A missense variant in the titin gene in Doberman pinscher dogs with familial dilated cardiomyopathy and sudden cardiac death.

The dog provides a large animal model of familial dilated cardiomyopathy for the study of important aspects of this common familial cardiovascular disease. We have previously demonstrated a form of canine dilated cardiomyopathy in the Doberman pinscher breed that is inherited as an autosomal dominant trait and is associated with a splice site variant in the pyruvate dehydrogenase kinase 4 (PDK4) gene, however, genetic heterogeneity exists in this species as well and not all affected dogs have the PDK4 variant. Whole genome sequencing of a family of Doberman pinchers with dilated cardiomyopathy and sudden cardiac death without the PDK4 variant was performed. A pathologic missense variant in the titin gene located in an immunoglobulin-like domain in the I-band spanning region of the molecule was identified and was highly associated with the disease (p < 0.0001). We demonstrate here the identification of a variant in the titin gene highly associated with the disease in this spontaneous canine model of dilated cardiomyopathy. This large animal model of familial dilated cardiomyopathy shares many similarities with the human disease including mode of inheritance, clinical presentation, genetic heterogeneity and a pathologic variant in the titin gene. The dog is an excellent model to improve our understanding of the genotypic phenotypic relationships, penetrance, expression and the pathophysiology of variants in the titin gene.

Impact of the canine double-deletion ß1 adrenoreceptor polymorphisms on protein structure and heart rate response to atenolol, a ß1-selective ß-blocker.

OBJECTIVE: ß-Adrenergic receptor antagonists are widely utilized for the management of cardiac diseases in dogs. We have recently identified two deletion polymorphisms in the canine adrenoreceptor 1 (ADRB1) gene.We hypothesized that canine ADRB1 deletions would alter the structure of the protein, as well as the heart rate response to the ß-adrenergic receptor antagonist, atenolol. The objectives of this study were to predict the impact of these deletions on the predicted structure of the protein and on the heart rate response to atenolol in a population of healthy adult dogs. METHODS: Eighteen apparently healthy, mature dogs with (11) and without (seven) ADRB1 deletions were evaluated. The heart rate of the dogs was evaluated with a baseline ambulatory ECG before and 14-21 days after atenolol therapy (1 mg/kg orally q12 h). Minimum, average, and maximum heart rates were compared between groups of dogs (deletions, controls) using an unpaired t-test and within each group of dogs using a paired t-test. The protein structure of ADRB1 was predicted by computer modeling. RESULTS: Deletions were predicted to alter the structure of the ADRB1 protein. The heart rates of the dogs with deletions were lower than those of the control dogs (the average heart rates were significantly lower). CONCLUSION: ADRB1 deletions appear to have structural and functional consequences. Individual genome-based treatment recommendations could impact the management of dogs with heart disease.

A splice site mutation in a gene encoding for PDK4, a mitochondrial protein, is associated with the development of dilated cardiomyopathy in the Doberman pinscher.

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.

Performance of Different Echocardiographic Measurements of Left Atrial Size in Dogs by Observers with Different Levels of Experience.

Assessment of left atrial (LA) sizes in dogs informs clinical staging, risk assessment, treatment decisions, and prognosis. The objective of this study was to assess the diagnostic performance of observers with different levels of experience measuring the LA with three different techniques. Echocardiographic images from 36 dogs with different degrees of left atrial enlargement (LAE) were retrospectively retrieved, anonymized and measured in a blinded fashion by a veterinary student, a first-year cardiology resident, a third-year cardiology resident, and two board-certified veterinary cardiologists. The LA to aortic root ratio (LA:Ao), LA antero-postero diameter indexed to body weight (LAiAPD) and left atrial area were measured. Inter- and intra-observer intraclass correlation coefficients (ICCs) were calculated for all three variables. Bland-Altman plots and accuracy in identification of LAE were calculated for the three least experienced observers using LA:Ao and LAiAPD. Intra- and interobserver ICCs were greater than 0.9 for every variable. The observer with least experience had significant positive bias and a tendency to overestimate larger measurements using LA:Ao, but not using LAiAPD. The accuracy of identification of LAE also increased with the increasing level of experience and was higher for LAiAPD compared to LA:Ao. Combining both methods for identification of LAE, further increased accuracy.

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.

Clinical characteristics of cats referred for evaluation of subclinical cardiac murmurs.

OBJECTIVES: The aim of this study was to define the clinical characteristics of cats referred for evaluation of subclinical cardiac murmurs, and, secondarily, to identify predictors of echocardiographic identification of cardiac disease. METHODS: One hundred and sixty-three apparently healthy cats with subclinical murmurs were retrospectively enrolled. Medical records of cats older than 1 year of age referred for the evaluation of subclinical murmurs were reviewed. Cats were considered healthy if clinical signs of systemic disease or cardiac disease were not reported and cats were not receiving cardiac medications. Logistic regression was used to identify clinical variables that predict echocardiographic identification of cardiac disease. RESULTS: One hundred and eight cats (66.3%) had echocardiographic evidence of cardiac disease with hypertrophic cardiomyopathy being the most common (80.5%). Left atrial enlargement was uncommon; in 90% of cats with echocardiographically identified cardiac disease, the left atrial aortic ratio from two-dimensional echocardiography was <1.51. Cats with cardiac disease were more likely to be male (P = 0.016), weigh more (P <0.01) and have a murmur of intensity ⩾3/6 (P = 0.019) than cats without cardiac disease. Murmur intensity ⩾grade 3/6 (P = 0.01) and male sex (P = 0.01) were independent predictors of echocardiographic evidence of cardiac disease in multivariable analysis. CONCLUSIONS AND RELEVANCE: The majority of cats referred for evaluation of subclinical cardiac murmurs have cardiac disease. Based on left atrial dimensions, cardiac disease is generally mild. Male sex and a loud cardiac murmur are associated with the identification of cardiac disease.

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.

Disease association and clinical assessment of feline pericardial effusion.

Records were reviewed from 83 cases to determine the main causes and clinical significance of feline pericardial effusion. The most common causes included hypertrophic cardiomyopathy with congestive heart failure, neoplasia, and systemic infection. Most cases had concurrent or secondary pleural effusion or pulmonary edema, with clinical signs of respiratory disease. However, several cases appeared to be affected solely by pericardial effusion rather than pulmonary pathology. Feline pericardial effusion remains an infrequent diagnosis, but its clinical relevance and association with severe cardiac and extracardiac disease warrant diagnostic evaluation.

Comparison of the mitral valve morphologies of Cavalier King Charles Spaniels and dogs of other breeds using 3D transthoracic echocardiography.

BACKGROUND: Myxomatous mitral valve disease (MMVD) is more prevalent in Cavalier King Charles Spaniels (CKCSs) compared to dogs of other breeds at a given age. Abnormal valvular stress is thought to contribute to the development and progression of MMVD, and a relationship exists between mitral valve (MV) morphology and stress acting on the valve. OBJECTIVES: To determine whether the MV morphology of healthy adult CKCSs differs from the morphology of healthy adult dogs of other breeds determined by RT-3DTTE. ANIMALS: Thirty-five healthy CKCSs and 41 healthy dogs of other breeds. METHODS: Prospective cross-sectional study. Dogs underwent physical examination, conventional echocardiography, and RT-3DTTE. RT-3DTTE datasets were analyzed using dedicated software for MV morphologic analysis. Morphologic variables were compared between CKCSs and dogs of other breeds. RESULTS: The MV of healthy CKCSs had a smaller annulus height (0.46 ± 0.11 vs. 0.56 ± 0.17; P = .0021), tenting height (0.26 ± 0.12 vs. 0.42 ± 0.18; P < .001), tenting area (0.42 ± 0.15 vs. 0.79 ± 0.34; P < .001), normalized tenting volume (0.09 [0.05-0.13] vs. 0.14 [0.10-0.20]; P < .001), and normalized area of the posterior leaflet (0.57 ± 0.15 vs. 0.66 ± 0.18; P = .016) compared to healthy dogs of other breeds; this results in CKCSs having a flatter MV with reduced tenting, compared to the MV of other breeds. CONCLUSIONS AND CLINICAL IMPORTANCE: These morphologic features could confer a mechanical disadvantage and play a role in the predisposition of CKCSs to the early development of MMVD.

Expression of distinct classes of titin isoforms in striated and smooth muscles by alternative splicing, and their conserved interaction with filamins.

While the role of titin as a sarcomeric protein is well established, its potential functional role(s) in smooth muscles and non-muscle tissues are controversial. We used a titin exon array to search for which part(s) of the human titin transcriptional unit encompassing 363 exons is(are) expressed in non-striated muscle tissues. Expression profiling of adult smooth muscle tissues (aorta, bladder, carotid, stomach) identified alternatively spliced titin isoforms, encompassing 80 to about 100 exons. These exons code for parts of the titin Z-disk, I-band and A-band regions, allowing the truncated smooth muscle titin isoform to link Z-disks/dense bodies together with thick filaments. Consistent with the array data, Western blot studies detected the expression of approximately 1 MDa smooth muscle titin in adult smooth muscles, reacting with selected Z-disc, I-band, and A-band titin antibodies. Immunofluorescence with these antibodies located smooth muscle titin in the cytoplasm of cultured human aortic smooth muscle cells and in the tunica media of intact adult bovine aorta. Real time PCR studies suggested that smooth muscle titins are expressed from a promoter located 35 kb or more upstream of the transcription initiation site used for striated muscle titin, driving expression of a bi-cistronic mRNA, coding 5' for the anonymous gene FL39502, followed 3' by titin, respectively. Our work showed that smooth muscle and striated muscle titins share in their conserved amino-terminal regions binding sites for alpha-actinin and filamins: Yeast two-hybrid screens using Z2-Zis1 titin baits identified prey clones coding for alpha-actinin-1 and filamin-A from smooth muscle, and alpha-actinin-2/3, filamin-C, and nebulin from skeletal muscle cDNA libraries, respectively. This suggests that the titin Z2-Zis1 domain can link filamins and alpha-actinin together in the periphery of the Z-line/dense bodies in a fashion that is conserved in smooth and striated muscles.

Developmental control of titin isoform expression and passive stiffness in fetal and neonatal myocardium.

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.

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.

Identification of striatin, a desmosomal protein, in the canine corneal epithelium.

Striatin is a scaffolding protein expressed in brain and cardiac tissues. In the heart, striatin has been localized to the region of the cardiac desmosome. A causal mutation within the gene encoding for this scaffolding protein has been described as the etiology for arrhythmogenic right ventricular cardiomyopathy, a disease of the cardiac desmosome, in a canine model. Hemidesmosomes are cell adhesion complexes located within the cornea where they anchor the corneal epithelium to the stroma at the basement membrane and participate in cell-signaling processes. Traditional cell adhesion desmosomes are also known to link the corneal epithelial cells together. We hypothesized that striatin may be found in the cornea localized to regions of either hemidesmosomes and/or desmosomes. Immunohistochemical evaluation was performed to evaluate for striatin labeling in normal canine cornea. Striatin was localized to the cytoplasmic region of corneal epithelial cells. The role of striatin in corneal disease warrants investigation.

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 titin's 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.

Fungal pericarditis and endocarditis secondary to porcupine quill migration in a dog.

A dog evaluated for acute onset of neurologic clinical signs was discovered to have a porcupine quill traversing the left atrium with fungal endocarditis. The dog had been quilled by a porcupine one month prior to presentation and had had several quills removed from the thoracic inlet and left dorsal shoulder areas. A new murmur was identified during the initial examination. Echocardiographic changes consistent with mitral valve endocarditis were identified, in addition to a linear, hyperechoic structure in the left atrium. A thoracic CT identified a possible mediastinal migrating foreign body tract. The foreign body was surgically removed and confirmed as a porcupine quill. Routine aerobic cultures of blood and pericardial samples resulted in growth of presumptive candidal organisms. PCR amplification and sequencing of samples from pericardial cultures identified the presence of a fungal organism, Lodderomyces elongisporus. The neurologic signs were attributed to a left-sided central vestibular lesion presumed secondary to an embolic event from infective endocarditis. After 3 months of antimicrobial and antifungal therapy the valvular changes had markedly improved and the clinical signs resolved. To the authors' knowledge, this is the first description of fungal endocarditis secondary to an intracardiac foreign body in a dog.

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.

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.

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.