Deel, E.D. van

eNOS and Oxidative Stress in the Remodeling Heart: a delicate balance (Doctoral Thesis)

2012-01-25T00:00:00Z

Cardiovascular disease has become the leading cause of death worldwide. Although typically characterized as a major burden in the Western world, heart related diseases are also rapidly expanding in developing countries. In the Netherlands, cardiovascular diseases are responsible for 30% of all deaths and are the main cause of death in woman. Paradoxically, improved treatment of cardiovascular diseases reduced acute mortality but greatly increased the number of patients suffering from insufficient cardiac pump function referred to as heart failure (HF). HF is the final common stage of all cardiac diseases and considerably increases the risk for morbidity and mortality. Moreover, the prevalence (proportion of the population affected by the disease) and incidence (number of new cases in a period of time) are still increasing. With a prevalence of over 23 million globally, HF has become a chronic disease epidemic. Large cohort studies like the Framingham Heart Study, the Rochester Epidemiologic Project in Olmsted County and the Rotterdam Study consistently show that the prevalence of HF is higher in men than in woman and substantially increases with age 6-10 reaching 1% in those 55-64 years of age and 13% in those aged over 75 years in the Rotterdam Study.6 Additionally race appears to play a role in the prevalence of HF as death rates of HF have shown to be higher in black than in white patients.

Exercise training does not improve cardiac function in compensated or decompensated left ventricular hypertrophy induced by aortic stenosis (Article)

2011-06-01T00:00:00Z

There is ample evidence that regular exercise exerts beneficial effects on left ventricular (LV) hypertrophy, remodeling and dysfunction produced by ischemic heart disease or systemic hypertension. In contrast, the effects of exercise on pathological LV hypertrophy and dysfunction produced by LV outflow obstruction have not been studied to date. Consequently, we evaluated the effects of 8 weeks of voluntary wheel running in mice (which mitigates post-infarct LV dysfunction) on LV hypertrophy and dysfunction produced by mild (mTAC) and severe (sTAC) transverse aortic constriction. mTAC produced ~ 40% LV hypertrophy and increased myocardial expression of hypertrophy marker genes but did not affect LV function, SERCA2a protein levels, apoptosis or capillary density. Exercise had no effect on global LV hypertrophy and function in mTAC but increased interstitial collagen, and ANP expression. sTAC produced ~ 80% LV hypertrophy and further increased ANP expression and interstitial fibrosis and, in contrast with mTAC, also produced LV dilation, systolic as well as diastolic dysfunction, pulmonary congestion, apoptosis and capillary rarefaction and decreased SERCA2a and ryanodine receptor (RyR) protein levels. LV diastolic dysfunction was likely aggravated by elevated passive isometric force and Ca2+-sensitivity of myofilaments. Exercise training failed to mitigate the sTAC-induced LV hypertrophy and capillary rarefaction or the decreases in SERCA2a and RyR. Exercise attenuated the sTAC-induced increase in passive isometric force but did not affect myofilament Ca2+-sensitivity and tended to aggravate interstitial fibrosis. In conclusion, exercise had no effect on LV function in compensated and decompensated cardiac hypertrophy produced by LV outflow obstruction, suggesting that the effect of exercise on pathologic LV hypertrophy and dysfunction depends critically on the underlying cause.

Left-ventricular remodeling after myocardial infarction is associated with a cardiomyocyte-specific hypothyroid condition (Article)

2011-02-01T00:00:00Z

Similarities in cardiac gene expression in hypothyroidism and left ventricular (LV) pathological remodeling after myocardial infarction (MI) suggest a role for impaired cardiac thyroid hormone (TH) signaling in the development of heart failure. Increased ventricular activity of the TH-degrading enzyme type 3 deiodinase (D3) is recognized as a potential cause. In the present study, we investigated the cardiac expression and activity of D3 over an 8-wk period after MI in C57Bl/6J mice. Pathological remodeling of the noninfarcted part of the LV was evident from cardiomyocyte hypertrophy, interstitial fibrosis, and impairment of contractility. These changes were maximal and stable from the first week onward, as was the degree of LV dilation. A strong induction of D3 activity was found, which was similarly stable for the period examined. Plasma T4 levels were transiently decreased at 1 wk after MI, but T3 levels remained normal. The high D3 activity was associated with increased D3 mRNA expression at 1 but not at 4 and 8 wk after MI. Immunohistochemistry localized D3 protein to cardiomyocytes. In vivo measurement of TH-dependent transcription activity in cardiomyocytes using a luciferase reporter assay indicated a 48% decrease in post-MI mice relative to sham-operated animals, and this was associated with a 50% decrease in LVtissue T3 concentration. In conclusion, pathological ventricular remodeling after MI in themouse leads to high and stable induction of D3 activity in cardiomyocytes and a local hypothyroid condition.

Dendritic cell function in transplantation arteriosclerosis is regulated by heme oxygenase 1 (Article)

2010-05-28T00:00:00Z

RATIONALE: Heme oxygenase (HO)1 is an important modulator of physiological function with cytoprotective properties. Although HO1 has previously been associated with an improved survival of the vascular allograft in rat models in response to pharmaceutical induction of HO1 the exact mechanism by which HO1 exerts it protective function remains to be elucidated. OBJECTIVE: We sought to define the role of HO1 in dendritic cells (DCs) function that governs the alloimmune response underlying the development of transplantation associated vasculopathy. METHODS AND RESULTS: Loss of HO1 in DCs or by small interfering RNA silencing resulted in major histocompatibility complex class II (MHCII) upregulation by CIITA-driven transcriptional regulation and by STAT1 (signal transducers and activators of transcription 1) phosphorylation. As a result, increased MHCII alloantigen presentation by HO1 DCs directed the primary T-cell response preferentially toward a CD4 T-cell, rather than a CD8 T-cell reaction. In a murine model for transplantation arteriosclerosis, adoptive transfer of HO1 DCs before allograft transplantation was indeed associated with pronounced intragraft CD4 T-cell infiltration and increased IgG deposition, suggestive of an accelerated development of vasculopathy toward the chronic phase. The role of HO1 in DC-mediated T cell activation was further validated by inhibition of endogenous HO1 in allograft recipients. Inhibition of HO1 in DCs aggravated transplant arteriosclerosis development, by increasing intima hyperplasia, and by activation of a CD4 T cells allograft response, mediated by MHCII upregulation. CONCLUSIONS: These findings demonstrate that HO1 plays an important role in the genetic regulation of the vascular alloimmune response elicited by DCs.

Early exercise training after myocardial infarction prevents contractile but not electrical remodelling or hypertrophy (Article)

2010-04-01T00:00:00Z

Aims: Exercise started early after myocardial infarction (MI) improves in vivo cardiac function and myofilament responsiveness to Ca2+. We investigated whether this represents partial or complete reversal of cellular remodelling. Methods and results: Mice with MI following left coronary ligation were given free access to a running wheel (MIEXE, N = 22) or housed sedentary (MISED, N = 18) for 8 weeks and compared with sedentary sham-operated animals (SHAM, N = 11). Myocytes were enzymatically isolated from the non-infarcted left ventricle. Myocytes in MI were significantly longer and even more so with exercise (165 ± 3 μm in MIEXEvs. 148 ± 3 μm in MISEDand 136 ± 2 μm in SHAM; P < 0.05, mean ± SEM); cell width was not different. Contraction was measured during electrical field stimulation at 1, 2, and 4 Hz. Unloaded cell shortening was significantly reduced in MISED(at 1 Hz, L/L0=4.4 ± 0.3% vs. 6.7 ± 0.4% in SHAM; P < 0.05, also at 2 and 4 Hz). Exercise restored cell shortening to SHAM values (MIEXE, L/L0=6.4 ± 0.5%). Membrane currents and [Ca2+]iwere measured via whole-cell patch clamping, with Fluo-3 as Ca2+indicator, all at 30°C. Ca2+transient amplitude, ICaLand sarcoplasmic reticulum Ca2+content were not different between the three groups. Diastolic Ca2+levels at 4 Hz were significantly elevated in MISEDonly, with a trend to increased spontaneous Ca2+release events (sparks). Action potential duration was increased and transient outward K+currents significantly reduced after MI; this was unaffected by exercise. Conclusions: Early voluntary exercise training after MI restores cell contraction to normal values predominantly because of changes in the myofilament Ca2+response and has a beneficial effect on diastolic Ca2+handling. However, the beneficial effect is not a complete reversal of remodelling as hypertrophy and loss of repolarizing K+currents are not affected.

Epicardial coronary artery Doppler: Validation in the animal model (Article)

2008-08-01T00:00:00Z

The aim of the study was to validate a newly-designed epicardial coronary artery Doppler probe and test its detection of changes in coronary blood flow velocity. Left anterior descending (LAD) coronary blood flow and flow velocity were evaluated in four pigs with a pericoronary transit time flow (TTF) probe and a newly-designed epicardial Doppler micro-probe. Four consecutive measurements were taken for each of the following conditions: basal, partial stenosis, occlusion, and reperfusion of the LAD. Mean TTF value (mlymin) was 23.2±6.6 in basal condition, 16.2±5.7 after partial LAD stenosis, 0.1±0.3 during LAD occlusion, and 67.4±23.3 at reperfusion (P<0.001). Similar patterns were recorded in terms of Doppler velocity (cmys) with values of 4.0±1.9 in basal condition, 3.5±2.3 after partial LAD stenosis, 0.5±1.4 during LAD occlusion, and 11.1±5.5 at reperfusion (P<0.001). No significant differences in both TTF and Doppler velocity were detected between basal condition and partial LAD stenosis (Psns). Epicardial coronary arterial Doppler represents a valuable tool to detect coronary arterial flow velocity in basal condition. Although changes in flow velocity are easily recorded after coronary occlusion and reperfusion, modifications after partial coronary stenosis are not clearly defined.

Extracellular superoxide dismutase protects the heart against oxidative stress and hypertrophy after myocardial infarction (Article)

2008-04-01T00:00:00Z

Extracellular superoxide dismutase (EC-SOD) contributes only a small fraction to total SOD activity in the heart but is strategically located to scavenge free radicals in the extracellular compartment. EC-SOD expression is decreased in myocardial-infarction (MI)-induced heart failure, but whether EC-SOD can abrogate oxidative stress or modify MI-induced ventricular remodeling has not been previously studied. Consequently, the effects of EC-SOD gene deficiency (EC-SOD KO) on left ventricular (LV) oxidative stress, hypertrophy, and fibrosis were studied in EC-SOD KO and wild-type mice under control conditions, and at 4 and 8 weeks after permanent coronary artery ligation. EC-SOD KO had no detectable effect on LV function in normal hearts but caused small but significant increases of LV fibrosis. At 8 weeks after MI, EC-SOD KO mice developed significantly more LV hypertrophy (LV mass increased 1.64-fold in KO mice compared to 1.35-fold in wild-type mice; p < 0.01) and more fibrosis and myocyte hypertrophy which was more prominent in the peri-infarct region than in the remote myocardium. EC-SOD KO mice had greater increases of nitrotyrosine in the peri-infarct myocardium, and this was associated with a greater reduction of LV ejection fraction, a greater decrease of sarcoplasmic or endoplasmic reticulum calcium2+ATPase, and a greater increase of atrial natriuretic peptide in the peri-infarct zone compared to wild-type mice. EC-SOD KO was associated with more increases of phosphorylated p38 (p-p38Thr180/Tyr182), p42/44 extracellular signal-regulated kinase (p-ErkThr202/Tyr204), and c-Jun N-terminal kinase (p-JNKThr183/Tyr185) both under control conditions and after MI, indicating that EC-SOD KO increases activation of mitogen-activated protein kinase signaling pathways. These findings demonstrate that EC-SOD plays an important role in protecting the heart against oxidative stress and infarction-induced ventricular hypertrophy.

Extracellular superoxide dismutase deficiency exacerbates pressure overload-induced left ventricular hypertrophy and dysfunction (Article)

2008-01-01T00:00:00Z

Extracellular superoxide dismutase (SOD) contributes only a small fraction to total SOD activity in the normal heart but is strategically located to scavenge free radicals in the extracellular compartment. To examine the physiological significance of extracellular SOD in the response of the heart to hemodynamic stress, we studied the effect of extracellular SOD deficiency on transverse aortic constriction (TAC)-induced left ventricular remodeling. Under unstressed conditions extracellular SOD deficiency had no effect on myocardial total SOD activity, the ratio of glutathione:glutathione disulfide, nitrotyrosine content, or superoxide anion production but resulted in small but significant increases in myocardial fibrosis and ventricular mass. In response to TAC for 6 weeks, extracellular SOD-deficient mice developed more severe left ventricular hypertrophy (heart weight increased 2.56-fold in extracellular SOD-deficient mice as compared with 1.99-fold in wild-type mice) and pulmonary congestion (lung weight increased 2.92-fold in extracellular SOD-deficient mice as compared with 1.84-fold in wild-type mice). Extracellular SOD-deficient mice also had more ventricular fibrosis, dilation, and a greater reduction of left ventricular fractional shortening and rate of pressure development after TAC. TAC resulted in greater increases of ventricular collagen I, collagen III, matrix metalloproteinase-2, matrix metalloproteinase-9, nitrotyrosine, and superoxide anion production. TAC also resulted in a greater decrease of the ratio of glutathione:glutathione disulfide in extracellular SOD-deficient mice. The finding that extracellular SOD deficiency had minimal impact on myocardial overall SOD activity but exacerbated TAC induced myocardial oxidative stress, hypertrophy, fibrosis, and dysfunction indicates that the distribution of extracellular SOD in the extracellular space is critically important in protecting the heart against pressure overload.

Vasomotor control in mice overexpressing human endothelial nitric oxide synthase (Article)

2007-08-01T00:00:00Z

Nitric oxide (NO) plays a key role in regulating vascular tone. Mice overexpressing endothelial NO synthase [eNOS-transgenic (Tg)] have a 20% lower systemic vascular resistance (SVR) than wild-type (WT) mice. However, because eNOS enzyme activity is 10 times higher in tissue homogenates from eNOS-Tg mice, this in vivo effect is relatively small. We hypothesized that the effect of eNOS overexpression is attenuated by alterations in NO signaling and/or altered contribution of other vasoregulatory pathways. In isoflurane-anesthetized open-chest mice, eNOS inhibition produced a significantly greater increase in SVR in eNOS-Tg mice compared with WT mice, consistent with increased NO synthesis. Vasodilation to sodium nitroprusside (SNP) was reduced, whereas the vasodilator responses to phosphodiesterase-5 blockade and 8-bromo-cGMP (8-Br-cGMP) were maintained in eNOS-Tg compared with WT mice, indicating blunted responsiveness of guanylyl cyclase to NO, which was supported by reduced guanylyl cyclase activity. There was no evidence of eNOS uncoupling, because scavenging of reactive oxygen species (ROS) produced even less vasodilation in eNOS-Tg mice, whereas after eNOS inhibition the vasodilator response to ROS scavenging was similar in WT and eNOS-Tg mice. Interestingly, inhibition of other modulators of vascular tone [including cyclooxygenase, cytochrome P-450 2C9, endothelin, adenosine, and Ca-activated K+channels] did not significantly affect SVR in either eNOS-Tg or WT mice, whereas the marked vasoconstrictor responses to ATP-sensitive K+and voltage-dependent K+channel blockade were similar in WT and eNOS-Tg mice. In conclusion, the vasodilator effects of eNOS overexpression are attenuated by a blunted NO responsiveness, likely at the level of guanylyl cyclase, without evidence of eNOS uncoupling or adaptations in other vasoregulatory pathways. Copyright

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.