Contributions of sustained INa and IKv43 to transmural heterogeneity of early repolarization and arrhythmogenesis in canine left ventricular myocytes

The roles of sustained components of I(Na) and I(Kv43) in shaping the action potentials (AP) of myocytes isolated from the canine left ventricle (LV) have not been studied in detail. Here we investigate the hypothesis that these two currents can contribute substantially to heterogeneity of early repolarization and arrhythmic risk. Quantitative data from voltage-clamp and expression profiling experiments were used to complete meaningful modifications to an existing "local control" model of canine midmyocardial myocyte excitation-contraction coupling for epicardial and endocardial cells. We include 1) heterogeneous I(Kv43), I(Ks), and I(SERCA) density; 2) modulation of I(Kv43) by Kv channel interacting protein type 2 (KChIP2) channel subunits; 3) a possible Ca(2+)-dependent open-state inactivation of I(Kv43); and 4) a sustained component of the inward Na(+) current, I(NaL). The resulting simulations illustrate ways in which KChIP2- and Ca(2+)-dependent control of I(Kv43) can result in a sustained outward current that can neutralize I(NaL) in a rate- and myocyte subtype-dependent manner. Both these currents appear to play significant roles in modulating AP duration and rate dependence in midmyocardial myocytes. Furthermore, an increased ratio of I(Kv43) to I(NaL) is capable of protecting epicardial myocytes from the early afterdepolarizations resulting from the SCN5A-I1768V mutation-induced increase in I(NaL). Experimentally observed transmural differences in Ca(2+) handling, including greater sarcoplasmic reticulum Ca(2+) content and faster Ca(2+) transient decay rates on the epicardium, were recapitulated in our simulations. By design, these models allow upward integration into organ models or may be used as a basis for further investigations into cellular heterogeneities.     

Beaked whale auditory evoked potential hearing measurements

Several mass strandings of beaked whales have recently been correlated with military exercises involving mid-frequency sonar highlighting unknowns regarding hearing sensitivity in these species. We report the hearing abilities of a stranded juvenile beaked whale (Mesoplodon europaeus) measured with auditory evoked potentials. The beaked whale’s modulation rate transfer function (MRTF) measured with a 40-kHz carrier showed responses up to an 1,800 Hz amplitude modulation (AM) rate. The MRTF was strongest at the 1,000 and 1,200 Hz AM rates. The envelope following response (EFR) input–output functions were non-linear. The beaked whale was most sensitive to high frequency signals between 40 and 80 kHz, but produced smaller evoked potentials to 5 kHz, the lowest frequency tested. The beaked whale hearing range and sensitivity are similar to other odontocetes that have been measured.     

Hematologic, biochemical, and cytologic findings from apparently healthy atlantic bottlenose dolphins (Tursiops truncatus) inhabiting the Indian River Lagoon, Florida, USA

The objective of this study was to establish reference baseline data for hematologic, biochemical, and cytologic findings in apparently healthy Atlantic bottlenose dolphins (Tursiops truncatus) inhabiting the Indian River Lagoon, Florida, USA. Sixty-two dolphins were captured, examined, and released during June 2003 and June 2004. Mean, standard deviation, and range were calculated for each parameter, and values for which published data were available, were close to or within the ranges previously reported for free-ranging bottlenose dolphins. No pathologic abnormalities were found in fecal and blowhole cytologic specimens. However, 24% (7/29) of the dolphins examined in 2003 had evidence of gastritis, which was graded as severe in 14% (4/29) of the cases. In 2004, only 4% (1/24) of dolphins sampled had evidence of mild or moderate gastritis; no severe inflammation was present. Dolphins with evidence of gastritis were 8 yr of age or older and predominantly male. Several statistically significant differences were found between males and females, between pregnant and nonpregnant animals, and between juveniles (<6 yr) and adults (> or =6 yr). However, the values remained within the established ranges for this species, and the differences were not likely to be of clinical significance.     

Actin polymerization stabilizes α4β1 integrin anchors that mediate monocyte adhesion

Leukocytes arrested on inflamed endothelium via integrins are subjected to force imparted by flowing blood. How leukocytes respond to this force and resist detachment is poorly understood. Live-cell imaging with Lifeact-transfected U937 cells revealed that force triggers actin polymerization at upstream α4β1 integrin adhesion sites and the adjacent cortical cytoskeleton. Scanning electron microscopy revealed that this culminates in the formation of structures that anchor monocyte adhesion. Inhibition of actin polymerization resulted in cell deformation, displacement, and detachment. Transfection of dominant-negative constructs and inhibition of function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. These included activation of Rap1, phosphoinositide 3-kinase γ isoform, and Rac but not Cdc42. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces.     

Kinase-dependent adhesion to fibronectin: regulation by calreticulin

We studied the phosphorylation (activation status) of c-Src and CaMKII in MEFs either wild type for calreticulin, calreticulin-null, or rescued with full-length calreticulin. We found that calreticulin-null cells were poorly spread on the substratum and formed few, if any, focal contacts. Fibronectin expression and deposition were lower in calreticulin-null MEFs compared to calreticulin-expressing cells, which also exhibited increased c-Src and CaMKII phosphorylation (activity). Plating MEFs on preformed fibronectin rescued the poor adhesive phenotype of calreticulin-null cells, and caused a decrease in c-Src Y418 phosphorylation (activity). c-Src inhibition caused the calreticulin-null MEFs to become well spread on the substratum and to make many prominent focal contacts. Calmodulin and CaMKII inhibition caused similar results, along with a notable increase in paxillin phosphorylation (activation). To test if the calcium storage function of calreticulin was responsible for these effects, we manipulated intracellular [Ca(2+)]. Lowering [Ca(2+)](ER) caused an increase in c-Src phosphorylation and a decrease in fibronectin abundance. Conversely, increasing [Ca(2+)] caused opposite effects. These results suggest that calreticulin regulates both the c-Src and calmodulin/CaMKII pathways, enabling cells to be better spread on the substratum by allowing greater fibronectin deposition and increased focal contact formation.     

Trypanosoma brucei BRCA2 acts in antigenic variation and has undergone a recent expansion in BRC repeat number that is important during homologous recombination

Antigenic variation in Trypanosoma brucei has selected for the evolution of a massive archive of silent Variant Surface Glycoprotein (VSG) genes, which are activated by recombination into specialized expression sites. Such VSG switching can occur at rates substantially higher than background mutation and is dependent on homologous recombination, a core DNA repair reaction. A key regulator of homologous recombination is BRCA2, a protein that binds RAD51, the enzyme responsible for DNA strand exchange. Here, we show that T. brucei BRCA2 has undergone a recent, striking expansion in the number of BRC repeats, a sequence element that mediates interaction with RAD51. T. brucei BRCA2 mutants are shown to be significantly impaired in antigenic variation and display genome instability. By generating BRCA2 variants with reduced BRC repeat numbers, we show that the BRC expansion is crucial in determining the efficiency of T. brucei homologous recombination and RAD51 localization. Remarkably, however, this appears not to be a major determinant of the activation of at least some VSG genes.     

Simple, defensible sample sizes based on cost efficiency

Summary .   The conventional approach of choosing sample size to provide 80% or greater power ignores the cost implications of different sample size choices. Costs, however, are often impossible for investigators and funders to ignore in actual practice. Here, we propose and justify a new approach for choosing sample size based on cost efficiency, the ratio of a study's projected scientific and/or practical value to its total cost. By showing that a study's projected value exhibits diminishing marginal returns as a function of increasing sample size for a wide variety of definitions of study value, we are able to develop two simple choices that can be defended as more cost efficient than any larger sample size. The first is to choose the sample size that minimizes the average cost per subject. The second is to choose sample size to minimize total cost divided by the square root of sample size. This latter method is theoretically more justifiable for innovative studies, but also performs reasonably well and has some justification in other cases. For example, if projected study value is assumed to be proportional to power at a specific alternative and total cost is a linear function of sample size, then this approach is guaranteed either to produce more than 90% power or to be more cost efficient than any sample size that does. These methods are easy to implement, based on reliable inputs, and well justified, so they should be regarded as acceptable alternatives to current conventional approaches.     

A role for decorin in the remodeling of myocardial infarction.

Because the small leucine-rich proteoglycan decorin has been implicated in regulation of collagen fibrillogenesis leading to proper extracellular matrix assembly, we hypothesized it could play a key role in cardiac fibrosis following myocardial infarction. In this study we ligated the left anterior descending coronary artery in wildtype and decorin-null mice to produce large infarcts in the anterior wall of the left ventricle. At early stages post-coronary occlusion the myocardial infarction size did not appreciably differ between the two genotypes. However, we found a wider distribution of collagen fibril sizes with less organization and loose packing in mature scar from decorin-null mice. Thus, we tested the hypothesis that these abnormal collagen fibrils would adversely affect post-infarction mechanics and ventricular remodeling. Indeed, scar size, right ventricular remote hypertrophy, and left ventricular dilatation were greater in decorin-null animals compared with wildtype littermates 14 days after acute myocardial infarction. Echocardiography revealed depressed left ventricular systolic function between 4 and 8 weeks post-ischemia in the decorin-null animals. These changes indicate that decorin is required for the proper fibrotic evolution of myocardial infarctions, and that its absence leads to abnormal scar tissue formation. This might contribute to aneurysmal ventricular dilatation, remote hypertrophy, and depressed ventricular function.     

Model study of ATP and ADP buffering, transport of Ca(2+) and Mg(2+), and regulation of ion pumps in ventricular myocyte.

We extended the model of the ventricular myocyte by Winslow et al. (Circ. Res 1999, 84:571-586) by incorporating equations for Ca(2+) and Mg(2+) buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na(+)-K(+) pump, sarcolemmal and sarcoplasmic Ca(2+) pumps). The results indicate that, under normal conditions, Ca(2+) binding by low-affinity ATP and diffusion of CaATP may affect the amplitude and time course of intracellular Ca(2+) signals. The model also suggests that a fall in ATP/ADP ratio significantly reduces sarcoplasmic Ca(2+) content, increases diastolic Ca(2+), lowers systolic Ca(2+), increases Ca(2+) influx through L-type channels, and decreases the efficiency of the Na(+)/Ca(2+) exchanger in extruding Ca(2+) during periodic voltage-clamp stimulation. The analysis suggests that the most important reason for these changes during metabolic inhibition is the down-regulation of the sarcoplasmic Ca(2+)-ATPase pump by reduced diastolic MgATP levels. High Ca(2+) concentrations developed near the membrane might have a greater influence on Mg(2+), ATP, and ADP concentrations than that of the lower Ca(2+) concentrations in the bulk myoplasm. The model predictions are in general agreement with experimental observations measured under normal and pathological conditions.