Thin filament regulatory protein targets and efficiency of dual gene transfer.

<p>(<b>A</b>) Schematic representation of the troponin subunits, troponin I (TnI), troponin T (TnT), and troponin C (TnC) and tropomyosin (Tm) the location of CM-linked mutations (gray circles) with in the secondary structure of troponin and tropomyosin. (<b>B</b>) Representative images (10X) of cultured adult rat cardiac myocytes co-transduced with the eGFP (200 MOI) and Lac Z genes (200 MOI) 3 days post gene transfer. Bottom panel is a 40X image of one myocyte expressing both eGFP and β-galactosidase. (<b>C</b>) Summary of the ratio of transduced to total myocytes in a field. Approximately 98% of myocytes are expressing both eGFP and β-galactosidase by day four in cultured myocytes co-transduced with adenovirus containing the eGFP and Lac Z genes. Data is presented as mean+SEM, n = 5 preparations.</p

Myocyte shortening and Ca²⁺ transient parameters.

<p>Wild type (WT) is the control group and consists of combined data from non-transduced, AdcTnI (WT), and AdcTnIFlag. A t-test indicated these data were not significantly different (P>0.32). Mechanical and Ca²⁺ transient data from each experimental and control group were collected simultaneous, thus the WT data represents the control group for each pairing. <i>1X4-way ANOVA</i> was used to statistically compare dual gene transfer pairings to single gene transfer and control groups. Values are represented as the mean ± SEM and Newman-Keuls post hoc comparisons defined as follows: (*) different from WT, (+) different from R193H cTnI, (#) different from A63V Tm or G159D cTnC, P<0.05, n = 19–64 myocytes derived from 5 different rat heart isolations. Shortening velocity is normalized to the peak shortening amplitude (Amp). Relaxation time is calculated from peak shortening to 50, 75, or 90% of relaxation. Key: BL =  baseline, Dep Vel  =  sarcomere length shortening velocity, RTP  =  relaxation time from peak, D Vel Ca²⁺  =  rate of rise of the Ca²⁺ transient, and DTP  =  Ca²⁺ transient decay time from peak.</p

Model of dual gene transfer incorporation of mutant Tm + cTnI and cTnC + cTnI combinations.

<p>(<b>A</b>) Native cTnI and αTm is shown in white. This model predicts that mutant Tm (green) has ordered incorporation starting at the pointed end and moving towards the Z-line, while the simultaneous transduction of mutant cTnI (red) will stochastically incorporate along the thin filament. As such double mutant regulatory units containing both mutant Tm and cTnI are located at the pointed end of the thin filament in this dual gene transfer approach. (<b>B</b>) Schematic depicting the predicted population of regulatory units that decorate the thin filament in this model system and in CM patients. Four populations of regulatory units consisting of wild type (WT), mutant cTnI alone, mutant Tm alone, and both mutant cTnI and TM (double mutant) are predicted to constitute the thin filament regulatory system as a whole. The relative proportion of units will be directly related to the percent replacement of native Tm and cTnI that in a patient will reach an even and random distribution overtime as the thin filaments turn-over. In our model system, TmA63V + cTnIR193H myocytes very few double mutant regulatory units are predicted as R193H mutant cTnI will be randomly distributed while only 10% should contain A63V Tm. (<b>C</b>) Native cTnI and cTnC are shown in white. This model predicts that like mutant cTnI (red), mutant cTnC (yellow) will have stochastic incorporation. Regulatory units containing both mutant cTnC and cTnI are randomly decorated along the entire length of the thin filament. The incorporation of mutant cTnC is predicted to follow cTnI. (<b>D</b>) Schematic depicting the predicted population of regulatory units that decorate the thin filament with this dual gene transfer approach. Four populations consisting of wild type (WT), mutant cTnI alone, mutant cTnC alone, and both mutant cTnI and cTnC (double mutant) regulatory units are predicted to constitute the thin filament regulatory system in our model and in CM patients. If mutant cTnC follows cTnI then the population of single mutant regulatory units should approach zero with double mutant and WT regulatory units being more prominent and in a 50∶50 distribution. The relative proportion of double mutant regulatory units is predicted to be directly related to the percent replacement of native cTnC and cTnI.</p

Intact single myocyte shortening from double activating mutations in αTm and cTnI.

<p>(<b>A</b>) Representative Western blot showing non-transduced (WT), R146G cTnI, and A63V Tm + R146G cTnI myocytes. Blots were probed with anti-Tm and anti-cTnI antibodies. (<b>B</b>) Sarcomere shortening transients from WT, A63V Tm, R146G cTnI, and A63V+R146G transduced myocytes. Traces were normalized to peak shortening to emphasize the mutant dependent slowing of relaxation. (<b>C</b>) Summary of 75% sarcomere relaxation time. Relaxation time was determined by calculating the difference from the time of peak shortening to 75% relaxation. Values are represented as the mean+SEM and Newman-Keuls post hoc comparisons defined as follows: (*) different from WT, (+) different from R146G cTnI, (#) different from A63V Tm, P<0.05, myocytes were derived from a minimum of 5 different rat heart isolations with n = 45 WT, n = 40 R146G, and n = 40 A63V+R146G myocytes.</p

Freshwater Availability and Water Fetching Distance Affect Child Health in Sub-Saharan Africa

Currently, more than two-thirds of the population in Africa must leave their home to fetch water for drinking and domestic use. The time burden of water fetching has been suggested to influence the volume of water collected by households as well as time spent on income generating activities and child care. However, little is known about the potential health benefits of reducing water fetching distances. Data from almost 200 000 Demographic and Health Surveys carried out in 26 countries were used to assess the relationship between household walk time to water source and child health outcomes. To estimate the causal effect of decreased water fetching time on health, geographic variation in freshwater availability was employed as an instrumental variable for one-way walk time to water source in a two-stage regression model. Time spent walking to a household’s main water source was found to be a significant determinant of under-five child health. A 15-min decrease in one-way walk time to water source is associated with a 41% average relative reduction in diarrhea prevalence, improved anthropometric indicators of child nutritional status, and a 11% relative reduction in under-five child mortality. These results suggest that reducing the time cost of fetching water should be a priority for water infrastructure investments in Africa

Working model of additive and neutralizing outcomes for double heterozygous genotypes based on the combine effects of allele-specific thin filament activation.

<p>In the models depicted in all panels the arrows represent the individual allele as a linear vector. The length of the arrow represent the magnitude of activation/deactivation of the thin filament caused by the mutant allele alone, which is classified as weak, moderate, or strong. The arrow's direction represents the allele's alteration of thin filament activation in which a rightward pointing arrow (positive direction) represents activation and a leftward pointing (negative direction) arrow represents deactivation. (A) Double Activating Allele Model: the positive sloping gradient represents the additive slowing of relaxation and heightened severity of diastolic dysfunction that occurs with the combination of two activating mutant alleles from different sarcomeric loci. The combination of two activating alleles is represented by adding two activating allele vectors together, which yields a higher degree of activation and thus more severe diastolic dysfunction. Some double activating allele combinations may cause the thin filament to become saturated thereby approaching a threshold of activation in which the combination of two vectors is not additive on a one to one basis. (B) Double Deactivating Allele Model: the negative sloping gradient represents the predicted additive consequence to accelerate relaxation and reduce contractility that occurs with the combination of two deactivating mutant alleles from different sarcomeric loci. The combination of two deactivating alleles is represented by adding two activating allele vectors together but in the negative direction, which yields a higher degree of deactivation and thus altered contractility with enhanced relaxation. Some double deactivating allele combinations may also result in saturation thereby approaching a threshold of deactivation. (C) Mixed Model of Activating + Deactivating Alleles: the combination of two alleles with different activation properties is depicted by the addition of a positive gradient and vector (activating allele) and a negative gradient and vector (deactivating allele). This combination results in mitigating effects, in which the primary phenotypes of each individual mutant are neutralized (represented by the trapezoid) when the opposing alleles are co-expressed.</p

Apoptosis Repressor with a CARD Domain (ARC) Restrains Bax-Mediated Pathogenesis in Dystrophic Skeletal Muscle

<div><p>Myofiber wasting in muscular dystrophy has largely been ascribed to necrotic cell death, despite reports identifying apoptotic markers in dystrophic muscle. Here we set out to identify the contribution of canonical apoptotic pathways to skeletal muscle degeneration in muscular dystrophy by genetically deleting a known inhibitor of apoptosis, apoptosis repressor with a card domain (Arc), in dystrophic mouse models. <i>Nol3</i> (Arc protein) genetic deletion in the dystrophic <i>Sgcd</i> or <i>Lama2</i> null backgrounds showed exacerbated skeletal muscle pathology with decreased muscle performance compared with single null dystrophic littermate controls. The enhanced severity of the dystrophic phenotype associated with <i>Nol3</i> deletion was caspase independent but dependent on the mitochondria permeability transition pore (MPTP), as the inhibitor Debio-025 partially rescued skeletal muscle pathology in <i>Nol3</i>^<i>-/-</i><i>Sgcd</i>^<i>-/-</i> double targeted mice. Mechanistically, <i>Nol3</i>^<i>-/-</i><i>Sgcd</i>^<i>-/-</i> mice showed elevated total and mitochondrial Bax protein levels, as well as greater mitochondrial swelling, suggesting that Arc normally restrains the cell death effects of Bax in skeletal muscle. Indeed, knockdown of Arc in mouse embryonic fibroblasts caused an increased sensitivity to cell death that was fully blocked in <i>Bax Bak1</i> (genes encoding Bax and Bak) double null fibroblasts. Thus Arc deficiency in dystrophic muscle exacerbates disease pathogenesis due to a Bax-mediated sensitization of mitochondria-dependent death mechanisms.</p> </div

Hand-to-Mouth Contacts Result in Greater Ingestion of Feces than Dietary Water Consumption in Tanzania: A Quantitative Fecal Exposure Assessment Model

Diarrheal diseases kill 1800 children under the age of five die each day, and nearly half of these deaths occur in sub-Saharan Africa. Contaminated drinking water and hands are two important environmental transmission routes of diarrhea-causing pathogens to young children in low-income countries. The objective of this research is to evaluate the relative contribution of these two major exposure pathways in a low-income country setting. A Monte Carlo simulation was used to model the amount of human feces ingested by children under five years old from exposure via hand-to-mouth contacts and stored drinking water ingestion in Bagamoyo, Tanzania. Child specific exposure data were obtained from the USEPA 2011 Exposure Factors Handbook, and fecal contamination was estimated using hand rinse and stored water fecal indicator bacteria concentrations from over 1200 Tanzanian households. The model outcome is a distribution of a child’s daily dose of feces via each exposure route. The model results show that Tanzanian children ingest a significantly greater amount of feces each day from hand-to-mouth contacts than from drinking water, which may help elucidate why interventions focused on water without also addressing hygiene often see little to no effect on reported incidence of diarrhea

Nol3^-/-Lama2^-/- mice have smaller skeletal muscles and more severe pathology.

<p>A, Muscle weights normalized to tibial length of gastrocnemius and quadriceps measured from WT, Nol3^-/-, Lama2^-/-, and Nol3^-/-Lama2^-/- mice. *P<0.05 vs WT; #P<0.05 vs Lama2^-/-; N=9 per group. B, Histological images taken at 200x of Masson’s trichrome stained sections of gastrocnemius, quadriceps and diaphragm from Lama2^-/- and Nol3^-/-Lama2^-/- mice.</p

Treatment with the MPTP inhibitor Debio-025 reduces skeletal muscle pathology in Nol3^-/-Sgcd^-/- mice.

<p>A, Muscle weights normalized to body weight of gastrocnemius and quadriceps, and B, body weight measurements of Sgcd^-/- and Nol3^-/-Sgcd^-/- mice treated with vehicle or Debio-025 for 4 weeks. C, Quantification of the time to exhaustion as assessed by involuntary treadmill running measured from vehicle or Debio-025 treated Nol3^-/- Sgcd^-/- mice. D, Quantitation of fibrosis and associated E, histological images taken at 100x of Masson’s trichrome stained sections in gastrocnemius and quadriceps muscles from vehicle or Debio-025 treated Sgcd^-/- and Nol3^-/-Sgcd^-/- mice. *P<0.05 vs vehicle; #P<0.05 vs Sgcd^-/- + vehicle; †P<0.05 vs Nol3^-/-Sgcd^-/- + vehicle; N=5-7 per group. Representative absorbance (540 nm) readings from skeletal muscle derived mitochondria demonstrating the amount of mitochondrial F, swelling in response to Ca²⁺ and G, shrinkage in response to PEG from the indicated genotypes of mice. </p