Biochemical and Mechanical Investigation of Cardiac Titin Isoforms

SUMMARY Background: Titin is a giant elastic protein of muscle sarcomeres. Titin molecules link the Z-disk with the M-line and have structural, elastic and signaling functions in myocytes. The primary structure determination of several titin isoforms and the mechanical characterization of different muscle tissues revealed that titin elasticity depends on the differential splicing of the titin spring region consisting of immunoglobulin-like domains, a so-called PEVK domain and larger unique sequence insertions like N2-B. The molecular weight of a titin isoform is correlated with its mechanical, spring-like properties: the smaller the isoform, the stiffer the spring. Heart muscles of mammalian organisms co-express two major classes of titin isoforms: the stiff N2B-titin and the compliant N2BA-titin. Sarcomeric stiffness is tuned by altering the expression ratio of N2BA:N2B titins, whereas the amount of total titin in a sarcomere likely is constant owing to stoichiometric constraints. Objectives of the study: 1) To determine the patterns of titin isoform expression in different muscle tissues using MDa-range high-resolution gel electrophoresis and Western blotting; 2) To understand the functional significance of the expression of various cardiac titin isoforms; 3) To look for variations in cardiac titin expression in diseased myocardium; 4) To establish conditions/factors determining different expression patterns of cardiac titin; 5) To understand consequences of pathological changes in titin protein expression for the heart. Methods and Results: N2BA to N2B titin isoform ratio was determined by loose-gel electrophoresis. The titin isoform ratio differed between: 1) Hearts from different mammalian species; 2) Various regions of the same heart; 3) Diseased and normal human hearts. Western blotting using sequence assigned anti-titin antibodies confirmed the identity of the titin bands. The N2BA proportion varied from ~5% in rat left ventricle to almost 70% in cow right ventricle. The N2BA:N2B ratio was generally higher in the right ventricle than in the corresponding plane of the left ventricle and decreased from the base to the apex of a given heart (assessed in goat and rabbit). Titin isoform expression was altered under disease conditions: human heart transplants due to coronary artery disease (CAD) exhibited an average N2BA:N2B ratio of 47:53, whereas normal donor hearts had a ratio of ~30:70. Increased expression of larger N2BA titin isoforms was also seen in failing myocardium of dilated cardiomyopathy (DCM) patients. Coexpression of N2BA-titin and N2B-titin in a sarcomere was demonstrated by immunofluorescence microscopy. A regular cross-striated staining pattern for titin on tissue sections of CAD-transplant hearts indicated uniform changes of titin expression instead of titin structural damage. The functional relevance of the observed changes in titin isoform expression was estimated in mechanical experiments on isolated myofibrils from human hearts. Diseased (CAD, DCM) human myofibrils expressing elevated N2BA proportions had lowered passive stiffness compared to non-failing human myofibrils. Thus, sarcomeres can modify their passive tension by adjusting the N2BA:N2B titin expression ratio. Failing human hearts, even if they are globally stiffened (collagen upregulated), have more compliant myofibrils than normal donor human hearts. Titin isoform switching was also studied in a rat model of myocardial infarction (ligature of left anterior descending coronary artery). Titin gels showed that 43% of diseased hearts displayed a distinct N2BA-titin band, compared to only 14% of the hearts of sham-operated control rats, suggesting an isoform switch had occurred in this heart failure model. Conclusions: An improved titin detection method by modified 2% SDS-polyacrylamide gel electrophoresis revealed the presence of multiple titin isoforms in different tissues. Results established that the elastic diversity of titin is altered in human heart disease and during development. The shift towards expression of more compliant titin isoforms in human heart failure alters mechanical properties of the cardiomyocytes, in particular the passive stiffness. The disease-induced shift in titin isoform ratio may also impair active contraction, e.g. by interfering with the ability of the heart to use the Frank-Starling mechanism

Noncoding RNAs in Lung Cancer Angiogenesis

Lung cancer is the major death-related cancer in both men and women, due to late diagnostic and limited treatment efficacy. The angiogenic process that is responsible for the support of tumor progression and metastasis represents one of the main hallmarks of cancer. The role of VEGF signaling in angiogenesis is well‐established, and we summarize the role of semaphorins and their related receptors or hypoxia‐related factors role as prone of tumor microenvironment in angiogenic mechanisms. Newly, noncoding RNA transcripts (ncRNA) were identified to have vital functions in miscellaneous biological processes, including lung cancer angiogenesis. Therefore, due to their capacity to regulate almost all molecular pathways related with altered key genes, including those involved in angiogenesis and its microenvironment, ncRNAs can serve as diagnosis and prognosis markers or therapeutic targets. We intend to summarize the latest progress in the field of ncRNAs in lung cancer and their relation with hypoxia‐related factors and angiogenic genes, with a particular focus on ncRNAs relation to semaphorins

Innovative Hybrid Materials with Improved Tensile Strength Obtained by 3D Printing

Barium titanate (BT) and barium strontium titanate (BST) are one of the most studied ferroelectric materials with excellent piezoelectric properties, which can be used to stimulate bone formation by applying an electrical field. It is known that this ceramic is biocompatible and can be used for medical applications. New hybrid materials based on BT and collagen and BST and collagen, with potential applications in bone reconstruction, are presented, emphasizing the potential of fabricating 3D structures by integrating hydrothermal synthesis with additive manufacturing. Designing such structures may take advantage of rheological characterization at single-molecule level for some elastic biopolymers like titin and collagen and their molecular dissection into structural motifs that independently contribute to the protein viscoelasticity. Atomic force spectroscopy measurements on synthetic polypeptides showed that a polypeptide chain containing Ig domain modules is protected against rupture at high stretch by Ig domain unfolding, an important mechanism for stress relaxation in titin molecules. This property may be exploited to enhance the tensile strength of a 3D structure by adding specific synthetic polypeptides to the composition of the printing paste

Titin-based contribution to shortening velocity of rabbit skeletal myofibrils

The shortening velocity of skeletal muscle fibres is determined principally by actomyosin cross-bridges. However, these contractile elements are in parallel with elastic elements, whose main structural basis is thought to be the titin filaments. If titin is stretched, it may contribute to sarcomere shortening simply because it can recoil ‘passively’. The titin-based contribution to shortening velocity (Vp) was quantified in single rabbit psoas myofibrils. Non-activated specimens were rapidly released from different initial sarcomere lengths (SLs) by various step amplitudes sufficient to buckle the myofibrils; Vp was calculated from the release amplitude and the time to slack reuptake. Vp increased progressively (upper limit of detection, ∼60 μm s−1 sarcomere−1) between 2.0 and 3.0 μm SL, albeit more steeply than passive tension. At very low passive tension levels already (< 1–2 mN mm−2), Vp could greatly exceed the unloaded shortening velocity measured in fully Ca2+-activated skinned rabbit psoas fibres. Degradation of titin in relaxed myofibrils by low doses of trypsin (5 min) drastically decreased Vp. In intact myofibrils, average Vp was faster, the smaller the release step applied. Also, Vp was much higher at 30 °C than at 15 °C (Q10: 2.0, 3.04 or 6.15, for release steps of 150, 250 or 450 nm sarcomere−1, respectively). Viscous forces opposing the shortening are likely to be involved in determining these effects. The results support the idea that the contractile system imposes a braking force onto the passive recoil of elastic structures. However, elastic recoil may aid active shortening during phases of high elastic energy utilization, i.e. immediately after the onset of contraction under low or zero load or during prolonged shortening from greater physiological SLs

Experimental Study on the Mechanical Behavior of Orthodontic Arches Exposed to the Environment in the Oral Cavity

Background. The arches used in orthodontic therapy are subject to increasing physical and chemical stresses. Purpose of the study: This in vitro experimental study aims to highlight and compare the main mechanical properties of orthodontic arches. Materials and Methods: We used 40 springs, 2 materials, 20 of Ni-Cr and 20 of Co-Cr, of different diameters, 0.7 mm 0.8 mm and 1.2 mm, subjected to the environment of artificial saliva and artificial saliva with cola for one month and two months, respectively. Five springs of each material were tested at different times: T0, before application in the oral cavity, then at time T1, T2, T3, T4. Three lengths of the lever arm were considered for bending forces acting on the springs (dental wires). These lengths were 15, 10 and 5 mm. The wires were tested under the action of bending forces on a Hans Schmidt HV 500N stand, obtaining the characteristics of the wires: deformation-force-time. Results: Graphical determinations show that the degree of deformation of the wires is influenced by the applied force, diameter and obviously by the immersion time, respectively by the type of solution in which the springs were immersed. Conclusions: The final degree of bending is higher for Co-Cr arcs than for Ni-Cr at all three dimensions

A Comprehensive Picture of Extracellular Vesicles and Their Contents. Molecular Transfer to Cancer Cells

Critical processes such as growth, invasion, and metastasis of cancer cells are sustained via bidirectional cell-to-cell communication in tissue complex environments. Such communication involves the secretion of soluble factors by stromal cells and/or cancer cells within the tumor microenvironment (TME). Both stromal and cancer cells have been shown to export bilayer nanoparticles: encapsulated regulatory molecules that contribute to cell-to-cell communication. These nanoparticles are known as extracellular vesicles (EVs) being classified into exosomes, microvesicles, and apoptotic bodies. EVs carry a vast repertoire of molecules such as oncoproteins and oncopeptides, DNA fragments from parental to target cells, RNA species (mRNAs, microRNAs, and long non-coding RNA), and lipids, initiating phenotypic changes in TME. According to their specific cargo, EVs have crucial roles in several early and late processes associated with tumor development and metastasis. Emerging evidence suggests that EVs are being investigated for their implication in early cancer detection, monitoring cancer progression and chemotherapeutic response, and more relevant, the development of novel targeted therapeutics. In this study, we provide a comprehensive understanding of the biophysical properties and physiological functions of EVs, their implications in TME, and highlight the applicability of EVs for the development of cancer diagnostics and therapeutics

The Connection between MicroRNAs and Oral Cancer Pathogenesis: Emerging Biomarkers in Oral Cancer Management

Oral cancer is a common human malignancy that still maintains an elevated mortality rate despite scientific progress. Tumorigenesis is driven by altered gene expression patterns of proto-oncogenes and tumor-suppressor genes. MicroRNAs, a class of short non-coding RNAs involved in gene regulation, seem to play important roles in oral cancer development, progression, and tumor microenvironment modulation. As properties of microRNAs render them stable in diverse liquid biopsies, together with their differential expression signature in cancer cells, these features place microRNAs at the top of promising biomarkers for diagnostic and prognostic values. In this review, we highlight eight expression levels and functions of the most relevant microRNAs involved in oral cancer development, progression, and microenvironment sustainability. Furthermore, we emphasize the potential of using these small RNA species as non-invasive biomarkers for the early detection of oral cancerous lesions. Conclusively, we highlight the perspectives and limitations of microRNAs as novel diagnostic tools, as well as therapeutic models