Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography.

Detailed knowledge of the ultrastructure of intracellular compartments is a prerequisite for our understanding of how cells function. In cardiac muscle cells, close apposition of transverse (t)-tubule (TT) and sarcoplasmic reticulum (SR) membranes supports stable high-gain excitation-contraction coupling. Here, the fine structure of this key intracellular element is examined in rabbit and mouse ventricular cardiomyocytes, using ultra-rapid high-pressure freezing (HPF, omitting aldehyde fixation) and electron microscopy. 3D electron tomograms were used to quantify the dimensions of TT, terminal cisternae of the SR, and the space between SR and TT membranes (dyadic cleft). In comparison to conventional aldehyde-based chemical sample fixation, HPF-preserved samples of both species show considerably more voluminous SR terminal cisternae, both in absolute dimensions and in terms of junctional SR to TT volume ratio. In rabbit cardiomyocytes, the average dyadic cleft surface area of HPF and chemically fixed myocytes did not differ, but cleft volume was significantly smaller in HPF samples than in conventionally fixed tissue; in murine cardiomyocytes, the dyadic cleft surface area was higher in HPF samples with no difference in cleft volume. In both species, the apposition of the TT and SR membranes in the dyad was more likely to be closer than 10 nm in HPF samples compared to CFD, presumably resulting from avoidance of sample shrinkage associated with conventional fixation techniques. Overall, we provide a note of caution regarding quantitative interpretation of chemically-fixed ultrastructures, and offer novel insight into cardiac TT and SR ultrastructure with relevance for our understanding of cardiac physiology.

Field study on bovine paratuberculosis using real-time PCR and liquid culture for testing environmental and individual fecal samples implemented in dairy cow management.

Bovine paratuberculosis (Johne's disease) is a bacterial, chronic, and wasting intestinal disease caused by Mycobacterium avium ssp. paratuberculosis (MAP). Johne's disease causes severe losses in dairy farm productivity and is also suspected to be a potential trigger for Crohn's disease in humans. The fecal-oral infection of MAP to neonates is recognized as an important within-herd transmission route. Our objective was to recommend diagnostic methods for herds with suspected paratuberculosis requiring fast results, as well as for herds with breeding programs or others that aim at being nonsuspected of paratuberculosis infection. We determined a period of 8 wk from sampling to diagnostic findings suitable for testing of cows during the dry period. We therefore tested environmental and individual fecal samples with one rapid and one highly sensitive diagnostic method. Environmental samples (boot swabs) were taken as a first step in 3 herds and tested using a DNA extraction protocol for feces and subsequent real-time PCR (referred to as fecal PCR). Additionally, cultivation in liquid medium for 6 wk was performed and verified with real-time PCR (referred to as liquid culture). Automation of DNA extraction based on magnetic beads and the PCR setup was performed with pipetting robots. As a result, we successfully detected MAP in boot swabs of all herds by both methods. In a second step, 245 individual fecal samples from the 3 herds were examined using also fecal PCR and liquid culture. The results obtained by fecal PCR were compared with detection of MAP using cultivation in liquid medium for 6 wk. Testing individual cows, we identified MAP-specific DNA in 53 fecal samples using the liquid culture. Using fecal PCR, we revealed 43 positive samples of which 39 also tested positive in the liquid culture, revealing MAP-positive cows in all 3 herds. The fecal PCR procedure allows rapid detection of MAP-specific DNA with 74% of the sensitivity of liquid culture. For the purpose of testing with maximal sensitivity, cultivation in liquid medium is recommended. Cultivation of MAP in liquid medium M7H9C means a significant time gain in comparison to cultivation on solid media, which requires twice as much time. Thus, this testing fits within the 6- to 8-wk dry period of gravid cows and provides test results before calving, a prerequisite to prevent fecal-oral transmission to newborn calves.

Detection of Mycobacterium avium subsp. paratuberculosis in faeces using different procedures of pre-treatment for real-time PCR in comparison to culture.

One of the most relevant aspects in the diagnosis of paratuberculosis (Johne's disease) in cattle is the availability of a method for the rapid and sensitive detection of Mycobacterium avium subsp. paratuberculosis (MAP) in order to facilitate the prompt removal of pathogen-shedding animals from a herd. To meet this requirement, methods for pre-treatment of bovine faecal samples and subsequent extraction of DNA for detection of MAP by real-time PCR were compared with MAP culture results. A total of 116 bovine faecal samples that showed weak (64.7%), moderate (18.1%) or strong (17.2%) growth of MAP on solid HEY medium were investigated. For PCR, supernatants, sediments or bacterial pellets were obtained from faecal samples by pre-treatment before extraction of MAP DNA based on silica membranes or magnetic particles. Samples then were tested by MAP IS900 and ISMav2 real-time PCR with an analytical sensitivity of 6 and 28 genome equivalents (GE) per mL, respectively. The best results were obtained by including a microfiltration step in the sample pre-treatment in combination with silica membrane-based mini-columns or magnetic particles for DNA extraction. This approach enhanced the detection rate of MAP in IS900 real-time PCR from 58.6% to 84.5% using silica membrane mini-columns and from 61.2% to 64.7% using magnetic particles.

Models of cardiac tissue electrophysiology: progress, challenges and open questions.

Models of cardiac tissue electrophysiology are an important component of the Cardiac Physiome Project, which is an international effort to build biophysically based multi-scale mathematical models of the heart. Models of tissue electrophysiology can provide a bridge between electrophysiological cell models at smaller scales, and tissue mechanics, metabolism and blood flow at larger scales. This paper is a critical review of cardiac tissue electrophysiology models, focussing on the micro-structure of cardiac tissue, generic behaviours of action potential propagation, different models of cardiac tissue electrophysiology, the choice of parameter values and tissue geometry, emergent properties in tissue models, numerical techniques and computational issues. We propose a tentative list of information that could be included in published descriptions of tissue electrophysiology models, and used to support interpretation and evaluation of simulation results. We conclude with a discussion of challenges and open questions.

A model of electrical conduction in cardiac tissue including fibroblasts.

Fibroblasts are abundant in cardiac tissue. Experimental studies suggested that fibroblasts are electrically coupled to myocytes and this coupling can impact cardiac electrophysiology. In this work, we present a novel approach for mathematical modeling of electrical conduction in cardiac tissue composed of myocytes, fibroblasts, and the extracellular space. The model is an extension of established cardiac bidomain models, which include a description of intra-myocyte and extracellular conductivities, currents and potentials in addition to transmembrane voltages of myocytes. Our extension added a description of fibroblasts, which are electrically coupled with each other and with myocytes. We applied the extended model in exemplary computational simulations of plane waves and conduction in a thin tissue slice assuming an isotropic conductivity of the intra-fibroblast domain. In simulations of plane waves, increased myocyte-fibroblast coupling and fibroblast-myocyte ratio reduced peak voltage and maximal upstroke velocity of myocytes as well as amplitudes and maximal downstroke velocity of extracellular potentials. Simulations with the thin tissue slice showed that inter-fibroblast coupling affected rather transversal than longitudinal conduction velocity. Our results suggest that fibroblast coupling becomes relevant for small intra-myocyte and/or large intra-fibroblast conductivity. In summary, the study demonstrated the feasibility of the extended bidomain model and supports the hypothesis that fibroblasts contribute to cardiac electrophysiology in various manners.

Influence of electrophysiological heterogeneity on electrical stimulation in healthy and failing human hearts.

The application of strong electrical stimuli is a common method used for terminating irregular cardiac behaviour. The study presents the influence of electrophysiological heterogeneity on the response of human hearts to electrical stimulation. The human electrophysiology was simulated using the ten Tusscher-Noble-Noble-Panfilov cell model. The anisotropic propagation of depolarisation in three-dimensional virtual myocardial preparations was calculated using bidomain equations. The research was carried out on different types of virtual cardiac wedge. The selection of the modelling parameters emphasises the influence of cellular electrophysiology on the response of the human myocardium to electrical stimulation. The simulations were initially performed on a virtual cardiac control model characterised by electrophysiological homogeneity. The second preparation incorporated the transmural electrophysiological heterogeneity characteristic of the healthy human heart. In the third model type, the normal electrophysiological heterogeneity was modified by the conditions of heart failure. The main currents responsible for repolarisation (Ito, IKs and IKI) were reduced by 25%. Successively, [Na+]i was increased by the regulation of the Na+-Ca2+ exchange function, and fibrosis was represented by decreasing electrical conductivity. Various electrical stimulation configurations were used to investigate the differences in the responses of the three different models. Monophasic and biphasic electrical stimuli were applied through rectangular paddles and needle electrodes. A whole systolic period was simulated. The distribution of the transmembrane voltage indicated that the modification of electrophysiological heterogeneity induced drastic changes during the repolarisation phase. The results illustrated that each of the heart failure conditions amplifies the modification of the response of the myocardium to electrical stimulation. Therefore a theoretical model of the failing human heart must incorporate all the characteristic features.

Hyperelastic description of elastomechanic properties of the heart: a new material law and its application.

Knowledge concerning passive mechanic cardiac properties is necessary to model behavior of whole hearts. Commonly, a continuum mechanics based description is chosen in conjunction with the finite element method. The aim of this work is to summarize, derive and evaluate hyperelastic material laws for inhomogeneous, anisotropic myocardium. Hence, different material laws were set up and their parameters were determined taking measurement data in literature into account. The material laws were compared from a theoretical and numerical point of view. Furthermore, the application of continuum mechanics based methods is evaluated concerning aspects of numerical solution and spatial discretisation. In further work the laws will be implemented and integrated in an existing software environment, which allows the calculation of deformations in complex geometries.

Modeling force development in the sarcomere in consideration of electromechanical coupling.

Models of the cellular force development simulate the contractive behavior of the sarcomere. In conjunction with electrophysiological models they can contribute to a better comprehension of physiology and pathologies. Aim of this study is to examine the coupling of cellular electrophysiological processes and force development. For that a graphical user interface was developed to simplify the parameterization and calculation of the models as well as to present the results graphically. A feedback mechanism is introduced to pay attention to close connections between force development and intracellular processes. On basis of various tests with different boundary conditions, new force models are developed, parameterized, validated and compared with models in literature. In future studies the results will be tested in multiple cell organization.

Comparison of macroscopic models of excitation and force propagation in the heart.

Computer aided simulations of the heart provide knowledge of phenomena, which are commonly neither visible nor measurable with current techniques. This knowledge can be applied e.g. in cardiologic diagnosis and therapy. A variety of models was created to reconstruct cardiac processes, e.g. electrical propagation and force development. In this work different macroscopic models were compared, i.e. models based on excitation-diffusion equations and cellular automata. The comparison was carried out concerning reconstruct-ability of cardiac phenomena, mathematical and biophysical foundation as well as computational expense. Particularly, the reconstruct-ability of electromechanic feedback mechanisms was examined. Perspectives for further developments and improvements of models were given.