ABSTRACT
INTRODUCTION: To accurately assess evidence from environmental and public health field trials, context and implementation details of the intervention must be weighed with trial results; yet these details are under and inconsistently reported for water, sanitation, and hygiene (WASH), limiting the external validity of the evidence. METHODS: To quantify the level of reporting of context and implementation in WASH evaluations, we conducted a scoping review of the 40 most cited evaluations of WASH interventions published in the last 10 years (2012-2022). We applied criteria derived from a review of existing reporting guidance from other sectors including healthcare and implementation science. We subsequently reviewed main articles, supplements, protocols, and other associated resources to assess thoroughness of context and implementation reporting. RESULTS: Of the final 25 reporting items we searched for, four-intervention name, approach, location, and temporality-were reported by all studies. Five items-theory, implementer qualifications, dose intensity, targeting, and measured fidelity-were not reported in over a third of reviewed articles. Only two studies (5%) reported all items in our checklist. Only 74% of items were found in the main article, while the rest were found in separate papers (7%) or not at all (19%). DISCUSSION: Inconsistent reporting of WASH implementation illustrates a major challenge in the sector. It is difficult to know what interventions are actually being evaluated and how to compare evaluation results. This inconsistent and incomplete implementation reporting limits the ability of programmers and policy makers to apply the available evidence to their contexts. Standardized reporting guidelines would improve the application of the evidence for WASH field evaluations.
Subject(s)
Hygiene , Sanitation , Hygiene/standards , Sanitation/standards , Sanitation/methods , Water Supply/standardsABSTRACT
INTRODUCTION: Despite evidence for the efficacy and effectiveness of hand hygiene in reducing the transmission of infectious diseases, there are gaps in global normative guidance around hand hygiene in community settings. The goal of this review is to systematically retrieve and synthesise available evidence on hand hygiene in community settings across four areas: (1) effective hand hygiene; (2) minimum requirements; (3) behaviour change and (4) government measures. METHODS AND ANALYSIS: This protocol entails a two-phased approach to identify relevant studies for multiple related systematic reviews. Phase 1 involves a broad search to capture all studies on hand hygiene in community settings. Databases, trial registries, expert consultations and hand searches of reference lists will be used to ensure an exhaustive search. A comprehensive, electronic search strategy will be used to identify studies indexed in PubMed, Web of Science, EMBASE, CINAHL, Global Health, Cochrane Library, Global Index Medicus, Scopus, PAIS Index, WHO IRIS, UN Digital Library and World Bank eLibrary published in English from January 1980 to March 2023. The outcome of phase 1 will be a reduced sample of studies from which further screening, specific to research questions across the four key areas can be performed. Two reviewers will independently assess each study for inclusion and disagreements will be resolved by a third reviewer. Quantitative and qualitative data will be extracted following best practices. We will assess all studies using the Mixed Method Appraisal Tool. All effect measures pertaining to review outcomes will be reported and a narrative synthesis of all studies will be presented including 'data-driven' descriptive themes and 'theory-driven' analytical themes as applicable. ETHICS AND DISSEMINATION: This systematic review is exempt from ethics approval because the work is carried out on published documents. The findings of the reviews will be disseminated in related peer-reviewed journals. PROSPERO REGISTRATION NUMBER: CRD42023429145.
Subject(s)
Hand Hygiene , Humans , Research Design , Systematic Reviews as TopicABSTRACT
Many water, sanitation, and hygiene (WASH) interventions target improvements in personal hygiene behaviors. Yet measuring personal hygiene behaviors is a challenge due to a lack of reliable, valid, objective, and simple-to-use approaches. The purpose of this study was to examine differences between two types of hygiene outcome measures and their ability to detect relationships between WASH-related behavioral factors and behaviors. We compared hygiene outcomes generated by the Quantitative Personal Hygiene Assessment Tool (qPHAT), which yields objective measures of cleanliness on an 11-point scale, and those generated by conventional, dichotomous indicators of cleanliness. We used cross-sectional data on hygiene outcomes related to facial and hand cleanliness collected during the Andilaye Trial, an impact evaluation of a community-based WASH intervention implemented in Amhara, Ethiopia. We fit multivariable models to examine associations between measures of children's facial and hand cleanliness, via both qPHAT and dichotomous indicators, and 1) household WASH conditions, 2) psychosocial factors, and 3) reported personal hygiene practices. The qPHAT-generated outcomes were able to detect relationships between intermediate behavioral factors and hygiene outcomes that dichotomous indicators were not, including associations with water insecurity and various psychosocial factors. qPHAT-generated outcomes were negatively associated with reported face washing practices, suggesting a bias in reported behaviors. Our study highlights the limitations of reported practices and dichotomous hygiene indicators and indicates that using more quantitative hygiene outcome measures, such as those generated by qPHAT, may reveal important intermediate factors that influence hygiene behavior and support improved monitoring and evaluation of interventions.
Subject(s)
Health Behavior , Hygiene , Child , Humans , Ethiopia , Cross-Sectional Studies , Sanitation , Water , Water SupplyABSTRACT
INTRODUCTION: Despite clear linkages between provision of clean water and improvements in child health, limited information exists about the health impacts of large water infrastructure improvements in low-income settings. Billions of dollars are spent annually to improve urban water supply, and rigorous evaluation of these improvements, especially targeting informal settlements, is critical to guide policy and investment strategies. Objective measures of infection and exposure to pathogens, and measures of gut function, are needed to understand the effectiveness and impact of water supply improvements. METHODS AND ANALYSIS: In the PAASIM study, we examine the impact of water system improvements on acute and chronic health outcomes in children in a low-income urban area of Beira, Mozambique, comprising 62 sub-neighbourhoods and ~26 300 households. This prospective matched cohort study follows 548 mother-child dyads from late pregnancy through 12 months of age. Primary outcomes include measures of enteric pathogen infections, gut microbiome composition and source drinking water microbiological quality, measured at the child's 12-month visit. Additional outcomes include diarrhoea prevalence, child growth, previous enteric pathogen exposure, child mortality and various measures of water access and quality. Our analyses will compare (1) subjects living in sub-neighbourhoods with the improved water to those living in sub-neighbourhoods without these improvements; and (2) subjects with household water connections on their premises to those without such a connection. This study will provide critical information to understand how to optimise investments for improving child health, filling the information gap about the impact of piped water provision to low-income urban households, using novel gastrointestinal disease outcomes. ETHICS AND DISSEMINATION: This study was approved by the Emory University Institutional Review Board and the National Bio-Ethics Committee for Health in Mozambique. The pre-analysis plan is published on the Open Science Framework platform (https://osf.io/4rkn6/). Results will be shared with relevant stakeholders locally, and through publications.
Subject(s)
Gastrointestinal Microbiome , Child , Female , Infant , Humans , Pregnancy , Mozambique , Cohort Studies , Prospective Studies , Water Supply , Ethics Committees, ResearchABSTRACT
Introduction: Despite clear linkages between provision of clean water and improvements in child health, limited information exists about the health impacts of large water infrastructure improvements in low-income settings. Billions of dollars are spent annually to improve urban water supply, and rigorous evaluation of these improvements, especially targeting informal settlements, is critical to guide policy and investment strategies. Objective measures of infection and exposure to pathogens, and measures of gut function, are needed to understand the effectiveness and impact of water supply improvements. Methods and analysis: In the PAASIM study, we examine the impact of water system improvements on acute and chronic health outcomes in children in a low-income urban area of Beira, Mozambique, comprising 62 sub-neighbourhoods and ~26 300 households. This prospective matched cohort study follows 548 mother-child dyads from late pregnancy through 12 months of age. Primary outcomes include measures of enteric pathogen infections, gut microbiome composition and source drinking water microbiological quality, measured at the child's 12-month visit. Additional outcomes include diarrhoea prevalence, child growth, previous enteric pathogen exposure, child mortality and various measures of water access and quality. Our analyses will compare (1) subjects living in sub-neighbourhoods with the improved water to those living in sub-neighbourhoods without these improvements; and (2) subjects with household water connections on their premises to those without such a connection. This study will provide critical information to understand how to optimise investments for improving child health, filling the information gap about the impact of piped water provision to low-income urban households, using novel gastrointestinal disease outcomes. Ethics and dissemination: This study was approved by the Emory University Institutional Review Board and the National Bio-Ethics Committee for Health in Mozambique. The pre-analysis plan is published on the Open Science Framework platform (https://osf.io/4rkn6/). Results will be shared with relevant stakeholders locally, and through publications.
Subject(s)
Humans , Pregnancy , Infant , Water Supply/methods , Pregnancy , Waterborne Diseases/transmission , Infant , Humans , Prospective Studies , Cohort Studies , Ethics Committees, Research , Gastrointestinal Microbiome , MozambiqueABSTRACT
Rapid urbanization, resulting in population growth within informal settlements, has worsened exclusion and inequality in access to water and sanitation (WASH) services in the poorest and most marginalized communities. In this study, we describe the heterogeneity in water service satisfaction and WASH access in low-income, peri-urban neighborhoods of Beira, Mozambique, and examine whether this heterogeneity can be explained by distance to water distribution mains. Using spatial statistics and regression analyses, we identify spatial heterogeneity in household WASH access, as well as consumer-reported satisfaction with water services (services, pressure, quality, and sufficient quantity). We find that as distance from the water main increased, both access to an improved water source at the household and satisfaction with water pressure decreases, and water supply intermittency increases, controlling for household density and socioeconomic status. The odds of a household having access to a water source at the household or on the compound decreases with every 100-meter increase in distance from a water main pipe (odds ratio [OR] 0.87, 95% confidence interval [CI]: 0.82, 0.92). Satisfaction with water services also decreases with every 100-meter increase in distance from a water main pipe (OR: 0.80; 95% CI: 0.69, 0.94). Days of availability in the past week decreases by a factor of 0.22 for every 100-meter increase in distance from the water main (95% CI: -0.29, -0.15). Findings from this study highlight the unequal household access to water and sanitation in urban informal settlements, even within low-income neighborhoods. Describing this heterogeneity of access to water services, sanitation, and satisfaction-and the factors influencing them-can inform stakeholders and guide the development of infrastructural solutions to reduce water access inequities within urban settings.
ABSTRACT
Behaviors related to water, sanitation, and hygiene (WASH) are key drivers of infectious disease transmission, and experiences of WASH are potential influencers of mental well-being. Important knowledge gaps exist related to the content and delivery of effective WASH programs and their associated health impacts, particularly within the contexts of government programs implemented at scale. We developed and tested a demand-side intervention called Andilaye, which aimed to change behaviors related to sanitation, personal hygiene, and household environmental sanitation. This theory-informed intervention was delivered through the existing Ethiopian Health Extension Programme (HEP). It was a multilevel intervention with a catalyzing event at the community level and behavior change activities at group and household levels. We randomly selected and assigned 50 kebeles (sub-districts) from three woredas (districts), half to receive the Andilaye intervention, and half the standard of care sanitation and hygiene programming (i.e., community-led total sanitation and hygiene [CLTSH]). We collected data on WASH access, behavioral outcomes, and mental well-being. A total of 1,589 households were enrolled into the study at baseline; 1,472 households (94%) participated in an endline assessment two years after baseline, and approximately 14 months after the initiation of a multi-level intervention. The intervention did not improve construction of latrines (prevalence ratio [PR]: 0.99; 95% CI: 0.82, 1.21) or handwashing stations with water (PR: 0.96; 95% CI: 0.72, 1.26), or the removal of animal feces from the compound (PR: 1.10; 95% CI: 0.95, 1.28). Nor did it impact anxiety (PR: 0.90; 95% CI: 0.72, 1.11), depression (PR: 0.83; 95% CI: 0.64, 1.07), emotional distress (PR: 0.86; 95% CI: 0.67, 1.09) or well-being (PR: 0.90; 95% CI: 0.74, 1.10) scores. We report limited impact of the intervention, as delivered, on changes in behavior and mental well-being. The effectiveness of the intervention was limited by poor intervention fidelity. While sanitation and hygiene improvements have been documented in Ethiopia, behavioral slippage, or regression to unimproved practices, in communities previously declared open defecation free is widespread. Evidence from this trial may help address knowledge gaps related to challenges associated with scalable alternatives to CLTSH and inform sanitation and hygiene programming and policy in Ethiopia and beyond. Trial registration: This trial was registered with clinicaltrials.gov (NCT03075436) on March 9, 2017.
ABSTRACT
BACKGROUND: Few countries are likely to achieve universal sanitation within the next decade as sustaining household sanitation coverage remains a critical challenge. This study aimed to investigate factors that may have supported or hindered sustainability of sanitation coverage 1-2 years after the completion of an integrated, area-wide sanitation program in 4 countries. METHODS: We conducted qualitative analyses to identify factors related to the sustainability of sanitation coverage in Bhutan, Kenya, Nepal, and Zambia, 2 years after completion of the Sustainable Sanitation and Hygiene for All program. From November 2019 to March 2020, we conducted focus group discussions and key informant interviews with community members, project implementers, and decision makers. We triangulated the qualitative findings with data from household surveys to characterize subnational sanitation coverage throughout implementation and 1-2 years after. RESULTS: Our data revealed behavioral, contextual, and service delivery factors that were related to the sustainability of sanitation improvements. Service delivery factors included follow-up hygiene promotion, access to construction materials, local government commitment postimplementation, functioning monitoring systems, private sector uptake of supply chain improvements, and capacity for innovation. Contextual and behavioral factors included poverty, soil type, road networks, social cohesion, desire for improved latrines, maintenance and cleaning, and knowledge of sanitation benefits. CONCLUSION: The presence or absence of sustainability factors identified through this research may have implications on where certain programmatic approaches will work and where adaptations may be required. By comparing sustainability factors with subnational slippage rates, we were able to illustrate how local service delivery systems may respond to barriers and enablers. Understanding the programmatic and contextual factors that either drive or hinder long-term sanitation coverage may allow for greater program impact through adapting implementation based on existing challenges in service delivery and context.
Subject(s)
Sanitation , Humans , Kenya , Nepal , Zambia , BhutanABSTRACT
There are considerable challenges to achieving the Sustainable Development Goals' target of universal access to basic sanitation in schools. Schools require safe, clean, and sex-segregated facilities for a large number of students. Robust and affordable solutions are needed to address the economic, spatial, social, institutional, and political factors which contribute to poor sanitary conditions in informal settlements. In 2015, we undertook a randomized controlled trial to assess the feasibility of private sector sanitation delivery (PSSD) in 20 primary schools, in informal settlements of Nairobi, Kenya. Our preliminary evaluation after one year of service delivery suggested that PSSD of urine-diverting dry latrines with routine waste collection and maintenance provided a feasible, lower-cost alternative to the government standard delivery (GSD) of cistern-flush toilets or ventilated improved pit latrines. We conducted a mixed-methods follow-up study to assess sanitation delivery over 3-4 years and investigate prevailing drivers and barriers that may influence the scalability of PSSD. The conditions of newly constructed and rehabilitated GSD facilities diminished quickly, reverting to the conditions of existing facilities, indicating lower sustainability compared to sanitation delivered from the private sector. Barriers in financial aspects related to the ongoing implementation of PSSD emerged, particularly among public schools, and few were able to pay for continued service. Our study demonstrates that the engagement of the private sector may lead to improvements in affordable, safely managed sanitation for schools and their students. Yet, to reach a sustained scale, additional guidance is needed on how to develop these partnerships, streamline procurement and contracting processes, and incorporate appropriate financing mechanisms.
Subject(s)
Private Sector , Sanitation , Follow-Up Studies , Humans , Kenya , Toilet FacilitiesABSTRACT
Personal hygiene practices, including facewashing and handwashing, reduce transmission of pathogens, but are difficult to measure. Using color theory principles, we developed and tested a novel metric that generates quantitative measures of facial and hand cleanliness, proxy indicators of personal hygiene practices. In this cross-sectional study, conventional qualitative cleanliness metrics (e.g., presence or absence of nasal and ocular discharge, dirt under nails or on finger pads and palms) were also recorded. We generated Gwet's agreement coefficients to determine the inter-rater reliability of novel and conventional metrics between various rating groups, where appropriate, including two non-blinded raters, non-blinded vs. blinded raters, three blinded raters, and blinded vs. computer raters. Inter-rater reliability of the novel metric was high across all rating groups, ranging from 0.98 (95% CI: 0.97, 0.99) to 0.90 (95% CI: 0.90, 0.91) for facial cleanliness, and 0.97 (95% CI: 0.96, 0.98) to 0.92 (95% CI: 0.91, 0.93) for hand cleanliness. Our novel metric generates more nuanced data than conventional qualitative metrics, and allows for quantifiable assessments of facial and hand cleanliness.
Subject(s)
Health Education , Hygiene , Adult , Child , Child, Preschool , Cross-Sectional Studies , Face , Female , Hand , Health Behavior , Humans , Male , Observer Variation , Reproducibility of Results , Single-Blind MethodABSTRACT
BACKGROUND: Unimproved water, sanitation, and hygiene (WASH) behaviors are key drivers of infectious disease transmission and influencers of mental well-being. While WASH is seen as a critical enabler of health, important knowledge gaps related to the content and delivery of effective, holistic WASH programming exist. Corresponding impacts of WASH on mental well-being are also underexplored. There is a need for more robust implementation research that yields information regarding whether and how community-based, demand-side interventions facilitate progressive and sustained adoption of improved sanitation and hygiene behaviors and downstream health impacts. The purpose of this protocol is to detail the rationale and design of a cluster-randomized trial evaluating the impact of a demand-side sanitation and hygiene intervention on sustained behavior change and mental well-being in rural and peri-urban Amhara, Ethiopia. METHODS: Together with partners, we developed a theoretically-informed, evidence-based behavioral intervention called Andilaye. We randomly selected and assigned 50 sub-districts (kebeles) from three purposively selected districts (woredas); half to receive the Andilaye intervention, and half the standard of care sanitation and hygiene programming (i.e., community-led total sanitation and hygiene [CLTSH]). During baseline, midline, and endline, we will collect data on an array of behavioral factors, potential moderators (e.g., water and sanitation insecurity, collective efficacy), and our primary study outcomes: sanitation and hygiene behaviors and mental well-being. We will perform a process evaluation to assess intervention fidelity and related attributes. DISCUSSION: While CLTSH has fostered sanitation and hygiene improvements in Ethiopia, evidence of behavioral slippage, or regression to unimproved practices in communities previously declared open defecation free exists. Other limitations of CLTSH, such as its focus on disgust, poor triggering, and over-saturation of Health Extension Workers have been documented. We employed rigorous formative research and practically applied social and behavioral theory to develop Andilaye, a scalable intervention designed to address these issues and complement existing service delivery within Ethiopia's Health Extension Program. Evidence from this trial may help address knowledge gaps related to scalable alternatives to CLTSH and inform sanitation and hygiene programming and policy in Ethiopia and beyond. TRIAL REGISTRATION: This trial was registered with clinicaltrials.gov ( NCT03075436 ) on March 9, 2017.
Subject(s)
Health Behavior , Health Promotion/organization & administration , Hygiene/standards , Mental Health/statistics & numerical data , Rural Population , Sanitation/standards , Suburban Population , Adult , Child , Child, Preschool , Ethiopia , Female , Humans , Infant , Male , Program Evaluation , Research Design , Rural Population/statistics & numerical data , Suburban Population/statistics & numerical dataSubject(s)
Anthelmintics/therapeutic use , Communicable Disease Control/legislation & jurisprudence , Helminthiasis/prevention & control , Public Health Administration , Soil/parasitology , Adolescent , Adult , Anthelmintics/administration & dosage , Anthelmintics/economics , Child , Female , Humans , Neglected Diseases/parasitology , Neglected Diseases/prevention & control , Time Factors , World Health OrganizationABSTRACT
BACKGROUND: There is debate over the effectiveness of deworming children against soil-transmitted helminthiasis (STH) to improve health outcomes, and current evidence may be limited in study design and generalizability. However, programmatic deworming continues throughout low and middle-income countries. METHODOLOGY AND PRINCIPAL FINDINGS: We performed an empirical evaluation of the relationship between deworming in pre-school age children (ages 1-4 years) within the previous 6 months, as proxy-reported by the mother, and health outcomes of weight, height, and hemoglobin. We used nationally representative cross-sectional data from 45 countries using the Demographic and Health Surveys (DHS) during the period 2005-2016. We used logistic regression with coarsened exact matching, fixed effects for survey and year, and person-level covariates. We included data on 325,115 children in 45 STH-endemic countries from 66 DHS surveys. Globally in STH-endemic countries, children who received deworming treatment were less likely to be stunted (1.2 percentage point decline from mean of 36%; 95% CI [-1.9, -0.5%]; p<0.001), but we did not detect consistent associations between deworming and anemia or weight. In sub-Saharan Africa, we found that children who received deworming treatment were less likely to be stunted (1.1 percentage point decline from mean of 36%; 95% CI [-2.1, -0.2%]; p = 0.01) and less likely to have anemia (1.8 percentage point decline from mean of 58%; 95% CI [-2.8, -0.7%]; p<0.001), but we did not detect consistent associations between deworming and weight. These findings were robust across multiple statistical models, and we did not find consistently measurable associations with data from non-endemic settings. CONCLUSIONS AND SIGNIFICANCE: Among pre-school age children, we detected a robust and consistent association between deworming and reduced stunting, with additional evidence for reduced anemia in sub-Saharan Africa. We did not find a consistent relationship between deworming and improved weight. This global empirical analysis provides evidence to support the deworming of pre-school age children.
Subject(s)
Anthelmintics/administration & dosage , Helminthiasis/drug therapy , Patient Outcome Assessment , Anemia/epidemiology , Anthelmintics/adverse effects , Anthelmintics/therapeutic use , Body Weight/drug effects , Child, Preschool , Cross-Sectional Studies , Female , Helminthiasis/parasitology , Helminthiasis/transmission , Humans , Infant , Logistic Models , Male , Nutritional Status , Regression Analysis , Soil/parasitology , Weight Gain/drug effectsABSTRACT
BACKGROUND: Two-pore K(+) channels have emerged as potential targets to selectively regulate cardiac cell membrane excitability; however, lack of specific inhibitors and relevant animal models has impeded the effort to understand the role of 2-pore K(+) channels in the heart and their potential as a therapeutic target. The objective of this study was to determine the role of mechanosensitive 2-pore K(+) channel family member TREK-1 in control of cardiac excitability. METHODS AND RESULTS: Cardiac-specific TREK-1-deficient mice (αMHC-Kcnk(f/f)) were generated and found to have a prevalent sinoatrial phenotype characterized by bradycardia with frequent episodes of sinus pause following stress. Action potential measurements from isolated αMHC-Kcnk2(f/f) sinoatrial node cells demonstrated decreased background K(+) current and abnormal sinoatrial cell membrane excitability. To identify novel pathways for regulating TREK-1 activity and sinoatrial node excitability, mice expressing a truncated allele of the TREK-1-associated cytoskeletal protein ßIV-spectrin (qv(4J) mice) were analyzed and found to display defects in cell electrophysiology as well as loss of normal TREK-1 membrane localization. Finally, the ßIV-spectrin/TREK-1 complex was found to be downregulated in the right atrium from a canine model of sinoatrial node dysfunction and in human cardiac disease. CONCLUSIONS: These findings identify a TREK-1-dependent pathway essential for normal sinoatrial node cell excitability that serves as a potential target for selectively regulating sinoatrial node cell function.
Subject(s)
Potassium Channels, Tandem Pore Domain/physiology , Sinoatrial Node/physiology , Action Potentials/physiology , Animals , Echocardiography , Mice , Mice, Knockout , Potassium Channels, Tandem Pore Domain/deficiencyABSTRACT
AIMS: Cardiac function depends on the highly regulated and co-ordinate activity of a large ensemble of potassium channels that control myocyte repolarization. While voltage-gated K(+) channels have been well characterized in the heart, much less is known about regulation and/or targeting of two-pore K(+) channel (K(2P)) family members, despite their potential importance in modulation of heart function. METHODS AND RESULTS: Here, we report a novel molecular pathway for membrane targeting of TREK-1, a mechano-sensitive K(2P) channel regulated by environmental and physical factors including membrane stretch, pH, and polyunsaturated fatty acids (e.g. arachidonic acid). We demonstrate that ß(IV)-spectrin, an actin-associated protein, is co-localized with TREK-1 at the myocyte intercalated disc, associates with TREK-1 in the heart, and is required for TREK-1 membrane targeting. Mice expressing ß(IV)-spectrin lacking TREK-1 binding (qv(4J)) display aberrant TREK-1 membrane localization, decreased TREK-1 activity, delayed action potential repolarization, and arrhythmia without apparent defects in localization/function of other cardiac potassium channel subunits. Finally, we report abnormal ß(IV)-spectrin levels in human heart failure. CONCLUSIONS: These data provide new insight into membrane targeting of TREK-1 in the heart and establish a broader role for ß(IV)-spectrin in organizing functional membrane domains critical for normal heart function.
Subject(s)
Myocardium/metabolism , Nerve Tissue Proteins/metabolism , Potassium Channels, Tandem Pore Domain/metabolism , Spectrin/metabolism , Animals , Cell Membrane/metabolism , Mice , Myocardium/cytologyABSTRACT
AIMS: Recent studies suggest that proarrhythmic effects of cardiac glycosides (CGs) on cardiomyocyte Ca(2+) handling involve generation of reactive oxygen species (ROS). However, the specific pathway(s) of ROS production and the subsequent downstream molecular events that mediate CG-dependent arrhythmogenesis remain to be defined. METHODS AND RESULTS: We examined the effects of digitoxin (DGT) on Ca(2+) handling and ROS production in cardiomyocytes using a combination of pharmacological approaches and genetic mouse models. Myocytes isolated from mice deficient in NADPH oxidase type 2 (NOX2KO) and mice transgenically overexpressing mitochondrial superoxide dismutase displayed markedly increased tolerance to the proarrhythmic action of DGT as manifested by the inhibition of DGT-dependent ROS and spontaneous Ca(2+) waves (SCW). Additionally, DGT-induced mitochondrial membrane potential depolarization was abolished in NOX2KO cells. DGT-dependent ROS was suppressed by the inhibition of PI3K, PKC, and the mitochondrial KATP channel, suggesting roles for these proteins, respectively, in activation of NOX2 and in mitochondrial ROS generation. Western blot analysis revealed increased levels of oxidized CaMKII in WT but not in NOX2KO hearts treated with DGT. The DGT-induced increase in SCW frequency was abolished in myocytes isolated from mice in which the Ser 2814 CaMKII phosphorylation site on RyR2 is constitutively inactivated. CONCLUSION: These results suggest that the arrhythmogenic adverse effects of CGs on Ca(2+) handling involve PI3K- and PKC-mediated stimulation of NOX2 and subsequent NOX2-dependent ROS release from the mitochondria; mitochondria-derived ROS then activate CaMKII with consequent phosphorylation of RyR2 at Ser 2814.
Subject(s)
Anti-Arrhythmia Agents/toxicity , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Digitoxin/toxicity , Myocytes, Cardiac/drug effects , Reactive Oxygen Species/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Animals , Arrhythmias, Cardiac/chemically induced , Calcium/metabolism , Male , Membrane Potential, Mitochondrial , Mice , Mice, Knockout , NADPH Oxidases/metabolism , Phosphorylation , Superoxide Dismutase/metabolismABSTRACT
In this study, we report the preparation of a novel microcapsule of ~ 100 µm with a liquid (as compared to solid-like alginate hydrogel) core and an alginate-chitosan-alginate (ACA) shell for encapsulation and culture of embryonic stem (ES) cells in the miniaturized 3D space of the liquid core. Murine R1 ES cells cultured in the microcapsules were found to survive (> 90%) well and proliferate to form either a single aggregate of pluripotent cells or embryoid body (EB) of more differentiated cells in each microcapsule within 7 days, dependent on the culture medium used. This novel microcapsule technology allows massive production of the cell aggregates or EBs of uniform size and controllable pluripotency, which is important for the practical application of stem cell based therapy. Moreover, the semipermeable ACA shell was found to significantly reduce immunoglobulin G (IgG) binding to the encapsulated cells by up to 8.2 times, compared to non-encapsulated cardiac fibroblasts, mesenchymal stem cells, and ES cells. This reduction should minimize inflammatory and immune responses induced damage to the cells implanted in vivo becasue IgG binding is an important first step of the undesired host responses. Therefore, the ACA microcapsule with selective shell permeability should be of importance to advance the emerging cell-based medicine.
ABSTRACT
Kinase/phosphatase balance governs cardiac excitability in health and disease. Although detailed mechanisms for cardiac kinase regulation are established, far less is known regarding cardiac protein phosphatase 2A (PP2A) regulation. This is largely due to the complexity of the PP2A holoenzyme structure (combinatorial assembly of three subunit enzyme from >17 subunit genes) and the inability to segregate "global" PP2A function from the activities of multiple "local" holoenzyme populations. Here we report that PP2A catalytic, regulatory, and scaffolding subunits are tightly regulated at transcriptional, translational, and post-translational levels to tune myocyte function at base line and in disease. We show that past global read-outs of cellular PP2A activity more appropriately represent the collective activity of numerous individual PP2A holoenzymes, each displaying a specific subcellular localization (dictated by select PP2A regulatory subunits) as well as local specific post-translational catalytic subunit methylation and phosphorylation events that regulate local and rapid holoenzyme assembly/disassembly (via leucine carboxymethyltransferase 1/phosphatase methylesterase 1 (LCMT-1/PME-1). We report that PP2A subunits are selectively regulated between human and animal models, across cardiac chambers, and even within specific cardiac cell types. Moreover, this regulation can be rapidly tuned in response to cellular activation. Finally, we report that global PP2A is altered in human and experimental models of heart disease, yet each pathology displays its own distinct molecular signature though specific PP2A subunit modulatory events. These new data provide an initial view into the signaling pathways that govern PP2A function in heart but also establish the first step in defining specific PP2A regulatory targets in health and disease.
Subject(s)
Myocardium/enzymology , Protein Phosphatase 2/metabolism , Animals , Base Sequence , DNA Primers , Dogs , Humans , Immunoprecipitation , Mice , Polymerase Chain Reaction , Protein Biosynthesis , Protein Phosphatase 2/genetics , Signal Transduction , Transcription, GeneticABSTRACT
BACKGROUND: Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked to potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite more than a decade of investigation. Posttranslational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel posttranslational modifications and disease. We recently identified a novel pathway for posttranslational regulation of the primary cardiac voltage-gated Na(+) channel (Na(v)1.5) by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). However, a role for this pathway in cardiac disease has not been evaluated. METHODS AND RESULTS: We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Na(v)1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Na(v)1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that the human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Na(v)1.5, resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel, resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large-animal model of acquired heart disease and in failing human myocardium. CONCLUSIONS: We identify the mechanism for 2 human arrhythmia variants that affect Na(v)1.5 channel activity through direct effects on channel posttranslational modification. We propose that the CaMKII phosphorylation motif in the Na(v)1.5 DI-DII cytoplasmic loop is a critical nodal point for proarrhythmic changes to Na(v)1.5 in congenital and acquired cardiac disease.
Subject(s)
Arrhythmias, Cardiac/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/physiology , NAV1.5 Voltage-Gated Sodium Channel/metabolism , Animals , Arrhythmias, Cardiac/enzymology , Arrhythmias, Cardiac/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Cells, Cultured , Cytoplasm/enzymology , Cytoplasm/genetics , Cytoplasm/metabolism , Dogs , Genetic Variation , HEK293 Cells , Humans , Mice , NAV1.5 Voltage-Gated Sodium Channel/genetics , Phosphorylation , Protein Processing, Post-Translational/geneticsABSTRACT
Ankyrins (ankyrin-R, -B, and -G) are adapter proteins linked with defects in metazoan physiology. Ankyrin-B (encoded by ANK2) loss-of-function mutations are directly associated with human cardiovascular phenotypes including sinus node disease, atrial fibrillation, ventricular tachycardia, and sudden cardiac death. Despite the link between ankyrin-B dysfunction and monogenic disease, there are no data linking ankyrin-B regulation with common forms of human heart failure. Here, we report that ankyrin-B levels are altered in both ischemic and non-ischemic human heart failure. Mechanistically, we demonstrate that cardiac ankyrin-B levels are tightly regulated downstream of reactive oxygen species, intracellular calcium, and the calcium-dependent protease calpain, all hallmarks of human myocardial injury and heart failure. Surprisingly, ß(II)-spectrin, previously thought to mediate ankyrin-dependent modulation in the nervous system and heart, is not coordinately regulated with ankyrin-B or its downstream partners. Finally, our data implicate ankyrin-B expression as required for vertebrate myocardial protection as hearts deficient in ankyrin-B show increased cardiac damage and impaired function relative to wild-type mouse hearts following ischemia reperfusion. In summary, our findings provide the data of ankyrin-B regulation in human heart failure, provide insight into candidate pathways for ankyrin-B regulation in acquired human cardiovascular disease, and surprisingly, implicate ankyrin-B as a molecular component for cardioprotection following ischemia.