O overreaching não funcional (NFOR) induzido pelo exercício excêntrico (EE) em modelo animal está associado com a diminuição de desempenho físico, dano no DNA (amostras de músculo esquelético e soro), estresse oxidativo (amostras de músculo esquelético e soro), inflamação crônica de baixo grau (amostras de músculo esquelético e soro) e prejuízo da via de sinalização da insulina (amostras de músculo esquelético). No entanto, as adaptações do músculo cardíaco em resposta ao estado de NFOR induzido ou não pela predominância do EE ainda não foram investigadas. Além disso, sabe-se que a mTOR (mammalian target of rapamycin) possui um efeito protetor no músculo cardíaco, suprimindo o aumento de citocinas pró-inflamatórias, que estão relacionadas à disfunções cardíacas. Assim, o presente estudo teve como objetivo comparar os efeitos do NFOR em declive com outros dois protocolos de mesmo volume e intensidade, mas realizados sem inclinação e em aclive, no conteúdo das proteínas relacionadas às vias moleculares inflamatória e hipertrófica, no conteúdo de fibrose intersticial e na expresão gênica em músculo cardíaco de camundongos. Os animais foram divididos em 6 grupos: Naíve (N; camundongos sedentários), Controle (C; camundongos sedentários submetidos aos testes físicos), Treinado (TR; camundongos submetidos ao protocolo de treinamento), Overtraining em declive (OTR/down; camundongos submetidos ao protocolo de OT com corrida na descida), Overtraining sem inclinação (OTR; camundongos submetidos ao protocolo de OT com corrida sem inclinação) e Overtraining em aclive (OTR/up; camundongos submetidos ao protocolo de OT com corrida na subida). Em relação aos parâmetros metabólicos, o grupo OTR/down apresentou menor variação de peso corporal na semana 8. Todos os grupos que passaram pelo protocolo de OT demonstraram queda de desempenho ao final da semana 8, aumento no conteúdo de tecido conjuntivo no ventrículo esquerdo e menor ativação da proteína AMPKalfa. O grupo OTR/down apresentou menor conteúdo da proteína mTOR e S6RP, e aumento na expressão do gene beta-MHC. Pode-se concluir que os protocolos de OT provocaram indícios de hipertrofia patológica no ventrículo esquerdo, sendo esse efeito mais pronunciado no grupo OTR/down. / Nonfunctional overreaching (NFOR) induced by eccentric exercise (EE) in animal model is associated with performance decrement, DNA damage (muscle and serum samples), oxidative stress (muscle and serum samples), low grade chronic inflammation (muscle and serum samples) and insulin signaling impairment (muscle and serum samples). However, the adaptations of cardiac muscle in response to NFOR induced or not induced by EE are unknown. In addition, the mammalian target of rapamycin (mTOR) has a protector effect in the cardiac muscle, suppressing the increase of the proinflammatory cytokines that is related to cardiac dysfunctions. Thus, the main aim of present study was to compare the effects of NFOR based on EE (downhill running) with other two protocols with similar intensity and volume, but performed in uphill and without inclination, on the protein contents related to hypertrophic and inflammatory signaling, on the content of interstitial fibrotic tissue and on genes expression in mice cardiac muscle. The animals were divided on six groups: Naïve (N; sedentary mice), Control (C; sedentary mice submitted to physical tests), Trained (TR; mice submitted to training protocol), Overtraining in downhill (OTR/down; mice submitted to the overtraining protocol in downhill), Overtraining without inclination (OTR; mice submitted to the overtraining protocol without inclination), and Overtraining in uphill (OTR/up; mice submitted to the overtraining protocol in uphill). Regarding metabolic parameters, OTR/down group presented reduced body weight variation at week 8. All OT groups presented performance drop at end of week 8, increased connective tissue content in left ventricle and reduced AMPKalpha activation. OTR/down group presented reduced mTOR and S6RP protein content, and increased betaMHC gene expression. The conclusion is that OT protocols provoked signs of pathological hypertrophy in left ventricle, being this effect more pronounced in OTR/down group.

Haemodialysis (HD) patients have a high mortality risk and most have echocardiographic evidence of abnormal cardiac structure or function. Markers, such as left ventricular hypertrophy (LVH), show association with adverse outcome in the general population and can aid in clinical decision making. The aim of this research was to explore the prognostic utility of established and novel two-dimensional (2DE) and three-dimensional transthoracic echocardiographic (RT3DE) techniques in HD patients. Adult maintenance HD patients from a single tertiary nephrology centre including satellite dialysis units were enrolled. Exclusion criteria were if patients were clinically unstable, unable to consent, or if required ambulance transportation for echocardiography visits. Consented patients underwent 2DE with speckle tracking (STE), RT3DE and VicorderTm measurements of pulse wave velocity (PWV) on a non-dialysis day, after the short inter-dialytic break. Clinical phenotype data, 3-month averaged blood results and dialysis prescriptions were obtained from the hospital electronic patient records. All patients screened were followed-up until death, renal transplantation, moving out of the region, or 16th November 2015. Regression analysis was used to assess the cross-sectional relationship between echocardiographic parameters. Relationship of echocardiographic parameters with outcome was assessed by Cox regression analysis. The first study explored whether patients recruited had similar characteristics and survival compared with patients who declined consent or who were excluded from the study. Patients who declined consent had an adjusted hazard ratio (HR) for all-cause mortality compared with recruited patients of 1.70, 95% confidence interval (CI) 1.10-2.52, and excluded patients had an adjusted HR of 1.30, 95% CI 0.75-2.25. Recruited patients may be a 'fitter' population and this needs to be considered when interpreting results. The second study reports that when global longitudinal strain (GLS) is combined in a multivariable model with PWV; PWV is superior to GLS in its association with mortality (adjusted HR 1.23, 95% CI 1.03-1.47 versus HR 1.00, 95% CI 0.86-1.17). When this analysis was repeated in a sub-group of patients with LVH, neither GLS nor PWV were associated with mortality, whilst both were prognostically significant in a preserved LVEF sub-group (PWV: HR 1.23, 95% CI 1.04-1.4 and GLS: HR 1.16, 95% CI 1.01-1.33). Therefore GLS has different prognostic implications in different patient sub-groups. The third study explored whether tissue motion mitral annular displacement (TMAD) measured by STE may be a more useful alternative to GLS as it measures strain but is quicker and less user-dependent. TMAD was closely correlated to GLS (r=-0.614, p<0.001), but had no prognostic power for mortality (adjusted HR 1.04,95% CI 0.91-1.19). The correlation between 2DE and RT3DE determined LV mass and volume measurements and the prognostic significance of RT3DE measurements were assessed. Although there was good correlation between 2DE and RT3DE LV volume measurements, 2DE overestimated LV mass compared to RT3DE. RT3DE measures gave no added prognostic value, and there were added difficulties in obtaining adequate images for RT3DE (35% of patients who had adequate 2D images). Furthermore, although RT3DE determined LV mechanical dyssynchrony index was prolonged in HD patients compared with published general population controls, it failed to show any prognostic significance (HR 2.16, 95% CI 0.96-4.89) for mortality, but was associated with hospitalisation for heart failure (HR 1.03, 95% CI 1.00-1.06). These results indicate that novel measurements of sub-clinical cardiac dysfunction have the potential to aid prognostication in this high risk population. Follow-up studies exploring the longitudinal change in these parameters is ongoing.

Cardiac hypertrophy and heart failure (HF) remains one of the major health problems in the UK and worldwide. However, advances in their management are limited because the underlying pathological mechanisms are not completely understood. Therefore, it is important to understand novel signalling pathways leading to HF. Myocardial hypertrophy is a crucial pathophysiological process that can lead to the development of HF. Signalling initiated by members of G-protein-coupled receptors (GPCRs) proteins plays an important role in mediating cardiac hypertrophy. One member of this family, the G-protein coupled receptor 99 (GPR99), may have a crucial role in the heart because it acts as a receptor for alpha-ketoglutarate, a metabolite that is elevated in heart failure patients. GPR99 is expressed in the heart, but its precise function during cardiac pathophysiological processes is unknown. The aim of this PhD study is to investigate the role of GPR99 during cardiac hypertrophy. In this study I used in vivo and in vitro approaches to investigate whether GPR99 is directly involved in mediating cardiac hypertrophy. Mice with genetic deletion of GPR99 (GPR99-/-) exhibited a significant increase in hypertrophy following two weeks of transverse aortic constriction (TAC) as indicated by heart weight/tibia length ratio (HW/TL). In addition, GPR99-/- mice displayed increased cardiomyocytes cross-sectional area (CSA) after TAC compared to wild-type (WT) littermates. Hypertrophic markers such as brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) were also elevated in GPR99-/- mice following TAC compared to WT mice. Although interstitial fibrosis was indistinguishable in both genotypes after TAC, a precursor of fibrosis, collagen, type III, alpha1 (COL3A1) was elevated in GPR99-/- mice compared to WT mice after TAC. The baseline cardiac function as indicated by ejection fraction (EF) and fractional shortening (FS) were reduced in GPR99-/- mice compared to WT littermates following TAC. Furthermore, left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), interventricular septum wall thickness (IVS) and posterior wall thickness at diastole (PW) indicated profound wall thickening and enlargement of the left ventricular (LV) chamber in GPR99-/- mice compared to WT littermates after TAC. In an attempt to examine the mechanism through which GPR99 signals during hypertrophy, I performed molecular analyses based on the data from yeast two hybrid screening showing that GPR99 interacted with COP9 signalosome element 5 (CSN5). Using immunoprecipitation assay, I found that GPR99 formed a ternary complex with CSN5 and non-receptor tyrosine kinase 2 (TYK2). TYK2 is known as a regulator of pro-hypertrophic molecules including signal transducer and activation of transcription 1 (STAT1) and STAT3. I found that the activation of these molecules was increased in GPR99-/- mice following TAC and correspondingly, adenovirus-mediated overexpression of GPR99 in neonatal rat cardiomyocytes (NRCM) blunted TYK2 phosphorylation. In conclusion, my study has identified GPR99 as a novel regulator of pathological hypertrophy via the regulation of the STAT pathway. Identification of molecules that can specifically activate or inhibit this receptor may be very useful in the development of a new therapeutic approach for cardiac hypertrophy in the future.

Cardiac hypertrophy and heart failure are affecting almost one million people in the UK alone. The available therapies of cardiac hypertrophy are for symptomatic treatment. Recently attention has been moved towards identification of novel drugs which intervene with signalling pathways involved in hypertrophy. To achieve this goal it was important to understand the role of genes involved in the development of cardiac hypertrophy. One of such genes is plasma membrane calcium ATPase isoform 4 (PMCA4). Although several studies which used genetically modified animal models suggested the involvement of PMCA4 during the development of cardiac hypertrophy, the actual role of PMCA4 is still unclear. In this study, we will clarify the role of PMCA4 during the development of cardiac hypertrophy using a novel PMCA4 specific inhibitor. Until now there is no known PMCA4 specific inhibitor so a library of 1280 medically optimised compounds was screened using a novel in vitro assay which measures the ATPase activity of PMCA4. The compound aurintricarboxylic acid (ATA) was identified, which inhibited PMCA4 ATPase activity with higher affinity (IC50= 100 nM) compared with related ATPases. In isolated neonatal rat cardiomyocytes, ATA showed dose dependent inhibition of phenylephrine-induced hypertrophy. In vivo studies showed that ATA (5mg/kg body weight/day IP) significantly reduced the development of pressure-overload induced hypertrophy in wild type mice following two weeks transverse aortic constriction (TAC). Echocardiography and haemodynamic analyses showed that ATA treatment significantly reduced the abnormal left ventricular remodelling after TAC compared with vehicle treatment. ATA treated TAC mice showed a significant reduction in the enlargement of heart weight/tibia length ratio as well as cardiomyocyte cross sectional surface area compared with vehicle treated TAC mice. A significant reduction in the expression of the hypertrophic markers ANP and BNP and, importantly, in the percentage of fibrosis was observed in ATA treated TAC mice compared with vehicle treated TAC mice. In addition, ATA treatment significantly reversed the already established pressure overload induced hypertrophy following three weeks TAC. ATA treatment to TAC mice led to a significant reduction in the expression of the bona fide calcineurin target MCIP1 and a reduction in NFAT phosphorylation level in vivo and NFAT transcriptional activity in vitro. ATA did not show a direct inhibition to the active form of calcineurin nor to the phosphatase activity of full length calcineurin.In conclusion, we have identified ATA as a novel and specific inhibitor to PMCA4 ATPase activity. Pharmacological inhibition of PMCA4 significantly reduces the hypertrophic response to pressure overload likely through inhibition of calcineurin/NFAT signalling.

Estudos têm mostrado que o treinamento de força de baixa intensidade com oclusão vascular (TFOV) tem apresentado resultados similares nos ganhos de força e hipertrofia comparado ao treinamento de força (TF) de alta intensidade. O objetivo deste estudo foi comparar os efeitos de três diferentes programas de TF nos ganhos de força e hipertrofia musculares e na expressão da miostatina (MSTN) e seus antagonistas. Para isso, vinte e nove jovens do sexo masculino, sem experiência em TF, foram recrutados e divididos randomicamente nos grupos: treinamento de força de baixa intensidade sem oclusão (BI), treinamento de força de baixa intensidade com oclusão (BIO) e treinamento de força de alta intensidade sem oclusão (AI). Os grupos BIO e BI treinaram com intensidade de 20% 1RM, enquanto o grupo AI treinou com intensidade de 80% 1RM. A ANOVA one way foi utilizada para testar as diferenças percentuais nos ganhos de força (1RM) e na área de secção transversa (AST) do músculo quadríceps femoral. O modelo misto para análise das medidas repetidas foi utilizado para testar as diferenças nas variáveis miostatina (MSTN), folistatina-3 (FLST-3), SMAD-7 e GASP-1 nos grupos BI, BIO e AI nas condições pré e pós-treinamento. Os resultados mostraram que os aumentos de força e hipertrofia musculares nos grupos BIO e AI foram similares, entretanto superiores ao grupo BI. Esses resultados podem ser atribuídos a maior diminuição na expressão da MSTN nos grupos BIO (45%) e AI (41%) comparados com o grupo BI (16%) e o aumento na expressão dos genes que antagonizam sua atividade (SMAD-7, FLST-3 e GASP-1). Podemos concluir que a inibição na atividade da MSTN dos grupos BIO e AI podem responder em parte a similaridade nos ganhos de força e hipertrofia entre os grupos e a diferença para o grupo BI / It has been demonstrated that low intensity training associated to vascular occlusion (LIO) promotes similar gains in strength and muscle mass when compared to high intensity strength training (HI). The aim of the present study was to evaluate the effect of three different training programs on skeletal muscle hypertrophy and atrophy related gene expression. Twenty nine young male, with no previous experience in strength training were randomly allocated in three groups: low intensity strength training (i.e. 20% - 1-RM) (LI); low intensity strength training associated to vascular occlusion (i.e. 20% - 1-RM) (LIO); high intensity strength training (HI) (i.e. 80% - 1-RM). One-way ANOVA was used to assess differences in % delta change values of 1-RM and cross sectional area (CSA) of the quadriceps femoris. Mixed model analysis was used to compare myostatin (MSTN), folistatyn-3 (FLST-3), SMAD-7 e GASP-1 changes between groups pre and post training. Results demonstrated similar increases in strength and muscle hypertrophy for LIO and HI groups. Moreover, such increases were significantly greater when compared to LI. These results may be, at least in part, explained by a significant decrease in MSTN mRNA expression in LIO (45%) and HI (41%) when compared to LI (16%); additionally, SMAD-7; FLST-3 and GASP-1 mRNA expression were significantly increased. In conclusion, LIO training promotes similar gains than HI training. The results may be explained by changes in MSTN and related genes mRNA expression

O overreaching não funcional (NFOR) induzido pelo exercício excêntrico (EE) em modelo animal está associado com a diminuição de desempenho físico, dano no DNA (amostras de músculo esquelético e soro), estresse oxidativo (amostras de músculo esquelético e soro), inflamação crônica de baixo grau (amostras de músculo esquelético e soro) e prejuízo da via de sinalização da insulina (amostras de músculo esquelético). No entanto, as adaptações do músculo cardíaco em resposta ao estado de NFOR induzido ou não pela predominância do EE ainda não foram investigadas. Além disso, sabe-se que a mTOR (mammalian target of rapamycin) possui um efeito protetor no músculo cardíaco, suprimindo o aumento de citocinas pró-inflamatórias, que estão relacionadas à disfunções cardíacas. Assim, o presente estudo teve como objetivo comparar os efeitos do NFOR em declive com outros dois protocolos de mesmo volume e intensidade, mas realizados sem inclinação e em aclive, no conteúdo das proteínas relacionadas às vias moleculares inflamatória e hipertrófica, no conteúdo de fibrose intersticial e na expresão gênica em músculo cardíaco de camundongos. Os animais foram divididos em 6 grupos: Naíve (N; camundongos sedentários), Controle (C; camundongos sedentários submetidos aos testes físicos), Treinado (TR; camundongos submetidos ao protocolo de treinamento), Overtraining em declive (OTR/down; camundongos submetidos ao protocolo de OT com corrida na descida), Overtraining sem inclinação (OTR; camundongos submetidos ao protocolo de OT com corrida sem inclinação) e Overtraining em aclive (OTR/up; camundongos submetidos ao protocolo de OT com corrida na subida). Em relação aos parâmetros metabólicos, o grupo OTR/down apresentou menor variação de peso corporal na semana 8. Todos os grupos que passaram pelo protocolo de OT demonstraram queda de desempenho ao final da semana 8, aumento no conteúdo de tecido conjuntivo no ventrículo esquerdo e menor ativação da proteína AMPKalfa. O grupo OTR/down apresentou menor conteúdo da proteína mTOR e S6RP, e aumento na expressão do gene beta-MHC. Pode-se concluir que os protocolos de OT provocaram indícios de hipertrofia patológica no ventrículo esquerdo, sendo esse efeito mais pronunciado no grupo OTR/down. / Nonfunctional overreaching (NFOR) induced by eccentric exercise (EE) in animal model is associated with performance decrement, DNA damage (muscle and serum samples), oxidative stress (muscle and serum samples), low grade chronic inflammation (muscle and serum samples) and insulin signaling impairment (muscle and serum samples). However, the adaptations of cardiac muscle in response to NFOR induced or not induced by EE are unknown. In addition, the mammalian target of rapamycin (mTOR) has a protector effect in the cardiac muscle, suppressing the increase of the proinflammatory cytokines that is related to cardiac dysfunctions. Thus, the main aim of present study was to compare the effects of NFOR based on EE (downhill running) with other two protocols with similar intensity and volume, but performed in uphill and without inclination, on the protein contents related to hypertrophic and inflammatory signaling, on the content of interstitial fibrotic tissue and on genes expression in mice cardiac muscle. The animals were divided on six groups: Naïve (N; sedentary mice), Control (C; sedentary mice submitted to physical tests), Trained (TR; mice submitted to training protocol), Overtraining in downhill (OTR/down; mice submitted to the overtraining protocol in downhill), Overtraining without inclination (OTR; mice submitted to the overtraining protocol without inclination), and Overtraining in uphill (OTR/up; mice submitted to the overtraining protocol in uphill). Regarding metabolic parameters, OTR/down group presented reduced body weight variation at week 8. All OT groups presented performance drop at end of week 8, increased connective tissue content in left ventricle and reduced AMPKalpha activation. OTR/down group presented reduced mTOR and S6RP protein content, and increased betaMHC gene expression. The conclusion is that OT protocols provoked signs of pathological hypertrophy in left ventricle, being this effect more pronounced in OTR/down group.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22361号 / 医博第4602号 / 新制||医||1043(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 上本 伸二, 教授 小西 靖彦, 教授 黒田 知宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

For decades, most scientists and practitioners have agreed that muscle hypertrophy also induces strength gains. However, a recent publication "The Problem of Muscle Hypertrophy: Revisited," questioned the mechanistic role that exercise-induced increases in muscle size have on the exercise-induced increases in strength (of force production), as well as the influence that exercise-induced increases in strength have on sports performance. Such suggestions undermine the important of certain aspects of strength and conditioning for sport. Specifically, if not acting as a mechanism for strength adaptation, it is unclear if there is a sports-related benefit to skeletal muscle hypertrophy. In addition, the authors argued that if strength has little impact on sports performance, strength and conditioning programs may be doing little more than delaying recovery from practicing the actual sport. This contention also indicates that hypertrophy should be avoided in nearly all scenarios because increased muscle size would be additional mass that must be overcome. The purpose of this special discussion is to allow for an in-depth scientific discussion of the experimental evidence for and against the position of Buckner et al. That exercise-induced increases in muscle size have little relevance on the exercise-induced increases in strength and thus, sport performance.

Skeletal muscle regeneration is a critical process that replaces damaged muscle fibers with new fibers. The regenerative process can be segmented into four main phases: necrosis, inflammation, regeneration, and maturation. While many of the key signaling molecules are known and characterized, there are still gaps in our understanding of how this process is regulated. While it is reported that growth arrest specific 6 (Gas6) and its receptor Axl are expressed in mature muscle tissue, nothing is known about the effect that Gas6 and Axl have on regulating skeletal muscle regeneration. In this study we investigated the regenerative process in a Gas6/Axl double knockout (dKO) mouse model. The tibialis anterior (TA) muscle was chemically injured with BaCl2 and allowed to recover for 3, 7, or 14 days. We investigated satellite cell (SC) activation and muscle growth. We found that the dKO injured muscle has fewer SCs at 3-days post-injury, but the percentage of mitotically active SCs were no different between WT and dKO injured muscle. Interestingly, basal and injured dKO muscle has an increased cross-sectional area compared to wild type in male mice. Together this may suggest that in the absence of Gas6/Axl signaling may lead to impaired regeneration and compensatory fiber hypertrophy. The mechanism behind the hypertrophy remains unknown, but ultimately our findings suggest that Gas6/Axl signaling has an effect on skeletal muscle regeneration.

Cardiovascular disease (CVD) is the leading cause of death worldwide. Pre-menopausal women have a lower incidence and severity of CVD compared to age-matched men. However, at the onset of menopause, CVD increases. A central feature in patients with CVD is excessive chronic sympathetic stimulation (CSS) of β-adrenergic receptors (β-AR’s). Clinical and animal studies show estrogen deficiency and age exacerbate cardiac β-AR signaling and contractile function. This led to the hypothesis that prolonged estrogen deficiency followed by CSS worsens left ventricular cardiac function and hypertrophy in the aged female heart. Female mice underwent bilateral ovariectomy or SHAM surgery at 2.5 months of age. At 12 months post-ovariectomy, mice were infused with Isoproterenol (400μg/kg/h) via mini-osmotic pumps for three days to induce CSS. This observation demonstrates prolonged estrogen deficiency worsens cardiac function and structure in aged female hearts. Thereby emphasizing the importance of clinical intervention and prevention for CVD in menopausal women.