The skeletal muscle fibre is a syncitium where each myonucleus regulates the gene products in a finite volume of cytoplasm i.e., the myonuclear domain (MND). A novel image analysis algorithm applied to confocal images, analyzing MND size and myonuclear spatial distribution in 3-dimensions in single skeletal muscle fibres has been used in this project. The goal was to explore the modulation of myonuclei count and MND size in response to muscle adaptation processes. The effects of ageing, gender, hormones, muscle hypertrophy and body size were investigated on MND size. A strong linear relationship was found between MND size and body size in the muscle fibres from mammals representing a 100,000-fold difference in body size. Independent of species, MND size was highly dependent on MyHC isoform type and mitochondrial contents of skeletal muscle fibres. In hypertrophic mice, a significant effect of MND size on specific force and myosin content was observed. This effect was muscle fibre type-specific and shows that the bigger MNDs in fast-twitch EDL muscle fibres are optimally tuned for force production while smaller MNDs in slow-twitch soleus muscle fibres have a much more dynamic range of hypertrophy without functional compromise. This indicates a critical volume individual myonuclei can support efficiently for a proportional gain in muscle fibre force and size. In human muscle fibres, spatial organization of myonuclei was affected by both ageing and MyHC isoform expression. In fibres expressing type I MyHC isoform, an increased MND size variability and myonuclear aggregates were observed in old age although average MND size was unchanged. In contrast, in type IIa fibres, the average MND size was smaller reflecting smaller size of muscle fibres. Those changes may influence the transcriptional activity per myonucleus and/or local cooperatively of myonuclei in a gender and muscle fibre-type specific manner. Finally, hormone replacement therapy was shown to negate menopause-related functional impairment in skeletal muscle fibres. The positive effect on force was due to quantitative effect in fibres expressing fast myosin isoform while the effect was both quantitative and qualitative in fibres expressing slow myosin isoform. The effect on MND size was fibre type dependent and was achieved by significantly reducing domain size in slow- but not the fast-twitch muscle fibres. Together, our data suggest that modulation of myonuclei count and MND size is a mechanism contributing to remodelling of skeletal muscle in muscle adaptation process. These findings should be considered when developing therapeutic approaches towards restoring muscle mass and strength in muscle wasting conditions.

Background: The aim of this thesis was to study transcription patterns and extracellular signalling of the hypertrophic heart to better understand the mechanisms initiating, controlling and maintaining cardiac hypertrophy. Cardiac hypertrophy is a risk factor for cardiovascular morbidity and mortality. Hypertrophy of the myocardium is a state, independent of underlying disease, where the myocardium strives to compensate for an increased workload. This remodelling of the heart includes physiological changes induced by a changed gene expression, alteration of the extracellular matrix and diverse cell-to-cell signalling. Shedding microvesicles and exosomes are membrane released vesicles derived from the plasma membrane, which can mediate messages between cells and induce various cell-related processes in target cells. Methods and materials: Two different microarray studies on different materials were performed. In the first study, cardiac myectomies from 8 patients with hypertrophic obstructive cardiomyopathy (HOCM) and 5 controls without cardiac disease were used. In the second study, myocardial tissue from 6 aorta ligated and 6 sham operated (controls) rats at three different time points (1, 6 and 42 days post-surgically) were analysed. To reveal differences in gene expression the materials were analyzed with Illumina whole genome microarray and multivariate data analysis (PCA and OPLS-DA). Cultured cardiomyocytes (HL-1) were incubated with and without growth factors (TGF-β2 or PDGF BB). Microvesicles and exosomes were collected and isolated after differential centrifugations and ultracentrifugations of the cell culture medium. The microvesicles and exosomes were characterized with dynamic light scattering (DLS), flow cytometry, western blot, electron microscopy and Illumina whole genome microarray. Results: The two different microarray studies revealed differentially expressed gene transcripts and groups of transcripts. When comparing HOCM patients to controls significant down-regulation of the MYH6 gene transcript and two immediate early genes (IEGs, EGR1 and FOS), as well as significant up-regulation of the ACE2, JAK2 and HDAC5 gene transcripts were found. In the rat model, 5 gene groups showed interesting clustering after multivariate data analysis (OPLS-DA) associated with the hypertrophic development: “Atherosclerosis”, “ECM and adhesion molecules”, “Fatty acid metabolism”, “Glucose metabolism” and “Mitochondria”. The shedding microvesicles were rounded vesicles, 40-300 nm in size and surrounded by a bilayered membrane. Chromosomal DNA sequences were identified in the microvesicles. The microvesicles could be taken up by fibroblasts resulting in an altered gene expression in the fibroblasts. The exosomes from cultured cardiomyocytes (incubated with TGF-β2 or PDGF BB) had an average diameter of 50-80 nm, similar to the unstimulated control exosomes. A large, for all cardiomyocyte derived exosomes, common pool of mRNA seems stable and a smaller pool varied in mRNA content according to treatment of the cardiomyocyte. Of the common mRNA about 14% were ribosomal, 14% were of unknown locus and 5% connected to the function of the mitochondria. Conclusions: The microarray studies showed that transcriptional regulation at a stable stage of the hypertrophic development is a balance of pro and anti hypertrophic mechanisms and that diverse gene groups are differently regulated at different time points in the hypertrophic progression. OPLS-DA is a very useful and powerful tool when analyzing gene expression data, especially in finding clusters of gene groups not seen with traditional statistics. The extracellular vesicle studies suggests that microvesicles and exosomes released from cardiomyocytes contain DNA and can be involved in events in target cells by facilitating an array of processes including gene expression changes. Different treatment of the cardiomyocyte influence the content of the exosome produced, indicating that the signal function of the exosome might vary according to the state of the cardiomyocyte. / Bakgrund: Syftet med den här avhandlingen var att studera transkriptions-mönster och extracellulär signalering vid hjärthypertrofi för att bättre förstå de mekanismer som startar, styr och underhåller tillväxten. Hjärthypertrofi, onormal tillväxt av hjärtmuskeln, är en riskfaktor för andra hjärt-kärlsjukdomar och dödlighet. Hypertrofi av hjärtmuskeln är ett tillstånd, oberoende av bakomliggande sjukdom, där hjärtmuskeln strävar efter att kompensera för ökad arbetsbelastning. Denna omställning av hjärtat innefattar fysiologiska förändringar orsakade av ett förändrat genuttryck, modifiering av miljön utanför cellen och ändrad cell-till-cell signalering. Mikrovesiklar och exosomer är små membranomslutna bubblor som frisätts från cellmembranet, ut i cellens omgivning. De kan förmedla budskap mellan celler och påverka olika processer i målceller. Metoder och material: Avhandlingen innefattar två olika microarraystudier på olika material. I den första studien användes hjärtbiopsier från 8 patienter med hypertrofisk obstruktiv kardiomyopati (HOCM) och 5 kontroller utan hjärtsjukdom. I det andra projektet användes hjärtvävnad från 6 aortaligerade och 6 skenopererade (kontroller) råttor vid tre olika tidpunkter (1, 6 och 42 dagar efter kirurgiskt ingrepp). För att påvisa skillnader i genuttryck analyserades proverna med Illumina helgenom microarray och multivariat dataanalys. Avhandlingens andra del innehåller två studier om mikrovesiklar och exosomer. Odlade hjärtmuskelceller (HL-1) stimulerades med tillväxt-faktorer (TGF-β2 eller PDGF BB) och ostimulerade celler användes som kontroll. Mikrovesiklar och exosomer renades fram med centrifugeringar och ultracentrifugering av cellodlingsmediet för att sedan karakteriseras med olika metoder för att studera storlek, ytmarkörer och innehåll. Illumina helgenom microarray användes för att studera microvesiklarnas och exosomernas mRNA innehåll. Resultat: I de två olika microarraystudierna hittades gentranskript och grupper av gentranskript som skiljde sig mellan kontroller och den hypertrofa hjärtvävnaden. När HOCM patientproverna jämfördes med kontroller hittades nedreglering av MYH6, EGR1 och FOS samt uppreglering av ACE2, JAK2 och HDAC5. Efter multivariat dataanalys av materialet från råtta, hittades 5 grupper av gentranskript med intressanta mönster som kunde kopplas till den hypertrofiska utvecklingen av hjärtmuskeln: "Ateroskleros", "ECM och adhesionsmolekyler", "Fettsyrametabolism", "Glukosmetabolis-men" och "Mitokondrien". Mikrovesiklarna hade en diameter på 40-300 nm och innehöll kromosomala DNA-sekvenser. När mikrovesiklarna överfördes till en annan celltyp (fibroblaster) resulterade det i ett förändrat genuttryck i fibroblasterna. Exosomer från hjärtmuskelcellerna som odlats med eller utan tillväxtfaktor hade en diameter på 50-80 nm. En stor pool av olika gentranskript var gemensam för alla exosomer oavsett stimulering eller ej. En mindre pool av gentranskript varierade i innehåll mellan de stimulerade och ostimulerade hjärtmuskelcellerna. I den gemensamma gentranskript poolen var ca 14 % ribosomala, ca 14 % var okända och ca 5 % var associerade till mitokondrien och dess funktion. Slutsats: Microarraystudierna visade att transkriptionsreglering i ett stabilt skede av hypertrofiutvecklingen är en balans mellan pro- och anti-hypertrofiska mekanismer och att olika gengrupper var olika reglerade vid olika tidpunkter i hjärtmuskeltillväxten. OPLS-DA är ett mycket användbart och kraftfullt verktyg när man analyserar genexpressionsdata, särskilt för att hitta grupper av gen-transkript som är svåra att upptäcka med traditionell statistik. Microvesikel- och exosomstudierna visade att mikrovesiklar och exosomer som frisätts från hjärtmuskelceller innehåller både DNA och RNA och kan vara inblandade i händelserna i målceller genom att underlätta en rad processer, inklusive ändringar av genuttryck. Olika stimulering av hjärtmuskelcellen kan påverka innehållet i exosomernas som produceras, vilket indikerar att exosomernas signalfunktion kan variera beroende på hjärtmuskelcellens tillstånd.

Left ventricular hypertrophy (LVH) and the insulin resistance syndrome are common conditions associated with a markedly increased cardiovascular risk. In a fairly large prospective longitudinal study of men from the general population, we found that an unfavorable serum fatty acid profile and components of the insulin resistance syndrome such as dyslipidemia, obesity and hypertension at age 50 predicted the prevalence of LVH at age 70. In cross-sectional analyses at age 70, several components of the insulin resistance syndrome were significantly related to left ventricular relative wall thickness and concentric remodeling, but less to LVH. Left ventricular relative wall thickness was inversely related to insulin sensitivity in skeletal muscle and borderline significantly directly related to insulin sensitivity in the myocardium in a healthy, normotensive sample of the cohort investigated with positron emission tomography, whereas left ventricular mass index was not related to myocardial or skeletal muscle insulin sensitivity. At age 70, echocardiographic LVH was related to a variety of common electrocardiographic diagnoses. In a prospective mortality analysis with baseline at age 70 and a median follow-up time of five years, echocardiographic and electrocardiographic LVH predicted mortality independently of each other and of other cardiovascular risk factors, implying that echocardiographic and electrocardiographic LVH in part carry different prognostic information. In summary, components of the insulin resistance syndrome predicted LVH twenty years later, but were cross-sectionally more related to increased left ventricular relative wall thickness and concentric remodeling. Echocardiographic and electrocardiographic LVH predicted mortality independently of each other and of components of the insulin resistance syndrome.

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.

Cardiac hypertrophy is a compensatory response to increased work load or stress on the heart, but over time can lead to heart failure and death. The molecular mechanisms underlying this disease are still not completely understood, however the Rho/Rho kinase pathway has been shown to play a role. N-[11C]-methyl-hydroxyfasudil, a PET radiotracer, binds to active Rho kinase and could be a possible tracer for hypertrophy. Hypertrophy was induced in vitro using the β-adrenergic receptor agonist isoproterenol to evaluate optimal Rho kinase activity. Rho kinase activity data was correlated to N-[11C]-methyl-hydroxyfasudil binding. Cardiac hypertrophy was verified with an increase in nuclear size (1.74 fold) and cell size (~2 fold), activation of hypertrophic signalling pathways, and increased Rho kinase activity (1.64 fold). This correlated to a 10.3% increase in N-[11C]-methyl-hydroxyfasudil binding. This data suggests that N-[11C]-methyl-hydroxyfasudil may be useful as a radiotracer for detecting cardiac hypertrophy and merits further in vivo investigation.

Pax3 protein is initially expressed in the dermomyotome of embryonic somites, which gives rise to skeletal muscle. Following myogenesis, Pax3 expression is mostly down-regulated and becomes restricted to a few satellite cells (SCs) of select mature muscles. SCs are activated to form new myonuclei during muscle hypertrophy, regeneration and repair. Intrafusal fibers of muscle spindles are thought to persist in a comparatively immature state as, unlike extrafusal fibers, they maintain small diameters, developmental myosins, Myf5 expression and high SC concentrations. This thesis tests the hypotheses that Pax3 expression is preferentially maintained in SCs of adult skeletal muscle spindles and can be augmented under conditions of SC activation. To study Pax3 through development, immunohistochemical techniques were used to identify SCs by their Pax7 expression, and analyze the proportion of SCs and myonuclei (MN) expressing Pax3 in chicken anterior latissimus dorsi (ALD) muscle excised at 9, 30, 62, and 145 days post-hatch. To induce SC activation, tenotomy was performed on the right ALD muscle of 138-day post-hatch chicks to induce compensatory hypertrophy of the ipsilateral synergistic posterior latissimus dorsi (PLD) muscle. The PLD was analyzed seven days after ALD tenotomy using similar immunohistochemical techniques. This is the first study to show Pax3 expressing SCs within intrafusal fibers of muscle spindles. This thesis demonstrates that throughout development there is a higher percentage of Pax3 expressing SCs within intrafusal fibers of muscle spindles than the surrounding extrafusal fibers that compose the bulk of the muscle. It is also revealed that the proportion of the SC population expressing Pax3 declines with age in both intrafusal and extrafusal fibers. Compensatory hypertrophy of the PLD resulted in a greater percentage of Pax3 expressing SCs in intrafusal and extrafusal fibers than under control conditions. The percentage of SCs expressing Pax3 after PLD overload was similar to that seen in young control muscle. The percentage of Pax3 expressing MN also increased after muscle overload to levels seen in young muscle. A disproportionate decrease in the proportion of SCs expressing Pax3 during development, and a disproportionate increase in the percentage of Pax3 positive SCs as a result of experimentally induced muscle hypertrophy, suggests that Pax3 expression in maturing muscle may be more than just a developmental vestige. Pax3 may be a factor in the activation and differentiation of SCs in maturing muscle.

The effects of an 8-week swim training program and a 3-week detraining period on the size and protein composition of the heart of female Wistar rats were examined. The animals were separated into three groups: trained, detrained, and sedentary control. The training program, 6 hours/day, 5 days/week, resulted in a significantly larger dry heart weight, a normal collagen concentration, and a nonsignificantly greater total protein content. The greatest changes occurred between 4 and 6 weeks of training. The mild physiologic hypertrophy was assumed to be typical, and therefore simply an enlargement of a normal heart with proportional increases in myocyte size, and connective and vascular tissue hyperplasia. The detraining period produced a return of the heart mass to normal by the end of 2 weeks of detraining. The total protein content and collagen concentration remained elevated, though nonsignificant. The results indicate the detrained heart is composed of more connective, vascular, and metabolically active tissue, all of which may be important in the detrained heart’s response to increased work.Ball State UniversityMuncie, IN 47306

In setting the AMDR for protein at 10-35% of daily energy, the Institute of Medicine acknowledged a lack of data regarding the safety of long-term intakes. The current study assessed the impact of chronic (17 months) protein consumption at the upper end of the AMDR on renal function, histology, and inflammation. Using plant and animal whole protein sources, female Sprague-Dawley rats (70 days old; n=8-11 at 4, 8, 12, or 17 mo.) were randomized to either a normal (NP; 15% of energy) or high protein (HP; 35% of energy) diet. Egg albumen and skim milk replaced carbohydrates in the HP diet. Diets were balanced for energy, fat, vitamins and minerals, and offered ad libitum. Renal function was analyzed by creatinine clearance and urinary protein levels. Glomerular hypertrophy, glomerulosclerosis and tubulointerstitial fibrosis were assessed on kidney sections. Kidney disease progression was determined by the measurement of transforming growth factor beta-1 (TGF-β1) and renal inflammation by the measurement of chemokines monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation normal T-cell expressed and secreted (RANTES). Rats consuming the HP compared to NP diet had ~17% higher kidney weights (P<0.0001) and ~4.8 times higher proteinuria (P<0.0001). There was a trend towards higher creatinine clearance with HP (P=0.055). Consistent with this, HP compared to NP rats had ~22% larger glomeruli (P<0.0001) and ~33% more glomerulosclerosis (P=0.0003). The HP diet had no significant effect on tubulointerstitial fibrosis and renal TGF-β1 levels and did not result in higher renal levels of MCP-1 and RANTES. In fact, per mg renal protein, HP rats had ~16% lower MCP-1 (P<0.0001) and ~34% lower levels of RANTES (P<0.0001) than NP. The absence of an increase in cytokine levels may be a reflection of the moderate changes in renal pathology observed in rats offered HP diets. These data in normal female rats suggest that protein intakes at the upper end of the AMDR are detrimental to kidney health in the long-term. While modest, this may have implications for individuals whose kidney function is compromised, especially given the prevalence of those unaware of their kidney disease within North America. / October 2007

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.

Evidence has shown that hypoxic hearts have greater heart/fetus mass ratio. However, it is still unclear if either hyperplasia or hypertrophy causes the relatively increased heart mass. Furthermore, the genes that might be involved in the process have not yet been identified. In the present study, the cardiac transcriptome was analyzed to identify differentially expressed genes related to hypoxia. Eggs were incubated for 15 and 19 days in two different environments, normoxic and hypoxic. Normalized microarray results were analyzed to isolate differentially expressed probes using the Affymetrix chip. Total RNA was also isolated from another set of fetuses incubated in the same conditions and used to perform a qPCR in order to confirm the microarray results. In the four groups (15N, 15H, 19N, 19H), some probes were differentially expressed. From the eggs incubated for 15 days, the microarray revealed five probes that were differentially expressed according to the criteria (p&lt;0.01 and absolute fold change FC&gt;2) in the two programs (PLIER &amp; RMA) used to normalize the data. From the eggs incubated up to 19 days, eight probes were differentially expressed in both programs. No further tests were performed on the 19 days fetuses since there was no significant difference in that group after incubation for the heart/fetus mass ratio. Apolipoprotein-A1, p22, similar to ENS-1 and b2 adrenergic receptor were further tested in qPCR (15 days sample). The differently expressed genes are linked to cell division and should be further studied to identify their function, especially the similar to ENS-1.