The conformational changes of a calcium transport ATPase were investigated with molecular dynamics (MD) simulations as well as fluorescence resonance energy transfer (FRET) measurements to determine the significance of a discrete structural element for regulation of the conformational dynamics of the transport cycle. Previous MD simulations indicated that a loop in the cytosolic domain of the SERCA calcium transporter facilitates an open-to-closed structural transition. To investigate the significance of this structural element, we performed additional MD simulations and new biophysical measurements of SERCA structure and function. Rationally designed mutations of three acidic residues of the loop decreased SERCA domain-domain contacts and increased domain-domain separation distances. Principal component analysis of MD simulations suggested decreased sampling of compact conformations upon N-loop mutagenesis. Deficits in headpiece structural dynamics were also detected by measuring intramolecular FRET of a Cer-YFP-SERCA construct (2-color SERCA). Compared with WT, the mutated 2-color SERCA shows a partial FRET response to calcium, whereas retaining full responsiveness to the inhibitor thapsigargin. Functional measurements showed that the mutated transporter still hydrolyzes ATP and transports calcium, but that maximal enzyme activity is reduced while maintaining similar calcium affinity. In live cells, calcium elevations resulted in concomitant FRET changes as the population of WT 2-color SERCA molecules redistributed among intermediates of the transport cycle. Our results provide novel insights on how the population of SERCA pumps responds to dynamic changes in intracellular calcium.
The type 2a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a key role in Ca2+ regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca2+ sensor R-CEPIA1er was used to measure the concentration of Ca2+ in the lumen of the endoplasmic reticulum (ER) ([Ca2+]ER) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca2+ release channel ryanodine receptor (RyR2) created a Ca2+ release/reuptake system that mimicked aspects of cardiac myocyte Ca2+ handling. SERCA2a function was quantified from the rate of [Ca2+]ER refilling after ER Ca2+ depletion; then, ER Ca2+ leak was measured after SERCA inhibition. ER Ca2+ uptake and leak were analyzed as a function of [Ca2+]ER to determine maximum ER Ca2+ uptake rate and maximum ER Ca2+ load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/[ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function. NEW & NOTEWORTHY The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) function in the cellular environment. The newly developed Ca2+ sensor R-CEPIA1er was used to successfully analyze Ca2+ uptake mediated by recombinant and native cardiac SERCA. These results demonstrate that this new approach can be used as a powerful tool to study new mechanisms of Ca2+ pump regulation.
Background and Aims High blood pressure and reduced aortic compliance are associated with increased atherosclerotic plaque accumulation in humans. Animal studies support these associations, but additional factors, such as fragmented elastic fibers, are present in most previous animal studies. Elastin heterozygous (Eln+/−) mice have high blood pressure and reduced aortic compliance, with no evidence of elastic fiber fragmentation and represent an appropriate model to directly investigate the effects of these factors on atherosclerosis Methods and Results Eln+/− and Eln+/+ mice were crossed with low density lipoprotein receptor knockout (Ldlr−/−) and wild-type (Ldlr+/+) mice and fed normal or Western diet (WD) for 16 weeks. We hypothesized that on WD, Eln+/−Ldlr−/− mice with high blood pressure and reduced aortic compliance would have increased atherosclerotic plaque accumulation compared to Eln+/+Ldlr−/− mice. We measured serum cholesterol and cytokine levels, blood pressure, aortic compliance, and plaque accumulation. Contrary to our hypothesis, we found that on WD, Eln+/−Ldlr−/− mice do not have increased plaque accumulation compared to Eln+/+Ldlr−/− mice. At the aortic root, there are no significant differences in plaque area between Eln+/−Ldlr−/− and Eln+/+Ldlr−/− mice on WD (p = .89), while in the ascending aorta, Eln+/−Ldlr−/− mice on WD have 29% less normalized plaque area than Eln+/+Ldlr−/− mice on WD (p = 0.009). Conclusion Using an atherogenic mouse model, we conclude that increased blood pressure and reduced aortic compliance are not direct causes of increased aortic plaque accumulation. We propose that additional insults, such as fragmentation of elastic fibers, are necessary to alter plaque accumulation.
Numerous diseases have been linked to genetic mutations that lead to reduced amounts or disorganization of arterial elastic fibres. Previous work has shown that mice with reduced amounts of elastin (Elnþ/2) are able to live a normal lifespan through cardiovascular adaptations, including changes in haemodynamic stresses, arterial geometry and arterial wall mechanics. It is not known if the timeline and presence of these adaptations are consistent in other mouse models of elastic fibre disease, such as those caused by the absence of fibulin-5 expression (Fbln52/2). Adult Fbln52/2 mice have disorganized elastic fibres, decreased arterial compliance and high blood pressure. We examined mechanical behaviour of the aorta in Fbln52/2 mice through early maturation when the elastic fibres are being assembled. We found that the physiologic circumferential stretch, stress and modulus of Fbln52/2 aorta are maintained near wild-type levels. Constitutive modelling suggests that elastin contributions to the total stress are decreased, whereas collagen contributions are increased. Understanding how collagen fibre structure and mechanics compensate for defective elastic fibres to meet the mechanical requirements of the maturing aorta may help to better understand arterial remodelling in human elastinopathies.
Rationale & Objective Persons with end-stage kidney disease receiving in-center maintenance hemodialysis may be at high risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure and severe outcomes with coronavirus disease 2019 (COVID-19). The objective of this study was to examine the correlation of SARS-CoV-2 positivity rate per capita and COVID-19–associated deaths with number of dialysis stations and demographics of residents within zip codes in Cook County, IL. Study Design Ecological analysis. Setting & Participants Data for SARS-CoV-2 test results and COVID-19–associated deaths during January 21 to June 15, 2020, among the 5,232,412 residents living within the 163 zip codes in Cook County, IL, were merged with demographic and income data from the US Census Bureau. The total number of positive test results in this population was 84,353 and total number of deaths was 4,007. Assessments Number of dialysis stations and stations per capita within a zip code were calculated. SARS-CoV-2–positive test results per capita were calculated as number of positive test results divided by the zip code population. COVID-19–associated deaths per capita were calculated as COVID-19 deaths among residents for a given zip code divided by the zip code population. Analytic Approach Spearman rank correlation coefficients were calculated to examine the correlation of SARS-CoV-2–positive tests per capita and COVID-19–associated deaths per capita with dialysis stations, demographics, and household poverty. To account for multiple testing, statistical significance was considered as P < 0.005. Results Among the 163 Cook County zip codes, there were 2,501 dialysis stations. Positive test results per capita were significantly associated with number of dialysis stations ( r = 0.25; 95% CI, 0.19 to 0.29; P < 0.005) but not with dialysis stations per capita ( r = 0.02; 95% CI, −0.03 to 0.08; P = 0.7). Positive test results per capita also correlated significantly with number of households living in poverty ( r = 0.57; 95% CI, 0.53-0.6; P < 0.005) and percentage of residents reporting Black race ( r = 0.28; 95% CI, 0.23-0.33; P < 0.005) and Hispanic ethnicity ( r = 0.68; 95% CI, 0.65-0.7; P < 0.001;). COVID-19–associated deaths per capita correlated significantly with the percentage of residents reporting Black race ( r = 0.24; 95% CI, 0.19-0.29; P < 0.005) and with percentage of households living in poverty ( r = 0.34; 95% CI, 0.29-0.38; P < 0.005). The association...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.