Background-The role of the giant protein titin in patients with heart failure is not well established. We investigated titin expression in patients with end-stage heart failure resulting from nonischemic dilated cardiomyopathy, in particular as it relates to left ventricular (LV) myocardial stiffness and LV function. Methods and Results-SDS-agarose gels revealed small N2B (stiff) and large N2BA (compliant) cardiac titin isoforms with a mean N2BA:N2B expression ratio that was significantly (PϽ0.003) increased in 20 heart failure patients versus 6 controls. However, total titin was unchanged. The coexpression ratio was highest in a subsample of patients with an impaired LV relaxation pattern (nϭ7), intermediate in those with pseudonormal filling (nϭ6), and lowest in the group with restrictive filling (nϭ7). Mechanical measurements on LV muscle strips dissected from these hearts (nϭ8) revealed that passive muscle stiffness was significantly reduced in patients with a high N2BA:N2B expression ratio. Clinical correlations support the relevance of these changes for LV function (assessed by invasive hemodynamics and Doppler echocardiography). A positive correlation between the N2BA:N2B titin isoform ratio and deceleration time of mitral E velocity, A wave transit time, and end diastolic volume/pressure ratio was found. These changes affect exercise tolerance, as indicated by the positive correlation between the N2BA:N2B isoform ratio and peak O 2 consumption (nϭ10). Upregulated N2BA expression was accompanied by increased expression levels of titin-binding proteins (cardiac ankyrin repeat protein, ankrd2, and diabetes ankyrin repeat protein) that bind to the N2A element of N2BA titin (studied in 13 patients). Conclusions-Total titin content was unchanged in end-stage failing hearts and the more compliant N2BA isoform comprised a greater percentage of titin in these hearts. Changes in titin isoform expression in heart failure patients with dilated cardiomyopathy significantly impact diastolic filling by lowering myocardial stiffness. Upregulation of titin-binding proteins indicates that the importance of altered titin expression might extend to cell signaling and regulation of gene expression.
Titin (also known as connectin) is a giant protein with a wide range of cellular functions, including providing muscle cells with elasticity. Its physiological extension is largely derived from the PEVK segment, rich in proline (P), glutamate (E), valine (V), and lysine (K) residues. We studied recombinant PEVK molecules containing the two conserved elements: Ϸ28-residue PEVK repeats and E-rich motifs. Single molecule experiments revealed that calcium-induced conformational changes reduce the bending rigidity of the PEVK fragments, and site-directed mutagenesis identified four glutamate residues in the E-rich motif that was studied (exon 129), as critical for this process. Experiments with muscle fibers showed that titin-based tension is calcium responsive. We propose that the PEVK segment contains E-rich motifs that render titin a calciumdependent molecular spring that adapts to the physiological state of the cell.T itin comprises a multifunctional myofilament system in muscle, with a single molecule spanning the half-sarcomere (1-5). A large portion of the molecule functions as a molecular spring that, when extended, develops force. This force underlies the passive muscle force, which maintains the structural integrity of the contracting sarcomere and influences the filling behavior of the heart (3). Physiological force levels are largely determined by extension of the proline-glutamate-valine-lysine (PEVK) rich segment (6, 7). PEVK-like sequences are found in many titin-like proteins among evolutionary divergent organisms (8-11). The PEVK region of the human titin gene contains 114 exons; most code for conserved Ϸ28-residue PEVK repeats and 10 are more complex and encode E-rich motifs (12, 13). Here, we studied recombinant proteins that contain both PEVK repeats and glutamate (E)-rich motifs. Previous work suggests that the PEVK segment binds calcium with high affinity, raising the possibility that the extensibility of the PEVK segment may be calcium regulated (14). Thus, an important goal was to examine the effect of calcium on the mechanical behavior of the PEVK segment. MethodsProteins. We engineered only PEVK sequences (''naked'' PEVK fragment) or PEVK sequences flanked by Ig-like domains (PEVK-Ig fusion fragment). Fragments were cloned, expressed, and purified by using routine methods (12, 15). All fragments were His-tagged at their amino terminus and, except when intended for structural analysis, had two Cys residues at the carboxyl terminus for covalent attachment to gold-coated substrates. Purified proteins were dialyzed into AB buffer (in mM; 25 Mops, pH 7.4͞150 KCl͞1 EGTA͞1 DTT), quick frozen, and stored at Ϫ80°C. For additional details, see Supporting Text, which is published as supporting information on the PNAS web site.Single Molecule Mechanics. Molecules were stretched by using an atomic force microscope specialized for stretching molecules (15, 16). Proteins (Ϸ100 g͞ml) were allowed to bind for Ϸ10 min to gold-coated microscope slides (precleaned glass slides and freshly cleaved mica were also...
Abstract-Developmental changes in contractile behavior are known to occur during fetal and postnatal heart development.In this study, we examined whether adaptations take place in titin. A range of species was used to evaluate titin isoform expression and altered function during cardiac muscle development. A novel titin exon microarray that allows all 363 titin exons to be monitored simultaneously was used for transcript studies. Results reveal expression of fetal titin isoforms, characterized by additional spring elements both in the tandem Ig and PEVK region of the molecule. At the protein level, the fetal cardiac isoform predominates in fetal and neonatal myocardium and gradually disappears during postnatal development with a time course that varies in different species. Passive myocardium, contrary to previous reports, was found to be less stiff in the neonate than in the adult. This can be explained by the unique spring composition of fetal cardiac titin expressed by the neonate. Changes in titin expression are likely to impact functional transitions and diastolic filling behavior during development of the heart. (Circ Res. 2004;94:505-513.)Key Words: diastole Ⅲ compliance Ⅲ filling Ⅲ connectin Ⅲ microarray L arge changes in hemodynamic load occur during the course of cardiac development and are known to be associated with changes in contractility due to alterations in isoform expression patterns of sarcomeric proteins. 1,2 Whether changes occur in passive tension of the myocardium is less well established. Research performed decades ago revealed that passive stiffness is highest in fetal myocardium and progressively decreases with age. 3,4 To our knowledge, this earlier work has not been followed up. Furthermore, the molecular basis of the adaptations in passive stiffness during cardiac development is also unknown, except that the total amount of collagen in fetal and adult myocardium appears unaltered. 5 Considering that titin is a major source of passive stiffness in adult myocardium, 6,7 we investigated titin's role in the developmental regulation of passive stiffness.Titin is a giant protein that spans the half sarcomere with an I-band segment that functions as a molecular spring, the elastic properties of which define the passive mechanical properties of the cardiac myocyte. 8 Titin is encoded by a single gene containing in humans 363 exons that are differentially spliced in the adult heart, creating the stiff N2B (short molecular spring) and more compliant N2BA (long molecular spring) isoforms. 9,10 These isoforms can be coexpressed in the same sarcomere, allowing passive stiffness to be adjusted anywhere in-between that of stiff sarcomeres that express only N2B titin and compliant sarcomeres that express N2BA titin. 11 Recent work revealed that differential splicing is subject to regulatory mechanisms that control entry to either N2B or N2BA splice-pathways. 7,12,13 For example, the adult canine myocardium coexpresses N2B and N2BA titins at a similar level, but in response to long-term tachy...
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease characterized by ventricular arrhythmias and sudden cardiac death. It is most frequently inherited as an autosomal dominant trait with incomplete and age-related penetrance and variable clinical expression. The human disease is most commonly associated with a causative mutation in one of several genes encoding desmosomal proteins.We have previously described a spontaneous canine model of ARVC in the boxer dog. We phenotyped adult boxer dogs for ARVC by performing physical examination, echocardiogram and ambulatory electrocardiogram. Genome-wide association using the canine 50k SNP array identified several regions of association, of which the strongest resided on chromosome 17. Fine-mapping and direct DNA sequencing identified an eight base pair deletion in the 3' untranslated region (UTR) of the striatin (STRN) gene on chromosome 17 in association with ARVC in the boxer dog. Evaluation of the secondary structure of the 3' UTR demonstrated that the deletion affects a stem loop structure of the mRNA and expression analysis identified a reduction in striatin mRNA. Dogs that were homozygous for the deletion had a more severe form of disease based on a significantly higher number of ventricular premature complexes. Immunofluorescence studies localized striatin to the intercalated disc region of the cardiac myocyte and co-localized it to three desmosomal proteins, plakophilin-2, plakoglobin and desmoplakin, all involved in the pathogenesis of ARVC in human beings.We suggest that striatin may serve as a novel candidate gene for human ARVC.
Familial dilated cardiomyopathy is a primary myocardial disease that can result in the development of congestive heart failure and sudden cardiac death. Spontaneous animal models of familial dilated cardiomyopathy exist and the Doberman pinscher dog is one of the most commonly reported canine breeds. The objective of this study was to evaluate familial dilated cardiomyopathy in the Doberman pinscher dog using a genome-wide association study for a genetic alteration(s) associated with the development of this disease in this canine model. Genome-wide association analysis identified an area of statistical significance on canine chromosome 14 (p(raw) = 9.999e-05 corrected for genome-wide significance), fine-mapping of additional SNPs flanking this region localized a signal to 23,774,190-23,781,919 (p = 0.001) and DNA sequencing identified a 16-base pair deletion in the 5' donor splice site of intron 10 of the pyruvate dehydrogenase kinase 4 gene in affected dogs (p < 0.0001). Electron microscopy of myocardium from affected dogs demonstrated disorganization of the Z line, mild to moderate T tubule and sarcoplasmic reticulum dilation, marked pleomorphic mitochondrial alterations with megamitochondria, scattered mitochondria with whorling and vacuolization and mild aggregates of lipofuscin granules. In conclusion, we report the identification of a splice site deletion in the PDK4 gene that is associated with the development of familial dilated cardiomyopathy in the Doberman pinscher dog.
Feline-specific amino acid changes in ABCG2 cause a functional defect of the transport protein in cats. This functional defect may be owing, in part, to defective cellular localization of feline ABCG2. Regardless, dysfunction of ABCG2 at the blood-retinal barrier likely results in accumulation of photoreactive fluoroquinolones in feline retina. Exposure of the retina to light would then generate reactive oxygen species that would cause the characteristic retinal degeneration and blindness documented in some cats receiving high doses of some fluoroquinolones. Pharmacological inhibition of ABCG2 in other species might result in retinal damage if fluoroquinolones are concurrently administered.
A comparative proteome analysis of human metaphase chromosomes between a typical epitheliallike cell, HeLa S3, and a lymphoma-type cell, BALL-1, was performed. One-dimensional (1-D) SDS-PAGE and radical-free and highly reducing two-dimensional electrophoresis (RFHR 2-DE) detected more than 200 proteins from chromosomes isolated from HeLa S3 cells, among which 189 proteins were identified by mass spectrometry (MS). Consistent with our recent four-layer structural model of a metaphase chromosome, all the identified proteins were grouped into four distinct levels of abundance. Both HeLa S3 and BALL-1 chromosomes contained specific sets of abundant chromosome structural and peripheral proteins in addition to less abundant chromosome coating proteins (CCPs). Furthermore, titin array analysis and a proteome analysis of the ultrahigh molecular mass region indicated an absence of titin with their molecular weight (MW) more than 1000 kDa. Consequently, the present proteome analyses together with previous information on chromosome proteins provide the comprehensive list of proteins essential for the metaphase chromosome architecture.
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