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A role for the PI3K/Akt/mTOR pathway in cardiac hypertrophy has been well documented. We reported that NFκB activation is needed for cardiac hypertrophy in vivo. To investigate whether both NFκB activation and PI3K/Akt/mTOR signaling participate in the development of cardiac hypertrophy, two models of cardiac hypertrophy, namely, induction in caAkt-transgenic mice and by aortic banding in mice, were employed. Rapamycin (2 mg/kg/daily), an inhibitor of the mammalian target of rapamycin, and the antioxidant pyrrolidine dithiocarbamate (PDTC; 120 mg/kg/daily), which can inhibit NFκB activation, were administered to caAkt mice at 8 weeks of age for 2 weeks. Both rapamycin and PDTC were also administered to the mice immediately after aortic banding for 2 weeks. Administration of either rapamycin or PDTC separately or together to caAkt mice reduced the ratio of heart weight/body weight by 21.54, 32.68, and 42.07% compared with untreated caAkt mice. PDTC administration significantly reduced cardiac NFκB activation by 46.67% and rapamycin significantly decreased the levels of p70S6K by 34.20% compared with untreated caAkt mice. Similar results were observed in aortic-banding-induced cardiac hypertrophy in mice. Our results suggest that both NFκB activation and the PI3K/Akt signaling pathway participate in the development of cardiac hypertrophy in vivo.

Objective: We have previously demonstrated that nuclear factor kappa B (NFκB) activation is needed for the development of cardiac hypertrophy in vivo. NFκB is a downstream transcription factor in the Toll-like receptor (TLR)-mediated signaling pathway; therefore, we investigated a role of TLR4 in cardiac hypertrophy in vivo. Methods: TLR4-deficient mice (C.C3H-Tlr4 lps-d, n = 6), wild-type (WT) genetic background mice (BALB/c, n = 6), TLR4-deleted strain (C57BL/10ScCr, n = 8), and WT controls (C57BL/10ScSn, n = 8) were subjected to aortic banding for 2 weeks. Age-matched surgically operated mice served as controls. In a separate experiment, rapamycin (2 mg/kg, daily) was administered to TLR4-deficient mice and WT mice immediately following aortic banding. The ratio of heart weight/body weight (HW / BW) was calculated, and cardiac myocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. NFκB binding activity and the levels of phospho-p70S6K in the myocardium were also examined. Results: Aortic banding significantly increased the ratio of HW / BW by 33.9% (0.601 ± 0.026 vs. 0.449 ± 0.004) and cell size by 68.4% in WT mice and by 10.00% (0.543 ± 0.011 vs. 0.495 ± 0.005) and by 11.8% in TLR4-deficient mice, respectively, compared with respective sham controls. NFκB binding activity and phospho-p70S6K levels were increased by 182.6% and 115.2% in aortic-banded WT mice and by 78.0% and 162.0% in aortic-banded TLR4-deficient mice compared with respective sham controls. In rapamycin-treated aortic-banded mice, the ratio of HW / BW was increased by 18.0% in WT mice and by 3.5% in TLR4-deficient mice compared with respective sham controls. Conclusion: Our results demonstrate that TLR4 is a novel receptor contributing to the development of cardiac hypertrophy in vivo and that both the TLR4-mediated pathway and PI3K/Akt/mTOR signaling are involved in the development of cardiac hypertrophy in vivo.

Background: Resistant hypertension (RH) is defined as blood pressure (BP) that remains elevated (>140/90mmHg) despite being treated with an antihypertensive regimen of 3 or more medications from different classes, including a long-acting calcium channel blocker, an angiotensin converting enzyme inhibitor or angiotension receptor blocker and a diuretic. The prevalence of RH in South Africa is currently unknown, however, clinical reports suggest that it is not rare. Patients with RH are significantly predisposed to cardiovascular (CV) diseases compared to patients with controlled BP. Consequences of RH include left ventricular hypertrophy, heart failure, ischaemic heart disease, chronic kidney disease leading to end-stage renal disease, stroke, vascular dementia, CV death and peripheral arterial disease. A proportion of patients with RH who never achieve BP control despite maximal medical treatment, represent a potentially novel and distinctive phenotype which is different from RH patients whose BP canbe controlled. Recognising and categorising such patients becomes the initial and crucial step in stratifying phenotypes and defining mechanisms of treatment resistance. Objectives: The aim of this study was to identify patients with resistant uncontrolled hypertension (RUH) and compare phenotypes in these patients to resistant controlled hypertensives (RCH). Methods: We enrolled 50 patients from the Groote Schuur Hospital Hypertension Clinic: a teriary referral hospital for RH. Patients on 4 or more antihypertensive medication including a diuretic, with BP< 140/90mmHg were considered RCH, and those with BP ≥ 140/90 considered RUH. Assessments included clinical examination, electrocardiography, echocardiography, applanation tonometry, serum biomarkers and cardiovascular magnetic resonance (CMR - which included biventricular volumes and function, myocardial strain, tissue characteristics and late gadolinium enhancement - LGE). Results: Thirty were diagnosed with RUH and twenty with RCH. Patients with RUH were more likely to have a longer duration since diagnosis of hypertension (10.5±10.7 vs. 3.6±3.4, p=0.02) and more likely to be on treatment that included an ACE-inhibitor (90% vs. 58%, p=0.01). As expected, patients with RUH had significantly higher systolic BP (155.6±21.6 vs. 137.8±16.5 mmHg, p< 0.001), diastolic BP (88.4±14.5 vs. 77.5±13.6 mmHg, p= 0.03), mean arterial BP (115.4±17.2 vs 101±15.3 mmHg, p= 0.004) and pulse pressure (67.3±14.2 vs. 60.1±12.4 mmHg, p=0.001). Further, RUH patients had significantly lower large artery elasticity (12.5±4 vs 14.7±3.8ml/mmHgx100, p=0.08) and lower small artery elasticity (4.1±2.1 vs. 6.9±3.6ml/mmHgx100, p< 0.001). RUH patients also had a higher systemic vascular resistance (1754±418.4 vs. 1363±371.5dyneXsecXcm-5, p=0.002). On CMR, RUH patients had lower right ventricular (RV) end-systolic and end-diastolic volumes (p=0.02), as well as higher indexed left ventricular mass (LVMI) (61.6±17.6 vs 52.9±13.9 g/m2 , p= 0.06). There were no differences in native T1, extracellular volume quantification and LGE volume fraction between RUH and RCH patients. Conclusions: Patients with RUH have a greater involvement and more severe CV phenotype, that is likely to result in increased CV morbidity and mortality, including greater target end organ damage as a result of vascular adaptations and concentric remodeling.

Physiological muscle adaptations due to resistance training are still not fully known. The rate and area of hypertrophy could drastically help or hinder athletic performance. The purpose of this study was to observe the changes in lean body mass (and related factors), relative allometrically scaled strength and absolute strength through an 11-week block periodized resistance training program. The subjects (n = 15) realized an increase in total body water (pre = 49.77Kg; post = 51.70Kg), lean body mass (pre = 67.98Kg; post = 70.63Kg), adjusted lean body mass (pre = 20.35Kg; post = 21.03Kg) and cross sectional area (pre = 32.73 cm2; post = 36.33cm2). Subjects (n= 15) were divided into either a strong (1 RM ≥ 1.75x body weight), moderate (1 RM = ≥ 1.25-1.74x body weight), or weak (1 RM < 1.25x body weight) group and data were analyzed in pre-post training. While all subjects showed gains in LBM and related factors, initial strength levels altered these adaptations. Subjects with a lower initial maximum strength level tended to make greater gains. However, due to the increase in total body water and relatively small increases in adjusted LBM, it appears, among this group, that little myofibrillar hypertrophy occurred during this short training period. These data suggest that greater accuracy for measures of alterations in LBM and related factors may require measures of total body water.

Alors que l’entrée SOC (store-operated Ca2+ entry) portée par les canaux calciques TRPCs (transient receptor potential canonical) et Orai1 est essentielle dans les cellules non-excitables, son rôle physiologique dans les cardiomyocytes adultes reste à élucider. Néanmoins, il est largement admis qu’une entrée SOC exacerbée dépendante des canaux TRPCs et de la protéine régulatrice STIM1 participe à la pathogenèse de l’hypertrophie et de l’insuffisance cardiaque (IC) par induction de voies pro-hypertrophiques telles que la CaMKII (Ca2+/calmoduline-dépendante kinase II ) et la calcineurine (CaN)/NFAT (Nuclear factor of activated T-cells). Au contraire, une inhibition fonctionnelle ou une extinction génique des canaux TRPCs et de la protéine STIM1 serait cardioprotectrice contre le stress hypertrophique. Cependant, le rôle physiopathologique des canaux calciques Orai1 dans le cœur reste, à ce jour, méconnu et débattu puisque son extinction in vitro présente un effet bénéfique contre l’hypertrophie des cardiomyocytes alors que son extinction in vivo présente des effets délétères avec le développement d’une cardiomyopathie dilatée. De plus amples investigations quant au rôle d’Orai1 dans la physiopathologie cardiaque apparaissent donc primordiales. De ce fait, les objectifs de ma thèse sont d’explorer le rôle de la signalisation calcique dépendante d’Orai1 dans le cœur dans des conditions physiologiques et pathologiques grâce à un modèle de souris transgéniques exprimant un mutant non fonctionnel d’Orai1, spécifiquement dans le cœur (dn-Orai1R91W/tTa) et un inhibiteur pharmacologique sélectif, le JPIII. Tout d’abord, nous montrons que les souris dn-Orai1R91W/tTa présentent une fonction cardiaque normale et une homéostasie calcique impliquée dans le couplage excitation-contraction conservée suggérant qu’Orai1 n’a pas de rôle majeur dans le coeur adulte en condition physiologique. Cependant, nous avons démontré une augmentation de l’expression et de l’activité d’Orai1 dans un modèle murin d’hypertrophie cardiaque induite par surcharge de pression, qui serait délétère pour la fonction ventriculaire. Au contraire, l’inhibition fonctionnelle d’Orai1 par manipulation génétique ou par l’outil pharmacologique (JPIII) semble protéger le coeur des dysfonctions ventriculaires au cours de l’hypertrophie. Cet effet bénéfique passerait par une restauration de l’homéostasie calcique et notamment par un maintien de l’expression de la pompe ATPase SERCA2a. Nous avons également mis en évidence que la voie de l’aldostérone/récepteurs aux minéralocorticoïdes modulait l’expression des canaux TRPC1, -C4, -C5 et notamment Orai1 via la protéine SGK1 (Serum and Glucocorticoid-regulated Kinase 1) dans les cardiomyocytes ventriculaires de rat nouveaux-nés. L’activation de cette voie de signalisation pourrait être à l’origine de la surexpression des canaux TRPCs/Orai1 retrouvée au cours de l’hypertrophie cardiaque. Ces travaux décrivent donc Orai1 comme une cible thérapeutique potentielle dans le traitement de l’hypertrophie cardiaque et de l’IC. / While the SOCE (store-operated Ca2+ entry), carried by TRPCs (transient receptor potential canonical) and Orai1 channels, is essential in non-excitable cells, its physiological role in adult cardiomyocytes remains elusive. Nevertheless, it is well established that exacerbated TRPCs/STIM1-dependent Ca2+ entry participates in the pathogenesis of hypertrophy and heart failure (HF) via the induction of pro-hypertrophic signaling pathways, such as CaMKII (Ca2+/calmodulin-kinase II) and calcineurin (CaN)/ NFAT (nuclear factor of activated T-cells). By contrast, functional inhibition or gene silencing of TRPCs and STIM1 is cardioprotective against hypertrophic insults. As for Orai1 Ca2+ channels, their pathophysiological roles in the heart remain unknown and under debate, since in vitro Orai1 silencing has a beneficial effect against cardiomyocyte hypertrophy, whereas in vivo silencing has deleterious effects with the development of dilated cardiomyopathy. Further investigations are necessary to determine the pathophysiological role of Orai1 in the heart. My thesis objectives are to explore the role of Orai1-dependent Ca2+ signaling in the heart under physiological and pathological conditions using a transgenic mouse model expressing a non functional mutant of Orai1, specifically in the heart (dn-Orai1R91W/tTa) and a selective pharmacological inhibitor, JPIII. First, we showed that dn-Orai1R91W/tTa mice have normal cardiac function and conserved Ca2+ homeostasis involved in the excitation-contraction coupling suggesting that Orai1 is not instrumental in regulating cardiac function under physiological conditions. However, we demonstrated an increased Orai1 expression and activity in a mouse model of cardiac hypertrophy induced by pressure overload, which is a maladaptive alteration involved in pathological ventricular dysfunction. By contrast, functional inhibition of Orai1 by genetic manipulation or by the pharmacological tool (JPIII) protects the heart from ventricular dysfunction after pressure overload-induced cardiac hypertrophy. This beneficial effect is related to a restoration of Ca2+ homeostasis and more specifically, is due to preserved ATPase SERCA2a pump expression. We also showed that the aldosterone/mineralocorticoid receptor signaling pathway modulates the expression of TRPC1, -C4, -C5 channels and also the Orai1 channels expression via the SGK1 (Serum and Glucocorticoid-regulated Kinase 1) protein, in neonatal rat ventricular cardiomyocytes. The activation of this signaling pathway could be the cause of the TRPCs/Orai1 channels overexpression found during cardiac hypertrophy. In conclusion, our studies highlighted that Orai1 Ca2+ channels could constitute potential therapeutic target in the treatment of cardiac hypertrophy and HF.

The muscle hypertrophy of lambs expressing the Callipyge phenotype is possibly linked to characteristics of their muscle satellite cells. Therefore, characteristics (proliferation, fusion %, and protein accretion) of cultured satellite cells isolated from the longissimus muscle of Callipyge (n = 3) and normal (n = 3) lambs were compared in this study. In the first experiment, we tested whether or not the lll proliferation rates differ for satellite cells isolated from Callipyge or normal sheep when cultured in the presence of different serum types (horse, normal lamb, or Callipyge lamb). The average population doubling time (PDT, h) during log phase growth was calculated for cells from each animal grown in each serum type. Population doubling time was not affected (P > .1) by the interaction of satellite cell type with serum type, or by satellite cell type. Unexpectedly, PDT was longer (P < .05) for satellite cells grown in Callipyge serum (22 h) than for cells grown in normal sheep serum (20 h) or horse serum (18 h). These results suggest that muscle hypertrophy of Callipyge lambs is not linked to intrinsic differences in satellite cell proliferation, although hypertrophy may be associated with a decreased proliferation induced by a factor in Callipyge serum. In the second experiment, we tested whether cell fusion, or protein accretion differ for cultured satellite cells isolated from Callipyge or normal sheep. DNA and protein were determined at 24, 48, 72, and 96 h after satellite cell cultures were induced to differentiate. Fusion percentage was determined in a Giemsa stained plate after 72 h in differentiation medium (Dulbecco's Modified Eagle Medium containing 1.5% of horse serum). Callipyge cultures tended (P = .14) to have higher fusion% than normal cultures exhibited, suggesting that muscle hypertrophy of Callipyge lambs may be linked to an increased tendency of satellite cells to fuse. Protein content (μg/well) and protein:DNA ratio (ng of protein/ng of DNA) were not affected by satellite cell type (P = .80 and P = .79, respectively). Thus, there was no evidence for a link between increased protein accretion and Callipyge hypertrophy.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21253号 / 医博第4371号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 浅野 雅秀, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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In 2018, 1 in every 10 infants born in the United States was born preterm. The majority of neonatal deaths and nearly a third of infant deaths that occur are linked to preterm birth. Preterm birth is initiated when the quiescent state of the uterus ends prematurely, leading to contractions and parturition beginning as early as 32 weeks, though the origins are not well understood. Tocolytics are pharmaceuticals utilized to postpone preterm labor, but currently only manage to prolong pregnancy for up to 48 hours and have not proven effective in completely preventing preterm delivery. To enable research and discovery of therapeutics with potential to better address preterm birth, the capability to study isolated cell processes of pregnant uterine tissue in vitro is needed. Our development of an in vitro model of the myometrium utilizing uterine myocytes - uterine smooth muscle cells (uSMCs) responsible for contractions - provides a platform to examine the cellular mechanisms of late-stage pregnancy potentially involved in preterm birth. In this thesis, we discuss the optimized culture of uterine SMCs on a flexible polydimethylsiloxane (PDMS) substrate functionalized using a cationic solution, Poly-L-lysine (PLL), followed by extracellular matrix (ECM) protein coating. Using the model we developed, we then exposed this elastic substrate with uterine SMCs to different strain rates in order to investigate the impact of mechanical strain parameters on uterine SMC hypertrophy in the uterus during late-stage pregnancy. It was found that PLL and ECM protein coatings significantly impact cell morphology and density in unstrained substrates. It was also observed that when exposed to strain conditions, strain significantly increased hypertrophic morphological traits in select conditions. These results indicate that both surface and mechanical properties of in vitro systems impact uterine SMC phenotype, offering further understanding of cellular pathways involved in the uterus under mechanical load. / 2024-05-31T00:00:00Z