Cardiovascular
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21753
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Browsing Cardiovascular by Author "Fudala, Rafal"
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Item 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(2014-03) Nagwekar, Janhavi; Duggal, Divya; Midde, Krishna; Kazmierczak, Katarzyna; Huang, Weiwen; Fudala, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Szczesna-Cordary, Danuta; Borejdo, JulianThe 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.Item THE K104E MUTATION OF THE MYOSIN REGULATORY LIGHT CHAIN ALTERS KINETICS AND DISTRIBUTION OF ORIENTATIONS OF CROSS-BRIDGES IN TRANSGENIC CARDIAC MYOFIBRILS(2014-03) Duggal, Divya; Nagwekar, Janhavi; Rich, Ryan; Huang, W.; Midde, Krishna; Fudala, Rafal; Gryczynski, Ignacy; Szczesna-Cordary, Danuta; Borejdo, JulianMy project aims at finding out the differences in a healthy and diseased heart. The disease I am focusing on is Familial Hypertrophic Cardiomyopathy(FHC), in particular, the K104E mutation, which is a mutation in the Regulatory Light Chain(RLC) of Myosin. The differences are established in terms of the rate of association and dissociation of myosin from actin, as well as the order of cross bridges. This study will help us to investigate the role of such mutations in causing FHC as well as offer us the opportunity to investigate other mutations causing this disease. Purpose (a): The purpose of my study is to examine the cross-bridge (XB) kinetics and the degree of order in contracting myofibrils from the ex-vivo left ventricles of transgenic (Tg) mice expressing Familial Hypertrophic Cardiomyopathy (FHC) Regulatory Light Chain (RLC) mutation K104E. Methods (b): 1. Myofibrils were prepared from the frozen hearts of Tg-WT mice and newly generated Tg-K104E mice. 2. Since the kinetics and degree of order are best studied when an individual cross bridge (XB) makes a significant contribution to the overall signal, the number of observed XBs was minimized to ~20 by sparsely labeling the Essential Light Chain(ELC) of myosin. Autofluorescence and photobleaching were minimized by labeling ELC with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule, SeTau 647. 3. Myofibrils were crosslinked with a cross linker prior to labeling. 4. Following labeling, fluorescence was measured by PicoQuant MT 200 inverse time-resolved fluorescence instrument coupled to Olympus IX 71 microscope. Results (c): We show that the K104E mutation, when compared with Wild Type (WT) ventricles, had significant effect on both the kinetics of the interaction between actin and myosin and on the degree of order of the myosin lever arm. In particular, the K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke, while decreasing the rate of XB dissociation from actin. Mutated XBs were significantly better ordered during steady-state contraction and during rigor but mutation had no effect on the degree of order in relaxed myofibrils. Conclusions (d): This implies that the mutated ventricle may be prone to decreased maximal tension and increased muscle relaxation time suggesting a potential for diastolic dysfunction in patients.