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Charles E Murry

Publications and source records attributed to Charles E Murry.

At least 19 recordsLinked to original sources
PubMed2006

rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice.

Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we show that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores expression of dystrophin in the respiratory, cardiac and limb musculature of these mice, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector-mediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy.

Animals↗
PubMed2006

Regeneration gaps: observations on stem cells and cardiac repair.

Substantial evidence indicates that cell transplantation can improve function of the infarcted heart. A surprisingly wide range of non-myogenic cell types improves ventricular function, suggesting that benefit may result in part from mechanisms that are distinct from true myocardial regeneration. While clinical trials explore cells derived from skeletal muscle and bone marrow, basic researchers are investigating sources of new cardiomyocytes, such as resident myocardial progenitors and embryonic stem cells. In this commentary, we briefly review the evolution of cell-based cardiac repair, discuss the current state of clinical research, and offer some thoughts on how newcomers can critically evaluate this emerging field.

Animals↗
PubMed2005

Extracardiac progenitor cells repopulate most major cell types in the transplanted human heart.

BACKGROUND: Extracardiac progenitor cells are capable of repopulating cardiomyocytes at very low levels in the human heart after injury. Here, we explored the extent of endothelial, smooth muscle, and Schwann cell chimerism in patients with sex-mismatched (female-to-male) heart transplants. METHODS AND RESULTS: Autopsy specimens from 5 patients and endomyocardial biopsies from 7 patients were used for this study. Endothelial, vascular smooth muscle, and Schwann cells were stained with antibodies against CD31 or Ulex europaeus lectin, smooth muscle alpha-actin, and S-100, respectively, and the Y chromosome was identified with in situ hybridization. Biopsy specimens from 1, 4, 6, and 12 months and 5 and 10 years after heart transplantation were evaluated. Y-positive cells were counted by conventional bright-field microscopy and confirmed by confocal microscopy. Endothelial cells showed the highest degree of chimerism, averaging 24.3+/-8.2% from extracardiac sources. Schwann cells showed the next highest chimerism, averaging 11.2+/-2.1%; vascular smooth muscle cells averaged 3.4+/-1.8%. All 3 cell types showed substantially higher chimerism than we previously observed for cardiomyocytes (0.04+/-0.05%). Endothelial chimerism was much higher in the microcirculation than in larger vessels. Analysis of serial endomyocardial biopsies revealed that high levels of endothelial chimerism occurred as early as 1 month after transplantation (22+/-6.6%) with no significant increases even up to 10 years after cardiac transplantation. CONCLUSIONS: Extracardiac progenitor cells are capable of repopulating most major cell types in the heart, but they do so with varying frequency. The signals for endothelial progenitor recruitment occur early and could relate to injury during allograft harvest or transplantation. The high degree of endothelial chimerism may have immune implications such as for myocardial rejection or graft vasculopathy.

Adult↗
PubMed2005

Proliferation of cardiomyocytes derived from human embryonic stem cells is mediated via the IGF/PI 3-kinase/Akt signaling pathway.

Cardiomyocytes from common experimental animals rapidly exit the cell cycle upon isolation, impeding studies of basic cell biology and applications such as myocardial repair. Here we examined proliferation of cardiomyocytes derived from human and mouse embryonic stem (ES) cells. While mouse ES cell-derived cardiomyocytes showed little proliferation, human cardiomyocytes were highly proliferative under serum-free conditions (15-25% BrdU+/sarcomeric actin+). The cells exhibited only a small serum dose-response, and proliferation gradually slowed with increasing differentiation of the cells. Neither cell density nor different matrix attachment factors affected cardiomyocyte proliferation. Blockade of phosphatidylinositol 3-kinase (PI 3-kinase) and Akt significantly reduced cardiomyocyte proliferation, whereas MEK inhibition had no effect. Antibody blocking of the insulin-like growth factor-1 (IGF-1) receptor significantly inhibited cardiomyocyte proliferation, while addition of IGF-1 or IGF-2 stimulated cardiomyocyte proliferation in a dose-dependent manner. Thus, cardiomyocytes derived from human ES cells proliferate extensively in vitro, and their proliferation appears to be mediated primarily via the PI 3-kinase/Akt signaling pathway, using the IGF-1 receptor as one upstream activator. This system should permit identification of regulatory pathways for human cardiomyocyte proliferation and may facilitate expansion of cardiomyocytes from human ES cells for therapeutic purposes.

Animals↗
PubMed2005

Formation of human myocardium in the rat heart from human embryonic stem cells.

Human embryonic stem cells (hESCs) offer the opportunity to replenish cells lost in the postinfarct heart. We explored whether human myocardium could be generated in rat hearts by injecting differentiated cardiac-enriched hESC progeny into the left ventricular wall of athymic rats. Although initial grafts were predominantly epithelial, noncardiac elements were lost over time, and grafts consisted predominantly of cardiomyocytes by 4 weeks. No teratomatous elements were observed. Engrafted cardiomyocytes were glycogen-rich and expressed expected cardiac markers including beta-myosin heavy chain, myosin light chain 2v, and atrial natriuretic factor. Heat-shock treatment improved graft size approximately threefold. The cardiac implants exhibited substantial angiogenesis, both recipient and graft derived. Importantly, there was greater proliferation in human cardiomyocytes than previously seen in rodent-derived cardiomyocytes: 14.4% of graft cardiomyocytes expressed the proliferation marker Ki-67, and 2.7% incorporated the thymidine analog BrdU 4 weeks after transplantation. This proliferation was associated with a sevenfold increase in graft size over the 4-week interval. Thus, hESCs can form human myocardium in the rat heart, permitting studies of human myocardial development and physiology and supporting the feasibility of their use in myocardial repair.

Animals↗
PubMed2005

Regenerating the heart.

Cell-based cardiac repair offers the promise of rebuilding the injured heart from its component parts. Work began with committed cells such as skeletal myoblasts, but recently the field has expanded to explore an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and both mouse and human embryonic stem cells. A related strategy for cardiac repair involves cell mobilization with factors such as cytokines. Translation of cell-based approaches to the clinic has progressed rapidly, and clinical trials using autologous skeletal myoblasts and bone marrow cells are under way. Many challenges remain before the vision of healing an infarct by muscle regeneration can be realized. Future research is likely to focus on improving our ability to guide the differentiation of stem cells, control their survival and proliferation, identify factors that mediate their homing and modulate the heart's innate inflammatory and fibrotic responses.

Animals↗
PubMed2004

NFATc3-induced reductions in voltage-gated K+ currents after myocardial infarction.

Reductions in voltage-activated K+ (Kv) currents may underlie arrhythmias after myocardial infarction (MI). We investigated the role of beta-adrenergic signaling and the calcineurin/NFAT pathway in mediating the reductions in Kv currents observed after MI in mouse ventricular myocytes. Kv currents were produced by the summation of 3 distinct currents: I(to), I(Kslow1), and I(Kslow2). At 48 hours after MI, we found a 4-fold increase in NFAT activity, which coincided with a decrease in the amplitudes of I(to), I(Kslow1), and I(Kslow2). Consistent with this, mRNA and protein levels of Kv1.5, 2.1, 4.2, and 4.3, which underlie I(Kslow1), I(Kslow2), and I(to), were decreased after MI. Administration of the beta-blocker metoprolol prevented the activation of NFAT and the reductions in I(to), I(Kslow1), and I(Kslow2) after MI. Cyclosporine, an inhibitor of calcineurin, also prevented the reductions in these currents after MI. Importantly, Kv currents did not change after MI in ventricular myocytes from NFATc3 knockout mice. Conversely, chronic beta-adrenergic stimulation or expression of an activated NFATc3 decreased Kv currents to a similar extent as MI. Taken together, these data indicate that NFATc3 plays an essential role in the signaling pathway leading to reduced I(to), I(Kslow1), and I(Kslow2) after MI. We propose that increased beta-adrenergic signaling after MI activates calcineurin and NFATc3, which decreases I(to), I(Kslow1), and I(Kslow2) via a reduction in Kv1.5, Kv2.1, Kv4.2, and Kv4.3 expression.

Action Potentials↗
PubMed2004

Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.

The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.

Animals↗
PubMed2004

Myogenic fusion of human bone marrow stromal cells, but not hematopoietic cells.

Following marrow transplantation in both patients and animals, cells containing donor nuclei have been detected in a variety of nonhematopoietic tissue. Whether this phenomenon represents transdifferentiation of marrow-derived cells, infiltration of blood cells, or cell fusion is still controversial. In muscle, where cell fusion occurs during normal myogenesis, fusion of marrow-derived cells with resident myotubes is a likely explanation. We tested 8 subpopulations of human bone marrow for their ability to fuse with mouse C2C12 myoblast cells. Relatively high fusion efficiency was observed with marrow stromal cells whereas hematopoietic cells, including populations enriched for stem cells, progenitor cells, and monocytes were refractory to fusion. Mouse myotubes containing human nuclei also contained transcripts for human muscle-specific genes. Injection in vivo of human stromal cells expressing green fluorescent protein (GFP) into the regenerating tibialis anterior muscle of nonobese diabetic-severe combined immunodeficient (NOD/SCID) beta 2m(-/-) mice resulted in regenerating mouse muscle fibers expressing GFP. These data suggest that marrow-derived cells contribute to myogenesis through fusion and that stromal cells are better fusion partners than hematopoietic cells.

Animals↗
PubMed2004

Evidence for fusion between cardiac and skeletal muscle cells.

Cardiomyoplasty with skeletal myoblasts may benefit cardiac function after infarction. Recent reports indicate that adult stem cells can fuse with other cell types. Because myoblasts are "fusigenic" cells by nature, we hypothesized they might be particularly likely to fuse with cardiomyocytes. To test this, neonatal rat cardiomyocytes labeled with LacZ and green fluorescent protein (GFP) were cocultured with unlabeled C2C12 myoblasts. After 3 days, we observed a small population of skeletal myotubes that expressed LacZ and GFP, indicating cell fusion. To test whether such fusion occurred in vivo, LacZ-expressing C2C12 myoblasts were grafted into normal nude mouse hearts. At 2 weeks after grafting, cells at the graft-host interface expressed both LacZ and cardiac-specific myosin light chain 2v (MLC2v). To test more definitively whether fusion between skeletal and cardiac muscle could occur, we used a Cre/lox reporter system that activated LacZ only upon cell fusion. When neonatal cardiomyocytes from -myosin heavy chain promoter (-MHC)-Cre mice were cocultured with myoblasts from floxed-lacZ reporter mice, LacZ was activated in a subset of cells, indicating cell fusion occurred in vitro. Finally, we grafted the floxed-lacZ myoblasts into normal hearts of -MHC-Cre+ and -MHC-Cre- mice (n=5 each). Hearts analyzed at 4 days and 1 week after transplantation demonstrated activation of LacZ when the skeletal muscle cells were implanted into hearts of -MHC-Cre+ mice, but not after implantation into -MHC-Cre- mice. These data indicate that skeletal muscle cell grafting gives rise to a subpopulation of skeletal-cardiac hybrid cells with a currently unknown phenotype. The full text of this article is available online at http://circres.ahajournals.org.

Animals↗
PubMed2004

Gene transfer of connexin43 into skeletal muscle.

Cellular cardiomyoplasty using skeletal myoblasts may be beneficial for infarct repair. One drawback to skeletal muscle cells is their lack of gap junction expression after differentiation, thus preventing electrical coupling to host cardiomyocytes. We sought to overexpress the gap junction protein connexin43 (Cx43) in differentiated skeletal myotubes, using retroviral, adenoviral, and plasmid-mediated gene transfer. All strategies resulted in overexpression of Cx43 in cultured myotubes, but expression of Cx43 from constitutive viral promoters caused significant death upon differentiation. Dye transfer studies showed that surviving myotubes contained functional gap junctions, however. Retrovirally transfected myoblasts did not express Cx43 after grafting into the heart, possibly due to promoter silencing. Adenovirally transfected myoblasts expressed abundant Cx43 after forming myotubes in cardiac grafts, but grafts showed signs of injury at 1 week and had died by 2 weeks. Interestingly, transfection of already differentiated myotubes with adenoviral Cx43 was nontoxic, implying a window of vulnerability during differentiation. To test this hypothesis, Cx43 was expressed from the muscle creatine kinase (MCK) promoter, which is active only after myocyte differentiation. The MCK promoter resulted in high levels of Cx43 expression in differentiated myotubes but did not cause cell death during differentiation. MCK-Cx43-transfected myoblasts formed viable cardiac grafts and, in some cases, Cx43-expressing myotubes were in close apposition to host cardiomyocytes, possibly allowing electrical coupling. Thus, high levels of Cx43 during skeletal muscle differentiation cause cell death. When, however, expression of Cx43 is delayed until after differentiation, using the MCK promoter, myotubes are viable and express gap junction proteins after grafting in the heart. This strategy may permit electrical coupling of skeletal and cardiac muscle for cardiac repair.

Adenoviridae↗
PubMed2003

Spatially organized layers of cardiomyocytes on biodegradable polyurethane films for myocardial repair.

Tissue engineering constructs should match the physical and mechanical properties of the native tissue. This implies that pliable scaffolds might be better suited for soft-tissue applications than rigid polymeric materials. In this study, we examined spatially organized cardiomyocyte cultures on biodegradable, elastomeric polyurethane films patterned by microcontact printing of laminin lanes. The resulting cardiomyocyte patterns on polyurethane displayed a similar morphology to those previously achieved for up to 7-10 days on other substrates, such as polystyrene dishes. However, the integrity of the cardiomyocyte patterns on thin, spin-cast or solvent-cast polyurethane films was retained for up to 4 weeks in culture. When additional cardiomyocytes (labeled with Cell Tracker reagents) were seeded onto the patterned cultures, secondary and tertiary cell populations aligned between and on top of the primary patterned cells to form a multilayered, organized tissue construct approximately 2-3 cell layers thick. In addition, dense, highly aligned monolayers of patterned cardiomyocytes were able to contract the thin, solvent-cast polyurethane films. These results indicate that elastomeric, biodegradable polyurethane films can serve as an appropriate scaffold material to support stably the engineering of spatially organized layers of cardiomyocytes in vitro. This approach may serve as a novel method for transplantation of organized cardiac tissue constructs to the heart for myocardial repair.

Animals↗
PubMed2003

Myofibroblast and endothelial cell proliferation during murine myocardial infarct repair.

Granulation tissue formation is a critical step in infarct repair, however, the kinetics of cell replication and the molecules that regulate this process are poorly understood. In uninjured mouse hearts and at 2 days post-infarction, very little DNA synthesis (measured by incorporation of a BrdU pulse) was detected in any cell type. Four days after permanent coronary occlusion, the rates of myofibroblast (smooth muscle alpha-actin and BrdU double-positive) and endothelial cell (CD31 and BrdU double-positive) proliferation were 15.4 +/- 1.1% and 2.9 +/- 0.5%, respectively. Most proliferating cells were located at the interface of the infarct and viable tissue. By 1 week, fibroblast and endothelial cell proliferation declined to 4.1 +/- 0.6% and 0.7 +/- 0.1%, respectively. In the 2-week infarct, the remaining necrosis had been phagocytosed, and fibroblast and endothelial cell proliferation were <0.5%. Although leukocytes were abundant throughout infarct repair, no significant proliferation was detected at any time in cells expressing CD45 or mac-3. Infarct size at 4 days was 38 +/- 5% of the left ventricle and contracted to 20 +/- 4% by 4 weeks. After 4 days, the chamber dilated to four times that of the control hearts and remained so for the duration of the time course. The vascular density (per mm(2)) declined from 3643 +/- 82 in control hearts to 2716 +/- 197 at 1 week and 1010 +/- 47 at 4 weeks post-myocardial infarction (MI). The average percent area occupied by vessels did not change significantly between the groups but the area/vessel ( micro m(2)) increased from 14.1 +/- 0.3 in control hearts to 16.9 +/- 1.9 at 1 week and 38.7 +/- 7.9 at 4 weeks post-MI. These data indicate that mitogens for fibroblasts and endothelial cells peak within 4 days of infarction in the mouse heart. This provides the basis for identifying the responsible molecules and developing strategies to alter wound repair and improve cardiac function.

Animals↗
PubMed2002

In vitro generation of differentiated cardiac myofibers on micropatterned laminin surfaces.

Cardiac muscle fibers consist of highly aligned cardiomyocytes containing myofibrils oriented parallel to the fiber axis, and successive cardiomyocytes are interconnected at their ends through specialized junctional complexes (intercalated disks). Cell culture studies of cardiac myofibrils and intercalated disks are complicated by the fact that cardiomyocytes become extremely flattened and exhibit disorganized myofibrils and diffuse intercellular junctions with neighboring cells. In this study we sought to direct the organization of cultured cardiomyocytes to more closely resemble that found in vivo. Lanes of laminin 5-50 microm wide were microcontact-printed onto nonadhesive (BSA-coated) surfaces. Adherent cardiomyocytes responded to the spatial constraints by forming elongated, rod-shaped cells whose myofibrils aligned parallel to the laminin lanes. Patterned cardiomyocytes displayed a striking, bipolar localization of the junction molecules N-cadherin and connexin43 that ultrastructurally resembled intercalated disks. When laminin lanes were widely spaced, each lane of cardiomyocytes beat independently, but with narrow-spacing cells bridged between lanes, yielding aligned fields of synchronously beating cardiomyocytes. Similar cardiomyocyte patterns were achieved on the biodegradable polymer PLGA, suggesting that patterned cardiomyocytes could be used in myocardial tissue engineering. Such highly patterned cultures could be used in cell biology and physiology studies, which require accurate reproduction of native myocardial architecture.

Animals↗
PubMed2002

Evidence for cardiomyocyte repopulation by extracardiac progenitors in transplanted human hearts.

Human myocardium has long been considered to have essentially no intrinsic regenerative capacity. Recent studies in rodent models, however, have suggested the presence of an extracardiac stem cell population, perhaps in bone marrow, that is capable of some reconstitution of cardiomyocytes after injury. To determine whether similar mechanisms exist in the human heart, we evaluated human female allograft hearts transplanted into male patients. The presence of Y chromosomes in cardiomyocytes would indicate these cells arose from the recipient, rather than the donor heart. We identified 5 male patients who had retained a female heart at least 9 months before death and necropsy. Remarkably, in each case, the transplanted heart contained a minute but readily detectable fraction of Y chromosome-positive cardiomyocytes. The mean percentage of cardiomyocytes arising from the host was estimated to be 0.04% with a median of 0.016%. Most Y-positive cardiomyocytes were associated with regions of acute rejection, suggesting such chimerism involves an injury event. Furthermore, the sole patient whose immediate cause of death was allograft rejection showed a much higher percentage of host-derived cardiomyocytes, up to 29% in local, 1-mm(2) "hot spots." Thus, adult humans have extracardiac progenitor cells capable of migrating to and repopulating damaged myocardium, but this process occurs at very low levels.

Cell Differentiation↗