Search

The ligand-activated transcription factor peroxisome proliferator activated receptor gamma (PPARgamma) is expressed in vascular endothelium where it exerts anti-inflammatory and antioxidant effects. However, its role in regulating vascular function remains undefined. We examined endothelial function in transgenic mice expressing dominant-negative mutants of PPARgamma under the control of an endothelial-specific promoter to test the hypothesis that endothelial PPARgamma plays a protective role in the vasculature. Under baseline conditions, responses to the endothelium-dependent agonist acetylcholine were not affected in either aorta or the basilar artery in vitro. In response to feeding a high-fat diet for 12 weeks, acetylcholine produced dilation that was markedly impaired in the basilar artery of mice expressing dominant-negative mutants, but not in mice expressing wild-type PPARgamma controlled by the same promoter. Unlike basilar artery, 12 weeks of a high-fat diet was not sufficient to cause endothelial dysfunction in the aorta of mice expressing dominant-negative PPARgamma, although aortic dysfunction became evident after 25 weeks. The responses to acetylcholine in basilar artery were restored to normal after treatment with a scavenger of superoxide. Baseline blood pressure was only slightly elevated in the transgenic mice, but the pressor response to angiotensin II was augmented. Thus, interference with PPARgamma in the endothelium produces endothelial dysfunction in the cerebral circulation through a mechanism involving oxidative stress. Consistent with its role as a fatty acid sensor, these findings provide genetic evidence that endothelial PPARgamma plays a critical role in protecting blood vessels in response to a high-fat diet.

62 hips in 57 patients who were all considered to be at risk for ectopic bone formation were randomly divided into two groups: one control group and one group who received single dose radiotherapy of 5 Gy at the operation field preoperatively. With a mean follow-up of 2.5 years, the incidence of heterotopic ossification in the control group was 16 of the 19 hips and in the irradiated group 6 of the 43 hips (p = 0.001).

River Bank Filtration (RBF) contributes ca. 7% (80 Mm3/a) to the national drinking water supply in the Netherlands, through a total of 26 well fields. These RBF well fields are classified on the basis of (1) the main driving mechanism of flow (polder or pump driven); (2) RBF periodicity (flow direction temporarily reversing or not), (3) type of riverbed (sand or gravel), and (4) type of contact of river with aquifer (with or without intercalated aquitard(s)).

Congestive heart failure is a debilitating disease in which the principal pathology is impaired ventricular contractility leading to diminished cardiac output, and previous work indicates that reduced contractility is based in part on the ratio of myosin heavy chain (MyHC) isoforms, α- and β-MyHC, expressed in the ventricles. Normal human ventricles express ∼10% of the fast α-MyHC on a background of the slower β-MyHC, while in failing hearts α-MyHC is reduced to virtually undetectable levels with complete replacement by β-MyHC. Data from permeabilized myocardial preparations suggests that this isoform switch may be partly responsible for reduced myocardial twitch force and pressure development by failing ventricles since β-MyHC is a slower motor protein, yet most experiments have used non-human myosins and experimental conditions in which preparations were steadily activated, thus little is known about the response of human myosins to a time-varying Ca2+ transient. To address these limitations, we recently developed a human 3D engineered cardiac tissue (hECT) system in which we can express recombinant human muscle myosin motors. Using commercially available cloning and adenoviral expression systems, α- or β-MyHC isoform expressing adenoviral particles were used to transduce human cardiomyocytes produced from human iPS cells and construct hECTs. Preparations displayed well-defined cellular structure with elongated morphology aligned in the direction of preparation shortening during electrical pacing, while histological analysis of hECT revealed appropriate protein expression and localization within the sarcomere. In response to a Ca2+ transient, the time-course of twitch force development was accelerated in hECT expressing α-MyHC compared to β-MyHC, while peak twitch force was greater in hECT expressing α-MyHC. These results demonstrate the relative contribution of myosin isoforms to myocardial twitch kinetics in human engineered cardiac tissue constructs expressing a stable background of myofibrillar proteins.

Cardiac myosin binding protein-C (cMyBPC) is a modular protein consisting of 11 domains whose precise function and sarcomeric arrangement are incompletely understood. Identification of hypertrophic cardiomyopathy (HCM)–causing missense mutations in cMyBPC has highlighted the significance of certain domains. Of particular interest is domain C5, an immunoglobulin-like domain with a cardiac-specific insert, which is of unknown function yet is the site of two HCM-causing missense mutations. To identify interactors with this region, a human cardiac cDNA library was screened in a yeast two-hybrid (Y2H) assay using the C5 sequence as bait. Screening >7×10 6 clones surprisingly revealed that domain C5 preferentially bound to clones encoding C-terminal fragments of cMyBPC; the interacting region was narrowed to domain C8 by deletion mapping. A surface plasmon resonance assay using purified recombinant cMyBPC domains was used to measure the affinity of C5 and C8 in vitro (K a =1×10 5 mol/L −1 ). This affinity was decreased about 2-fold by the HCM mutation R654H, and by at least 10-fold by the mutation N755K. Further Y2H assays also demonstrated specific binding between domains C7 and C10 of cMyBPC. Based on these novel interactions, and previous biochemical and structural data, we propose that cMyBPC molecules trimerize into a collar around the thick filament, with overlaps of domains C5-C7 of one cMyBPC with C8-C10 of another. We speculate that this interaction may be dynamically formed and released, thereby restricting or favoring cross-bridge formation, respectively. We suggest that the HCM mutations act by altering the cMyBPC collar, indicating its importance in thick filament structure and regulation.

Truncation mutations in cardiac myosin binding protein-C (cMyBP-C) are prevalent causes of hypertrophic cardiomyopathy (HCM). Mounting evidence suggest that truncated cMyBP-C causes HCM through haploinsufficiency. Mouse models in which cMyBP-C is ablated (KO), or truncated, show varying degrees of systolic and diastolic dysfunction and alterations in contractile kinetics. Phenotypic variation observed in these models may, however, be related to secondary adaptations and hypertrophic remodeling, rather than the direct effect of cMyBP-C depletion.In order to study the effects of cMyBP-C ablation on contractility in the absence of hypertrophic remodeling, we produced engineered cardiac tissue (ECT) from wild type (WT) and (KO) neonatal mouse cardiomyocytes.Expression levels of several hypertrophic markers were similar in WT and KO ECT, indicating a lack of hypertrophic remodeling. Contraction (50.0±1.1ms vs. 42.9±1.9ms; p=0.002) and relaxation (41.3±0.8ms vs. 29.2±1.7; p

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that plays a critical role in metabolism. Thiazolidinediones, high-affinity PPARgamma ligands used clinically to treat type II diabetes, have been reported to lower blood pressure and provide other cardiovascular benefits. Some mutations in PPARgamma (PPARG) cause type II diabetes and severe hypertension. Here we tested the hypothesis that PPARgamma in vascular muscle plays a role in the regulation of vascular tone and blood pressure. Transgenic mice expressing dominant-negative mutations in PPARgamma under the control of a smooth-muscle-specific promoter exhibit a loss of responsiveness to nitric oxide and striking alterations in contractility in the aorta, hypertrophy and inward remodeling in the cerebral microcirculation, and systolic hypertension. These results identify PPARgamma as pivotal in vascular muscle as a regulator of vascular structure, vascular function, and blood pressure, potentially explaining some of the cardioprotective effects of thiazolidinediones.

Summary Hypertrophic cardiomyopathy (HCM) is an autosomal dominantly inherited disease of the cardiac sarcomere, caused by numerous mutations in genes encoding protein components of this structure. Mutation carriers are at risk of sudden cardiac death, mostly as adolescents or young adults. The reproductive disadvantage incurred may explain both the global occurrence of diverse independent HCM-associated mutations and the rare reports of founder effects within populations. We have investigated whether this holds true for two South African subpopulations, one of mixed ancestry and one of northern-European descent. Previously, we had detected three novel mutations—Ala797Thr in the β-myosin heavy-chain gene ( βMHC ), Arg92Trp in the cardiac troponin T gene ( cTnT ), and Arg645His in the myosin-binding protein C gene ( MyBPC )—and two documented βMHC mutations (Arg403Trp and Arg249Gln). Here we report three additional novel mutations—Gln499Lys in βMHC and Val896Met and Δc756 in MyBPC —and the documented βMHC Arg719Gln mutation. Seven of the nine HCM-causing mutations arose independently; no conclusions can be drawn for the remaining two. However, the βMHC Arg403Trp and Ala797Thr and cTnT Arg92Trp mutations were detected in another one, eight, and four probands, respectively, and haplotype analysis in families carrying these recurring mutations inferred their origin from three common ancestors. The milder phenotype of the βMHC mutations may account for the presence of these founder effects, whereas population dynamics alone may have overridden the reproductive disadvantage incurred by the more lethal, cTnT Arg92Trp mutation.