Waard, M.C. de

Age-related motor neuron degeneration in DNA repair-deficient Ercc1 mice (Article)

2010-10-01T00:00:00Z

Degeneration of motor neurons contributes to senescence-associated loss of muscle function and underlies human neurodegenerative conditions such as amyotrophic lateral sclerosis and spinal muscular atrophy. The identification of genetic factors contributing to motor neuron vulnerability and degenerative phenotypes in vivo are therefore important for our understanding of the neuromuscular system in health and disease. Here, we analyzed neurodegenerative abnormalities in the spinal cord of progeroid Ercc1Δ/- mice that are impaired in several DNA repair systems, i.e. nucleotide excision repair, interstrand crosslink repair, and double strand break repair. Ercc1Δ/- mice develop age-dependent motor abnormalities, and have a shortened life span of 6-7 months. Pathologically, Ercc1Δ/- mice develop widespread astrocytosis and microgliosis, and motor neuron loss and denervation of skeletal muscle fibers. Degenerating motor neurons in many occasions expressed genotoxic-responsive transcription factors p53 or ATF3, and in addition, displayed a range of Golgi apparatus abnormalities. Furthermore, Ercc1Δ/- motor neurons developed perikaryal and axonal intermediate filament abnormalities reminiscent of cytoskeletal pathology observed in aging spinal cord. Our findings support the notion that accumulation of DNA damage and genotoxic stress may contribute to neuronal aging and motor neuron vulnerability in human neuromuscular disorders.

Lentiviral gene therapy of murine hematopoietic stem cells ameliorates the Pompe disease phenotype (Article)

2010-07-01T00:00:00Z

Pompe disease (acid α-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector-mediated expression of human acid α-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid α-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid α-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue.

Detrimental effect of combined exercise training and eNOS overexpression on cardiac function after myocardial infarction (Article)

2009-05-01T00:00:00Z

It has been reported that exercise after myocardial infarction (MI) attenuates left ventricular (LV) pump dysfunction by normalization of myofilament function. This benefit could be due to an exercise-induced upregulation of endothelial nitric oxide synthase (eNOS) expression and activity. Consequently, we first tested the hypothesis that the effects of exercise after MI can be mimicked by elevated eNOS expression using transgenic mice with overexpression of human eNOS (eNOSTg). Both exercise and eNOSTg attenuated LV remodeling and dysfunction after MI in mice and improved cardiomyocyte maximal force development (Fmax). However, only exercise training restored myofilament Ca2+-sensitivity and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a protein levels and improved the first derivative of LV pressure at 30 mmHg. Conversely, only eNOSTg improved survival. In view of these partly complementary actions, we subsequently tested the hypothesis that combining exercise and eNOSTg would provide additional protection against LV remodeling and dysfunction after MI. Unexpectedly, the combination of exercise and eNOSTg abolished the beneficial effects on LV remodeling and dysfunction of either treatment alone. The latter was likely due to perturbations in Ca2+ homeostasis, as myofilament Fmax actually increased despite marked reductions in the phosphorylation status of several myofilament proteins, whereas the exercise-induced increases in SERCA2a protein levels were lost in eNOSTg mice. Antioxidant treatment with N-acetylcysteine or supplementation of tetrahydrobiopterin and L-arginine prevented these detrimental effects on LV function while partly restoring the phosphorylation status of myofilament proteins and further enhancing myofilament Fmax. In conclusion, the combination of exercise and elevated eNOS expression abolished the cardioprotective effects of either treatment alone after MI, which appeared to be, at least in part, the result of increased oxidative stress secondary to eNOS "uncoupling."

Myofilament dysfunction in cardiac disease from mice to men (Article)

2008-12-01T00:00:00Z

In healthy human myocardium a tight balance exists between receptor-mediated kinases and phosphatases coordinating phosphorylation of regulatory proteins involved in cardiomyocyte contractility. During heart failure, when neurohumoral stimulation increases to compensate for reduced cardiac pump function, this balance is perturbed. The imbalance between kinases and phosphatases upon chronic neurohumoral stimulation is detrimental and initiates cardiac remodelling, and phosphorylation changes of regulatory proteins, which impair cardiomyocyte function. The main signalling pathway involved in enhanced cardiomyocyte contractility during increased cardiac load is the β-adrenergic signalling route, which becomes desensitized upon chronic stimulation. At the myofilament level, activation of protein kinase A (PKA), the down-stream kinase of the β-adrenergic receptors (β-AR), phosphorylates troponin I, myosin binding protein C and titin, which all exert differential effects on myofilament function. As a consequence of β-AR down-regulation and desensitization, phosphorylation of the PKA-target proteins within the cardiomyocyte may be decreased and alter myofilament function. Here we discuss involvement of altered PKA-mediated myofilament protein phosphorylation in different animal and human studies, and discuss the roles of troponin I, myosin binding protein C and titin in regulating myofilament dysfunction in cardiac disease. Data from the different animal and human studies emphasize the importance of careful biopsy procurement, and the need to investigate localization of kinases and phosphatases within the cardiomyocyte, in particular their co-localization with cardiac myofilaments upon receptor stimulation.

Cardioprotective effects of exercise training: the importance of nitric oxide (Doctoral Thesis)

2008-09-10T00:00:00Z

Left ventricular remodeling after myocardial infarction is a compensatory mechanism, which serves to restore left ventricular pump function to normal levels. Despite the apparent appropriateness of the process, left ventricular remodeling after myocardial infarction is an independent risk factor for the development of angina pectoris and heart failure. In contrast, physiological left ventricular remodeling produced by regular exercise training is associated with a decreased risk for heart failure. This PhD-project focuses on the physiological, cellular and molecular basis for the differences in myocardial remodeling between exercise-induced and pathological left ventricular remodeling. In addition, we investigate how regular exercise training favorably influences the myocardial abnormalities associated with pathological left ventricular remodeling, in particular, the role of an increased nitric oxide production in the beneficial effects of exercise training. For this purpose, studies are performed in wild-type mice but also eNOS transgenic or knock-out mice. Specifically we test the hypothesis that eNOS overexpression mimics exercise training and that eNOS knock-out abrogates the beneficial effects of exercise training in mice with a myocardial infarction.

A novel model of cryoinjury-induced myocardial infarction in the mouse: a comparison with coronary artery ligation. (Article)

2005-09-01T00:00:00Z

Mouse myocardial infarction (MI) models are frequently used research tools. The most commonly applied model is coronary artery ligation. However, coronary ligation often gives rise to apical aneurysmatic infarcts of variable size. Other infarct models include cryoinfarction, which produces reproducible infarcts of the anterior wall. Thus far, this model has not been extensively described in mice. Therefore, we developed a murine cryoinfarction model and compared it with coronary ligation. Studies were performed under isoflurane anesthesia with a follow-up of 4 and 8 wk. Cryoinfarction was induced using a 2- or 3-mm cryoprobe. Two-dimensional guided M-mode echocardiography was used to assess fractional shortening and left ventricular (LV) dimensions at baseline and end point. At end point, hemodynamics were assessed using a 1.4-Fr Millar catheter. Pressure-diameter relations were constructed by combining echocardiography and hemodynamic data. Histological and morphometric analyses of infarct and remote areas were performed. At 4 wk, 3-mm cryoinfarction resulted in decreased LV fractional shortening as well as decreased global LV contractility and relaxation, which was comparable with coronary ligation. No adverse remodeling was observed at this time point, in contrast with the ligation model. However, progressive LV remodeling occured between 4 and 8 wk after cryoinfarction with a further decline in hemodynamic parameters and LV pump function. Histologically, cryoinfarction resulted in highly reproducible, transmural, cone-shaped infarcts with reperfusion at the macrovascular level. These results indicate that the cryoinfarction model represents the anterior myocardial infarct with modest adverse remodeling and may thus be representative for infarcts encountered in clinical practice.

Functional expression of endothelial nitric oxide synthase fused to green fluorescent protein in transgenic mice (Article)

2003-01-01T00:00:00Z

The activity of endothelial nitric oxide synthase (eNOS) is subject to complex transcriptional and post-translational regulation including the association with several proteins and variations in subcellular distribution. In the present study we describe a transgenic mouse model expressing eNOS fused to green fluorescent protein (GFP), which allows the study of localization and regulation of eNOS expression. We tested the functionality of eNOS in the eNOS-GFP mice. Expression of eNOS was restricted to the endothelial lining of blood vessels in various tissues tested, without appreciable expression in non-endothelial cells. Activity of the enzyme was confirmed by assaying the conversion of L-arginine to L-citrulline. NO production in isolated vessels was increased in transgenic mice when compared to non-transgenic control animals (4.88 +/- 0.59 and 2.48 +/- 0.47 micro mol/L NO, respectively, P < 0.005). Both the mean aortic pressure and the pulmonary artery pressure were reduced in eNOS-GFP mice (both approximately 30%, P < 0.05). Plasma cholesterol levels were also slightly reduced ( approximately 20%, P < 0.05). In conclusion, eNOS-GFP mice express functional eNOS and provide a unique model to study regulation of eNOS activity or eNOS-mediated vascular events, including response to ischemia, response to differences in shear stress, angiogenesis and vasculogenesis, and to study the subcellular distribution in relation with functional responses to these events.

Reduction of blood pressure, plasma cholesterol, and atherosclerosis by elevated endothelial nitric oxide. (Article)

2002-12-13T00:00:00Z

In the vascular system, nitric oxide is generated by endothelial NO synthase (eNOS). NO has pleiotropic effects, most of which are believed to be atheroprotective. Therefore, it has been argued that patients suffering from cardiovascular disease could benefit from an increase in eNOS activity. However, increased NO production can cause oxidative damage, cell toxicity, and apoptosis and hence could be atherogenic rather than beneficial. To study the in vivo effects of increased eNOS activity, we created transgenic mice overexpressing human eNOS. Aortic blood pressure was approximately 20 mm Hg lower in the transgenic mice compared with control mice because of lower systemic vascular resistance. The effects of eNOS overexpression on diet-induced atherosclerosis were studied in apolipoprotein E-deficient mice. Elevation of eNOS activity decreased blood pressure ( approximately 20 mm Hg) and plasma levels of cholesterol (approximately 17%), resulting in a reduction in atherosclerotic lesions by 40%. We conclude that an increase in eNOS activity is beneficial and provides protection against atherosclerosis.