Advancing Treatments for Feline Hypertrophic Cardiomyopathy: The Role of Animal Models and Targeted Therapeutics.

Feline HCM is the most common cardiovascular disease in cats, leading to devastating outcomes, including congestive heart failure (CHF), arterial thromboembolism (ATE), and sudden death. Evidence demonstrating long-term survival benefit with currently available therapies is lacking. Therefore, it is imperative to explore intricate genetic and molecular pathways that drive HCM pathophysiology to inspire the development of novel therapeutics. Several clinical trials exploring new drug therapies are currently underway, including those investigating small molecule inhibitors and rapamycin. This article outlines the key work performed using cellular and animal models that has led to and continues to guide the development of new innovative therapeutic strategies.

Exploration of Mediators Associated with Myocardial Remodelling in Feline Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) affects both humans and cats and exhibits considerable interspecies similarities that are exemplified by underlying pathological processes and clinical presentation to the extent that developments in the human field may have direct relevance to the feline disease. Characteristic changes on histological examination include cardiomyocyte hypertrophy and interstitial and replacement fibrosis. Clinically, HCM is characterised by significant diastolic dysfunction due to a reduction in ventricular compliance and relaxation associated with extracellular matrix (ECM) remodelling and the development of ventricular hypertrophy. Studies in rodent models and human HCM patients have identified key protein mediators implicated in these pathological changes, including lumican, lysyl oxidase and TGF-ß isoforms. We therefore sought to quantify and describe the cellular location of these mediators in the left ventricular myocardium of cats with HCM and investigate their relationship with the quantity and structural composition of the ECM. We identified increased myocardial content of lumican, LOX and TGF-ß2 mainly attributed to their increased expression within cardiomyocytes in HCM cats compared to control cats. Furthermore, we found strong correlations between the expressions of these mediators that is compatible with their role as important components of cellular pathways promoting remodelling of the left ventricular myocardium. Fibrosis and hypertrophy are important pathological changes in feline HCM, and a greater understanding of the mechanisms driving this pathology may facilitate the identification of potential therapies.

Rhythm disturbances associated with lidocaine administration in four dogs with supraventricular tachyarrhythmias.

OBJECTIVE: To describe arrhythmias associated with administration of lidocaine in dogs treated for supraventricular tachyarrhythmias. CASE SUMMARIES: Four dogs with recent-onset supraventricular tachyarrhythmias: 3 dogs had atrial fibrillation (AF), and 1 had focal atrial tachycardia (FAT), which was thought to be AF at the time of assessment. The substrate of the supraventricular tachyarrhythmia was considered to be due to primary cardiomyopathy in 1 dog, high vagal tone in 2 dogs, and the change in hemodynamics from heavy sedation in 1 dog. Pharmacological cardioversion using lidocaine was only successful in the 2 dogs with vagally mediated AF. In these 2 cases, lidocaine administration resulted in a paroxysmal atrial flutter that was self-limiting and quickly led to sinus rhythm within 10 seconds in 1 dog but did not change over a 5-minute interval and required additional boluses in another dog. In the latter case, the dog showed severe bradycardia for 17.5 seconds prior to achieving sinus rhythm. The 2 unsuccessful cases both developed ventricular arrhythmias shortly after the lidocaine administration, with 1 case degenerating into ventricular fibrillation and cardiac arrest. NEW OR UNIQUE INFORMATION PROVIDED: Arrhythmias associated with lidocaine should be considered when treating dogs with supraventricular tachyarrhythmia.

Immunohistological Evaluation of Von Willebrand Factor in the Left Atrial Endocardium and Atrial Thrombi from Cats with Cardiomyopathy.

Aortic thromboembolism (ATE) occurs in cats with cardiomyopathy and often results in euthanasia due to poor prognosis. However, the underlying predisposing mechanisms leading to left atrial (LA) thrombus formation are not fully characterised. von Willebrand Factor (vWF) is a marker of endothelium and shows increased expression following endothelial injury. In people with poor LA function and LA remodelling, vWF has been implicated in the development of LA thrombosis. In this study we have shown (1) the expression of endocardial vWF protein detected using immunohistofluorescence was elevated in cats with cardiomyopathy, LA enlargement (LAE) and clinical signs compared to cats with subclinical cardiomyopathy and control cats; (2) vWF was present at the periphery of microthrombi and macrothrombi within the LA where they come into contact with the LA endocardium and (3) vWF was integral to the structure of the macrothrombi retrieved from the atria. These results provide evidence for damage of the endocardial endothelium in the remodelled LA and support a role for endocardial vWF as a pro-thrombotic substrate potentially contributing to the development of ATE in cats with underlying cardiomyopathy and LAE. Results from this naturally occurring feline model may inform research into human thrombogenesis.

Clinical features and outcome of dogs and cats with bidirectional and continuous right-to-left shunting patent ductus arteriosus.

BACKGROUND: Studies describing the clinical progression of animals with reverse patent ductus arteriosus (PDA) are lacking. OBJECTIVES: To describe the signalment, presenting signs, echocardiographic features, and survival in a group of dogs and cats with bidirectional and continuous right-to-left PDA. ANIMALS: Forty-six client-owned animals included, comprising 43 dogs and 3 cats with bidirectional or continuous right-to-left PDA. METHODS: Retrospective multicenter study. Medical records and echocardiographic findings reviewed from animals diagnosed with bidirectional or continuous right-to-left PDA. Impact of ductal morphology, spectral Doppler flow profile, PCV, sildenafil treatment at presentation, sildenafil dose, severity of pulmonary hypertension, general anesthesia with or without surgery and the presence of right-sided congestive heart failure (R-CHF) on crude mortality rate were evaluated via Mantel-Cox log rank comparison of Kaplan-Meier survival curves. Univariable and multivariable Cox proportional hazards analysis was performed, and hazard ratio (HR) (95% confidence intervals [CI]) was presented. RESULTS: Hindlimb collapse was the most common presenting sign in dogs (n = 16). Clinical signs in cats were variable. Median survival time was 626 days in dogs (range 1-3628 days). Dogs with R-CHF had a shorter median survival time (58 days vs 1839 days, P = .03). Dogs treated with sildenafil at initial presentation survived longer (1839 days vs 302 days, P = .03), which was the only independent predictor of survival (HR 0.35, CI 0.15-0.86, P = 0.021). CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs and cats with reverse PDA have a variable clinical presentation and prognosis. Survival time was longer in animals prescribed sildenafil at diagnosis. Dogs with R-CHF at presentation have a worse overall outcome.

Increased insulin-like growth factor 1 concentrations in a retrospective population of non-diabetic cats diagnosed with hypertrophic cardiomyopathy.

OBJECTIVES: The aim of the study was to document whether a proportion of non-diabetic cats with left ventricular hypertrophy (LVH) previously diagnosed with hypertrophic cardiomyopathy (HCM) have elevated circulating insulin-like growth factor 1 (IGF-1) concentrations. METHODS: A retrospective analysis of residual blood samples obtained at the time of echocardiographic diagnosis of HCM from a population of 60 non-diabetic cats were analysed for circulating IGF-1 concentrations using a validated radioimmunoassay and compared with a control group of 16 apparently healthy cats without LVH. Clinical and echocardiographic data for cats with an IGF-1 level >1000 ng/ml were compared with those with an IGF-1 level <800 ng/ml. RESULTS: In total, 6.7% (95% confidence interval 1.8-16.2%) of cats with HCM had an IGF-1 level >1000 ng/ml. The prevalence of an IGF-1 level >1000 ng/ml in the control group was zero. CONCLUSIONS AND RELEVANCE: A small proportion of non-diabetic cats previously diagnosed with HCM had an IGF-1 concentration at a level that has been associated with feline hypersomatotropism (fHS) in the diabetic cat population. Further prospective research is required to confirm or refute the presence of fHS in non-diabetic cats with LVH and increased IGF-1.

Echocardiographic, morphometric and biomarker changes in female cats followed from 6 to 24 months of life.

OBJECTIVES: The aim of the study was to evaluate cardiac size and early growth through echocardiographic, body weight (BW), body condition score (BCS), morphometric and biomarker changes in cats followed from 6 to 24 months of age. METHODS: Twenty-four female European shorthair colony cats were evaluated at birth for BW and at 6, 12, 18 and 24 months of age for BW, BCS, head length (HL) and head width (HW), N-terminal pro B-type natriuretic peptide (NT-proBNP), insulin-like growth factor-1 (IGF-1) and echocardiographic measurements. RESULTS: BCS, HW, left ventricular free wall in diastole, left atrium diameter and aortic diameter increased significantly between 6 and 12 months, while BW, HL and interventricular septum in diastole increased significantly between 6, 12 and 18 months, and BW decreased significantly between 18 and 24 months. NT-proBNP decreased significantly between 6 and 12 months. IGF-1 increased significantly between 6 and 12 months but decreased significantly between 12 and 18 months. CONCLUSIONS AND RELEVANCE: This study prospectively evaluated changes in echocardiographic measurements, BW, BCS, HL, HW, IGF-1 and NT-proBNP in cats during the first 2 years of life. Results show a comparable change over time for different variables. These findings contribute to the understanding of a possible relationship between cardiac measures and body size from young age through to adulthood.

Blood taurine concentrations in dogs with exocrine pancreatic insufficiency.

OBJECTIVE: To determine whether blood taurine concentrations in dogs with exocrine pancreatic insufficiency (EPI) were lower than the reference interval (200 to 350 nmol/mL) or the cutoff used to indicate taurine deficiency (< 150 nmol/mL). ANIMALS: 18 dogs with clinical or presumptive subclinical EPI with residual blood samples available for taurine concentration analysis. PROCEDURES: Dogs were classified as having clinical EPI if they had a serum trypsin-like immunoreactivity concentration of < 2.0 µg/L and presumptive subclinical EPI if they had a concentration of 2.0 to 5.0 µg/L. Archived, frozen blood samples stored in EDTA were submitted for measurement of taurine concentration with an automated high-performance liquid chromatography amino acid analyzer. Medical record data were examined for associations with blood taurine concentration. RESULTS: None of the 18 dogs had a blood taurine concentration < 150 nmol/mL. Two dogs had a concentration < 200 nmol/mL. No clinical signs, physical examination findings, or serum biochemical abnormalities were associated with blood taurine concentration. Eleven of the 17 dogs for which diet histories were available were not receiving a diet that met recommendations of the World Small Animal Veterinary Association Global Nutrition Committee. CONCLUSIONS AND CLINICAL RELEVANCE: A low blood taurine concentration was noted in a small subset of dogs with EPI. Additional research is needed to determine whether EPI was the primary cause of this low concentration. Findings suggested the importance of obtaining complete diet histories and ensuring dietary requirements are sufficiently met in dogs with EPI. (Am J Vet Res 2020;81:958-963).

Large Animal Models in Regenerative Medicine and Tissue Engineering: To Do or Not to Do.

Rapid developments in Regenerative Medicine and Tissue Engineering has witnessed an increasing drive toward clinical translation of breakthrough technologies. However, the progression of promising preclinical data to achieve successful clinical market authorisation remains a bottleneck. One hurdle for progress to the clinic is the transition from small animal research to advanced preclinical studies in large animals to test safety and efficacy of products. Notwithstanding this, to draw meaningful and reliable conclusions from animal experiments it is critical that the species and disease model of choice is relevant to answer the research question as well as the clinical problem. Selecting the most appropriate animal model requires in-depth knowledge of specific species and breeds to ascertain the adequacy of the model and outcome measures that closely mirror the clinical situation. Traditional reductionist approaches in animal experiments, which often do not sufficiently reflect the studied disease, are still the norm and can result in a disconnect in outcomes observed between animal studies and clinical trials. To address these concerns a reconsideration in approach will be required. This should include a stepwise approach using in vitro and ex vivo experiments as well as in silico modeling to minimize the need for in vivo studies for screening and early development studies, followed by large animal models which more closely resemble human disease. Naturally occurring, or spontaneous diseases in large animals remain a largely untapped resource, and given the similarities in pathophysiology to humans they not only allow for studying new treatment strategies but also disease etiology and prevention. Naturally occurring disease models, particularly for longer lived large animal species, allow for studying disorders at an age when the disease is most prevalent. As these diseases are usually also a concern in the chosen veterinary species they would be beneficiaries of newly developed therapies. Improved awareness of the progress in animal models is mutually beneficial for animals, researchers, human and veterinary patients. In this overview we describe advantages and disadvantages of various animal models including domesticated and companion animals used in regenerative medicine and tissue engineering to provide an informed choice of disease-relevant animal models.

The gut microbiome in dogs with congestive heart failure: a pilot study.

Compromised gut health and dysbiosis in people with heart failure has received a great deal of attention over the last decade. Whether dogs with heart failure have a similar dysbiosis pattern to what is described in people is currently unknown. We hypothesised that dogs with congestive heart failure have quantifiable dysbiosis compared to healthy dogs that are similar in sex and age. A total of 50 dogs (15 healthy dogs and 35 dogs with congestive heart failure) were prospectively recruited, and their faecal gut microbiome was assessed using 16S rRNA sequencing (Illumina MiSeq platform). There was no significant change in the microbial diversity and richness in dogs with congestive heart failure. However, there was an increase in abundance of Proteobacteria in the congestive heart failure group (p = 0.014), particularly due to an increase in the family Enterobacteriaceae (p = 0.002) and Escherichia coli (p = 0.033). We conclude that dogs with congestive heart failure have dysbiosis, and we show additional trends in our data suggesting that dogs may have a similar pattern to that described in people. The results of this study provide useful preliminary information and raise the possibility that dogs represent a clinically relevant animal model of dysbiosis in people with heart failure.

Erratum to "Inter-observer variability for cardiac ultrasound measurements in cats repeated at different time points in early adult life" [5C (June 2018) 44-46].

[This corrects the article DOI: 10.1016/j.vas.2018.03.002.].

Biomarker changes with systolic anterior motion of the mitral valve in cats with hypertrophic cardiomyopathy.

BACKGROUND: N-terminal pro B-type natriuretic peptide (NT-proBNP) and cardiac troponin-I (cTnI) are biomarkers commonly evaluated in cats with suspected heart disease. Many cats with hypertrophic cardiomyopathy (HCM) have systolic anterior motion of the mitral valve (SAM), but its influence on circulating NT-proBNP or cTnI concentrations is currently unknown. HYPOTHESIS/OBJECTIVES: Cats with HCM and SAM (HCMSAM+ ) have higher NT-proBNP and cTnI concentrations than do cats with HCM but without SAM (HCMSAM- ). ANIMALS: One hundred forty cats with HCM: 70 with SAM and 70 without SAM. METHODS: Retrospective case-to-case study. Cats were recruited if diagnosed with HCM by echocardiography and results were available for NT-proBNP or cTnI concentrations or both. Cats with SAM were matched to those without SAM for clinical presentation, left atrial (LA) size and left ventricular (LV) fractional shortening. RESULTS: A total of 119 NT-proBNP and 123 cTnI results were available. The HCMSAM+ cats had higher median concentrations than did HCMSAM- cats for NT-proBNP (729 pmoL/L; interquartile range [IQR], 275-1467 versus 65 pmoL/L; IQR, 25-271; P < .001) and cTnI (0.27 ng/mL; IQR, 0.10-0.81 versus 0.07 ng/mL; IQR, 0.01-0.43; P = .002). In general linear models for both NT-proBNP and cTnI, the independent explanatory variables were SAM, congestive heart failure, maximal LV wall thickness, and LA size. CONCLUSIONS AND CLINICAL IMPORTANCE: For cats with HCM and equivalent LA size and LV systolic function, those with SAM had higher NT-proBNP and cTnI concentrations than did those without SAM. Presence of SAM should be considered when interpreting biomarker concentrations in cats with HCM.

Inducing Pluripotency in the Domestic Cat (Felis catus).

Domestic cats suffer from a range of inherited genetic diseases, many of which display similarities with equivalent human conditions. Developing cellular models for these inherited diseases would enable drug discovery, benefiting feline health and welfare as well as enhancing the potential of cats as relevant animal models for translation to human medicine. Advances in our understanding of these diseases at the cellular level have come from the use of induced pluripotent stem cells (iPSCs). iPSCs can differentiate into virtually any cell type and can be derived from adult somatic cells, therefore overcoming the ethical implications of destroying embryos to obtain embryonic stem cells. No studies, however, report the generation of iPSCs from domestic cats [feline iPSCs (fiPSCs)]. Feline adipose-derived fibroblasts were infected with amphotropic retrovirus containing the coding sequences for human Oct4, Sox2, Klf4, cMyc, and Nanog. Isolated iPSC clones were expanded on inactivated mouse embryonic fibroblasts in the presence of feline leukemia inhibitory factor (fLIF). Retroviral delivery of human pluripotent genes gave rise to putative fiPSC colonies within 5-7 days. These iPS-like cells required fetal bovine serum and fLIF for maintenance. Colonies were domed with refractile edges, similar to mouse iPSCs. Immunocytochemistry demonstrated positive staining for stem cell markers: alkaline phosphatase, Oct4, Sox2, Nanog, and SSEA1. Cells were negative for SSEA4. Expression of endogenous feline Nanog was confirmed by quantitative polymerase chain reaction. The cells were able to differentiate in vitro into cells representative of the three germ layers. These results confirm the first generation of induced pluripotent stem cells from domestic cats. These cells will provide valuable models to study genetic diseases and explore novel therapeutic strategies.

Speckle tracking echocardiography in cats with preclinical hypertrophic cardiomyopathy.

BACKGROUND: Cats with hypertrophic cardiomyopathy (HCM) have decreased left ventricular (LV) longitudinal deformation detected by mitral annular plane systolic excursion (MAPSE) and speckle tracking echocardiography. People with preclinical HCM have decreased systolic LV longitudinal and radial strain (S) and strain rate (SR), with preserved circumferential S and SR. HYPOTHESIS/OBJECTIVES: Cats with preclinical HCM have decreased systolic LV deformation compared to normal cats. ANIMALS: Seventy-three client-owned cats with (n = 37) and without (n = 36) preclinical HCM. METHODS: Retrospective echocardiographic study. Left and right ventricular longitudinal S and SR, LV radial and circumferential S and SR were calculated by STE. Left ventricular mass was also calculated. Correlation between STE variables and LV hypertrophy was determined and receiver-operating characteristic (ROC) curves were plotted for prediction of HCM. RESULTS: Cats with HCM had smaller absolute longitudinal S (-14.8 ± 3.3% vs -19.7 ± 2.7%, P < .001), longitudinal SR (-2.36 ± 0.62 vs -2.95 ± 0.68 second-1 , P < .001), radial S (46.2 ± 21.3% vs 66.7 ± 17.6%, P < .001), and radial SR (5.60 ± 2.08 vs 6.67 ± 1.8 second-1 , P < .001) compared to healthy controls. No difference was observed for circumferential S and SR. Cats with HCM had greater LV mass (13.2 ± 3.7 g vs 8.6 ± 2.7 g, P < .001). The ROC with the greatest area under the curve (AUC) for the identification of HCM (0.974) was plotted from a logistic regression equation combining LV mass, MAPSE at the free wall, and LV internal diameter in diastole (LVIDd). CONCLUSIONS AND CLINICAL IMPORTANCE: Cats with preclinical HCM have decreased long axis and radial deformation. Decreased longitudinal deformation and decreased LVIDd are factors that would support a diagnosis of HCM.

Cardiosphere-derived cells suppress allogeneic lymphocytes by production of PGE2 acting via the EP4 receptor.

Cardiosphere-derived cells (CDCs) are a cardiac progenitor cell population, which have been shown to possess cardiac regenerative properties and can improve heart function in a variety of cardiac diseases. Studies in large animal models have predominantly focussed on using autologous cells for safety, however allogeneic cell banks would allow for a practical, cost-effective and efficient use in a clinical setting. The aim of this work was to determine the immunomodulatory status of these cells using CDCs and lymphocytes from 5 dogs. CDCs expressed MHC I but not MHC II molecules and in mixed lymphocyte reactions demonstrated a lack of lymphocyte proliferation in response to MHC-mismatched CDCs. Furthermore, MHC-mismatched CDCs suppressed lymphocyte proliferation and activation in response to Concanavalin A. Transwell experiments demonstrated that this was predominantly due to direct cell-cell contact in addition to soluble mediators whereby CDCs produced high levels of PGE2 under inflammatory conditions. This led to down-regulation of CD25 expression on lymphocytes via the EP4 receptor. Blocking prostaglandin synthesis restored both, proliferation and activation (measured via CD25 expression) of stimulated lymphocytes. We demonstrated for the first time in a large animal model that CDCs inhibit proliferation in allo-reactive lymphocytes and have potent immunosuppressive activity mediated via PGE2.

Time spent with cats is never wasted: Lessons learned from feline acromegalic cardiomyopathy, a naturally occurring animal model of the human disease.

BACKGROUND: In humans, acromegaly due to a pituitary somatotrophic adenoma is a recognized cause of increased left ventricular (LV) mass. Acromegalic cardiomyopathy is incompletely understood, and represents a major cause of morbidity and mortality. We describe the clinical, echocardiographic and histopathologic features of naturally occurring feline acromegalic cardiomyopathy, an emerging disease among domestic cats. METHODS: Cats with confirmed hypersomatotropism (IGF-1>1000ng/ml and pituitary mass; n = 67) were prospectively recruited, as were two control groups: diabetics (IGF-1<800ng/ml; n = 24) and healthy cats without known endocrinopathy or cardiovascular disease (n = 16). Echocardiography was performed in all cases, including after hypersomatotropism treatment where applicable. Additionally, tissue samples from deceased cats with hypersomatotropism, hypertrophic cardiomyopathy and age-matched controls (n = 21 each) were collected and systematically histopathologically reviewed and compared. RESULTS: By echocardiography, cats with hypersomatotropism had a greater maximum LV wall thickness (6.5mm, 4.1-10.1mm) than diabetic (5.9mm, 4.2-9.1mm; Mann Whitney, p<0.001) or control cats (5.2mm, 4.1-6.5mm; Mann Whitney, p<0.001). Left atrial diameter was also greater in cats with hypersomatotropism (16.6mm, 13.0-29.5mm) than in diabetic (15.4mm, 11.2-20.3mm; Mann Whitney, p<0.001) and control cats (14.0mm, 12.6-17.4mm; Mann Whitney, p<0.001). After hypophysectomy and normalization of IGF-1 concentration (n = 20), echocardiographic changes proved mostly reversible. As in humans, histopathology of the feline acromegalic heart was dominated by myocyte hypertrophy with interstitial fibrosis and minimal myofiber disarray. CONCLUSIONS: These results demonstrate cats could be considered a naturally occurring model of acromegalic cardiomyopathy, and as such help elucidate mechanisms driving cardiovascular remodeling in this disease.

Pericardial effusion associated with systemic inflammatory disease in seven dogs (January 2006 - January 2012).

Pericardial effusion (PE) is reported in dogs as a consequence of neoplasia, primary cardiac disease or as an idiopathic condition. We describe seven dogs with systemic inflammatory disease, PE without tamponade and increased cardiac troponin I concentrations. Echocardiographic findings and adjunctive testing did not identify other known causes of PE. Resolution of the PE was documented in five of seven dogs in which follow-up echocardiography was performed, often after anti-inflammatory therapy. Resolution of PE was associated with normalisation of cardiac troponin I levels. Clinical signs had not recurred in six dogs with follow-up for more than 12 months and up to 7 years. These findings suggest an association between systemic inflammation and PE in dogs.

Inter-observer variability for cardiac ultrasound measurements in cats repeated at different time points in early adult life.

A high degree of accuracy is required when using echocardiography to diagnose hypertrophic cardiomyopathy (HCM) in cats, as variation in measurements of 0.5 mm may affect classification of individuals as 'abnormal'. This study in adult cats examined at different time points inter-observer variability between two Board certified echocardiographers in veterinary cardiology. Twenty-four female European shorthair cats were examined at 12, 18 and 24 months of age by observer 1. Two dimensional (2D) echocardiographic images were collected in conscious cats to measure left ventricular, aortic and left atrial dimensions. Measurements were repeated by observer 2 on stored images, and analysed for effect of time, observer and time-observer interaction. Based on end-diastolic left ventricular wall thickness, cats were diagnosed as 'normal' or 'abnormal'. Linear mixed models (generalized when appropriate) were performed. A significant difference between observers was found for all septal (IVSd) and free wall (LVFWd) thickness measurements and left ventricular internal diameters but not for aortic or left atrial measurements. All measurement coefficients of variation (CV) were < 10%. The CV for IVSd was higher than the CV for LVFWd. There was a significant effect of time on IVSd, aortic measurements and left ventricular internal diameter measurements. No significant time-observer interaction was found for any parameter. Diagnosis of cats as 'abnormal' (>5 mm in cats > 6 kg bodyweight) was significantly different between observers for IVSd but not LVFWd. Caution is warranted when diagnosing as 'abnormal' or interpreting small changes based on IVSd, due to significant inter-observer differences in this measurement.

Cryopreservation of canine cardiosphere-derived cells: Implications for clinical application.

The clinical application of cardiosphere-derived cells (CDCs) to treat cardiac disease has gained increasing interest over the past decade. Recent clinical trials confirm their regenerative capabilities, although much remains to be elucidated about their basic biology. To develop this new treatment modality, in a cost effective and standardized workflow, necessitates the creation of cryopreserved cell lines to facilitate access for cardiac patients requiring urgent therapy. Cryopreservation may however lead to alterations in cell behavior and potency. The aim of this study was to investigate the effect of cryopreservation on canine CDCs. CDCs and mesenchymal stem cells (MSCs) isolated from five dogs were characterized. CDCs demonstrated a population doubling time that was unchanged by cryopreservation (fresh vs. cryopreserved; 57.13 ± 5.27 h vs. 48.94 ± 9.55 h, P = 0.71). This was slower than for MSCs (30.46 h, P < 0.05). The ability to form clones, self-renew, and commit to multiple lineages was unaffected by cryopreservation. Cryopreserved CDCs formed larger cardiospheres compared to fresh cells (P < 0.0001). Fresh CDCs showed a high proportion of CD105+ (89.0% ± 4.98) and CD44+ (99.68% ± 0.13) cells with varying proportions of CD90+ (23.36% ± 9.78), CD34+ (7.18% ± 4.03) and c-Kit+ (13.17% ± 8.67) cells. CD45+ (0.015% ± 0.005) and CD29+ (2.92% ± 2.46) populations were negligible. Increasing passage number of fresh CDCs correlated with an increase in the proportion of CD34+ and a decrease in CD90+ cells (P = 0.003 and 0.03, respectively). Cryopreserved CDCs displayed increased CD34+ (P < 0.001) and decreased CD90+ cells (P = 0.042) when compared to fresh cells. Overall, our study shows that cryopreservation of canine CDCs is feasible without altering their stem characteristics, thereby facilitating their utilization for clinical trials. © 2017 International Society for Advancement of Cytometry.

Investigations into the Sarcomeric Protein and Ca2+-Regulation Abnormalities Underlying Hypertrophic Cardiomyopathy in Cats (Felix catus).

Hypertrophic cardiomyopathy (HCM) is the most common single gene inherited cardiomyopathy. In cats (Felix catus) HCM is even more prevalent and affects 16% of the outbred population and up to 26% in pedigree breeds such as Maine Coon and Ragdoll. Homozygous MYBPC3 mutations have been identified in these breeds but the mutations in other cats are unknown. At the clinical and physiological level feline HCM is closely analogous to human HCM but little is known about the primary causative mechanism. Most identified HCM causing mutations are in the genes coding for proteins of the sarcomere. We therefore investigated contractile and regulatory proteins in left ventricular tissue from 25 cats, 18 diagnosed with HCM, including a Ragdoll cat with a homozygous MYBPC3 R820W, and 7 non-HCM cats in comparison with human HCM (from septal myectomy) and donor heart tissue. Myofibrillar protein expression was normal except that we observed 20-44% MyBP-C haploinsufficiency in 5 of the HCM cats. Troponin extracted from 8 HCM and 5 non-HCM cat hearts was incorporated into thin filaments and studied by in vitro motility assay. All HCM cat hearts had a higher (2.06 ± 0.13 fold) Ca2+-sensitivity than non-HCM cats and, in all the HCM cats, Ca2+-sensitivity was not modulated by troponin I phosphorylation. We were able to restore modulation of Ca2+-sensitivity by replacing troponin T with wild-type protein or by adding 100 µM Epigallocatechin 3-gallate (EGCG). These fundamental regulatory characteristics closely mimic those seen in human HCM indicating a common molecular mechanism that is independent of the causative mutation. Thus, the HCM cat is a potentially useful large animal model.