Gq-activated fibroblasts induce cardiomyocyte action potential prolongation and automaticity in a three-dimensional microtissue environment.

Cardiac fibroblasts (CFs) are known to regulate cardiomyocyte (CM) function in vivo and in two-dimensional in vitro cultures. This study examined the effect of CF activation on the regulation of CM electrical activity in a three-dimensional (3-D) microtissue environment. Using a scaffold-free 3-D platform with interspersed neonatal rat ventricular CMs and CFs, Gq-mediated signaling was selectively enhanced in CFs by Gαq adenoviral infection before coseeding with CMs in nonadhesive hydrogels. After 3 days, the microtissues were analyzed by signaling assay, histological staining, quantitative PCR, Western blots, optical mapping with voltage- or Ca2+-sensitive dyes, and microelectrode recordings of CF resting membrane potential (RMPCF). Enhanced Gq signaling in CFs increased microtissue size and profibrotic and prohypertrophic markers. Expression of constitutively active Gαq in CFs prolonged CM action potential duration (by 33%) and rise time (by 31%), prolonged Ca2+ transient duration (by 98%) and rise time (by 65%), and caused abnormal electrical activity based on depolarization-induced automaticity. Constitutive Gq activation in CFs also depolarized RMPCF from -33 to -20 mV and increased connexin 43 and connexin 45 expression. Computational modeling confers that elevated RMPCF and increased cell-cell coupling between CMs and CFs in a 3-D environment could lead to automaticity. In conclusion, our data demonstrate that CF activation alone is capable of altering action potential and Ca2+ transient characteristics of CMs, leading to proarrhythmic electrical activity. Our results also emphasize the importance of a 3-D environment where cell-cell interactions are prevalent, underscoring that CF activation in 3-D tissue plays a significant role in modulating CM electrophysiology and arrhythmias.NEW & NOTEWORTHY In a three-dimensional microtissue model, which lowers baseline activation of cardiac fibroblasts but enables cell-cell, paracrine, and cell-extracellular matrix interactions, we demonstrate that selective cardiac fibroblast activation by enhanced Gq signaling, a pathophysiological trigger in the diseased heart, modulates cardiomyocyte electrical activity, leading to proarrhythmogenic automaticity.

Functional scaffold-free 3-D cardiac microtissues: a novel model for the investigation of heart cells.

To bridge the gap between two-dimensional cell culture and tissue, various three-dimensional (3-D) cell culture approaches have been developed for the investigation of cardiac myocytes (CMs) and cardiac fibroblasts (CFs). However, several limitations still exist. This study was designed to develop a cardiac 3-D culture model with a scaffold-free technology that can easily and inexpensively generate large numbers of microtissues with cellular distribution and functional behavior similar to cardiac tissue. Using micromolded nonadhesive agarose hydrogels containing 822 concave recesses (800 µm deep × 400 µm wide), we demonstrated that neonatal rat ventricular CMs and CFs alone or in combination self-assembled into viable (Live/Dead stain) spherical-shaped microtissues. Importantly, when seeded simultaneously or sequentially, CMs and CFs self-sorted to be interspersed, reminiscent of their myocardial distribution, as shown by cell type-specific CellTracker or antibody labeling. Microelectrode recordings and optical mapping revealed characteristic triangular action potentials (APs) with a resting membrane potential of -66 ± 7 mV (n = 4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100 ± 30 ms and 18.0 ± 1.9 cm/s, respectively (n = 5 each). The presence of CFs led to a twofold AP prolongation in heterogenous microtissues (CM-to-CF ratio of 1:1). Importantly, Ba(2+)-sensitive inward rectifier K(+) currents and Ca(2+)-handling proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, were detected in CM-containing microtissues. Furthermore, cell type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we developed a novel scaffold-free cardiac 3-D culture model with several advancements for the investigation of CM and CF function and cross-regulation.

Traversing the ovine cervix - a challenge for cryopreserved semen and creative science.

This review brings together research findings on cervical relaxation in the ewe and its pharmacological stimulation for enhancement of the penetration needed for transcervical insemination and embryo transfer. On the basis that the success of artificial insemination is the percentage of ewes lambing, a review is made of recent research aimed at understanding and minimising the sub-lethal effects of freezing and thawing on the viability of spermatozoa, their membrane integrity and their ability to migrate through cervical mucus, as these characteristics have a major influence on fertility, particularly when semen is deposited, artificially, in the os cervix. Milestones of achievement are given for transcervical intrauterine insemination, embryo recovery and transfer and the birth of lambs of pre-determined sex, firstly following intracytoplasmic sperm injection, then laparoscopic intrauterine insemination using highly diluted flow-cytometrically sorted fresh semen and subsequently by os cervix insemination using sexed semen that had been frozen and thawed. Diversity of research endeavour (applied, cellular, molecular), research discipline (anatomy, histology, immunology, endocrinology) and research focus (cell, tissue, organ, whole animal) is embraced within the review as each has significant contributions to make in advancing recent scientific findings from the laboratory into robust on-farm transcervical insemination and embryo transfer techniques.

Risk factors for asthma incidence. A review of recent prospective evidence.

AIM: The aim of this study is to determine what factors have been shown, in prospective studies, to predict the incidence of asthma. METHODS: We performed a systematic review of peer-reviewed literature from 1994 to 2004 to determine what factors predict the development of asthma in both children and adults. This search strategy yielded 40 studies, with 36 providing some estimate of asthma incidence for the total sample and or a specific subgroup. RESULTS: Annual estimated incidence of physician-diagnosed asthma ranged from 0.6 to 29.5 per 1000 persons. Risk factors for incident asthma among children included: male sex, atopic sensitization, parental history of asthma, early-life stressors and infections, obesity, and exposure to indoor allergens, tobacco smoke and outdoor pollutants. Risk factors for adult-onset asthma included female sex, airway hyperresponsiveness, lifestyle factors, and work-related exposures. CONCLUSION: Risk factors for asthma include both modifiable and nonmodifiable ones, and they vary between children and adults. This review of prospective evidence supports tobacco and smoke avoidance as an intervention for the primary prevention of childhood asthma. During adolescence and adulthood, targeting lifestyle factors like obesity and smoking or reducing occupational exposures are the best opportunities for asthma prevention. Before specific public health recommendations can be made, however, additional longitudinal research is needed to better characterize target populations and identify appropriate settings for multifaceted asthma interventions.

Lambing rates and litter sizes following intrauterine or cervical insemination of frozen/thawed semen with or without oxytocin administration.

Intrauterine insemination by laparoscopy is required to achieve acceptable lambing rates in ewes when using frozen semen but the procedure has evoked welfare concerns. Oxytocin has been used to dilate the cervix as a means of accessing the uterus during conventional cervical insemination, but its effect on fertility is not well documented. Three hundred crossbred ewes were synchronised in estrus and randomly allocated to one of three insemination procedures using frozen/thawed semen containing 400 x 10(6)/ml progressively motile sperm: single cervical (0.2 ml), multiple cervical (4 x 0.05 ml) or laparoscopic (0.05 ml per uterine horn). The effects of each insemination procedure on lambing rate (percentage of treated ewes lambing) and litter size (lambs per ewe lambing) were tested with and without oxytocin (10 IU given i.m.) prior to fixed-time insemination. Oxytocin did not permit complete cervical penetration in any ewes and neither lambing rate nor litter size was influenced by the number of inseminations. Lambing percentages were 69 and 42 (P < 0.01) for the laparoscopic and cervical insemination methods, respectively, and oxytocin reduced these to 58 (NS) and 10 (P < 0.001) percent, respectively. Corresponding litter sizes for ewes not receiving oxytocin were 1.91 and 1.51 and for those receiving oxytocin, 1.83 and 1.41 (laparoscopic versus cervical, P < 0.02). Thus, in the absence of complete cervical penetration at insemination, 10 IU oxytocin decreased the number of ewes lambing but had no effect on their litter size.

Effect of diet and GnRH administration on post-partum ovarian cyclicity in autumn-lambing ewes.

Using autumn-lambing ewes, this study investigated (i) the effects of diet on gonadotrophin secretion and responsiveness of the hypothalamic-pituitary-ovarian axis to exogenous GnRH during the early post-partum period; and (ii) whether ovulation prior to completion of uterine involution results in an increased incidence of aberrant ovarian cycles. Thirty-two ewes rearing 1.9+/-0.12 lambs were equally allocated to two dietary treatments at lambing (22 October +/-0.2 day). Diets comprised ad libitum hay and 1.5 kg per ewe per day of one of two concentrates (11.5 MJ ME, 195 g CP per kg) containing 300 g kg(-1) cracked maize grain (M) or 300 g kg(-1) sugar beet pellets (S). Half of the ewes on each diet (G) received 25 i.v. injections of 250 ng GnRH in 2 ml 0.9% saline at 2 h intervals from days 12-14 post-partum while remaining ewes (N) were monitored for the resumption of spontaneous ovarian cyclicity. Blood samples were obtained from all ewes throughout the study (lambing to 18 December) for measurement of circulating hormone concentrations and the uteri and ovaries of all ewes were examined via laparoscopy on day 21 post-partum. There were no effects of dietary treatment on ewe daily live weight loss, lamb daily live weight gain or the immediate post-partum increase in circulating FSH concentrations. Diet did not affect insulin concentrations or LH pulse frequency on day 12 post-partum but LH pulse amplitude was lower in ewes fed concentrate M compared to concentrate S (1.4+/-0.10 versus 1.7+/-0.12 ng ml(-1), respectively, P<0.05) and this was associated with an increased interval to the resumption of spontaneous ovarian cycles (35+/-3.1 versus 26+/-2.1 day, respectively, P<0.05). Administration of exogenous GnRH increased (P<0.05) the proportion of ewes on both diets that ovulated within 20 days of parturition and advanced the onset of ovarian cyclicity in ewes fed concentrate M by 9.5 days (significance of interaction, P<0.05). Four ewes, all of which ovulated before day 22 post-partum, had extended luteal activity while in remaining ewes, duration of the first luteal phase was inversely related to the time of first ovulation (r(2)=0.16, P<0.05). Results demonstrate that (i) the onset of ovarian cyclicity is influenced by diet and can be advanced by administration of exogenous GnRH; and (ii) ovulation during the early post-partum period is associated with an increased incidence of extended luteal activity.

Interleukin 8 in the cervix of non-pregnant ewes.

Studies of cervical artificial insemination of ewes at hormone-synchronized oestrus indicate that the cervix remains relatively impenetrable to semen, in contrast to naturally breeding animals. During parturition the inflammatory response plays an important part in cervical dilation and possibly, to a lesser extent, in the non-pregnant cervix at oestrus to facilitate the transcervical transport of semen. The expression of interleukin 8 (IL-8), a pro-inflammatory cytokine in the ovine cervix, has been mapped and quantified, using semi-quantitative in situ hybridization, to ascertain the role played by inflammation in the ovine cervix during natural and artificially induced oestrous cycles. IL-8 gene expression was observed in both the luminal epithelium and fibroblastic cells of the cervix. The presence of IL-8 was confirmed using immunohistochemistry. IL-8 gene expression in the luminal epithelium varied throughout the oestrous cycle and was highest at oestrus and at day 5 of the oestrous cycle. In ewes artificially induced to ovulate, either by the withdrawal of progesterone pessaries after treatment for 12 days, or by two i.m. injections of prostaglandin 9 days apart, IL-8 gene expression at oestrus was significantly lower than it was at natural oestrus. Insemination increased IL-8 gene expression in progesterone-synchronized ewes. These data support the hypothesis that IL-8-induced inflammation is important in normal cervical function and that this process is inhibited during artificial synchronization of the oestrous cycle and is increased by exposure to semen.

Clinical applications of wall-stress analysis in the pediatric intensive care unit.

OBJECTIVE: To determine whether serial, noninvasive assessment of afterload, contractility, and Doppler-derived cardiac output reliably detects variations in cardiac function in unstable pediatric patients. DESIGN: Prospective, blinded clinical trial. SETTING: The pediatric intensive care unit at Massachusetts General Hospital. PATIENTS: Fourteen critically ill pediatric patients. INTERVENTIONS: Pediatric patients meeting criteria for hemodynamic instability underwent serial echocardiograms every 6 hrs until they met exit criteria, generating 75 studies. MEASUREMENTS AND MAIN RESULTS: Shortening fraction, cardiac index (CI), end-systolic wall stress (ESWS), and corrected velocity of circumferential shortening (Vcfc) were measured in each patient. Data points were plotted as a stress-velocity relationship, compared with published normal values, then correlated with changes in vital signs and pharmacologic interventions. Fourteen of 16 patients who were enrolled completed the study. A strong negative correlation between ESWS and Vcfc was confirmed (p < .001). As an internal measure of validity, Vcfc had a strong positive correlation with CI measurements (p = .012). An increase in dopamine infusion was associated with a fall in ESWS (p = .02), an increase in Vcfc (p = .03), and an increase in the CI as measured by Doppler (p = .035). The infusion of dopamine above renal perfusion levels moved patients from zones of normal or compensated contractility for afterload on a modified stress-velocity relationship to a zone of high contractility for afterload. Urine output was the only clinical index of cardiac function that had a significant correlation with the echocardiographic indices. Hemodynamically unstable patients followed similar patterns of deterioration and recovery on the modified stress-velocity graph. All surviving patients returned to a normal or compensated zone. CONCLUSIONS: Wall-stress analysis of cardiac function is easily and safely performed on mechanically ventilated pediatric patients with the production of consistently high-quality data. For internal validity, Vcfc and CI measurements were correlated and were strongly positive. Wall-stress indices reliably detected patient deterioration, recovery, and response to changes in dopamine infusion. Patients who failed to return to areas of normal or compensated levels of contractility and afterload did poorly in this study. Noninvasive measures of afterload and contractility appear useful for monitoring cardiac function of critically ill children in an intensive care setting.

Exercise reduces daily fatigue in women with breast cancer receiving chemotherapy.

PURPOSE: Cancer treatment-related fatigue is the most prevalent and distressing symptom of cancer therapy. Interventions to minimize fatigue are needed. The purpose of this study was to examine the relationship between exercise and fatigue over the first three cycles of chemotherapy in women receiving either cyclophosphamide, methotrexate, and fluorouracil (CMF) or doxorubicin and cyclophosphamide (AC) for breast cancer. METHODS: Seventy-two newly diagnosed women with breast cancer were instructed in a home-based moderate-intensity exercise intervention. Measures of functional ability, energy expenditure, and fatigue were obtained at baseline and posttest. Subjects maintained daily records of four types of fatigue, and exercise duration, intensity, and type. RESULTS: Exercise significantly reduced all four levels of fatigue (P < 0.01). As the duration of exercise increased, the intensity of fatigue declined (P < 0.01). There was a significant carry-over effect of exercise on fatigue, but the effect lasted only 1 d. The level of fatigue at study entry was not associated with number of days of exercise or amount of exercise a woman engaged in. CONCLUSIONS: The impact of exercise on fatigue was significant and suggests the effectiveness of a low- to moderate-intensity regular exercise program in maintaining functional ability and reducing fatigue in women with breast cancer receiving chemotherapy.

Structural analysis of Pick's disease-derived and in vitro-assembled tau filaments.

Pick's and Alzheimer's diseases are distinct neurodegenerative disorders both characterized in part by the presence of intracellular filamentous tau protein inclusions. The tight bundles of paired helical filaments (PHFs) of tau protein found in Alzheimer's disease (AD) differ from the tau filaments of Pick's disease in their morphology, distribution, and pathological structure as identified by silver impregnation. The filaments of Pick's disease are loosely arranged in pathognomonic spherical inclusions found in ballooned neurons, whereas the tau pathology of AD is classically described as a triad of neuropil threads, neurofibrillary tangles, and dystrophic neurites surrounding and invading plaques. In this study we used the high-resolution technique of scanning transmission electron microscopy to characterize and compare the filaments found in Pick's disease with those found in AD. In addition, we determined the mass/nm length and density of arachidonic acid-induced in vitro-assembled filaments. Three morphologically distinct populations of Pick's filaments were identified but each was indistinguishable from AD-PHFs in mass/nm length and density. Filaments assembled in vitro from single isoforms were similar in mass/nm length, but less dense than AD-PHFs and Pick's disease filaments. Finally, we provide clear structural evidence that a PHF, whether found in disease or assembled in vitro, is composed of two distinct intertwined filaments.

Concept, development, administration, and analysis of a certifying examination in echocardiography for physicians.

In 1993, the American Society of Echocardiography appointed a committee to develop an objective examination in echocardiography. In 1995, a pilot of this examination was administered, with operational examinations offered each year from 1996 to 1999. This report describes the development of the examination, including its underlying philosophy, the test itself, and the scoring process, and includes results from the first 4 examinations. To date, 1266 physicians have taken the examination, and roughly 60% of those have passed. The number of echocardiograms performed or interpreted each week had the largest effect on examination scores; the effects of both the amount of training and the practice discipline were small but significant. The evolution of the original committee and new directions for the testing organization are also discussed.

Oxidative regulation of fatty acid-induced tau polymerization.

Alzheimer's disease (AD) is characterized by the presence of amyloid-positive senile plaques and tau-positive neurofibrillary tangles. Aside from these two pathological hallmarks, a growing body of evidence indicates that the amount of oxidative alteration of vulnerable molecules such as proteins, DNA, and fatty acids is elevated in the brains of AD patients. It has been hypothesized that the elevated amounts of protein oxidation could lead directly to the formation of neurofibrillary tangles through a cysteine-dependent mechanism. We have tested this hypothesis in an in vitro system in which tau assembly is induced by fatty acids. Using sulfhydryl protective agents and site-directed mutagenesis, we found that cysteine-dependent oxidation of the tau molecule is not required for its polymerization and may even be inhibitory. However, by adjusting the oxidative environment of the polymerization reaction through the addition of a strong antioxidant or through the addition of an oxidizing system consisting of iron, adenosine diphosphate, and ascorbate, we found that oxidation does play a major role in our in vitro paradigm. The results indicated that fatty acid oxidation, the amount of which is found to be elevated in AD patients, can facilitate the polymerization of tau. However, "overoxidation" of the fatty acids can inhibit the process. Therefore, we postulate that specific fatty acid oxidative products could provide a direct link between oxidative stress mechanisms and the formation of neurofibrillary tangles in AD.

In vitro polymerization of tau protein monitored by laser light scattering: method and application to the study of FTDP-17 mutants.

Tau polymerization into the filaments that compose neurofibrillary tangles is seminal to the development of many neurodegenerative diseases. It is therefore important to understand the mechanisms involved in this process. However, a consensus method for monitoring tau polymerization in vitro has been lacking. Here we demonstrate that illuminating tau polymerization reactions with laser light and measuring the increased scattering at 90 degrees to the incident beam with a digital camera results in data that closely approximate the mass of tau polymer formation in vitro. The validity of the technique was demonstrated over a range of tau concentrations and through multiple angle scattering measurements. In addition, laser light scattering data closely correlated with quantitative electron microscopy measurements of the mass of tau filaments. Laser light scattering was then used to measure the efficiency with which the mutant tau proteins found in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) form filamentous structures. Several of these mutant proteins display enhanced polymerization in the presence of arachidonic acid, suggesting a direct role for these mutations in tau the filament formation that characterizes FTDP-17.

Closure of patent foramen ovale for paradoxical emboli: intermediate-term risk of recurrent neurological events following transcatheter device placement.

OBJECTIVES: We report the largest and the longest follow-up to date of patients who underwent transcatheter patent foramen ovale (PFO) closure for paradoxical embolism. BACKGROUND: Closure of a PFO has been proposed as an alternative to anticoagulation in patients with presumed paradoxical emboli. METHODS: Data were collected for patients following PFO closure with the Clamshell, CardioSEAL or Buttoned Devices at two institutions. RESULTS: There were 63 patients (46 +/- 18 years) with a follow-up of 2.6 +/- 2.4 years. Fifty-four (86%) had effective closure of the foramen ovale (trivial or no residual shunt by echocardiography) while seven (11%) had mild and two (3%) had moderate residual shunting. There were four deaths (leukemia, pulmonary embolism, sepsis following a hip fracture and lung cancer). There were four recurrent embolic neurological events following device placement: one stroke and three transient events. The stroke occurred in a 56-year-old patient six months following device placement. A follow-up transesophageal echocardiogram showed a well seated device without residual shunting. Two of the four events were associated with suboptimal device performance (one patient had a significant residual shunt and a second patient had a "friction lesion" in the left atrial wall associated with a displaced fractured device arm). The risk of recurrent stroke or transient neurological event following device placement was 3.2% per year for all patients. CONCLUSION: Transcatheter closure of PFO is an alternative therapy for paradoxical emboli in selected patients. Improved device performance may reduce the risk of recurrent neurological events. Further studies are needed to identify patients most likely to benefit from this intervention.

Differential assembly of human tau isoforms in the presence of arachidonic acid.

Six tau isoforms arise from the alternative splicing of a single gene in humans. Insoluble, filamentous deposits of tau protein occur in a number of neurodegenerative diseases, and in some of these diseases, the deposition of polymers enriched in certain tau isoforms has been documented. Because of these findings, we have undertaken studies on the efficacy of fatty acid-induced polymerization of the individual tau isoforms found in the adult human CNS. The polymerization of each tau isoform in the presence of two concentrations of arachidonic acid indicated that isoforms lacking N-terminal exons e2 and e3 formed small, globular oligomers that did not go on to elongate into straight (SF) or paired helical (PHF) filaments under our buffer conditions. The polymerization of all isoforms containing e2 or e2 and e3 occurred readily at a high arachidonic acid concentration. Conversely, at a lower arachidonic acid concentration, only tau isoforms containing four microtubule binding repeats assembled well. Under all buffer conditions employed, filaments formed from three of the isoforms containing e2 and e3 resembled SFs in morphology but began to form PHF-like structures following extended incubation at 37 degrees C. These results indicate that polymerization of the intact tau molecule may be facilitated by e2 and e3. Moreover, tau isoforms containing three versus four microtubule binding repeats display different assembly properties depending on the solvent conditions employed.

Fatigue patterns observed in patients receiving chemotherapy and radiotherapy.

The purpose of this study was to describe the patterns of cancer-related fatigue (CRF) and vigor in patients receiving chemotherapy or radiation therapy. Five studies that measured fatigue and vigor with the Profile of Mood States were used to describe the pattern of CRF and vigor during and after both types of treatment. Repeated-measures ANOVA was used to determine differences over time in each study. Results demonstrate different patterns of CRF for patients receiving chemotherapy and radiation therapy. Chemotherapy-related CRF peaks in the days after chemotherapy, whereas radiation therapy-related CRF gradually accumulates over the course of treatment. The CRF associated with both forms of treatment gradually declines over time. The prevalence, intensity, and persistence of CRF during treatment and for months after treatment is complete make this symptom one that cannot be ignored.

Ligand-dependent tau filament formation: implications for Alzheimer's disease progression.

The mechanism through which arachidonic acid induces the polymerization of tau protein into filaments under reducing conditions was characterized through a combination of fluorescence spectroscopy and electron microscopy. Results show that polymerization follows a ligand-mediated mechanism, where binding of arachidonic acid is an obligate step preceding tau-tau interaction. Homopolymerization begins with rapid (on the order of seconds) nucleation, followed by a slower elongation phase (on the order of hours). Although essentially all synthetic filaments have straight morphology at early time points, they interact with thioflavin-S and monoclonal antibody Alz50 much like authentic paired helical filaments, suggesting that the conformation of tau protein is similar in the two filament forms. Over a period of days, synthetic straight filaments gradually adopt paired helical morphology. These results define a novel pathway of tau filament formation under reducing conditions, where oxidation may contribute to final paired helical morphology, but is not a necessary prerequisite for efficient nucleation or elongation of tau filaments.