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Abstract: Mechanical properties of single cells are of increasing interest both from a fundamental cell biological perspective and in the context of disease diagnostics. In this respect, atomic force microscopy (AFM) has become a powerful tool for imaging and assessing mechanical properties of biological samples. However, while these tests are typically carried out on chemically fixed cells, the most important data is that on living cells. The present study applies AFM technique to assess the Young's modulus of two cell lines: mouse embryonic fibroblasts (NIH/3T3) and human epithelial cancer cells (SW-13). Both living cells and those fixed with paraformaldehyde were investigated. This analysis quantifies the difference between Young's modulus for these two conditions and provides a coefficient to relate them. Knowing the relation between Young's modulus of living and fixed cells, allows carrying out and comparing data obtained during steady-state measurements on fixed cells that are more frequently available in the clinical and research settings and simpler to maintain and probe. [Copyright &y& Elsevier]

Mechanical stimulation appears to be a critical modulator for many aspects of biology, both of living tissue and cells. The cell-stretcher, a novel device for the mechanical uniaxial stimulation of populations of cells, is described. The system is based on a variable stroke cam-lever-tappet mechanism which allows the delivery of cyclic stimuli with frequencies of up to 10 Hz and deformation between 1% and 20%. The kinematics is presented and a simulation of the dynamics of the system is shown, in order to compute the contact forces in the mechanism. The cells, following cultivation and preparation, are plated on an ad hoc polydimethylsiloxane membrane which is then loaded on the clamps of the cell-stretcher via force-adjustable magnetic couplings. In order to show the viability of the experimentation and biocompatibility of the cell-stretcher, a set of two in vitro tests were performed. Human epithelial carcinoma cell line A431 and Adult Mouse Ventricular Fibroblasts (AMVFs) from a dual reporter mouse were subject to 0.5 Hz, 24 h cyclic stretching at 15% strain, and to 48 h stimulation at 0.5 Hz and 15% strain, respectively. Visual analysis was performed on A431, showing definite morphological changes in the form of cellular extroflections in the direction of stimulation compared to an unstimulated control. A cytometric analysis was performed on the AMVF population. Results show a post-stimulation live-dead ratio deviance of less than 6% compared to control, which proves that the environment created by the cell-stretcher is suitable for in vitro experimentation. [ABSTRACT FROM AUTHOR]

Atomic force microscopy (AFM) for single-cell force spectroscopy (SCFS) and Poisson statistic were used to analyze the detachment work recorded during the removal of gold-covered microspheres from cardiac fibroblasts. The effect of Cytochalasin D, a disruptor of the actin cytoskeleton, on cell adhesion was also tested. The adhesion work was assessed using a Poisson analysis also derived from single-cell force spectroscopy retracting curves. The use of Poisson analysis to get adhesion work from AFM curves is quite a novel method, and in this case, proved to be effective to study the short-range and long-range contributions to the adhesion work. This method avoids the difficult identification of minor peaks in the AFM retracting curves by creating what can be considered an average adhesion work. Even though the effect of actin depolymerisation is well documented, its use revealed that control cardiac fibroblasts (CT) exhibit a work of adhesion at least 5 times higher than that of the Cytochalasin treated cells. However, our results indicate that in both cells short-range and long-range contributions to the adhesion work are nearly equal and the same heterogeneity index describes both cells. Therefore, we infer that the different adhesion behaviors might be explained by the presence of fewer membrane adhesion molecules available at the AFM tip–cell interface under circumstances where the actin cytoskeleton has been disrupted. [ABSTRACT FROM AUTHOR]

Abstract: AFM was used to collect the whole force–deformation cell curves. They provide both the elasticity and adhesion behavior of mouse primary cardiac fibroblasts. To confirm the hypothesis that a link exists between the membrane receptors and the cytoskeletal filaments causing therefore changing in both elasticity and adhesion behavior, actin-destabilizing Cytochalsin D was administrated to the fibroblasts. From immunofluorescence observation and AFM loading/unloading curves, cytoskeletal reorganization as well as a change in the elasticity and adhesion was indeed observed. Elasticity of control fibroblasts is three times higher than that for fibroblasts treated with 0.5μM Cytochalasin. Moreover, AFM loading–unloading curves clearly show the different mechanical behavior of the two different cells analyzed: (i) for control cells the AFM cantilever rises during the dwell time while cells with Cytochalasin fail to show such an active resistance; (ii) the maximum force to deform control cells is quite higher and as far as adhesion is concern (iii) the maximum separation force, detachment area and the detachment process time are much larger for control compared to the Cytochalasin treated cells. Therefore, alterations in the cytoskeleton suggest that a link must exist between the membrane receptors and the cytoskeletal filaments beneath the cellular surface and inhibition of actin polymerization has effects on the whole cell mechanical behavior as well as adhesion. [Copyright &y& Elsevier]

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited genetic myocardial disease characterized by fibrofatty replacement of the myocardium and a predisposition to cardiac arrhythmias and sudden death. We evaluated the cardiomyopathy gene titin (TTN) as a candidate ARVC gene because of its proximity to an ARVC locus at position 2q32 and the connection of the titin protein to the transitional junction at intercalated disks.Methods and Results: All 312 titin exons known to be expressed in human cardiac titin and the complete 3' untranslated region were sequenced in 38 ARVC families. Eight unique TTN variants were detected in 7 families, including a prominent Thr2896Ile mutation that showed complete segregation with the ARVC phenotype in 1 large family. The Thr2896IIe mutation maps within a highly conserved immunoglobulin-like fold (Ig10 domain) located in the spring region of titin. Native gel electrophoresis, nuclear magnetic resonance, intrinsic fluorescence, and proteolysis assays of wild-type and mutant Ig10 domains revealed that the Thr2896IIe exchange reduces the structural stability and increases the propensity for degradation of the Ig10 domain. The phenotype of TTN variant carriers was characterized by a history of sudden death (5 of 7 families), progressive myocardial dysfunction causing death or heart transplantation (8 of 14 cases), frequent conduction disease (11 of 14), and incomplete penetrance (86%).Conclusions: Our data provide evidence that titin mutations can cause ARVC, a finding that further expands the origin of the disease beyond desmosomal proteins. Structural impairment of the titin spring is a likely cause of ARVC and constitutes a novel mechanism underlying myocardial remodeling and sudden cardiac death. [ABSTRACT FROM AUTHOR]

Dilated cardiomyopathy is a disease of unknown cause characterized by dilation and impaired function of one or both ventricles. Most cases are believed to be sporadic, although familial forms have been detected. The familial form has been estimated to have a relative frequency of about 25%. Since, except for familial history, familial form has no other characteristics that could help in classifying the two diseases, the estimate of the frequency of the familial form should take into account a possible misclassification error. In our study, 100 cases were randomly selected in a prospective series of 350 patients. Out of them, 28 index cases were included in the analysis: 12 were known to be familial, and 88 were believed to be sporadic. After extensive clinical examination of the relatives, 3 patients supposed to have a sporadic form were found to have a familial form. 13 cases had a confirmed sporadic disease. Models in the Log-Linear Product class (LLP) have been used to separate classification errors from underlying patterns of disease incidence. The most conservative crude estimate of the misclassification error is 16.1% (CI 0.22- 23.27%), which leads to a crude estimate of the frequency of the familiar form of about 60%. An estimate of the disease frequency, adjusted for taking into consideration the sampling plan, is 40.93% (CI 32.29-44.17%). The results are consistent with the hypothesis that genetic factors are still underestimated, although they represent a major cause of the disease. [ABSTRACT FROM AUTHOR]

An abstract of the study "Familial dilated cardiomyopathy: an international registry," by E. Carniel et al, is presented.

An abstract of the study "Evaluation of systolic and diastolic dysfunction in hypertrophic cardiomyopathy with echocardiography," by E. Carniel and colleagues is presented.

An abstract of the article "Is the long term outcome of familial dilated cardiomyopathy different with respect to sporadic forms," by A. Di Lenarda and colleagues is presented.

An abstract of the study "Alpha-Myosin Heavy Chain Mutations Associated With Dilated and Hypertrophic Cardiomyopathy," by E. Carniel and colleagues is presented.