Browsing by Subject "hypertrophy"
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(2023)Heart failure is a global health issue that can result from various factors, one of which is myocardial infarction. The adult human heart has limited regenerative capacity to cover the loss of cardiomyocytes after myocardial infarction with new cardiomyocytes. The main responses to the loss of cardiomyocytes are fibrotic scar formation and the hypertrophy of remaining cardiomyocytes. Prolonged hypertrophy eventually leads to heart failure. Current treatments for heart failure only relieve the symptoms. Inducing cardiac regeneration could be one possible way to prevent and treat heart failure. Thus, to develop medical treatments that enhance the regenerative capacity, a comprehensive understanding of precise cellular mechanisms behind heart regeneration is crucial. The objective of this study was to establish a high-content analysis method for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) utilizing the Cell Painting assay to identify and categorize morphological alterations induced by various compounds in hiPSC-CMs. To evaluate the morphological impacts, dozens or even hundreds of cell features were measured at the same time. hiPSC-CMs were exposed to two hypertrophy inducers, endothelin-1 and angiotensin II, and to doxorubicin, which is known to be a cardiotoxic compound. In addition, the effects of a GATA4- targeting compound, C-2021, on hiPSC-CMs were examined. C-2021, was expected to have antihypertrophic effect on the cells. Previously used methods, proBNP staining and qPCR, were used to validate the novel method. According to proBNP staining and qPCR, endothelin-1 induced cardiomyocyte hypertrophy greater than angiotensin II. Compound C-2021 did not show statistically significant antihypertrophic properties after hypertrophic stimuli, but some tendency the alleviate the hypertrophy was noticed. Moreover, by utilizing different data processing programs a novel analysis method was developed. With this method, the different treatment groups were clustered based on the morphological alterations caused by compounds exposures. The hiPSC-CMs exposed to endothelin-1, angiotensin II or doxorubicin showed a different morphological profile compared to the control group hiPSC-CMs. Compound C-2021 was also observed to affect cell morphology. However, the data analysis still needs improvements in order to detect which cell features these compounds affect.
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(2021)Respirometry is a polarographic method that provides insights into mitochondrial respiratory capacity – specifically to electron transport chain (ETC) complexes I to V –, mitochondrial integrity and energy metabolism. The limitation of the respiratory measurements has been that it requires freshly isolated mitochondria or tissue sample. Long-term preservation of mitochondrial function in frozen samples has been a considerable challenge, since the membrane integrity of the mitochondria is lost during the freezing process. Thus, samples do not display coupled respiration. However, previous studies have found that despite coupled respiration is impaired the individual ETC complexes and the ability of ETC supercomplexes to consume oxygen are not destroyed due to freezing and thawing. On the basis of this knowledge, recently published article presented a novel protocol that overcomes the damages caused by freeze-thaw cycles. The protocol also enables respiration measurement of ETC complexes I-IV by using Seahorse XF96 Extracellular flux analyzer. In this MSc thesis I modified and optimized the aforementioned protocol for Oroboros O2k high- resolution respirometry using frozen skeletal muscle samples. In addition, this study provides an optimized sample preparation protocol for frozen muscle samples and respiration measurement. The new method broadens the possibilities within mitochondrial respiration studies since Oroboros O2k high-resolution respirometry records results with high sensitivity without limiting the number of substrates used. The possibility to use frozen samples reduces research costs, simplifies logistics and enables retrospective studies with previously stored frozen tissue samples. I also utilized the optimized respiration measurement protocol to study metabolic effects of combined gene therapy in skeletal muscle. This gene therapy mimics the positive effects of exercise by inducing skeletal muscle growth and angiogenesis. The mimicking effect was induced by systemic delivery of adeno-associated viral vectors encoding pro-myostatin and VEGF-B. In previous studies inhibition of myostatin has been connected to compromised oxidative capacity and vascular rarefaction. In contrast, VEGF-B has demonstrated to induce angiogenesis in several tissues. Thus, my hypothesis was that combination gene therapy would result in better mitochondrial function than pro-myostatin alone. Results from this study indicate that moderate inhibition of myostatin signaling by pro-myostatin using rAAV vectors could provide enhancements in ETC function when it is induced independently or combined with rAAV-VEGF-B. This result lays a solid foundation for future research and could provide a new therapeutic option against muscle loss and related metabolic diseases.
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