MYOSIN REGULATORY LIGHT CHAIN A13T MUTATION ASSOCIATED WITH CARDIAC HYPERTROPHY IMPOSES DIFFERENCES ON KINETICS AND SPATIAL DISTRIBUTIONS OF CROSS-BRIDGES IN HEALTHY AND DISEASED VENTRICLES

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dc.creatorNagwekar, Janhavi
dc.creatorDuggal, Divya
dc.creatorMidde, Krishna
dc.creatorKazmierczak, Katarzyna
dc.creatorHuang, Weiwen
dc.creatorFudala, Rafal
dc.creatorGryczynski, Ignacy
dc.creatorGryczynski, Zygmunt
dc.creatorSzczesna-Cordary, Danuta
dc.creatorBorejdo, Julian
dc.date.accessioned2019-08-22T19:47:01Z
dc.date.available2019-08-22T19:47:01Z
dc.date.issued2014-03
dc.date.submitted2014-02-03T12:10:25-08:00
dc.descriptionResearch Appreciation Day Award Winner - 2014 Cardiovascular Research Institute - 2nd Place Graduate Student
dc.description.abstractThe study is performed at protein and myofibrilar level to identify key sub-steps of ATP induced XB cycle deregulation in FHC. Humans being heterozygous (50%) for FHC, our experiments with 10% penetrance in the mice will analyze subtle changes in the XB mechanisms to which humans have 40% more chances to develop those defects. This project may also provide insights into other RLC mutations (e.g. R58Q, E22K, D166V, P95A) that may involve the same alterations. The project aims to identify drugs to alter specific rate constants, affect ordering of XBs or reverse A13T effect and thus treating patients with personalized therapy. Purpose (a): Muscle is organized into regular periodic thick myosin and thin actin filaments. Myosin tails interact with each other to form a tight coiled coil rod, and the heads protrude out to interact with actin. Myosin head referred to as cross-bridge has ATPase activity and actin binding domain. The tail has a site (Regulatory Light Chain (RLC) domain) which when mutated at A13T site cause myosin heads to bind slowly to the actin molecules affecting the overall ATPase cycle and power strokes necessary for a muscle to contract in the process. Methods (b): Rabbit ventricle muscle is the source of sample for experiments in this project. Glycerinated muscle bundles were homogenized and myofibrils were extracted. Myofibrils were labeled with 0.1 nM rhodamine-phalloidin (RP) + 10 μM unlabeled-phalloidin (UP) in Ca2+-rigor solution in the the ratio of 1:100,000 fluorescent to non-fluorescent phalloidin to ensure 1 in ~105 actin monomers carry a fluorophore. Labeled myofibrils were analyzed for error of the mean of polarized fluorescence to determine kinetic rate constants in the ATPase cycle and distribution of orientations emanating from myosin cross-bridges. Results (c): Histograms were plotted from the polarized fluorescence data and the Full Width at Half Maximum (FWHM) of the mean was calculated. The mean polarization of a contracting WT myofibril was -0.176±0.018 and that of contracting A13T Mutated myofibril was -0.247±0.017. Significant differences in rate constants k1, k2 and k3 of the ATPase cycle were observed with WT values being 325±34, 0.16±0.03, 0.32±0.08 and A13T mutated values being 54±80*, 0.25±0.04, 0.57±0.12 respectively. On comparing the peaks of the fit of the data, peak 1 assumed to be the pre power stroke was lost in the A13T mutated myofibrils while peak 2 (post power stroke) almost remained constant in both muscle types. Conclusions (d): The study suggest that the functional differences between ventricles containing WT myosin and myosin in which the RLC contains the A13T mutation are caused by a change in the rate of binding of myosin cross-bridges to the thin filaments. Differences in the polarization, FWHM and peaks indicate that pre-power strokes are necessary for myosin cross-bridges and that any alterations in its functions may lead to cardiomyopathy.
dc.identifier.urihttps://hdl.handle.net/20.500.12503/26976
dc.language.isoen
dc.subjectCardiac hypertrophy
dc.subjectRegulatory Light Chain and Kinetic Study
dc.titleMYOSIN REGULATORY LIGHT CHAIN A13T MUTATION ASSOCIATED WITH CARDIAC HYPERTROPHY IMPOSES DIFFERENCES ON KINETICS AND SPATIAL DISTRIBUTIONS OF CROSS-BRIDGES IN HEALTHY AND DISEASED VENTRICLES
dc.typeposter
dc.type.materialtext

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