Caveolae in ventricular myocytes are required for stretch-dependent conduction slowing.

Mechanical stretch of cardiac muscle modulates action potential propagation velocity, causing potentially arrhythmogenic conduction slowing. The mechanisms by which stretch alters cardiac conduction remain unknown, but previous studies suggest that stretch can affect the conformation of caveolae in myocytes and other cell types. We tested the hypothesis that slowing of action potential conduction due to cardiac myocyte stretch is dependent on caveolae. Cardiac action potential propagation velocities, measured by optical mapping in isolated mouse hearts and in micropatterned mouse cardiomyocyte cultures, decreased reversibly with volume loading or stretch, respectively (by 19±5% and 26±4%). Stretch-dependent conduction slowing was not altered by stretch-activated channel blockade with gadolinium or by GsMTx-4 peptide, but was inhibited when caveolae were disrupted via genetic deletion of caveolin-3 (Cav3 KO) or membrane cholesterol depletion by methyl-ß-cyclodextrin. In wild-type mouse hearts, stretch coincided with recruitment of caveolae to the sarcolemma, as observed by electron microscopy. In myocytes from wild-type but not Cav3 KO mice, stretch significantly increased cell membrane capacitance (by 98±64%), electrical time constant (by 285±149%), and lipid recruitment to the bilayer (by 84±39%). Recruitment of caveolae to the sarcolemma during physiologic cardiomyocyte stretch slows ventricular action potential propagation by increasing cell membrane capacitance.

Spatial Multi-omics Reveals the Role of the Wnt Modulator, Dkk2, in Palatogenesis'.

Multiple genetic and environmental etiologies contribute to the pathogenesis of cleft palate, which is the most common of the inherited disorders of the craniofacial complex. Insights into the molecular mechanisms regulating osteogenic differentiation and patterning in the palate during embryogenesis are limited and needed for the development of innovative diagnostics and cures. This study used the Pax9-/- mouse model with a consistent phenotype of cleft secondary palate to investigate the role of Pax9 in the process of palatal osteogenesis. Although prior research has identified the upregulation of Wnt pathway modulators Dkk1 and Dkk2 in Pax9-/- palate mesenchyme, limitations of spatial resolution and technology restricted a more robust analysis. Here, data from single-nucleus transcriptomics and chromatin accessibility assays validated by in situ highly multiplex targeted single-cell spatial profiling technology suggest a distinct relationship between Pax9+ and osteogenic populations. Loss of Pax9 results in spatially restricted osteogenic domains bounded by Dkk2, which normally interfaces with Pax9 in the mesenchyme. Moreover, the loss of Pax9 leads to a disruption in the normal osteodifferentiaion of palatal osteogenic mesenchymal cells. These results suggest that Pax9-dependent Wnt signaling modulators influence osteogenic programming during palate formation, potentially contributing to the observed cleft palate phenotype.

Evaluation of the humoral immune response in BALB/c mice immunized with a naked DNA vaccine anti-methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is the major pathogen involved in nosocomial infections, leading to high rates of morbidity and mortality in hospitals worldwide. The methicillin resistance occurs due to the presence of an additional penicillin-binding protein, PBP2a, which has low affinity for beta-lactam antibiotics. In the past few years, vancomycin has been the only antibiotic option for treatment of infections caused by multiresistant MRSA; however, reports of vancomycin-resistant strains have generated great concerns regarding the treatment to overcome these infections. In the present study, we report preliminary results regarding the humoral immune response generated in BALB/c mice by two different doses of naked DNA vaccine containing an internal region, comprising the serine-protease domain, of the PBP2a of MRSA. The immunization procedure consisted of four immunizations given intramuscularly within 15-day intervals. Blood was collect weekly and anti-PBP2a-specific antibodies were screened by ELISA. BALB/c mice immunized with DNA vaccine anti-PBP2a have shown higher antibody titers mainly after the fourth immunization, and intriguingly, no correlation between the humoral immune response and DNA dose was observed. Our results suggest that the DNA vaccine anti-PBP2a induced an immune response by production of specific antibodies anti-MRSA in a non-dose-dependent manner, and it could represent a new and valuable approach to produce specific antibodies for passive immunization to overcome MRSA infections.

Cardiac-directed adenylyl cyclase expression improves heart function in murine cardiomyopathy.

BACKGROUND: We tested the hypothesis that increased cardiac myocyte adenylyl cyclase (AC) content increases cardiac function and response to catecholamines in cardiomyopathy. METHODS AND RESULTS: Transgenic mice with cardiac-directed expression of AC type VI (ACVI) were crossbred with mice with cardiomyopathy induced by cardiac-directed Gq expression. Gq mice had dilated left ventricles, reduced heart function, decreased cardiac responsiveness to catecholamine stimulation, and impaired beta-adrenergic receptor (betaAR)-dependent and AC-dependent cAMP production. Gq/AC mice showed improved basal cardiac function in vivo (P=0.01) and ex vivo (P<0.0005). When stimulated through the betaAR, cardiac responsiveness was increased (P=0.02), and cardiac myocytes showed increased cAMP production in response to isoproterenol (P=0.03) and forskolin (P<0.0001). CONCLUSIONS: Increasing myocardial ACVI content in cardiomyopathy restores cAMP-generating capacity and improves cardiac function and responsiveness to betaAR stimulation.

Intracoronary delivery of adenovirus encoding adenylyl cyclase VI increases left ventricular function and cAMP-generating capacity.

BACKGROUND: We tested the hypothesis that intracoronary injection of a recombinant adenovirus encoding adenylyl cyclase type VI (AC(VI)) would increase cardiac function in pigs. METHODS AND RESULTS: Left ventricular (LV) dP/dt and cardiac output in response to isoproterenol and NKH477 stimulation were assessed in normal pigs before and 12 days after intracoronary delivery of histamine followed by intracoronary delivery of an adenovirus encoding lacZ (control) or AC(VI) (1.4x10(12) vp). Animals that had received AC(VI) gene transfer showed increases in peak LV dP/dt (average increase of 1267+/-807 mm Hg/s; P=0.0002) and cardiac output (average increase of 39+/-20 mL. kg(-1). min(-1); P<0.0001); control animals showed no changes. Increased LV dP/dt was evident 6 days after gene transfer and persisted for at least 57 days. Basal heart rate, blood pressure, and LV dP/dt were unchanged, despite changes in cardiac responsiveness to catecholamine stimulation. Twenty-three hour ECG recordings showed no change in mean heart rate or ectopic beats and no arrhythmias. LV homogenates from animals receiving AC(VI) gene transfer showed increased AC(VI) protein content (P=0.0007) and stimulated cAMP production (P=0.0006), confirming transgene expression and function; basal LV AC activity was unchanged. Increased cAMP-generating capacity persisted for at least 18 weeks (P<0.0002). CONCLUSIONS: Intracoronary injection of a recombinant adenovirus encoding AC provides enduring increases in cardiac function.

Antagonism of platelet aggregation by 13-azaprostanoic acid in acute myocardial ischemia and sudden death.

The effects of 13-azaprostanoic acid (13-APA) were studied during acute myocardial ischemia in cats and in rabbit sudden death induced by sodium arachidonate (Na-Ar). To more clearly define the mechanism of action of 13-APA, we also examined its effects on isolated cat and rabbit coronary arteries, in vitro aggregation of cat and rabbit platelet-rich plasma (PRP) and circulating rabbit platelet count measured in vivo. 13-APA provided minimal protection during myocardial ischemia in cats, partially reversing ischemia-induced ST segment elevations by 3-5 hours after coronary artery occlusion. However, 13-APA was ineffective in inhibiting the rise in plasma creatine kinase (CK) activity or the loss of CK from ischemic myocardial tissue. 13-APA (1.0 - 100 microM) did not inhibit contraction of cat coronary arteries produced by a stable thromboxane A2 analog. However, 13-APA (100 microM) inhibited aggregation of cat PRP induced by AA (1.0 microM). 13-APA also provided significant protection against sudden death induced by Na-Ar in rabbits. While this agent was ineffective in reducing vasoconstriction of rabbit coronary arteries or inhibiting platelet aggregation in response to 500 microM AA, aggregation of rabbit PRP by 250 microM AA was completely inhibited. AA injection produced a significant decrease in circulating platelet count in vehicle-treated rabbits. However, 13-APA reduced the decrease in circulating platelet count in rabbits which survived AA injection during the 13-APA infusion. These results indicate that antagonism of thromboxane A2 receptors in platelets may be an important feature in protecting against sudden death. The difference in sensitivities of vascular and platelet thromboxane receptors as well as the accessability of 13-APA to these receptors may explain the lack of protection of 13-APA in myocardial ischemia.

Potentiation of leukotriene formation in pulmonary and vascular tissue.

Leukotriene (LT) release from vascular and pulmonary tissue was assessed by a radioimmunoassay for peptide leukotrienes (i.e., LTC4, LTD4 and LTE4). The calcium ionophore A-23187 at 1-3 micrograms/ml and platelet activating factor (PAF) at 10 micrograms/ml produced marked formation of peptide leukotrienes in minced cat pulmonary tissue. This was also confirmed by bioassay of the incubates in isolated perfused cat coronary arteries. Rat pulmonary tissue was comparable to cat with regard to LT production, but guinea-pig lung produced about 30-50% less on a weight basis. In addition, aortic and coronary artery vessel walls produced significant amounts of LTs. The time course for maximal leukotriene production occurred at 45-60 min of incubation at 37 degrees C in both the radioimmunoassay and the bioassay. Cat coronary artery constricted markedly to LTC4 or LTD4 (30-40 mm Hg) and to the lung or blood vessel incubate. This constriction was virtually totally blocked by the leukotriene antagonist FPL-55712, but not by the thromboxane receptor antagonist, pinane thromboxane A2, the alpha-adrenergic receptor antagonist, phenoxybenzamine, or the angiotensin receptor antagonist, saralasin. Thus, pulmonary and vascular tissue produce leukotrienes that appear to exert coronary constrictor effects on specific leukotriene receptors. These results indicate that the ischemia of shock and anaphylaxis may be accentuated by the release of peptide leukotrienes.

Effects of lipoxygenase inhibitors in arachidonate-induced sudden death.

Both BW 755c, a cyclo-oxygenase and lipoxygenase inhibitor, and nordihydroguaiaretic acid (NDGA), a selective lipoxygenase inhibitor, were tested for their protection against arachidonate-induced sudden death in rabbits. 100% survival was seen with BW 755c (1 mg kg-1), while NDGA showed 0 and 17% survival (2 mg kg-1 and 4 mg kg-1). BW 755c prevented 12-fold increase in plasma thromboxane B2 concentrations and the formation of pulmonary artery thrombi normally seen with arachidonate-induced sudden death, while NDGA showed no such protective effect. Radioimmunoassay of rabbit plasma for leukotrienes (LTC4, LTD4 and LTE4) indicated that they do not accumulate in blood in the model and BW 755c had no effect, suggesting that the deleterious effects seen are caused by cyclo-oxygenase pathway metabolites such as thromboxane A2, but not by lipoxygenase pathway products such as leukotrienes.

Occupational genotoxicity risk evaluation through the comet assay and the micronucleus test.

The micronucleus (MN) test and the alkaline single cell gel or comet assay were applied to exfoliated cells of the buccal mucous in order to evaluate the genotoxic risk associated with occupational exposure of 10 storage battery renovation workers, and 10 car painters, with age matched controls, in Pelotas, RS, in southern Brazil. In the MN test, 2000 exfoliated buccal cells were analyzed for each individual, while 100 cells were examined in the comet assay. In the comet test, both comet tail length and a damage index were calculated. Highly significant effects of occupational exposure were found with both the MN test and the comet assay (P<0.001). The comet assay was found to be rapid, of simple visualization, and it is a sensitive technique for measuring and analyzing DNA damage in human cells.

Determination of monensin in high-moisture cattle rations by liquid chromatography with postcolumn derivatization.

An existing liquid chromatographic method using postcolumn derivatization has been used extensively to quantitate monensin in animal feeds. Because of the relatively high moisture content of many cattle feed rations, some modifications were made to this method. Several sample-processing steps were evaluated to determine optimum sample-processing procedure. The sample weight/sample diluent ratio was modified, and method linearity was validated for the lower monensin concentrations anticipated in high-moisture cattle rations. The accuracy and precision of data generated at these lower concentrations were also determined. Because of the high moisture content of these rations, data analysis for this method required correction of feed potency for loss on drying. With these modifications, monensin can be accurately determined in high-moisture cattle rations.

Promoting safe motherhood in the community: the case for strategies that include men.

Although a decade has now passed since the launching of the Safe Motherhood Initiative, maternal mortality continues to be the health indicator showing the greatest disparity between developed and developing countries. Recently revised WHO and UNICEF figures indicate that an estimated 90% of the 585,000 worldwide maternal deaths that occur each year take place in sub-Saharan Africa and Asia. In terms of the lifetime risk of maternal death, this disparity remains striking: 1 in 12 women in parts of sub-Saharan Africa, compared with 1 in 4,000 women in Northern Europe. In addition, for every woman who dies, an estimated 16-17 will suffer from pregnancy-related complications. Research suggests that, in addition to biomedical interventions and the strengthening of health care services, improving awareness of obstetric complications among members of a pregnant woman's immediate and wider social network is an important step in improving her chances of survival when such complications occur. Many of the interventions implemented so far have focused exclusively on improving women's knowledge and practices as they relate to maternal health issues. Nevertheless, it is now increasingly being recognised that the actions required to achieve improvements in reproductive health outcomes in general, and maternal health in particular, should involve communities in the process and encourage men's active participation. Despite this, very few studies on risk perceptions or interventions to raise community awareness of obstetric risk factors, their complications and their consequences have targeted men. The present article argues for the development and testing of risk awareness interventions, which, in addition to women, target men in their familial and social roles within communities and as workers within health care services as a means of improving maternal health outcomes.

Compartmentation of G-protein-coupled receptors and their signalling components in lipid rafts and caveolae.

G-protein-coupled receptors (GPCRs) and post-GPCR signalling components are expressed at low overall abundance in plasma membranes, yet they evoke rapid, high-fidelity responses. Considerable evidence suggests that GPCR signalling components are organized together in membrane microdomains, in particular lipid rafts, enriched in cholesterol and sphingolipids, and caveolae, a subset of lipid rafts that also possess the protein caveolin, whose scaffolding domain may serve as an anchor for signalling components. Caveolae were originally identified based on their morphological appearance but their role in compartmentation of GPCR signalling has been primarily studied by biochemical techniques, such as subcellular fractionation and immunoprecipitation. Our recent studies obtained using both microscopic and biochemical methods with adult cardiac myocytes show expression of caveolin not only in surface sarcolemmal domains but also at, or close to, internal regions located at transverse tubules/sarcoplasmic reticulum. Other results show co-localization in lipid rafts/caveolae of AC (adenylyl cyclase), in particular AC6, certain GPCRs, G-proteins and eNOS (endothelial nitric oxide synthase; NOS3), which generates NO, a modulator of AC6. Existence of multiple caveolin-rich microdomains and their expression of multiple modulators of signalling strengthen the evidence that caveolins and lipid rafts/caveolae organize and regulate GPCR signal transduction in eukaryotic cells.

Studies on the mechanism of leukotriene induced coronary artery constriction.

Leukotriene (LT) C4, D4, and E4 at concentrations of 10 to 100 ng/ml were found to be potent coronary artery constrictors in the perfused cat coronary artery and perfused rat heart. In contrast, LTB4, was essentially inactive. The coronary constrictor effect of leukotrienes was not related to thromboxane release, but rather appeared to be due to a calcium mediated activation of specific leukotriene receptors.

Thromboxane A2 and peptidoleukotrienes contribute to the myocardial ischemia and contractile dysfunction in response to intracoronary infusion of complement C5a in pigs.

Intracoronary infusions of activated complement C5a result in myocardial ischemia, contractile dysfunction, and leukocyte accumulation. The hypothesis was tested that the generation of the coronary vasoconstrictors, thromboxane A2 and the 5-lipoxygenase leukotrienes (LTC4 and LTD4), contributes to the C5a-induced decrease in coronary blood flow and contractile function. The left anterior descending coronary artery in anesthetized swine was cannulated and servo pump-perfused with arterial blood at constant pressure and measured flow. Regional subendocardial contractile function was assessed with sonomicrometry. The interventricular vein was cannulated for sampling of coronary venous blood for leukocyte count. The responses in left anterior descending coronary artery blood flow and percent segment shortening to intracoronary infusions of LTC4 (1 microgram), LTD4 (1 microgram), thromboxane agonist U46619 (7.5 micrograms), and C5a (500 ng) were assessed before and after 1) LTD4/LTE4 receptor blockade with leukotriene receptor blocker LY171883 (10 mg/kg i.v.) (n = 5), 2) thromboxane A2/prostaglandin H2 receptor blockade with thromboxane receptor blocker BM13505 (2 mg/kg i.v.) (n = 5), and 3) combined thromboxane and leukotriene receptor blockade (n = 5). In the absence of receptor blockade, intracoronary C5a decreased coronary flow (50-60%) and regional segment function (60-70%) compared with the preinfusion levels. This was accompanied by a fall in coronary venous blood leukocyte levels by 5-6 x 10(6) cells/ml in the absence of alterations in arterial blood leukocyte count. Intracoronary injections of LTD4, LTC4, or U46619 also resulted in prompt decreases in coronary blood flow (50-60%) and segment function (70-80%) from preinfusion levels. Leukotriene receptor blockade with LY171883 abolished these responses to LTD4 and LTC4. Administration of LY171883 also attenuated (p less than 0.05) the myocardial response to C5a; coronary flow and segment function decreased by approximately 28% from preinfusion levels. Thromboxane receptor blockade with BM13505 eliminated the response in coronary flow and segment function to intracoronary U46619. Similar to LY171883, administration of BM13505 blunted (p less than 0.05) the C5a-induced decreases in coronary flow and contractile function, which fell by approximately 20-25% from the preinfusion level. After the combined LTD4/LTE4 receptor and thromboxane A2/prostaglandin H2 receptor blockade, intracoronary C5a resulted in little change in both coronary blood flow and segment shortening. In contrast to the flow and function effects, the C5a-induced myocardial leukocyte extraction was not decreased by leukotriene and/or thromboxane receptor blockade.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered minimal coronary resistance to antegrade reflow after chronic coronary artery occlusion in swine.

We examined coronary pressure-flow relations after chronic coronary artery occlusion induced by placement of an ameroid occluder on the left circumflex coronary artery in swine. An acute open-chest procedure was performed in nine pigs 27 +/- 2 days (mean +/- SEM) after surgical placement of the ameroid occluder, and in eight nonoperated control pigs. Coronary vascular resistances were measured during maximal coronary vasodilation with adenosine. Minimal coronary resistance was assessed before and after cannulation and extracorporeal perfusion of the left circumflex coronary artery distal to the site of the ameroid occluder in pigs from the ameroid group and in a similar site in control pigs. Minimal coronary resistance to antegrade reflow in the left circumflex region was decreased significantly in ameroid pigs compared with control pigs (0.06 +/- 0.01 vs. 0.26 +/- 0.03 mm Hg.min.100 g/ml, p less than 0.001, respectively). Decreased minimal coronary vascular resistance was present transmurally in the left circumflex region of ameroid pigs. Altered vascular resistance occurred only in myocardium distal to the ameroid occluder since the nonoccluded left anterior descending region in ameroid pigs had minimal coronary resistance similar to that of the same region from control pigs (0.23 +/- 0.03 vs. 0.19 +/- 0.02 mm Hg.min.100 g/ml). Thus altered minimal coronary vascular resistance occurs and probably reflects vascular proliferation and/or vascular alterations which result in an increased total cross-sectional area of the vasculature in the myocardium distal to the occlusion.

Outpatient surgery in pediatric urology.

Indications for ambulatory pediatric urological surgery have been broadened to include most inguinal and scrotal surgery, many endoscopic procedures and distal hypospadias repairs with or without chordee or urethroplasty. We have reviewed a 1-year experience with the 440 outpatient urological procedures performed at Children's Hospital of Michigan in 1984, and found a low (3.4 per cent) incidence of postoperative hospitalization and only a single complication. We conclude that outpatient surgery in well selected patients and procedures is safe, timely and economical.

The pig as a model for myocardial ischemia and exercise.

The pig has been well characterized as an appropriate model for the study of coronary physiology, the coronary collateral circulation and exercise physiology. We compared both Yucatan miniature swine and young farm pigs in experiments involving myocardial ischemia, infarction and exercise. The Yucatan pig was vigorous, docile and proved to be an appropriate model of coronary physiology and exercise in man. The exercise capacity of the Yucatan pig was greater than that of the similar weight Hampshire pig, apparently because of the higher hematocrit and larger heart size. Both breeds were able to increase their maximal oxygen consumption (VO2 max) by approximately 25% after 10 weeks of training. Experiments measuring maximal coronary capacity suggest that the vascular capacity was similar to that of man, but less than that of the dog. Acute occlusion of the coronary artery in pigs infarcted most of the tissue of the vascular bed at risk. The collateral circulation of the pig is less than one fourth that of the dog and is similar to that of man. Slow occlusion of the left circumflex coronary artery produces an ischemic vascular bed which is collaterally dependent with only 5% infarction. Collateral flow is sufficient to meet resting conditions, but during exercise, severe ischemia is unmasked. This ischemia is present for up to 16 weeks following occlusion. The observation of limited infarction in conjunction with limited collateral vessel development suggests that this is a good model for investigating the growth and development of coronary collateral circulation in man.

Coronary collateral reserve during exercise induced ischemia in swine.

We determined coronary collateral vasodilator reserve during exercise-induced ischemia in 17 mini-swine. We induced coronary collateral development in the left circumflex bed by placing an ameroid occluder on that artery. Four weeks later we studied the animals at rest and during exercise (EX) eliciting heart rates (HR) of 240 and 265 beats/min. We measured myocardial blood flow with microspheres and myocardial function by wall thickness sonomicrometry gauges. At matched exercise HRs we treated the animals with nifedipine (10 micrograms/kg IV) (EXN 10), nifedipine (100 micrograms/kg IV), (EXN 100), and adenosine infusion (1.2 mg/min/kg) EXAD. EXN 10 did not significantly alter hemodynamics compared to EX but EXN 100 and EXAD both decreased blood pressure significantly (p less than 0.05). Ischemic endocardial/nonischemic endocardial flow ratios and collateral resistance served as indices of vasodilator reserve. In the ischemic zone exercise reduced vasodilator reserve to 24 +/- 3% in the endocardium and 64 +/- 7% in the epicardium. Neither EXN 10 nor EXAD improved exercise-induced ischemia measured either as flow or function. However EXN 100 improved function during exercise-induced ischemia without improving coronary collateral flow. We conclude there is no additional coronary flow reserve during exercise-induced ischemia in the collateral dependent bed of the pig a few days after occlusion that can be recruited. Large doses of nifedipine improve function by direct action on the myocardium or by reducing afterload. The lack of development and deep myocardial distribution of the coronary collateral vessels in the pig may be an important factor of why these nifedipine responses differ from those reported in species which have primarily large epicardial coronary collaterals.