Sunshine Lahmers

Altered Titin Expression, Myocardial Stiffness, and Left Ventricular Function in Patients With Dilated Cardiomyopathy

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 (P <0.003) 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 (n=7), intermediate in those with pseudonormal filling (n=6), and lowest in the group with restrictive filling (n=7). Mechanical measurements on LV muscle strips dissected from these hearts (n=8) 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 O2 consumption (n=10). 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.

Calcium-dependent molecular spring elements in the giant protein titin

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: ≈28-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 calcium-dependent molecular spring that adapts to the physiological state of the cell.

Genome-wide association identifies a deletion in the 3' untranslated region of striatin in a canine model of arrhythmogenic right ventricular cardiomyopathy.

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 8-bp deletion in the 3′ untranslated region (UTR) of the Striatin 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.

A comparative proteome analysis of human metaphase chromosomes isolated from two different cell lines reveals a set of conserved chromosome-associated proteins

A comparative proteome analysis of human metaphase chromosomes between a typical epithelial‐like 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 ultra‐high 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.

Molecular genetic basis for fluoroquinolone-induced retinal degeneration in cats.

Objectives Distribution of fluoroquinolones to the retina is normally restricted by ABCG2 at the blood–retinal barrier. As the cat develops a species-specific adverse reaction to photoreactive fluoroquinolones, our goal was to investigate ABCG2 as a candidate gene for fluoroquinolone-induced retinal degeneration and blindness in cats. Methods Feline ABCG2 was sequenced and the consensus amino acid sequence was compared with that of 10 other mammalian species. Expression of ABCG2 in feline retina was assessed by immunoblot. cDNA constructs for feline and human ABCG2 were constructed in a pcDNA3 expression vector and expressed in HEK-293 cells, and ABCG2 expression was analyzed by western blot and immunofluorescence. Mitoxantrone and BODIPY–prazosin efflux measured by flow cytometry and a phototoxicity assay were used to assess feline and human ABCG2 function. Results Four feline-specific (compared with 10 other mammalian species) amino acid changes in conserved regions of ABCG2 were identified. Expression of ABCG2 on plasma membranes was confirmed in feline retina and in cells transfected with human and feline ABCG2, although some intracellular expression of feline ABCG2 was detected by immunofluorescence. Function of feline ABCG2, compared with human ABCG2, was found to be deficient as determined by flow cytometric measurement of mitoxantrone and BODIPY-prazosin efflux and enrofloxacin-induced phototoxicity assays. Conclusion 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.

Familial subvalvular aortic stenosis in golden retrievers: inheritance and echocardiographic findings

OBJECTIVES To describe the echocardiographic findings and pedigree analysis of golden retrievers with subvalvular aortic stenosis. METHODS Seventy-three golden retrievers were evaluated by auscultation and echocardiography. A subcostal continuous-wave Doppler aortic velocity ê2·5 m/s and presence of a left basilar systolic ejection murmur were required for diagnosis of subvalvular aortic stenosis. Three echocardiographic characteristics were recorded: evidence of aortic insufficiency, subvalvular ridge or left ventricular hypertrophy. A disease status score was calculated by totalling the number of echocardiographic -characteristics per subject. RESULTS Thirty-two of 73 dogs were affected and their aortic velocities were as follows: range 2·5 to 6·8 m/s, median 3·4 m/s and standard deviation 1·2 m/s. Echocardiographic characteristics of 32 affected dogs were distributed as follows: left ventricular hypertrophy 12 of 32, aortic insufficiency 20 of 32 and subvalvular ridge 20 of 32. Disease status score ranged from 0 to 3 with a median of 2. There was a statistically significant correlation between aortic velocity and disease status score (r=0·644, P<0·0001). Subvalvular aortic stenosis was observed in multiple generations of several families and appears familial. CLINICAL SIGNIFICANCE Subvalvular aortic stenosis in the golden retriever is familial. Severity of stenosis correlates well with cumulative presence of echocardiographic characteristics (left ventricular hypertrophy, subvalvular ridge and aortic insufficiency).

Synovial T-Cell Lymphoma of the Stifle in a Dog

A 6-year-old, 43-kg, spayed female rottweiler was presented for a 1-month history of progressive, left hind-limb lameness. Upon physical examination, a cranial drawer sign and joint distention were present in the left stifle. Radiographically, the stifle had evidence of effusion, remodeling of the patella, and an enlarged popliteal lymph node. Marked synovial thickening and an intact cranial cruciate ligament were noted during surgery. Despite finding a nonspecific, mixed inflammatory response on joint fluid cytopathology, histopathology demonstrated T-cell lymphoma of the synovium. Lameness may be the sole presenting clinical sign in canine lymphoma.

Resolution of sustained narrow complex ventricular tachycardia and tachycardia-induced cardiomyopathy in a Quarter Horse following quinidine therapy.

Sustained narrow-QRS tachycardia of three months duration and left ventricular systolic dysfunction were identified in a fifteen-year-old Quarter Horse. No underlying cause for the tachyarrhythmia was found and no predisposing structural cardiac lesions were evident by echocardiography. Intravenous diltiazem and lidocaine were administered without achieving successful conversion of the arrhythmia. Oral quinidine therapy converted the tachyarrhythmia to sinus rhythm. Ventricular systolic dysfunction and chamber dilatation subsequently resolved. As with other species, echocardiographic features of dilated cardiomyopathy can be tachycardia-induced and may resolve following successful control of heart rate and rhythm.

Adaptations in titin's spring elements in normal and cardiomyopathic hearts.

The giant elastic protein titin contains an extensible segment that underlies the majority of physiological passive muscle stiffness. The extensible segment comprises mechanically distinct and serially-linked spring elements: the tandem Ig segments, the PEVK and the cardiac-specific N2B unique sequence. Under physiological conditions the tandem Ig segments are likely to largely consist of folded Ig domains whereas the N2B unique sequence and PEVK are largely unfolded and behave as wormlike chains with different persistence lengths. The mechanical characteristics of titins extensible region may be tuned to match changing mechanical demands placed on muscle, using mechanisms that operate at different time scales and that include post-transcriptional and post-translational processes.

Identification of DNA variants in the canine beta-1 adrenergic receptor gene.

Beta-adrenergic receptor antagonists are utilized for the management of several cardiac diseases in the dog. In humans the beneficial effects of beta-adrenergic receptor antagonists are variable and are associated with a genetic variability in the beta one adrenergic receptor gene (ADRB1). To determine if DNA variants were present in the canine ADRB1 gene, DNA from five breeds of dogs was evaluated. Two deletions were identified within the region of the gene that encodes the cytoplasmic tail of ADRB1. The functions of this region are not well understood although it is important in differentiating subtypes of adrenergic receptors and may be associated with control of receptor downregulation. The functional consequences of these identified variants deserve further study.