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Biomedical subjects

Loren J Field

Publications and source records attributed to Loren J Field.

At least 19 recordsLinked to original sources
PubMed2006

Expression of a mutant p193/CUL7 molecule confers resistance to MG132- and etoposide-induced apoptosis independent of p53 or Parc binding.

p193/CUL7 is an E3 ubiquitin ligase initially identified as an SV40 Large T Antigen binding protein. Expression of a dominant interfering variant of mouse p193/CUL7 (designated 1152stop) conferred resistance to MG132- and etoposide-induced apoptosis in U2OS cells. Immune precipitation/Western analyses revealed that endogenous p193/CUL7 formed a complex with Parc (a recently identified parkin-like ubiquitin ligase) and p53. Apoptosis resistance did not result from 1152stop-mediated disruption of the endogenous p193/CUL7 binding partners. Moreover, 1152stop molecule did not directly bind to endogenous p193/CUL7, Parc or p53. These data suggested a role for p193/CUL7 in the regulation of apoptosis independently of p53 and Parc activity.

Animals↗
PubMed2006

Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth.

Postnatal cardiac hypertrophies have traditionally been classified into physiological or pathological hypertrophies. Both of them are induced by hemodynamic load. Cardiac postnatal hypertrophic growth is regarded as a part of the cardiac maturation process that is independent of the cardiac working load. However, the functional significance of this biological event has not been determined, mainly because of the difficulty in creating an experimental condition for testing the growth potential of functioning heart in the absence of hemodynamic load. Recently, we generated a novel transgenic mouse model (alphaMHC-BMP10) in which the cardiac-specific growth factor bone morphogenetic protein 10 (BMP10) is overexpressed in postnatal myocardium. These alphaMHC-BMP10 mice appear to have normal cardiogenesis throughout embryogenesis, but develop to smaller hearts within 6 weeks after birth. alphaMHC-BMP10 hearts are about half the normal size with 100% penetrance. Detailed morphometric analysis of cardiomyocytes clearly indicated that the compromised cardiac growth in alphaMHC-BMP10 mice was solely because of defect in cardiomyocyte postnatal hypertrophic growth. Physiological analysis further demonstrated that the responses of these hearts to both physiological (e.g. exercise-induced hypertrophy) and pathological hypertrophic stimuli remain normal. In addition, the alphaMHC-BMP10 mice develop subaortic narrowing and concentric myocardial thickening without obstruction by four weeks of age. Systematic analysis of potential intracellular pathways further suggested a novel genetic pathway regulating this previously undefined cardiac postnatal hypertrophic growth event. This is the first demonstration that cardiac postnatal hypertrophic growth can be specifically modified genetically and dissected out from physiological and pathological hypertrophies.

Animals↗
PubMed2006

The cardiomyocyte cell cycle.

Many forms of cardiac disease are characterized by cardiomyocyte death due to necrosis, apoptosis and/or oncosis. Recently, the notion of promoting cardiac regeneration as a means to replace damaged heart tissue has engendered considerable interest. One approach to accomplish heart muscle regeneration entails promoting cardiomyocyte cell cycle activity in the surviving myocardium. Genetically modified mice have provided useful model systems to test the efficacy of specific pathways to promote cardiomyocyte proliferation in normal and diseased hearts. For example, expression of a heart-restricted dominant interfering version of p193 (an E3 ubiquitin ligase also known as Cul7) resulted in an induction of cardiomyocyte cell cycle activity at the infarct border zone and ventricular septum 4 weeks after permanent coronary artery occlusion. A concomitant reduction in hypertrophic cardiomyocyte growth was also observed in this model, suggesting that cell cycle activation partially counteracted the adverse ventricular remodelling that occurs post-infarction. In other studies, targeted expression of cyclin D2 promoted cardiomyocyte cell cycle activity in adult hearts. The level of cardiomyocyte cell cycle activity increased after myocardial infarction, ultimately resulting in a marked increase in cardiomyocyte number and a concomitant regression of infarct size. Collectively, these data suggest that modulation of cardiomyocyte cell cycle activity can be exploited to promote regenerative growth in injured hearts.

Animals↗
PubMed2006

Cardiac regeneration: repopulating the heart.

Many forms of pediatric and adult heart disease result from a deficiency in cardiomyocyte number. Through repopulation of the heart with new cardiomyocytes (that is, induction of regenerative cardiac growth), cardiac disease potentially can be reversed, provided that the newly formed myocytes structurally and functionally integrate in the preexisting myocardium. A number of approaches have been utilized to effect regenerative growth of the myocardium in experimental animals. These include interventions aimed at enhancing the ability of cardiomyocytes to proliferate in response to cardiac injury, as well as transplantation of cardiomyocytes or myogenic stem cells into diseased hearts. Here we review efforts to induce myocardial regeneration. We also provide a critical review of techniques currently used to assess cardiac regeneration and functional integration of de novo cardiomyocytes.

Animals↗
PubMed2006

Cell-based approaches for cardiac repair.

Many forms of cardiovascular disease are associated with cardiomyocyte loss via necrosis and/or apoptosis. The cumulative loss of contractile cells ultimately results in diminished cardiac function. Numerous approaches have been employed to reduce the rate of cardiomyocyte loss, or alternatively, to repopulate the heart with new cardiomyocytes. Strategies aimed at repopulating the heart include cardiomyocyte cell therapy, myogenic stem cell therapy, and cell cycle activation therapy. All three approaches are based on the assumption that the de novo cardiomyocytes will participate in a functional syncytium with the surviving myocardium. This review will discuss the current status of interventions aimed at repopulating the heart with functional cardiomyocytes.

Animals↗
PubMed2005

Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control.

It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5 x 10(6) cells/mL after 9 days of differentiation. Approximately 30%-40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (alphaMHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28 x 10(9) cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications.

Animals↗
PubMed2005

Cardiomyocyte cell cycle activation ameliorates fibrosis in the atrium.

MHC-TGFcys33ser transgenic mice have elevated levels of active transforming growth factor (TGF)-beta1 in the myocardium. Previous studies have shown that these animals develop atrial, but not ventricular, fibrosis. Here we show that atrial fibrosis was accompanied with cardiomyocyte apoptosis. Although similar levels of cardiomyocyte apoptosis were present in the right and left atria of MHC-TGFcys33ser hearts, the extent of fibrosis was more pronounced in the right atrium. Thus, additional factors influence the degree of atrial fibrosis in this model. Tritiated thymidine incorporation studies revealed cardiomyocyte cell cycle activity in left atrial cardiomyocytes, but not in right atrial cardiomyocytes. These observations suggested that cardiomyocyte cell cycle activation ameliorated the severity of atrial fibrosis. To directly test this hypothesis, MHC-TGFcys33ser mice were crossed with MHC-cycD2 mice (which have constitutive cardiomyocyte cell cycle activity in the right atrium). Mice inheriting both transgenes exhibited right atrial cardiomyocyte cell cycle activity and a concomitant reduction in the severity of right atrial fibrosis, despite the presence of a similar level of cardiomyocyte apoptosis as was observed in mice inheriting the MHC-TGFcys33ser transgene alone. These data support the notion that cardiomyocyte cell cycle induction can antagonize fibrosis in the myocardium.

Animals↗
PubMed2004

Targeted expression of cyclin D2 results in cardiomyocyte DNA synthesis and infarct regression in transgenic mice.

Restriction point transit and commitment to a new round of cell division is regulated by the activity of cyclin-dependent kinase 4 and its obligate activating partners, the D-type cyclins. In this study, we examined the ability of D-type cyclins to promote cardiomyocyte cell cycle activity. Adult transgenic mice expressing cyclin D1, D2, or D3 under the regulation of the alpha cardiac myosin heavy chain promoter exhibited high rates of cardiomyocyte DNA synthesis under baseline conditions. Cardiac injury in mice expressing cyclin D1 or D3 resulted in cytoplasmic cyclin D accumulation, with a concomitant reduction in the level of cardiomyocyte DNA synthesis. In contrast, cardiac injury in mice expressing cyclin D2 did not alter subcellular cyclin localization. Consequently, cardiomyocyte cell cycle activity persisted in injured hearts expressing cyclin D2, ultimately resulting in infarct regression. These data suggested that modulation of D-type cyclins could be exploited to promote regenerative growth in injured hearts.

Age Factors↗
PubMed2004

Expression of mutant p193 and p53 permits cardiomyocyte cell cycle reentry after myocardial infarction in transgenic mice.

Previous studies have demonstrated that expression of p193 and p53 mutants with dominant-interfering activities renders embryonic stem cell-derived cardiomyocytes responsive to the growth promoting activities of the E1A viral oncoproteins. In this study, the effects of p53 and p193 antagonization on cardiomyocyte cell cycle activity in normal and infarcted hearts were examined. Transgenic mice expressing the p193 and/or the p53 dominant-interfering mutants in the heart were generated. Transgene expression had no effect on cardiomyocyte cell cycle activity in uninjured adult hearts. In contrast expression of either transgene resulted in a marked induction of cardiomyocyte cell cycle activity at the infarct border zone at 4 weeks after permanent coronary artery occlusion. Expression of the p193 dominant-interfering mutant was also associated with an induction of cardiomyocyte DNA synthesis in the interventricular septa of infarcted hearts. A concomitant and marked reduction in hypertrophic cardiomyocyte growth was observed in the septa of hearts expressing the p193 dominant-interfering transgene, suggesting that cell cycle activation might partially counteract the adverse ventricular remodeling that occurs after infarction. Collectively these data suggest that antagonization of p193 and p53 activity relaxes the otherwise stringent regulation of cardiomyocyte cell cycle reentry in the injured adult heart.

Amino Acid Substitution↗
PubMed2004

Increased vulnerability to atrial fibrillation in transgenic mice with selective atrial fibrosis caused by overexpression of TGF-beta1.

Studies on patients and large animal models suggest the importance of atrial fibrosis in the development of atrial fibrillation (AF). To investigate whether increased fibrosis is sufficient to produce a substrate for AF, we have studied cardiac electrophysiology (EP) and inducibility of atrial arrhythmias in MHC-TGFcys33ser transgenic mice (Tx), which have increased fibrosis in the atrium but not in the ventricles. In anesthetized mice, wild-type (Wt) and Tx did not show significant differences in surface ECG parameters. With transesophageal atrial pacing, no significant differences were observed in EP parameters, except for a significant decrease in corrected sinus node recovery time in Tx mice. Burst pacing induced AF in 14 of 29 Tx mice, whereas AF was not induced in Wt littermates (P<0.01). In Langendorff perfused hearts, atrial conduction was studied using a 16-electrode array. Epicardial conduction velocity was significantly decreased in the Tx RA compared with the Wt RA. In the Tx LA, conduction velocity was not significantly different from Wt, but conduction was more heterogeneous. Action potential characteristics recorded with intracellular microelectrodes did not reveal differences between Wt and Tx mice in either atrium. Thus, in this transgenic mouse model, selective atrial fibrosis is sufficient to increase AF inducibility.

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

Allogeneic stem cell-derived "repair unit" therapy and the barriers to clinical deployment.

The development of cellular therapies has been seriously hampered by the paucity of cells available for grafting from living or cadaver donors. This sourcing problem can be resolved, at least in theory, by generating tissue-specific cells from autologous or allogeneic stem cells. Nonetheless, there are formidable barriers to the use of stem cells in the clinic. This review focuses on identifying and surmounting these barriers.

Animals↗
PubMed2004

Spontaneous and evoked intracellular calcium transients in donor-derived myocytes following intracardiac myoblast transplantation.

Skeletal myoblast transplantation is a potential treatment for congestive heart failure. To study the functional activity of both donor and host myocytes following transplantation, skeletal myoblasts expressing an enhanced green fluorescent protein (EGFP) transgene were transplanted into hearts of nontransgenic recipients, and changes in intracellular calcium concentration ([Ca2+]i) were monitored in donor and host cells. While the vast majority of donor-derived myocytes were observed to be functionally isolated from the host myocardium, a small population of donor myocytes exhibited action potential-induced calcium transients in synchrony with adjacent host cardiomyocytes. In many cases, the durations of these [Ca2+]i transients were heterogeneous compared with those in neighboring host cardiomyocytes. In other studies, EGFP-expressing donor myoblasts were transplanted into the hearts of adult transgenic recipient mice expressing a cardiomyocyte-restricted beta-gal reporter gene. A small population of myocytes was observed to express both reporter transgenes, indicating that the transplanted myoblasts fused with host cardiomyocytes at a very low frequency. These cells also expressed connexin43, a component of gap junctions. Thus engraftment of skeletal myoblasts generated spatial heterogeneity of [Ca2+]i signaling at the myocardial/skeletal muscle interface, most likely as a consequence of fusion events between donor myoblasts and host cardiomyocytes.

Action Potentials↗
PubMed2004

Modulation of the cardiomyocyte cell cycle in genetically altered animals.

Many forms of cardiovascular disease are associated with cardiomyocyte loss via apoptosis and/or necrosis. Although there is currently debate regarding the level at which adult cardiomyocytes can reenter the cell cycle and proliferate, it is clear that the intrinsic regenerative growth capacity is insufficient to reverse the progression to failure in badly injured hearts. The ability to reactivate cardiomyocyte proliferation in damaged hearts might permit regenerative growth, provided that the nascent cells are able to participate in a functional syncytium with the surviving myocardium. In this review, techniques commonly used to monitor cardiomyocyte cell cycle activity in normal and injured hearts are discussed. In addition, several genetic models are described wherein the expression of fundamental cell cycle regulatory proteins has been altered in cardiomyocytes.

Animals↗
PubMed2003

Myocyte and myogenic stem cell transplantation in the heart.

Cellular transplantation is emerging as a potential mechanism with which to augment myocyte number in diseased hearts. To date a number of cell types have been shown to successfully engraft into the myocardium, including fetal, neonatal, and embryonic stem cell-derived cardiomyocytes, skeletal myoblasts, and stem cells with apparent cardiomyogenic potential. Here we provide a review of studies wherein myocytes or stem cells with myogenic potential have been transplanted into the heart. In addition, issues pertaining to the tracking and functional consequences of cell transplantation are discussed.

Animals↗
PubMed2003

Physiological coupling of donor and host cardiomyocytes after cellular transplantation.

Cellular transplantation has emerged as a potential approach to treat diseased hearts. Although cell transplantation can affect global heart function, it is not known if this results directly via functional integration of donor myocytes or indirectly via enhanced revascularization and/or altered postinjury remodeling. To determine the degree to which donor cardiomyocytes are able to functionally integrate with the host myocardium, fetal transgenic cardiomyocytes expressing enhanced green fluorescent protein were transplanted into the hearts of nontransgenic adult mice. Two-photon molecular excitation laser scanning microscopy was then used to simultaneously image cellular calcium transients in donor and host cells within the intact recipient hearts. Calcium transients in the donor cardiomyocytes were synchronous with and had kinetics indistinguishable from those of neighboring host cardiomyocytes. These results strongly suggest that donor cardiomyocytes functionally couple with host cardiomyocytes and support the notion that transplanted cardiomyocytes can form a functional syncytium with the host myocardium.

Animals↗