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Physiological adaptations were investigated following a strength-endurance (S-E) block prescribed with accentuated eccentric loading (AEL) or traditional resistance training (TRAD). Recreationally trained participants (n = 11 males, 6 females, age = 23.2 ± 4.2 yrs, body mass (BM) = 81.3 ± 22.2 kg, height = 172.1 ± 10 cm) completed a four-week block of concurrent resistance, sprint, and change of direction training. Participants were assigned one of two training conditions, AEL (n = 9) or TRAD (n = 8). Training was identical, except AEL performed 110% eccentric overloading every 1st, 3rd, 5th, 7th, and 9th repetition during back squat (BS) and bench press (BP). Body composition, summated muscle size (ACSAsum) and thickness (MTsum), regional ACSA and MT, and region-specific fascicle angle (FA) and length (FL) were assessed pre- (PRE) and post-training (POST). External work was calculated and exercise displacement was measured to determine the mechanical stimulus provided. Physiological variables were analyzed using multiple mixed analysis of variance (ANOVA). External work and displacement were analyzed with independent Welch’s t-tests. A statistically significant main effect of Time was observed for ACSAsum and ICW (p < 0.05); however, there were no statistically significant Time x Condition interaction effects observed for any dependent variable (p > 0.05). Time x Length interaction effects also failed to reach statistical significance for regional ACSA or regional MT (p > 0.05). Moreover, Time x Position interaction effects were not statistically significant for regional MT (p > 0.05). There were also no statistically significant interaction effects observed for regional FA or FL (p > 0.05). Differences in external work did not reach statistical significance (p > 0.05). A four-week S-E training block, performed with or without AEL, increases muscle size, but results in only minor architectural alterations. Additionally, AEL appears to induce unique region-specific hypertrophy. In contrast, TRAD seems to induce greater increases in ICW, potentially indicating greater sarcoplasmic hypertrophy. Interestingly, 110% eccentric overloading did not lead to statistically greater work performed, although differences may be practically significant when allometrically scaled. Researchers and practitioners should examine region-specific musculoskeletal adaptations, when possible, to more accurately assess training effects.

<p>There is a prevalent belief that acute hormone responses to resistance exercise mediate adaptations in skeletal muscle hypertrophy; however, there is little supporting evidence. We conducted studies to examine the relationship between acute hormonal increases after resistance exercises and subsequent changes in muscle anabolism.</p> <p>We tested the hypothesis that exercise-induced responses of anabolic hormones—growth hormone (GH) and testosterone—would enhance rates of myofibrillar protein synthesis (MPS) after an acute bout of resistance exercise, and would augment muscle hypertrophy after training. We concluded, however, that resistance exercise-induced increases in putative anabolic hormones do not enhance MPS or hypertrophy.</p> <p>We also examined whether rates of MPS would be attenuated in women (compared with men) after resistance exercise, due to their lack of post-exercise testosteronemia. We reported similar increases in MPS in men and women; post-exercise testosterone responses in women, which were 45-fold lower than men, did not attenuate elevations in MPS.</p> <p>Collectively, our work leads to the conclusion that the acute rise in hormones such as testosterone and GH has very little bearing on MPS and hypertrophy responses to resistance exercise. Instead, the rise in these hormones appears to be a non-specific response to exercise stress rather than a response that is important for muscle anabolism. Contrary to widely used principles, our data suggests that exercise programs should not be designed based on nuances in the post-exercise hormonal milieu. Alternatively, understanding local mechanotransduction, which is directly linked to muscle fibre loading, will reveal the processes that drive human exercise-mediated muscle hypertrophy.</p> / Doctor of Philosophy (PhD)

Damage to the hyaline articular cartilage that cushions joints is exceedingly common worldwide, whether caused by traumatic injuries or degenerative pathologies that can lead to the onset of osteoarthritis. Clinically, cartilage lesions are treated with surgical procedures that attempt to restore the architecture of the hyaline tissue. Unfortunately, the current treatment options often result in the undesired formation of fibrocartilage, a type of cartilage with mechanical properties that are inferior to those of hyaline cartilage. The ability to withstand constant mechanical load is the primary function of articular cartilage, and therefore, critical to restore. The field of cartilage tissue engineering aims to address the limitations of current treatment options by generating restorative tissue with cartilaginous protein composition and concomitant mechanical competency of native hyaline cartilage. Efforts to recapitulate functional cartilage often include approaches that start with cells seeded on a scaffold. Scaffolds are employed to provide the mechanical structure while cells execute the formation of the extracellular matrix. Furthermore, adult mesenchymal stem cells (MSCs) are used in combination with chondrocytes, the single cell type of cartilage, to enhance chondrogenic composition. Given the possible adult MSC sources, such as bone marrow, adipose tissue or the synovial membrane, it is important to select the source that will yield maximum cartilage differentiation. However, the multi-lineage differentiation capacity of MSCs is also their intrinsic limitation. Stem cells undergoing differentiation to cartilage formation can transition into bone, a process known as hypertrophy, which yields changes in chondrocyte function and subsequent undesired deposition of mineralized matrix instead of a normal chondral matrix. The overarching hypothesis of this thesis is that using cartilage-specific MSCs from the synovial membrane, a tissue adjacent to the articular surface, will generate cartilage with superior properties when compared to tissue derived from other cell sources. This hypothesis was tested in the following four aims: First, to assess the in vivo response of tissue engineered cartilage generated from gold standard bone marrow-derived MSCs in a preclinical minipig model; second, to compare the chondrogenic capacity of synovial MSCs and bone marrow MSCs in scaffold-free and scaffold-based engineered cartilage; third, to challenge scaffold-based engineered cartilage with a hypertrophic environment and evaluate the response; and fourth, to explore the use of a hypoxia-simulating agent for the enhancement of chondrogenic differentiation. Together these studies contribute to the identification of an optimal cell source for cartilage tissue engineering to be used in translational preclinical models. / Bioengineering

Pathological hypertrophy leads to cardiac dysfunction and heart failure. It is not clearly defined how this process occurs in the cardiomyocyte, or how the pathology can be effectively treated. There are numerous processes that lead to pathological hypertrophy. We developed two models to study pathological hypertrophy and the role that Ca2+ plays. In one model, we administered clinical doses of the leukemia therapeutic drug imatinib to neonatal ventricular cardiomyocytes. This drug has recently been found to be cardiotoxic, and we set out to understand if Ca2+ is involved. In the second model, we developed mice with overexpression of the Ca2+ entrance channel, the L-type calcium channel (LTCC), which leads to pathological hypertrophy over time. We instituted a chronic exercise regimen on these mice to learn if physiological hypertrophy can ameliorate detrimental aspects of pathological hypertrophy. After cardiomyocytes were treated with imatinib, they expressed enhanced Ca2+ activity. Levels of atrial natriuretic peptide (ANP) were up, signifying pathological hypertrophy. We determined that Ca2+ was activating Calcineurin, leading to translocation of nuclear factor of activated T-cells (NFAT) into the nucleus, resulting in hypertrophy. This activity was blocked by Ca2+ and Calcineurin inhibitors. We concluded that imatinib causes Ca2+ induced pathological hypertrophy. When mice with LTCC overexpression were exercised, they exhibited enhanced cardiac function. They also had thicker septal walls and increased chamber diameter, hallmarks of physiological hypertrophy. Heart weight to body weight ratio was significantly higher after exercise. When isolated hearts were administered ischemia/reperfusion injury, the exercised hearts showed a significant improvement in recovery compared to sedentary LTCC overexpressed hearts. Calcium activity was enhanced at the cardiomyocyte level in both mouse lines of exercised mice. In conclusion, hearts with a pathological hypertrophic phenotype can enhance function and achieve cardioprotection through chronic exercise. / Physiology

Pathological cardiac stressors, including persistent hypertension or damage from ischemic heart disease, induce a chronic demand for enhanced contractile performance of the heart. The cytosolic calcium (Ca2+) transient that regulates myocyte contraction must be persistently increased in disease states in order to maintain cardiac output to sustain the metabolic requirements of the body. Associated with this enhanced intracellular Ca2+ ([Ca2+]i) state is pathological cardiac myocyte hypertrophy, which results in large part from the activation of Ca2+-dependent activation of calcineurin (Cn)-nuclear factor of activated T cells (NFAT) signaling. The puzzling feature of this hypertrophic signaling is that the cytosolic [Ca2+] that controls contractility appears to be separate from the [Ca2+] which activates Cn-NFAT signaling. The overarching theme of this dissertation is to explore the source and spatial constraints of pathological hypertrophic signaling Ca2+ and to investigate how it is possible that sensitive and finely tuned Ca2+-dependent signaling pathways are regulated in the background of massive Ca2+ fluctuations that oscillate between 100nM and upwards of 1-2&#956;M during each cardiac contractile cycle. L-type Ca2+ channels (LTCCs) are a major source of Ca2+ entry in cardiac myocytes and are known to play an integral role in the initiation of myocyte excitation contraction-coupling (EC-coupling). We performed a number of experiments to show that a small population of LTCCs reside outside of EC-coupling domains within caveolin (Cav-3) signaling microdomains where they provide a local source of Ca2+ to activate Cn-NFAT signaling. We designed a Cav-targeted LTCC blocker that could eliminate Cn-NFAT activation but did not reduce myocyte contractility. The activity of Cav-targeted LTCCs could also be upregulated to enhance hypertrophic signaling without affecting contractility. Therefore, we believe that caveolae-localized LTCCs do not participate in EC-coupling, but instead act locally to control the coordinated activation of Cn-NFAT signaling that drives pathological remodeling. Transient Receptor Potential (TRP) channels are also thought to provide a source of Ca2+ for activation of hypertrophic signaling. The canonical family of TRP channels (TRPC) is expressed at low levels in normal adult cardiac tissue, but these channels are upregulated in disease conditions which implicates them as stress response molecules that could potentially provide a platform for hypertrophic Ca2+ signaling. We show evidence that TRPC channel abundance and function increases in cardiac stress conditions, such as myocardial infarction (MI), and that these channels are associated with hypertrophic responses, likely through a Ca2+ microdomain effect. While we found that TRPC channels housed in caveolae membrane microdomains provides a source of [Ca2+] for induction of cardiac hypertrophy, this effect also requires interplay with LTCCs. We also found that TRPC channels have negative effects on cardiac contractility, which we believe are due to local activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) and subsequent modulation of ryanodine receptors (RyRs). Further, we found that inhibiting TRPC channels in a mouse model of MI led to increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling, as well as increased survival. Collectively, the data presented in this dissertation provides comprehensive evidence that Ca2+ regulation of Cn-NFAT signaling and resultant pathological hypertrophy can be coordinated by spatially localized and regulated Ca2+ channels. The compartmentalization of LTCCs and TRPC channels in caveolae membrane microdomains along with pathological hypertrophy signaling effectors makes for an attractive explanation for how Ca2+-dependent signaling pathways are regulated under conditions of continual Ca2+ transients that mediate cardiac contraction during each heart beat. Elucidation of additional Ca2+ signaling microdomains in adult cardiac myocytes will be important in more comprehensively resolving how myocytes differentiate between signaling versus contractile Ca2+. / Molecular and Cellular Physiology

Cell therapy is a novel potential approach to treat many diseases including diabetes. Embryonic stem cells have been examined in various diabetic and non-diabetic heart studies. However, the role of pancreas transcription factor 1 alpha (ptfla) over expressing embryonic stem (ES) cells has not been defined. We hypothesize that transplanted over expressing ptfla-ES cells in streptozotocin (STZ) induced diabetic mice will attenuate cardiac hypertrophy, fibrosis, and improve cardiac function. In this investigation we divided C57/bl6 mice into three groups: Control, STZ, and STZ + ptflaES cells. Diabetes was induced with STZ (lO0mg/kg, body weight), with two separate injections on day 1 (D1) and D2. Following STZ injections, mice were transplanted with 1.2 million ptfla-ES cells in three days. Control group received normal saline. After injections, animals were examined for glucose levels, cardiac hypertrophy, fibrosis, and heart function. Our data shows that glucose levels were significantly increased following STZ injections, suggesting diabetes, and this increase was reversed with transplanted ptfl a-ES cell. Our H&E qualitative data suggest that there was increase in cardiac hypertrophy in STZ-induced diabetic animals compared with control. Moreover, Massan's trichrome staining shows increased amount of cardiac fibrosis in STZ-induced diabetic animals compared with control. This data suggests that animals have developed diabetic cardiomyopathy. Interestingly, the increased cardiac hypertrophy and fibrosis was attenuated in the animals transplanted with ptfl a-ES cells. Furthermore, cardiac function examined by echocardiography was reduced in the STZ treated animals which was reversed following ptfla-ES cell treatment. In conclusion, our data suggests that