Thesis (Doctoral)--Department of Clinical Pharmacology, Lund University Hospital, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Glucocorticoids are growth-inhibiting steroids. They have been reported to reduce muscle growth by reducing protein synthesis. However, their actions on muscle protein degradation remain equivocal. Glucocorticoids have been reported to transiently increase muscle protein degradation, to not affect this process and to reduce muscle protein degradation. Reasons for these conflicting reports are not understood but may be related to species, glucocorticoid doses and route and duration of administration. Dexamethasone is a synthetic glucocorticoid and is not rapidly metabolized in vivo. As an inflammatory agent, dexamethasone is more potent than natural glucocorticoids. The objective of this study was to clarify the role in molecular regulation of calpain expression as a proteolytic catalyst linked to initiation of myofibrillar protein degradation by using glucocorticoid-dependent changes in muscle protein degradation as a model. A secondary objective of investigating molecular mechanisms responsible for regulation of calpain expression was conducted by examining effects of a synthetic glucocorticoid --dexamethasone on calpains and calpastatin activities and steady-state mRNA concentrations encoding these proteins. Female New Zealand White rabbits (1.8-2.1 kg) were treated with 1 mg dexamethasone/kg BW/day for 0 day, 1 day, 2 days or 4 days by daily subcutaneous injection. Cranial biceps femoris were taken for analysis of muscle protein concentration, muscle RNA concentration, ribosomal capacity, N'-methylhistidine (NMH) concentration and calpains and calpastatin activities. Because glucocorticoids may mediate their actions indirectly via other hormones, temporal effects of dexamethasone on plasma T3 and T4 concentrations were also examined. Dexamethasone transiently decreased (P < .05) final body weight and total body weight gain in the 1-day dexamethasone-treated rabbits, but food intake was maintained in both control and dexamethasone-treated rabbits (P > .05). Muscle protein concentration was unaffected (P > . 05) by dexamethasone, while dexamethasone decreased (P < .05) muscle RNA concentration in the 4-day dexamethasone-treated rabbits and tended to decrease ribosomal capacity (P > . 05) gradually as duration of dexamethasone treatment increased. Although urinary NMH excretion, which serves as an index of myofibrillar protein degradation, was not affected by dexamethasone (P > .05), the ratio of urinary NMH excretion to urinary creatinine output was reduced significantly (P < .05) by 4 days of dexamethasone treatment compared to 1 day of dexamethasone treatment. Also, muscle NMH concentration was reduced (P < .05) by dexamethasone treatment. These data indicate that dexamethasone treatment may have reduced muscle protein degradation. Calpain I, calpain II and calpastatin activities were not affected by dexamethasone (P > . 05) although both calpain I and calpain II activities tended to decrease and calpastatin activity had a tendency to increase as duration of dexamethasone treatment increased. Maximum effects of dexamethasone on both calpains, urinary NMH excretion and muscle NMH concentration were detected following 2 days of administration. These results indicated that the temporal decrease in rabbit skeletal muscle protein degradation by dexamethasone was related to calpains and calpastatin. mRNA concentrations encoding calpain I increased (P < .05) in the 1-day dexamethasone-treated rabbits, while mRNA concentrations encoding calpain II decreased (P < .05). These results imply that dexamethasone can affect calpain I and calpain II gene expression in an opposing manner (up-regulation and down-regulation). Plasma T₃ concentration but not plasma T₄ concentration was significantly reduced (P < . 05) by dexamethasone treatment. Because T₃ stimulates myofibrillar protein degradation, its lower concentration in dexamethasone-treated rabbits may account for the apparent reduction in protein degradation caused by dexamethasone. In this study, the observed effects of dexamethasone on muscle protein degradation may be the combination of direct and indirect responses. Thus, in vitro studies will be needed in order to clarify the direct effects of dexamethasone on muscle protein degradation. / Graduation date: 1991

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Thesis (Ph.D.) - University of Glasgow, 2007. / Ph.D. thesis submitted to the Faculty of Medicine, Dept. of Developmental Medicine, University of Glasgow. Includes bibliographical references. Print version also available.

Thesis (M.S.)--University of Wisconsin--Madison, 1983. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Glucocorticoids - cortisol and corticosterone - are steroid hormones synthesised in the adrenal gland that are important mediators of the stress response. Glucocorticoids are also vital in development to aid in organ maturation. Endogenous glucocorticoid levels rapidly rise before birth in all mammals to promote fetal organ maturation. Because preterm birth occurs before this natural rise in glucocorticoid levels, pregnant women at risk of preterm delivery are administered synthetic glucocorticoids to mature the fetal lung and aid neonatal survival. Mice that globally lack the glucocorticoid receptor (GR) die at birth, attributed to lung immaturity. Effects on tissues other than the lung remain less well characterised. Previous work has shown endogenous glucocorticoid action is also essential to mature the mouse fetal heart. Mice globally lacking GR have small, functionally and structurally immature hearts. Mice with tissue-specific deletion of GR in cardiomyocytes and vascular smooth muscle cells (SMGRKO mice; generated using Sm22α-Cre) also have an increased risk of death around the time of birth, suggesting that glucocorticoid maturation of the cardiovascular system is important for neonatal survival. GR expression within the fetal mouse heart initiates at E10.5 but GR in the myocardium is not activated and localised to the nucleus until E15.5. This suggests that mice can respond to glucocorticoid from E10.5. Here, it was hypothesised that antenatal glucocorticoid exposure, prior to the increase in endogenous glucocorticoid levels, would advance fetal heart maturation and this will depend on cardiovascular GR. To investigate the effects of antenatal glucocorticoid treatment on fetal heart maturation in mid-gestation and identify effects mediated by GR, mice with a conditional deletion of GR in cardiomyocytes and vascular smooth muscle cells were studied (SMGRKO mice). Pregnant mice received dexamethasone (dex) in the drinking water from E12.5-E15.5. Levels of Fkbp5 mRNA (a marker of glucocorticoid action) were unchanged between control and SMGRKO mice at E15.5 or following dex treatment. This suggested a lack of response to dex treatment. However, liquid chromatography mass spectrometry measurement confirmed the presence of dex and its active metabolite 6- hydroxydexamethasone (6OHDex) in livers of E15.5 fetuses from dex treated dams (fetal: Dex 0.46 ± 0.1 ng/g, 6OHDex 13.6 ± 0.35 ng/g; dam: Dex 7.96 ± 3.65 ng/g, 6OHDex 4.75 ± 1.2 ng/g). Livers of fetuses exposed to dex had lower levels of the naturally occurring active glucocorticoid, corticosterone, compared to vehicle treated fetuses. This suggests HPA axis suppression in dex exposed fetuses. Maternal liver showed no significant difference in corticosterone levels between dex and vehicle treated mice, suggesting that whilst dex suppressed the HPA axis in fetuses, it did not in the dams. To determine any persistent effects of early antenatal dex treatment on fetal heart, a later time point in gestation, E17.5, was also assessed. At E17.5, 2-days following cessation of dex treatment, dex and its metabolites were undetectable in the fetal and maternal liver. However, corticosterone levels remained reduced in fetal liver at E17.5 in dex exposed animals (vehicle treated: 4.31 ± 0.47 ng/g, Dex treated: 1.72 ± 0.42 ng/g, p < 0.01), whilst levels in the dam liver did not differ from vehicle treated controls. This suggests prolonged HPA axis suppression following dex treatment, which reduced the natural late-gestation rise in glucocorticoids required for fetal organ maturation. To determine whether early antenatal dex treatment could advance fetal heart function, Doppler imaging with a Vevo 770 high frequency ultrasound imager was used. Isovolumetric contraction time, isovolumetric relaxation time and ejection time of the left ventricle were unaltered by dex treatment. However, at E15.5 the mitral deceleration index (MDI), a measure of diastolic function that takes into account loading conditions, was 1.5 fold lower in vehicle treated SMGRKO mice than control (Cre-) littermates (p < 0.05). This reduction in SMGRKO mice suggests glucocorticoids are required within the fetal cardiomyocytes and/or vascular smooth muscle cells to mature the diastolic function of the fetal heart. Dex exposure had no effect on MDI in SMGRKO fetuses, but reduced the MDI by 1.5 fold in control mice to similar levels as in SMGRKO mice (p < 0.05). RNA analysis revealed a trend (p=0.09) for reduced levels of Nr3c1 mRNA (encoding GR) in hearts of E15.5 control (Cre-) fetuses following dex treatment. Although this requires confirmation at the level of GR protein, this finding together with the lack of induction of the GR target, Fkbp5, suggests dex may cause glucocorticoid resistance through down-regulation of GR. At E17.5, 2-days following cessation of dex there were no changes in systolic parameters and the reduction in MDI found at E15.5, following dex, had normalised. Litter size was reduced (close to a 50% reduction) at E17.5 in dex treated mice. This was similar between SMGRKO and control fetuses. The cause of death was not established, but potentially could be due to the reduction in the natural rise in glucocorticoids at E17.5, previously shown to be important for fetal heart maturation. It is therefore possible that mice with more immature hearts may die before reaching E17.5. RNA analysis was undertaken to determine any mechanistic alterations following dex treatment, which could support fetal heart functional alterations found at E15.5. In contrast to expectation, dex also decreased expression of mRNA encoding the calcium handling proteins SERCA2a, NCX1, and CaV1.2 in E15.5 fetal mouse hearts in both control and SMGRKO mice (p < 0.05), compared with the respective vehicle treated mice. These proteins had previously shown to be induced by glucocorticoid action in cardiomyocytes. However, the similar down-regulation in both genotypes indicates this effect is not dependent on GR in cardiomyocytes. Lowered SERCA2a activity following dex treatment could contribute to the changes in MDI observed in control mice. Similarly, Scnn1a and Kcnj12 mRNA levels, previously found to be induced by glucocorticoids in cardiomyocytes, were down-regulated in the E15.5 fetal heart in vivo following dex. Collectively, these data are consistent with glucocorticoid resistance or down-regulation of glucocorticoid action in E15.5 fetal hearts following dex administration. Mutations in KCNJ12 are associated with long QT syndrome, which is characterised by a delayed repolarisation of the heart following each contraction. An altered relaxation of the fetal heart found in control mice following dex could therefore be due to a prolongation of the cardiac action potential, particularly with a delayed repolarisation, because of lower Kcnj12 expression. At E17.5, there were no significant differences in expression of calcium handling genes or ion channel mRNAs between genotypes or following earlier dex exposure. Thus, effects of dex on mRNA expression level may not persist, which could account for the lack of functional changes observed 2-days following cessation of treatment. Because effects seen in vivo with dex treatment were contrary to those predicted, and to further investigate the effect of dex upon calcium content, an in vitro model of primary fetal E15.5 cardiomyocytes was used. Cardiomyocytes were treated with dex for 24 hours and effects on membrane potential voltage changes and calcium transients measured. Following dex, isolated fetal cardiomyocytes showed an elongated repolarisation phase of the action potential (untreated: 120.45 ± 13.81 ms, Dex: 142.34 ± 12.97 ms, p < 0.01), and duration of calcium transients (untreated: 103.31 ± 13.78 ms, Dex: 120.43 ± 23.36 ms, p < 0.05). This assessment of fetal cardiomyocytes was preliminary work to aid in the understanding of mechanisms of fetal heart functional alterations associated with glucocorticoid regulation. The results suggest glucocorticoids may be important in regulating calcium levels. In summary, dex treatment in mice from E12.5-E15.5 did not advance fetal heart maturation. It reduced litter size at E17.5, irrespective of whether GR was expressed in cardiomyocytes or not. The normal late-gestation increase in endogenous glucocorticoid levels in the fetus was reduced by dex, even after treatment finished. / The suppression of corticosterone levels following antenatal dex may reduce maturation of the heart at E15.5 and could be responsible for the reduction in litter size. Downregulation of GR in the fetal heart, may be a mechanism that results in glucocorticoid resistance following antenatal dex treatment, which could explain the lack of beneficial effects of antenatal dex upon fetal heart maturation in these experiments in mice.

No description available.

Glucocorticoid production by the adrenal gland is regulated by hypothalamicpituitary- adrenal (HPA) axis activity. Within cells, glucocorticoid levels are modulated by 11β-hydroxysteroid dehydrogenase (11β-HSD), which interconverts active and intrinsically inert glucocorticoids. Glucocorticoids have widespread physiological effects and, in the cardiovascular system, they play a crucial role in heart development and maturation, blood pressure control, and myocardial calcium cycling. Mice which are unable to regenerate the physiological glucocorticoid, corticosterone, from 11-dehydrocorticosterone due to deletion of the type 1 11β-HSD isozyme (11β-HSD1) have previously been shown to have smaller, lighter hearts but unaltered systolic function. Moreover, a single nucleotide polymorphism (SNP) in the Hsd11b1 gene has been associated with reduced left ventricular mass in humans, suggesting a role for 11β-HSD1 in regulating cardiac size. After myocardial infarction (MI), 11β-HSD1 deficient mice have an augmented inflammatory response, increased numbers of pro-reparative alternatively-activated macrophages in the heart, enhanced peri-infarct angiogenesis and improved cardiac function compared to C57BL/6 controls. However, the role of ‘cardiovascular’ 11β-HSD1 in the development of these phenotypes, both basally and after MI, is unknown. It was hypothesised that ‘cardiovascular’ 11β-HSD1 deficiency would result in smaller hearts, and that this selective deletion would lead to altered calcium handling protein expression and diastolic abnormalities. Furthermore, it was hypothesised that ‘cardiovascular’ 11β-HSD1 deletion would reproduce the beneficial post-MI phenotype previously seen in global 11β-HSD1 deficient mice. The first aim was to characterise the cardiac phenotype of mice with global deletion of 11β-HSD1 (DelI mice), and mice in which deletion is restricted to cardiomyocytes and vascular smooth muscle cells (SMAC mice). SMAC mice have ‘floxed’ 11β- HSD1 alleles and a Cre recombinase transgene inserted into the Sm22α gene. Sm22α is expressed in vascular smooth muscle cells, and transiently in cardiomyocytes during development. Thus, Cre expression in these cells results in deletion of exon three of the Hsd11b1 gene and gives rise to a non-functional protein. Controls for DelI mice were C57BL/6 mice, and controls for SMAC mice were their Cre- littermates. DelI, but not SMAC, mice have smaller, lighter hearts, which may be explained by their shorter cardiomyocytes measured following isolation using a Langendorff preparation. Cardiomyocyte cross-sectional area is unchanged. In vivo measurement of cardiac function using ultrasound imaging showed systolic function is comparable between DelI mice and SMAC mice and their respective controls. However, there is mild diastolic dysfunction in both DelI and SMAC mice, characterised by reduced E wave deceleration and an increased mitral valve deceleration time. This phenotype occurred following pharmacological inhibition of 11β-HSD1, by administration of UE2316, a selective 11β-HSD1 inhibitor, to adult C57BL6/SJL mice. While ventricular collagen content is unaltered in DelI, SMAC and UE2316-treated mice compared to their respective controls, expression of sarcoplasmic reticulum Ca2+ ATPase (SERCA) is reduced, suggesting that altered calcium handling, rather than changes in stiffness, may underlie this phenotype. The second aim was to determine whether the beneficial acute outcomes seen previously in 11β-HSD1 deficient mice after MI could be reproduced by selective cardiovascular deletion of the enzyme. Seven days after MI, compared to Cre- littermate controls, SMAC mice have similar peri-infarct angiogenesis, total macrophage and alternatively-activated macrophage infiltration into the heart, infarct size, ventricular dilatation and systolic function. This suggests 11β-HSD1 deletion in another cell type, or types, is responsible for the phenotype seen in global 11β-HSD1 deficient mice. The final aim was to assess the impact of global 11β-HSD1 deficiency and ‘cardiovascular’ 11β-HSD1 deletion on the development of heart failure, using magnetic resonance imaging to determine structure and function. Eight weeks after MI, mice globally deficient in 11β-HSD1 have attenuated expression of ANP and β- MHC, RNA markers of heart failure, and show attenuated pulmonary oedema, reduced chamber dilatation, preserved systolic function and smaller infarcts compared to control. None of these parameters are altered in SMAC mice relative to control. In conclusion, the data presented in this thesis shows that cardiovascular 11β-HSD1 influences physiological cardiac function, potentially through regulation of calcium handling. 11β-HSD1 in other cells influences cardiomyocyte length, resulting in smaller hearts in its absence. Despite this, global 11β-HSD1 deficiency prevents heart failure development after MI, suggesting that pharmacological inhibition of 11β-HSD1 may be of benefit in treating this condition. Cardiovascular 11β-HSD1 does not, however, account for the changes in infarct healing or remodelling associated with this beneficial outcome, therefore these effects must be related to 11β-HSD1 deficiency elsewhere, such as fibroblasts or myeloid cells.