Calcium handling in zebrafish ventricular myocytes

The zebrafish is an important model for the study of vertebrate cardiac development with a rich array of genetic mutations and biological reagents for functional interrogation. The similarity of the zebrafish (Danio rerio) cardiac action potential with that of humans further enhances the relevance of this model. In spite of this, little is known about excitation-contraction coupling in the zebrafish heart. To address this issue, adult zebrafish cardiomyocytes were isolated by enzymatic perfusion of the cannulated ventricle and were subjected to amphotericin-perforated patch-clamp technique, confocal calcium imaging, and/or measurements of cell shortening. Simultaneous recordings of the voltage dependence of the L-type calcium current (I(Ca,L)) amplitude and cell shortening showed a typical bell-shaped current-voltage (I-V) relationship for I(Ca,L) with a maximum at +10 mV, whereas calcium transients and cell shortening showed a monophasic increase with membrane depolarization that reached a plateau at membrane potentials above +20 mV. Values of I(Ca,L) were 53, 100, and 17% of maximum at -20, +10, and +40 mV, while the corresponding calcium transient amplitudes were 64, 92, and 98% and cell shortening values were 62, 95, and 96% of maximum, respectively, suggesting that I(Ca,L) is the major contributor to the activation of contraction at voltages below +10 mV, whereas the contribution of reverse-mode Na/Ca exchange becomes increasingly more important at membrane potentials above +10 mV. Comparison of the recovery of I(Ca,L) from acute and steady-state inactivation showed that reduction of I(Ca,L) upon elevation of the stimulation frequency is primarily due to calcium-dependent I(Ca,L) inactivation. In conclusion, we demonstrate that a large yield of healthy atrial and ventricular myocytes can be obtained by enzymatic perfusion of the cannulated zebrafish heart. Moreover, zebrafish ventricular myocytes differed from that of large mammals by having larger I(Ca,L) density and a monophasically increasing contraction-voltage relationship, suggesting that caution should be taken upon extrapolation of the functional impact of mutations on calcium handling and contraction in zebrafish cardiomyocytes.     

Association mapping for wood quality and growth traits in Eucalyptus globulus ssp. globulus Labill identifies nine stable marker-trait associations for seven traits

The moderate to high levels of nucleotide diversity and low linkage disequilibrium found in many forest tree species make them ideal candidates for association mapping. Here, we report candidate gene-based association mapping results for complex wood quality and growth traits in Eucalyptus globulus Labill. ssp. globulus, the most widely grown eucalypt in temperate regions of the world. Ninety-eight single nucleotide polymorphisms (SNPs) from 20 wood quality candidate genes were assayed in a discovery population consisting of 385 trees sourced from a provenance-progeny trial. Twenty-five selected SNPs with significant associations (P?.     

Organization and evolution of transposable elements along the bread wheat chromosome 3B

Background

The 17 Gb bread wheat genome has massively expanded through the proliferation of transposable elements (TEs) and two recent rounds of polyploidization. The assembly of a 774 Mb reference sequence of wheat chromosome 3B provided us with the opportunity to explore the impact of TEs on the complex wheat genome structure and evolution at a resolution and scale not reached so far.

Results

We develop an automated workflow, CLARI-TE, for TE modeling in complex genomes. We delineate precisely 56,488 intact and 196,391 fragmented TEs along the 3B pseudomolecule, accounting for 85% of the sequence, and reconstruct 30,199 nested insertions. TEs have been mostly silent for the last one million years, and the 3B chromosome has been shaped by a succession of bursts that occurred between 1 to 3 million years ago. Accelerated TE elimination in the high-recombination distal regions is a driving force towards chromosome partitioning. CACTAs overrepresented in the high-recombination distal regions are significantly associated with recently duplicated genes. In addition, we identify 140 CACTA-mediated gene capture events with 17 genes potentially created by exon shuffling and show that 19 captured genes are transcribed and under selection pressure, suggesting the important role of CACTAs in the recent wheat adaptation.

Conclusion

Accurate TE modeling uncovers the dynamics of TEs in a highly complex and polyploid genome. It provides novel insights into chromosome partitioning and highlights the role of CACTA transposons in the high level of gene duplication in wheat.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0546-4) contains supplementary material, which is available to authorized users.     

Functional Assessment of Cardiac Responses of Adult Zebrafish (Danio rerio) to Acute and Chronic Temperature Change Using High-Resolution Echocardiography

The zebrafish (Danio rerio) is an important organism as a model for understanding vertebrate cardiovascular development. However, little is known about adult ZF cardiac function and how contractile function changes to cope with fluctuations in ambient temperature. The goals of this study were to: 1) determine if high resolution echocardiography (HRE) in the presence of reduced cardiodepressant anesthetics could be used to accurately investigate the structural and functional properties of the ZF heart and 2) if the effect of ambient temperature changes both acutely and chronically could be determined non-invasively using HRE in vivo. Heart rate (HR) appears to be the critical factor in modifying cardiac output (CO) with ambient temperature fluctuation as it increases from 78 ± 5.9 bpm at 18°C to 162 ± 9.7 bpm at 28°C regardless of acclimation state (cold acclimated CA– 18°C; warm acclimated WA– 28°C). Stroke volume (SV) is highest when the ambient temperature matches the acclimation temperature, though this difference did not constitute a significant effect (CA 1.17 ± 0.15 μL at 18°C vs 1.06 ± 0.14 μl at 28°C; WA 1.10 ± 0.13 μL at 18°C vs 1.12 ± 0.12 μl at 28°C). The isovolumetric contraction time (IVCT) was significantly shorter in CA fish at 18°C. The CA group showed improved systolic function at 18°C in comparison to the WA group with significant increases in both ejection fraction and fractional shortening and decreases in IVCT. The decreased early peak (E) velocity and early peak velocity / atrial peak velocity (E/A) ratio in the CA group are likely associated with increased reliance on atrial contraction for ventricular filling.     

Spatial and Temporal Control of Hyperthermia Using Real Time Ultrasonic Thermal Strain Imaging with Motion Compensation,Phantom Study

Mild hyperthermia has been successfully employed to induce reversible physiological changes that can directly treat cancer and enhance local drug delivery. In this approach, temperature monitoring is essential to avoid undesirable biological effects that result from thermal damage. For thermal therapies, Magnetic Resonance Imaging (MRI) has been employed to control real-time Focused Ultrasound (FUS) therapies. However, combined ultrasound imaging and therapy systems offer the benefits of simple, low-cost devices that can be broadly applied. To facilitate such technology, ultrasound thermometry has potential to reliably monitor temperature. Control of mild hyperthermia was previously achieved using a proportional-integral-derivative (PID) controller based on thermocouple measurements. Despite accurate temporal control of heating, this method is limited by the single position at which the temperature is measured. Ultrasound thermometry techniques based on exploiting the thermal dependence of acoustic parameters (such as longitudinal velocity) can be extended to create thermal maps and allow an accurate monitoring of temperature with good spatial resolution. However, in vivo applications of this technique have not been fully developed due to the high sensitivity to tissue motion. Here, we propose a motion compensation method based on the acquisition of multiple reference frames prior to treatment. The technique was tested in the presence of 2-D and 3-D physiological-scale motion and was found to provide effective real-time temperature monitoring. PID control of mild hyperthermia in presence of motion was then tested with ultrasound thermometry as feedback and temperature was maintained within 0.3°C of the requested value.     

Characterization of zebrafish (Danio rerio) NCX4: a novel NCX with distinct electrophysiological properties

Members of the Na+/Ca2+ exchanger (NCX) family are important regulators of cytosolic Ca2+ in myriad tissues and are highly conserved across a wide range of species. Three distinct NCX genes and numerous splice variants exist in mammals, many of which have been characterized in a variety of heterologous expression systems. Recently, however, we discovered a fourth NCX gene (NCX4), which is found exclusively in teleost, amphibian, and reptilian genomes. Zebrafish (Danio rerio) NCX4a encodes for a protein of 939 amino acids and shows a high degree of identity with known NCXs. Although knockdown of NCX4a activity in zebrafish embryos has been shown to alter left-right patterning, it has not been demonstrated that NCX4a functions as a NCX. In this study, we 1) demonstrated, for the first time, that this gene encodes for a novel NCX; 2) characterized the tissue distribution of zebrafish NCX4a; and 3) evaluated its kinetic and transport properties. While ubiquitously expressed, the highest levels of NCX4a expression occurred in the brain and eyes. NCX4a exhibits modest levels of Na+-dependent inactivation and requires much higher levels of regulatory Ca2+ to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from Na+-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the Na+-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 microM exchanger inhibitory peptide. We demonstrated that a new putative member of the NCX gene family, NCX4a, encodes for a NCX with unique functional properties. These data will be useful in understanding the role that NCX4a plays in embryological development as well as in the adult, where it is expressed ubiquitously.     

Effect of temperature on the structure of trout troponin C

Adaptation for life at different temperatures can cause changes in many aspects of an organism. One example is the expression of different protein isoforms in species adapted to different temperatures. The calcium regulatory protein cardiac troponin C (cTnC), from rainbow trout (Oncorhynchus mykiss), is a good model for studying temperature effects, both because of its low physiological temperature and because mammalian cTnC, extensively studied at higher temperatures, can be used for comparison. We determined the structure and studied the backbone dynamics of the regulatory domain of trout cardiac troponin C (ScNTnC) with one Ca(2+) bound at 7 and 30 degrees C, using nuclear magnetic resonance spectroscopy (NMR). The overall fold of the regulatory domain of trout cTnC at both temperatures is similar to the regulatory domain of mammalian (human, bovine, and porcine isoform) cTnC bound to one Ca(2+). By comparing the trout structures at the two temperatures, we identify differences between the positions of the helices flanking the calcium binding loops, and the overall structure at 7 degrees C is more compact than that at 30 degrees C. The structure at 7 degrees C is more similar to the mammalian cTnC, which was determined at 30 degrees C, indicating that they have the same conformation at their respective physiological temperatures. The dynamic properties of the regulatory domain of trout cTnC are similar at the two temperatures that were used in these studies.     

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca2+ affinity of site II

Cardiac myofibrilsisolated from trout heart have been demonstrated to have a highersensitivity for Ca²⁺ than mammalian cardiac myofibrils.Using cardiac troponin C (cTnC) cloned from trout and mammalian hearts,we have previously demonstrated that this comparatively highCa²⁺ sensitivity is due, in part, to trout cTnC (ScTnC)having twice the Ca²⁺ affinity of mammalian cTnC (McTnC)over a broad range of temperatures. The amino acid sequence of ScTnC is92% identical to McTnC. To determine the residues responsible for thehigh Ca²⁺ affinity, the function of a number of ScTnC andMcTnC mutants was characterized by monitoring an intrinsic fluorescentreporter that monitors Ca²⁺ binding to site II (F27W). Theremoval of the COOH terminus (amino acids 90-161) from ScTnC andMcTnC maintained the difference in Ca²⁺ affinity betweenthe truncated cTnC isoforms (ScNTnC and McNTnC). The replacement ofGln²⁹ and Asp³⁰ in ScNTnC with thecorresponding residues from McNTnC, Leu and Gly, respectively, reducedCa²⁺ affinity to that of McNTnC. These results demonstratethat Gln²⁹ and Asp³⁰ in ScTnC are required forthe high Ca²⁺ affinity of site II.