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. Key Words: cells Ⅲ biopsy Ⅲ electrophysiology Ⅲ myocardial infarction Ⅲ myocytes W e sought to develop a clinically applicable method for the isolation and expansion of adult stem cells capable of regenerating myocytes and vessels and improving function in the injured heart. Given recent evidence that the adult mammalian heart contains endogenous, cardiac-committed stem cells, 1-5 we began with cardiac tissue as our stem cell source, postulating that cardiac-derived cells might be particularly well-suited for myocardial regeneration. Percutaneous endomyocardial biopsy specimens were utilized as a convenient, minimally invasive tissue source. 6,7 We began with the observation that cardiac surgical biopsy specimens in culture yield spherical multicellular clusters dubbed "cardiospheres." 8 Cardiospheres resemble neurospheres 9 in that they are derived from primary tissue culture and contain many proliferative cells that express stem cell-related antigens, as well as other cells undergoing spontaneous cardiac differentiation. 8 We modified the original culture method to improve efficiency and added a postcardiosphere expansion step to obtain reasonable numbers of cells (cardiosphere-derived cells [CDCs]) for transplantation from the small specimens in a timely manner. Editori...

Previous studies have shown familial aggregation of insulin resistance and nonalcoholic-fatty-liver-disease (NAFLD). Therefore, we aimed to examine whether family history of diabetes-mellitus (DM) is associated with nonalcoholic steatohepatitis (NASH) and fibrosis in patients with NAFLD. This is a cross-sectional analysis in participants of the NAFLD Database Study and PIVENS Trial who had available data on family history of DM. 1069 patients (63% women) with mean age of 49.6 (± 11.8) years and BMI of 34.2 (± 6.4) kg/m2, were included. 30% had DM and 56% had family history of DM. Both personal history of DM and family history of DM were significantly associated with NASH with an odds ratio (OR) of 1.93 (95% CI, 1.37–2.73; p-value <0.001) and 1.48 (95% CI, 1.11–1.97; P=0.01), and any fibrosis with an OR of 3.31 (95% CI, 2.26–4.85; p-value <0.001) and 1.66 (95% CI, 1.25–2.20; P<0.001), respectively. When the models were adjusted for age, sex, BMI, ethnicity, and metabolic traits, the association between diabetes and family history of DM, with NASH showed an increased adjusted-OR of 1.76 (95% CI, 1.13–2.72, p-value <0.001) and 1.34 (95% CI, 0.99–1.81; P=0.06), respectively, and with any fibrosis with an significant adjusted-OR of 2.57 (95% CI, 1.61–4.11; p-value < 0.0001) and 1.38 (95% CI, 1.02–1.87; P=0.04), respectively. After excluding patients with personal history of diabetes, family history of DM was significantly associated with presence of NASH and any fibrosis with adjusted OR of 1.51 (95% CI, 1.01–2.25; P=0.04), and 1.49 (95% CI, 1.01–2.20; P=0.04), respectively. Conclusions: Diabetes is strongly associated with risk of NASH, fibrosis and advanced fibrosis. Family history of diabetes especially among non-diabetics is associated with NASH and fibrosis in NAFLD.

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 K ATP 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 K ATP 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 K ATP channels and associated functions. D elivery of metabolic signals to intracellular compartments is a critical determinant of cellular homeostasis. In particular, efficient communication between cellular energetics and membrane metabolic sensors is required for regulation of cell excitability and associated functions (1, 2). Plasmalemmal ATPsensitive K ϩ (K ATP ) channels, formed by the Kir6.2 potassium channel and the sulfonylurea receptor (SUR), are unique nucleotide sensors that adjust membrane potential in response to intracellular metabolic oscillations (2-5). Transition of the SUR subunit from the ATP to the ADP-liganded state promotes K ϩ permeation through Kir6.2 and defines K ATP channel activity (5-7). However, the mechanism that facilitates nucleotide exchange in the K ATP channel environment and promotes coupling of membrane electrical events with cellular metabolic pathways remains unknown.Cellular phosphotransfer reactions catalyze nucleotide exchange facilitating communication between sites of ATP generation and ATP utilization (8-11). In this way, the phosphotransfer enzyme adenylate kinase (AK) amplifies metabolic signals and promotes intracellular phosphoryl transfer by catalyzing the reaction ATP ϩ AMP 7 2ADP (12, 13). Adenylate kinase has a distinct signaling role in setting the cellular response to stress through activation of AMP-dependent processes (12-15). Deletion of the major AK isoform (AK1) results in disturbed muscle energetic economy and decreased tolerance to metabolic stress (14, 15). Mutations in AK compromise nucleotide export from mitochondria (16), as well as the function of ATP-binding cassette transporters (17). Conversely, stimulation of AK phosphotransfer promotes nucleotide-dependent membrane functions (18,19). However, the actual significance of AK phosphotransfer in communicating energetic signals to membrane metabolic sensors, such as the K ATP cha...

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 ␤-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 ␤-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. (Circulation. 2008;117:1555-1562.)

Objectives To quantify acute myocardial retention of cardiac-derived stem cells (CDCs) and evaluate different delivery methods using 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 CDCs derived from syngeneic, male Wistar Kyoto (WK) rats were labeled with 18FDG and injected intramyocardially into the ischemic region of female WK rats following permanent left coronary artery ligation. The effects of fibrin glue, bradycardia (adenosine) and cardiac arrest were examined. 18FDG PET was performed for quantification of cell retention. Quantitative PCR for the male-specific SRY gene was performed to validate the PET results. Results Myocardial retention of cells suspended in PBS 1 hr after delivery was 17.6±11.5% by PCR and 17.8±7.3% by PET. When CDCs were injected immediately following 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 following epicardial application of fibrin glue at the injection site (37.5±8.2%). PCR revealed a significant increase in 3 week cell engraftment in the fibrin glue animals (22.1±18.6% vs 5.3±3.1%, for fibrin glue and PBS respectively). Conclusions In vivo PET permits accurate measurement of CDC retention early after intramyocardial delivery. Sealing injection sites with fibrin glue or lowering ventricular rate by adenosine may be clinically translatable methods for improving stem cell engraftment in a beating heart.

BackgroundIt 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 FindingsHere 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/SignificanceContradicting 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.

Abstract-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.

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.