Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the “gold-standard” in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.     

Ex Vivo Assessment of Contractility,Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca²⁺ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca²⁺ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.     

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain,Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized. The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed.     

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available ^1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important ^{7, 14, 30, 39}. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP ³⁹. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds³⁹. In addition to infarct staining, we tested cardiac troponin I (cTnI) as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction^{6, 8, 30, 40, 41}. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.     

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-α 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-α 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.     

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.     

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.     

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes ¹.Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension ². Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases ³. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention ⁴. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery^5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography^5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.     

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner''s time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems^1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control⁹. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.     

A hydrophilic microenvironment in the substrate-translocating groove of the YidC membrane insertase is essential for enzyme function

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.     

CO2 starvation experiments provide support for the carbon-limited hypothesis on the evolution of CAM-like behaviour in Isoëtes

Background and AimsCrassulacean acid metabolism (CAM) is an adaptation to increase water use efficiency in dry environments. Similar biochemical patterns occur in the aquatic lycophyte genus Isoëtes. It has long been assumed and accepted that CAM-like behaviour in these aquatic plants is an adaptation to low daytime carbon levels in aquatic ecosystems, but this has never been directly tested.MethodsTo test this hypothesis, populations of Isoëtes engelmannii and I. tuckermanii were grown in climate-controlled chambers and starved of atmospheric CO₂ during the day while pH was measured for 24 h.Key ResultsWe demonstrate that terrestrial plants exposed to low atmospheric CO₂ display diel acidity cycles similar to those in both xerophytic CAM plants and submerged Isoëtes.ConclusionsDaytime CO₂ starvation induces CAM-like nocturnal acid accumulation in terrestrial Isoëtes, substantiating the hypothesis that carbon starvation is a selective pressure for this physiological behaviour.     

Impact of estradiol on insulin signaling in the rat heart

It is well known that variation in the concentration of estrogens affects insulin action. In this study we examine the impact of estradiol (E2) on insulin signaling in the rat heart. Ovariectomized female rats were treated with E2 6 h prior to analysis of basal protein and mRNA content of insulin signaling molecules, and additionally with insulin 30 min before the experiment to delineate E2 effects on phosphorylations and molecular associations relevant for insulin signaling. The results show that E2 decreased insulin receptor (IR) tyrosine phosphorylation, while it did not alter IR protein and mRNA content. E2 administration did not change IR substrate 1 (IRS‐1) protein content and tyrosine phosphorylation, while decreased mRNA content and increased its association with the p85 subunit of phosphatidylinositol 3‐kinase (PI3K). E2 decreased protein and mRNA content of IR substrate 2 (IRS‐2), while did not change IRS‐2 tyrosine phosphorylation and IRS‐2 association with p85. The increase of IRS‐1/p85 is accompanied by increase of p85 protein and mRNA levels, and by stimulation of protein kinase B (Akt) Ser⁴⁷³ phosphorylation. In contrast, Akt protein and mRNA content were not changed. In summary, although in some aspects cardiac insulin signaling is obviously improved by E2 treatment (increase of p85 mRNA and protein levels, enhancement of IRS‐1/p85 association and Ser⁴⁷³Akt phosphorylation), the observed decrease of IR tyrosine phosphorylation, IRS‐2 protein content, and IRSs mRNA contents, suggest very complex interplay of beneficial and suppressive effects of E2, both genomic and non‐genomic, in regulation of heart insulin signaling. Copyright © 2009 John Wiley & Sons, Ltd.     

Influence of the variation of geometrical and topological traits on light interception efficiency of apple trees: sensitivity analysis and metamodelling for ideotype definition

Background and Aims

The impact of a fruit tree''s architecture on its performance is still under debate, especially with regard to the definition of varietal ideotypes and the selection of architectural traits in breeding programmes. This study aimed at providing proof that a modelling approach can contribute to this debate, by using in silico exploration of different combinations of traits and their consequences on light interception, here considered as one of the key parameters to optimize fruit tree production.

Methods

The variability of organ geometrical traits, previously described in a bi-parental population, was used to simulate 1- to 5-year-old apple trees (Malus × domestica). Branching sequences along trunks observed during the first year of growth of the same hybrid trees were used to initiate the simulations, and hidden semi-Markov chains previously parameterized were used in subsequent years. Tree total leaf area (TLA) and silhouette to total area ratio (STAR) values were estimated, and a sensitivity analysis was performed, based on a metamodelling approach and a generalized additive model (GAM), to analyse the relative impact of organ geometry and lateral shoot types on STAR.

Key Results

A larger increase over years in TLA mean and variance was generated by varying branching along trunks than by varying organ geometry, whereas the inverse was observed for STAR, where mean values stabilized from year 3 to year 5. The internode length and leaf area had the highest impact on STAR, whereas long sylleptic shoots had a more significant effect than proleptic shoots. Although the GAM did not account for interactions, the additive effects of the geometrical factors explained >90% of STAR variation, but much less in the case of branching factors.

Conclusions

This study demonstrates that the proposed modelling approach could contribute to screening architectural traits and their relative impact on tree performance, here viewed through light interception. Even though trait combinations and antagonism will need further investigation, the approach opens up new perspectives for breeding and genetic selection to be assisted by varietal ideotype definition.     

Effect of Bursaphelenchus xylophilus on the Assimilation and Translocation of 14 C in Pinus sylvestris

The effect of wound, wound + water, wound + Bursaphelenchus xylophilus culture filtrate, or wound + lethal B. xylophilus doses on the assimilation and translocation of ¹⁴C by 8-month-old Pinus sylvestris seedlings was tested. In two separate experiments, pine seedlings were exposed to 28.35 μCi of ¹⁴CO₂ for 20 minutes below or above (to the pine shoot leader) the point of nematode inoculation. After 2 and 4 hours of dark adaptation, 80% ethanol soluble ¹⁴C tissue extracts were determined by liquid scintillation counting. Nematode infection significantly (P = 0.05) decreased ¹⁴C assimilation. Treatments translocated less than 6% of the total amount of the fixed ¹⁴C and translocation generally decreased with increasing size of nematode inoculum. However, infected pines translocated a greater proportion of the amount of ¹⁴C fixed per gram of exposed nematode-plant tissue than did the control pines. The lower levels of photoassimilate entering the plant system probably resulted in a reduced metabolic capacity in B. xylophilus-infected pine seedlings. The effect on photosynthesis could be one of the key factors leading to death of pines through starvation, and it is possible that it was preceded by an effect on related physiological processes such as water uptake.     

Effect of Thymol on the spontaneous contractile activity of the smooth muscles

Effects of Thymol on the spontaneous contractile activity (SCA) have been found in in vitro experiments with circular smooth-muscle strips (SMAs) from guinea pig stomach and vena portae. Thymol was found to possess an agonistic effect on the alpha(1)-, alpha(2)- and beta-adrenergic receptors. Its spasmolytic effect is registered at doses higher than 10(-6)M. Thymol in a dose of 10(-4)M inhibits 100% the SCA of the SMAs and reduces the excitatory effect of 10(-5)M ACH to 35%. It is assumed that Thymol has an analgesic effect through its action on the alpha(2)-adrenergic receptors of the nerve cells. By influencing the beta-adrenergic receptors in the adipose cells, it is possible to induce increased synthesis of fatty acids and glycerol, which is a prerequisite for increased heat release.     

Thermal transitions in serum amyloid A in solution and on the lipid: implications for structure and stability of acute-phase HDL

Serum amyloid A (SAA) is an acute-phase protein that circulates mainly on plasma HDL. SAA interactions with its functional ligands and its pathogenic deposition in reactive amyloidosis depend, in part, on the structural disorder of this protein and its propensity to oligomerize. In vivo, SAA can displace a substantial fraction of the major HDL protein, apoA-I, and thereby influence the structural remodeling and functions of acute-phase HDL in ways that are incompletely understood. We use murine SAA1.1 to report the first structural stability study of human plasma HDL that has been enriched with SAA. Calorimetric and spectroscopic analyses of these and other SAA-lipid systems reveal two surprising findings. First, progressive displacement of the exchangeable fraction of apoA-I by SAA has little effect on the structural stability of HDL and its fusion and release of core lipids. Consequently, the major determinant for HDL stability is the nonexchangeable apoA-I. A structural model explaining this observation is proposed, which is consistent with functional studies in acute-phase HDL. Second, we report an α-helix folding/unfolding transition in SAA in the presence of lipid at near-physiological temperatures. This new transition may have potentially important implications for normal functions of SAA and its pathogenic misfolding.     

Impact of apolipoprotein A1- or lecithin:cholesterol acyltransferase-deficiency on white adipose tissue metabolic activity and glucose homeostasis in mice

High density lipoprotein (HDL) has attracted the attention of biomedical community due to its well-documented role in atheroprotection. HDL has also been recently implicated in the regulation of islets of Langerhans secretory function and in the etiology of peripheral insulin sensitivity. Indeed, data from numerous studies strongly indicate that the functions of pancreatic β-cells, skeletal muscles and adipose tissue could benefit from improved HDL functionality. To better understand how changes in HDL structure may affect diet-induced obesity and type 2 diabetes we aimed at investigating the impact of Apoa1 or Lcat deficiency, two key proteins of peripheral HDL metabolic pathway, on these pathological conditions in mouse models. We report that universal deletion of apoa1 or lcat expression in mice fed western-type diet results in increased sensitivity to body-weight gain compared to control C57BL/6 group. These changes in mouse genome correlate with discrete effects on white adipose tissue (WAT) metabolic activation and plasma glucose homeostasis. Apoa1-deficiency results in reduced WAT mitochondrial non-shivering thermogenesis. Lcat-deficiency causes a concerted reduction in both WAT oxidative phosphorylation and non-shivering thermogenesis, rendering lcat^?/? mice the most sensitive to weight gain out of the three strains tested, followed by apoa1^?/? mice. Nevertheless, only apoa1^?/? mice show disturbed plasma glucose homeostasis due to dysfunctional glucose-stimulated insulin secretion in pancreatic β-islets and insulin resistant skeletal muscles. Our analyses show that both apoa1^?/? and lcat^?/? mice fed high-fat diet have no measurable Apoa1 levels in their plasma, suggesting no direct involvement of Apoa1 in the observed phenotypic differences among groups.