Troponin I protein kinase C phosphorylation sites and ventricular function

OBJECTIVE: Cardiac Troponin I (cTnI) phosphorylation by protein kinase C (PKC) results in a reduction of maximal actomyosin ATPase activity, an effect that is more marked at higher levels of calcium (Ca2+) and is likely to reduce active force development. We postulated that there would be greater Ca2+-dependent changes in ventricular function in hearts of cTnI transgenic (TG) mice expressing mutant troponin I lacking PKC sites compared to wild-type (WT). METHODS: We studied left ventricular function in isolated perfused hearts over a wide range of left ventricular volumes (Frank-Starling relationships) and mechanical restitution at three levels of perfusate Ca2+ (1.5, 2.5, and 3.5 mM). Manganese-enhanced magnetic resonance imaging (MRI) was used to study in-vivo sarcolemmal Ca2+ influx. The phosphorylation status of cTnI was examined by western blot analysis. RESULTS: Systolic contractile function in TG mice was altered in a calcium-dependent manner such that ventricular contractility was significantly greater in TG mice only at 3.5 mM perfusate Ca2+. The relaxation process and passive mechanical properties were unaltered in TG mice. Mechanical restitution parameters were abnormal in TG mice only at 1.5 mM perfusate Ca2+. In-vivo MRI data demonstrated up to 48% reduction in Mn2+-induced contrast enhancement, indicating reduced sarcolemmal Ca2+ influx. Western blot analysis indicated increased cTnI phosphorylation in TG mice. CONCLUSIONS: (1) TG mice exhibit calcium-dependent positive inotropy without slowed relaxation and this phenotype is mitigated by concomitant (compensatory) changes of reduced intracellular Ca2+ and increased phosphorylation of remaining cTnI sites. (2) The contractile phenotype in TG mice can be interpreted as an amplification of the normal response to changes in cellular Ca2+ observed in WT mice. Thus, PKC phosphorylation sites on cTnI play a role in attenuating contractile responses to changes in intracellular Ca2+.     

Nonuniform distribution of glucosinolates in Arabidopsis thaliana leaves has important consequences for plant defense

The spatial distribution of plant defenses within a leaf may be critical in explaining patterns of herbivory. The generalist lepidopteran larvae, Helicoverpa armigera (the cotton bollworm), avoided the midvein and periphery of Arabidopsis thaliana rosette leaves and fed almost exclusively on the inner lamina. This feeding pattern was attributed to glucosinolates because it was not evident in a myrosinase mutant that lacks the ability to activate glucosinolate defenses by hydrolysis. To measure the spatial distribution of glucosinolates in A. thaliana leaves at a fine scale, we constructed ion intensity maps from MALDI-TOF (matrix assisted laser desorption/ionization-time of flight) mass spectra. The major glucosinolates were found to be more abundant in tissues of the midvein and the periphery of the leaf than the inner lamina, patterns that were validated by HPLC analyses of dissected leaves. In addition, there were differences in the proportions of the three major glucosinolates in different leaf regions. Hence, the distribution of glucosinolates within the leaf appears to control the feeding preference of H. armigera larvae. The preferential allocation of glucosinolates to the periphery may play a key role in the defense of leaves by creating a barrier to the feeding of chewing herbivores that frequently approach leaves from the edge.     

Mapping of atrial activation during sustained atrial fibrillation in dogs with rapid ventricular pacing induced heart failure: evidence for a role of driver regions

INTRODUCTION: Dogs with rapid ventricular pacing (RVP)-induced congestive heart failure (CHF) have inducible atrial tachycardia, flutter, and fibrillation (AF). We tested the hypothesis that rapid atrial activation in multiple regions and at different rates is responsible for sustained AF in this CHF model. METHODS AND RESULTS: We studied 12 episodes of sustained (>10 minutes) AF induced in 12 dogs with CHF produced by 3-6 weeks of RVP at 230 beats/minute. High-density mapping of AF was performed using 382 unipolar atrial electrograms recorded simultaneously from epicardial electrodes on the right (RA) and left atria (LA) and Bachmann's bundle. AF mechanisms were based on Fast Fourier Transform (FFT) analysis and activation sequence mapping. A driver was defined as a rapid stable activation region with a single dominant frequency peak in FFT analysis. During AF, three FFT and activation patterns were seen: (1) a single LA driver (7.8 +/- 1.1 Hz) near the pulmonary veins (PVs) with irregular activation in the rest of the atria (n = 4); (2) simultaneous, multisite, biatrial drivers at differing frequencies (LA vs RA dominant frequency gradient: 1.3 +/- 0.8 Hz) near the PVs (8.4 +/- 0.3 Hz) and high RA (8.5 +/- 1.5 Hz) (n = 7); and (3) biatrial irregular activation with multiple and/or broadband frequency peaks without a dominant frequency. (LA: 7.1-11.4 Hz; RA: 5.9-7.7 Hz) (n = 1). Atrial drivers had either a focal activation pattern or were due to a macroreentrant circuit around the PVs. CONCLUSIONS: In this CHF model, FFT analysis and activation sequence mapping demonstrate that sustained AF is characterized by single and multiple, stable LA and RA drivers with predominant sources in the PVs and high RA causing fibrillatory conduction.     

Multilayer three-dimensional super resolution imaging of thick biological samples

Recent advances in optical microscopy have enabled biological imaging beyond the diffraction limit at nanometer resolution. A general feature of most of the techniques based on photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) has been the use of thin biological samples in combination with total internal reflection, thus limiting the imaging depth to a fraction of an optical wavelength. However, to study whole cells or organelles that are typically up to 15 μm deep into the cell, the extension of these methods to a three-dimensional (3D) super resolution technique is required. Here, we report an advance in optical microscopy that enables imaging of protein distributions in cells with a lateral localization precision better than 50 nm at multiple imaging planes deep in biological samples. The approach is based on combining the lateral super resolution provided by PALM with two-photon temporal focusing that provides optical sectioning. We have generated super-resolution images over an axial range of ≈10 μm in both mitochondrially labeled fixed cells, and in the membranes of living S2 Drosophila cells.     

Cardiovascular system during the postpartum state in women with a history of preeclampsia

In subjects with previous preeclampsia, differences in cardiovascular and/or blood biochemical parameters are present in the nonpregnant state, and a simultaneous assessment of multiple derived indices better differentiates between women with or without previous preeclampsia. We examined 18 previous preeclamptic and 50 previous uncomplicated pregnancies, ≈16 months postpartum. Cardiovascular assessment included the following: (1) systemic hemodynamics and mechanics (Doppler echocardiography, tonometry, and oscillometric sphygmomanometry); (2) endothelial function (plethysmography); (3) left ventricular properties (echocardiography); and (4) blood biochemical analyses. Compared to women with previous uncomplicated pregnancies, previous preeclamptics had higher mean (80±1 versus 86±3 mm Hg; P=0.04) and diastolic (64±1 versus 68±2 mm Hg; P=0.04) pressures and total vascular resistance (1562±37 versus 1784±114 dyne · s/cm(5); P=0.03). Systolic blood pressure, arterial compliance, and left ventricular properties were not different. Although heart-to-femoral pulse wave velocity was not different, heart-to-brachial pulse wave velocity tended to be faster in previous preeclamptics (374±8 versus 404±20 cm/s; P=0.06). Stress-induced increase in forearm blood flow was less in previous preeclamptics (245%±21% versus 136%±22%; P=0.01), indicating impaired endothelial function. No significant differences were observed in markers of endothelial activation, dyslipidemia, or oxidative stress; previous preeclamptics tended to have higher glucose level (58.7±1.9 versus 95±5.2 mg/dL; P=0.06). Logistic regression analysis indicated that a simultaneous evaluation of multiple derived indices better discriminated between the 2 groups. The differences in the previous preeclamptic group are in directions known to be associated with greater cardiovascular disease risk later in life.     

Effects of Relaxin on Arterial Dilation,Remodeling, and Mechanical Properties

Administering relaxin to conscious rats and humans elicits systemic and renal vasodilation. The molecular mechanisms vary according to the duration of relaxin exposure—so-called “rapid” (within minutes) or “sustained” (hours to days) vasodilatory responses—both being endothelium-dependent. Rapid responses are mediated by G_αi/o protein coupling to phosphoinositol-3 kinase/Akt (protein kinase B)–dependent phosphorylation and activation of nitric oxide synthase. Sustained responses are mediated by vascular endothelial and placental growth factors, as well as increases in arterial gelatinase activity. Thus, after hours or days of relaxin treatment, respectively, arterial MMP-9 or MMP-2 hydrolyze “big” endothelin (ET) at a gly-leu bond to form ET_1-32, which in turn activates the endothelial ET_B receptor/nitric oxide vasodilatory pathway. Administration of relaxin to conscious rats also increases global systemic arterial compliance and passive compliance of select isolated blood vessels such as small renal arteries (SRA). The increase in SRA passive compliance is mediated by both geometric remodeling (outward) and compositional remodeling (decreased collagen). Relaxin-induced geometric remodeling has also been observed in brain parenchymal arteries, and this remodeling appears to be via the activation of peroxisome proliferator–activated receptor-γ. Given the vasodilatory and arterial remodeling properties of relaxin, the hormone may have therapeutic potential in the settings of abnormal pregnancies, heart failure, and pathologies associated with stiffening of arteries.     

Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue

Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 µm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images with better sectioning and contrast in thick scattering samples such as Caenorhabditis elegans embryos, Drosophila melanogaster larval salivary glands, and mouse liver tissue.Fluorescence microscopy is an invaluable tool for biologists. Protein distributions in cells have an interesting structure down to the nanometer scale, but features smaller than 200–300 nm are blurred by diffraction in widefield and confocal fluorescence microscopes. Superresolution techniques like photoactivated localization microscopy (1), stochastic optical reconstruction microscopy (2), or stimulated emission depletion (STED) (3) microscopy allow the imaging of details beyond the limit imposed by diffraction, but usually trade acquisition speed or straightforward sample preparation. And although STED can provide resolution down to 40 nm, STED-specific fluorophores are recommended and it often requires light intensities that are orders of magnitude above widefield and confocal microscopy. On the other hand, structured illumination microscopy (SIM) (4) gives twice the resolution of a conventional fluorescence microscope with light intensities on the order of widefield microscopes and can be used with most common fluorophores. SIM uses contributions from both the excitation and emission point spread functions (PSFs) to substantially improve the transverse resolution and is generally performed by illuminating the sample with a set of sharp light patterns and collecting fluorescence on a multipixel detector, followed by image processing to recover superresolution detail from the interaction of the light pattern with the sample. A related technique, image scanning microscopy (ISM), uses a scanned diffraction-limited spot as the light pattern (5, 6). Multifocal SIM (MSIM) parallelizes ISM by using many excitation spots (7), and has been shown to produce optically sectioned images with ∼145-nm lateral and ∼400-nm axial resolution at depths up to ∼50 µm and at ∼1 Hz imaging frequency. In MSIM, images are excited with a multifocal excitation pattern, and the resulting fluorescence in the multiple foci are pinholed, locally scaled, and summed to generate an image [multifocal-excited, pinholed, scaled, and summed (MPSS)] with root 2-improved resolution relative to widefield microscopy, and improved sectioning compared with SIM due to confocal-like pinholing. Deconvolution is applied to recover the final MSIM image which has a full factor of 2 resolution improvement over the diffraction limit.MSIM works well in highly transparent samples (such as zebrafish embryos), but performance degrades in light scattering samples (such as the Caenorhabditis elegans embryo). Imaging in scattering samples can be improved by two-photon microscopy (8) and although the longer excitation wavelength reduces the resolution in nondescanned detection configurations, this can be partially offset by descanned detection and the addition of a confocal pinhole into the emission path. Whereas the nondescanned mode collects the most signal, the addition of a pinhole in the emission path of a point-scanning system can improve resolution when the pinhole is closed (9). In practice this is seldom done for biological specimens because signal-to-noise decays as the pinhole diameter decreases (9–11).SIM is an obvious choice in improving resolution without a dramatic loss in signal-to-noise, but the high photon density needed for efficient two-photon excitation is likely difficult to achieve in the typical widefield SIM configuration. This has led to other methods, such as line scanning (12) to achieve better depth penetration than confocal microscopy and up to twofold improvements in axial resolution (but with only ∼20% gain in lateral resolution). Multiphoton Bessel plane illumination (13) achieved an anisotropic lateral resolution of 180 nm (only in one direction) but requires an instrument design with two objectives in an orthogonal configuration. Cells and embryos can be readily imaged, but the multiaxis design may hinder the intravital imaging of larger specimens. Here, a combination of multiphoton excitation with MSIM is shown to improve both lateral and axial resolutions twofold compared with conventional multiphoton imaging while improving the sectioning and contrast of MSIM in thick, scattering samples.     

Selective aldosterone blockade suppresses atrial tachyarrhythmias in heart failure

INTRODUCTION: Renin-angiotensin-aldosterone system activation may be involved in the pathogenesis of atrial arrhythmias in congestive heart failure (CHF). The effects of aldosterone blockade on atrial tachyarrhythmias have not been evaluated. This study's aim was to determine whether selective aldosterone blockade suppresses atrial tachyarrhythmia inducibility and modifies atrial electrical and/or structural remodeling in a canine model of rapid ventricular pacing (RVP)-induced CHF. METHODS AND RESULTS: Dogs were assigned randomly to treatment with oral placebo or eplerenone (50 mg/day) and divided into four groups: two sham-operated (no RVP) and two RVP groups. After 5 weeks of no RVP or RVP at 230 beats/min along with concurrent placebo or eplerenone treatment, dogs underwent electrophysiologic and echocardiographic studies. Sustained atrial tachyarrhythmia inducibility (>10-minute duration), atrial effective refractory periods (ERPs), systolic and diastolic function, and left atrial and left ventricular (LV) chamber sizes were assessed. Placebo-treated RVP dogs developed CHF with LV systolic and diastolic dysfunction, left atrial and LV enlargement, increased atrial ERPs, and inducible sustained atrial tachyarrhythmias. Eplerenone treatment in RVP dogs significantly suppressed sustained atrial tachyarrhythmia inducibility, nonuniformly prolonged atrial ERPs and attenuated LV diastolic dysfunction without modifying left atrial or LV dilation or ejection fractions in CHF. Isoproterenol (2-4 microg/min) reversed eplerenone's atrial antiarrhythmic and ERP prolonging effects in CHF. Eplerenone did not alter atrial ERPs in sham (no RVP) dogs without CHF. CONCLUSIONS: Eplerenone suppresses inducibility of sustained atrial tachyarrhythmias, selectively prolongs atrial ERPs, and attenuates LV diastolic remodeling in RVP-induced CHF. Aldosterone blockade may be a promising new approach for atrial tachyarrhythmia prevention in CHF.     

Determinants of Intima-Media Thickness in the Young: The ALSPAC Study

ObjectivesThis study characterized the determinants of carotid intima-media thickness (cIMT) in a large (n > 4,000) longitudinal cohort of healthy young people age 9 to 21 years.BackgroundGreater cIMT is commonly used in the young as a marker of subclinical atherosclerosis, but its evolution at this age is still poorly understood.MethodsAssociations between cardiovascular risk factors and cIMT were investigated in both longitudinal (ages 9 to 17 years) and cross-sectional (ages 17 and 21 years) analyses, with the latter also related to other measures of carotid structure and stress. Additional use of ultra-high frequency ultrasound in the radial artery at age 21 years allowed investigation of the distinct layers (i.e., intima or media) that may underlie observed differences.ResultsFat-free mass (FFM) and systolic blood pressure were the only modifiable risk factors positively associated with cIMT (e.g., mean difference in cIMT per 1-SD increase in FFM at age 17: 0.007 mm: 95% confidence interval [CI]: 0.004 to 0.010; p < 0.001), whereas fat mass was negatively associated with cIMT (difference: ?0.0032; 95% CI: 0.004 to ?0.001; p = 0.001). Similar results were obtained when investigating cumulative exposure to these factors throughout adolescence. An increase in cIMT maintained circumferential wall stress in the face of increased mean arterial pressure when increases in body mass were attributable to increased FFM, but not fat mass. Risk factor?associated differences in the radial artery occurred in the media alone, and there was little evidence of a relationship between intimal thickness and any risk factor.ConclusionsSubtle changes in cIMT in the young may predominantly involve the media and represent physiological adaptations as opposed to subclinical atherosclerosis. Other vascular measures may be more appropriate for the identification of arterial disease before adulthood.