBD2 reduced ATPase activity and produced channels with increased sensitivity to ATP. KATP channel openers, which bind to SUR, promoted ATPase activity in purified sarcolemma. At higher concentrations, openers reduced ATPase activity, possibly through stabilization of MgADP at the channel site. K1348A and D1469N mutations attenuated the effect of openers on KATP channel activity. Opener‐induced channel activation was also inhibited by the creatine kinase/creatine phosphate system that removes ADP from the channel complex. Thus, the KATP channel complex functions not only as a K+ conductance, but also as an enzyme regulating nucleotide‐dependent channel gating through an intrinsic ATPase activity of the SUR subunit. Modulation of the channel ATPase activity and/or scavenging the product of the ATPase reaction provide novel means to regulate cellular functions associated with KATP channel opening.—Bienengraeber, M., Alekseev, A. E., Abraham, M. R., Carrasco, A. J., Moreau, C., Vivaudou, M., Dzeja, P. P., Terzic, A. ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex. FASEB J. 14, 1943–1952 (2000)

Ectopic Expression of the Sodium-Iodide Symporter Enables Imaging of Transplanted Cardiac Stem Cells In Vivo by Single-Photon Emission Computed Tomography or Positron Emission Tomography

OBJECTIVES We examined the sodium-iodide symporter (NIS), which promotes in vivo cellular uptake of technetium 99m ((99m)Tc) or iodine 124 ((124)I), as a reporter gene for cell tracking by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. BACKGROUND Stem cells offer the promise of cardiac repair. Stem cell labeling is a prerequisite to tracking cell fate in vivo. METHODS The human NIS complementary deoxyribonucleic acid was transduced into rat cardiac-derived stem cells (rCDCs) using lentiviral vectors. Rats were injected intramyocardially with up to 4 million NIS(+)-rCDCs immediately after left anterior descending coronary artery ligation. Dual isotope SPECT (or PET) imaging was performed, using (99m)Tc (or (124)I) for cell detection and thallium 201 (or ammonia 13) for myocardial delineation. In a subset of animals, high resolution ex vivo SPECT scans of explanted hearts were obtained to confirm that in vivo signals were derived from the cell injection site. RESULTS NIS expression in rCDCs did not affect cell viability and proliferation. NIS activity was verified in isolated transduced cells by measuring (99m)Tc uptake. NIS(+) rCDCs were visualized in vivo as regions of (99m)Tc or (124)I uptake within a perfusion deficit in the SPECT and PET images, respectively. Cells could be visualized by SPECT up to 6 days post-injection. Ex vivo SPECT confirmed that in vivo (99m)Tc signals were localized to the cell injection sites. CONCLUSIONS Ectopic NIS expression allows noninvasive in vivo stem cell tracking in the myocardium, using either SPECT or PET. The general approach shows significant promise in tracking the fate of transplanted cells participating in cardiac regeneration, given its ability to observe living cells using clinically applicable imaging modalities.