Investigation of coagulation and proteomics profiles in symptomatic feline hypertrophic cardiomyopathy and healthy control cats.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a crucial heart disease in cats. The clinical manifestations of HCM comprise pulmonary edema, dyspnea, syncope, arterial thromboembolism (ATE), and sudden cardiac death. D-dimer and prothrombin time (PT) are powerful biomarkers used to assess coagulation function. Dysregulation in these two biomarkers may be associated with HCM in cats. This study aims to assess D-dimer levels, PT, and proteomic profiling in healthy cats in comparison to cats with symptomatic HCM. RESULTS: Twenty-nine client-owned cats with HCM were enrolled, including 15 healthy control and 14 symptomatic HCM cats. The D-dimer concentration and PT were examined. Proteomic analysis was conducted by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In symptomatic cats, D-dimer levels were statistically significantly higher (mean ± SEM: 372.19 ng/ml ± 58.28) than in healthy cats (mean ± SEM: 208.54 ng/ml ± 10.92) with P-value of less than 0.01, while PT was statistically significantly lower in symptomatic cats (mean ± SEM: 9.8 s ± 0.15) compared to healthy cats (mean ± SEM: 11.08 s ± 0.23) with P-value of less than 0.0001. The proteomics analysis revealed upregulation of integrin subunit alpha M (ITGAM), elongin B (ELOB), and fibrillin 2 (FBN2) and downregulation of zinc finger protein 316 (ZNF316) and ectonucleoside triphosphate diphosphohydrolase 8 (ENTPD8) in symptomatic HCM cats. In addition, protein-drug interaction analysis identified the Ras signaling pathway and PI3K-Akt signaling pathway. CONCLUSIONS: Cats with symptomatic HCM have higher D-dimer and lower PT than healthy cats. Proteomic profiles may be used as potential biomarkers for the detection and management of HCM in cats. The use of D-dimer as a biomarker for HCM detection and the use of proteomic profiling for a better understanding of disease mechanisms remain to be further studied in cats.

PKD1 gene mutation and ultrasonographic characterization in cats with renal cysts.

Background: Polycystic kidney disease (PKD) has a complex phenotype partly explained by genetic variants related to this disease. Ultrasonography is a promising approach for defining clinical signs. This study aimed to assess kidney characteristics in cats with Polycystin-1 (PKD1) gene mutations and wild-type cats. Kidney characteristics were identified by ultrasonography. Methods: A total of 108 cats of variable breeds aged an average of 37.01±3.50 months were included. Blood examination and biochemical tests were evaluated. For cystic formation, renal ultrasound was performed. The PKD1 gene mutation was identified via polymerase chain reaction (PCR) and DNA sequencing. Matrix correlation and effectiveness of ultrasound for PKD1 mutation detection were determined. Results: The results showed that 19.44% of cats had PKD1 mutations, a high prevalence in Persian and Persian-related breed cats. Our results demonstrated the characteristics of kidneys in wild-type cats and cats with gene mutations. Based on ultrasonography results, there was an association between cats with gene mutations and cyst formation. The findings indicated that ultrasound did not detect cysts in cats aged 4-36 months, supporting the evidence that PKD1 gene mutations may not be present. This study found high sensitivity and renal specificity ultrasound for PKD1 heterozygous mutation. Moreover, cystic formation via renal ultrasound showed an increased risk for PKD1 mutation 2,623 times compared to normal kidneys. Conclusions: Ultrasonographic examination, coupled with genetic investigations, may help to clarify the phenotypic variability of PKD1. The phenotypic profile of PKD1 will guide therapeutic outcomes and reduce the prevalence of PKD morbidity and mortality in cats.

Echocardiography and MALDI-TOF Identification of Myosin-Binding Protein C3 A74T Gene Mutations Involved Healthy and Mutated Bengal Cats.

This study aimed to identify the potential peptide candidates and expected proteins associated with MYBPC3-A74T gene mutations in Bengal cats and determine if peptidome profiles differ between healthy controls and cats with MYBPC3-A74T gene mutations. All animals were evaluated using echocardiography. DNA was isolated and followed by the screening test of MYBPC3 gene mutation. The MALDI-TOF mass spectrometry was conducted for analyzing the targeted peptide and protein patterns. The expected protein candidates were searched for within the NCBI database. Our results demonstrated that the MYBPC3-A74T gene mutation was dominant in Bengal cats but not in domestic shorthair cats. Correlations between baseline characteristics and echocardiographic parameters were discovered in Bengal cats. Mass spectrometry profiles of the candidate proteins were suspected to accompany the cat with the MYBPC3-A74T gene mutation, involving integral protein-membrane, organization of nucleus, DNA replication, and ATP-binding protein. Therefore, MYBPC3-A74T gene mutations occur frequently in Bengal cat populations. The high incidence of homozygotes for the mutation supports the causal nature of the MYBPC3-A74T mutation. In addition, peptidomics analysis was established for the first time under this condition to promise a complementary technique for the future clinical diagnosis of the MYBPC3-A74T mutation associated with physiological variables and cardiac morphology in cats.

Prevalence of cardiac myosin-binding protein C3 mutations in Maine Coon cats with hypertrophic cardiomyopathy.

Background and Aim: Hypertrophic cardiomyopathy (HCM) is a common heart problem that affects many cats. Although cats with HCM are symptomatic, some die suddenly or develop congestive heart failure. Therefore, this study aimed to estimate the prevalence of myosin-binding protein C3 (MYBPC3), A31P, and A74T polymorphisms in Maine Coon cats to assess risk factors for diagnosing HCM in cats. Materials and Methods: Forty-nine Maine Coon cats of at least 10 months of age were enrolled in this study. First, clinical parameters, such as heart rate, systolic blood pressure, and echocardiography, were evaluated. Then, polymerase chain reaction, followed by DNA sequencing, was conducted using specific primers for amino acid substitutions caused by genetic variants of MYBPC3-A31P and -A74T polymorphisms. Results: Investigations showed that the prevalence of MYBPC3-A31P and -A74T mutations in this study was 16.33% and 24.45%, respectively. Moreover, HCM in cats with MYBPC3-A31P and A74T mutations increased with age, body weight, high heart rate, and prolonged isovolumic relaxation time. Conclusion: Therefore, we propose that Maine Coon cats develop HCM due to multiple genetic factors and underlying clinical characteristics in individual cats. Furthermore, relaxation time assessments can be a sensitive technique for HCM screening during its preclinical phase and can help identify the risk of developing HCM. However, further studies are warranted to evaluate the effect of MYBPC3 mutations on the phenotypic expression of HCM.

Development of a Loop-Mediated Isothermal Amplification Assay Coupled With a Lateral Flow Dipstick Test for Detection of Myosin Binding Protein C3 A31P Mutation in Maine Coon Cats.

Background: Myosin-binding protein C3 A31P (MYBPC3-A31P) missense mutation is a genetic deviation associated with the development of hypertrophic cardiomyopathy (HCM) in Maine Coon cats. The standard detection of the MYBPC3-A31P mutation is complicated, time-consuming, and expensive. Currently, there has been a focus on the speed and reliability of diagnostic tools. Therefore, this study aimed to develop a loop-mediated isothermal amplification assay (LAMP) coupled with a lateral flow dipstick (LFD) test to detect MYBPC3-A31P mutations in Maine Coon cats. Materials and Methods: Fifty-five Maine Coon cats were enrolled in this study, and blood samples were collected. MYBPC3-A31P was genotyped by DNA sequencing. Primers for LAMP with a LFD test were designed. The optimal conditions were determined, including temperature and time to completion for the reaction. The sensitivity of A31P-LAMP detection was compared between agarose gel electrophoresis (the standard method) and the LFD test. The A31P-LAMP-LFD test was randomly performed on seven cats (four with the A31P mutation and three wild-type cats). Results: The A31P-LAMP procedure was able to distinguish between cats with MYBPC3-A31P wild-type cats and MYBPC3-A31P mutant cats. The LAMP reactions were able to be completed in 60 min at a single temperature of 64◦C. Moreover, this study demonstrated that A31P-LAMP coupled with the LFD test allowed for A31P genotype detection at a lower DNA concentration than agarose gel electrophoresis. Discussions: This new A31P-LAMP with a LFD test is a successful and reliable assay with a rapid method, cost-effectiveness, and low requirements for sophisticated equipment for the detection of MYBPC3-A31P mutations. Thus, this assay has excellent potential and can be recognized as a novel screening test for hypertrophic cardiomyopathy associated with MYBPC3-A31P mutations in felines.

Analysis of the Serum Peptidomics Profile for Cats With Sarcomeric Gene Mutation and Hypertrophic Cardiomyopathy.

Background: Hypertrophic cardiomyopathy (HCM) has a complex phenotype that is partly explained by genetic variants related to this disease. The serum peptidome profile is a promising approach to define clinically relevant biomarkers. This study aimed to classify peptide patterns in serum samples between cats with sarcomeric gene mutations and normal cats. Materials and Methods: In the total serum samples from 31 cats, several essential proteins were identified by peptidomics analysis. The 5,946 peptides were differentially expressed in cats with sarcomeric gene mutations compared with cats without mutations. Results: Our results demonstrated characteristic protein expression in control cats, Maine Coon cats, and Maine Coon cats with gene mutations. In cats with gene mutations, peptide expression profiling showed an association with three peptides, Cytochrome 3a132 (CYP3A132), forkhead box O1 (FOXO1), and ArfGAP, with GTPase domains, ankyrin repeats, and PH domain 2 (AGAP2). Discussion: The serum peptidome of cats with mutations might provide supporting evidence for the dysregulation of metabolic and structural proteins. Genetic and peptidomics investigations may help elucidate the phenotypic variability of HCM and treatment targets to reduce morbidity and mortality of HCM in cats.

A Cardiac Protection of Germinated Brown Rice During Cardiopulmonary Bypass Surgery and Simulated Myocardial Ischemia.

PURPOSE: The potential cardio-protective property of germinated brown rice (GBR) has been revealed by ameliorating risk factors related to cardiovascular diseases. This study hypothesized that the combination of GBR and cardioplegic solution could protect the cardiomyocytes exposed to simulated ischemic reperfusion injury in vitro study and preserve cardiac function during cardiopulmonary bypass surgery in animal models. METHODS: Primary porcine cardiomyocytes were isolated and experimented cell viability against simulated ischemic reperfusion injury. In a cardiac surgical animal model, six pigs were randomly assigned to receive the two types of cardioplegic solution: i) St. Thomas cardioplegic solution (20 cc/kg); and ii) St. Thomas cardioplegic solution plus GBR (1 mg/kg). During open-heart surgery, the aorta was cross-clamped for 20 minutes, followed by reperfusion for 1 hour. Cardiopulmonary bypass parameters were recorded until the end of the procedure. Furthermore, hemodynamic parameters and arterial blood gas characteristics of animals among groups were monitored at different time points, including baseline before cardiopulmonary bypass (T1), during cardiopulmonary bypass (T2), during aortic clamp on (T3), and aortic clamp off (T4). RESULTS: Primarily, GBR cotreatment with cardioplegic solution essentially resulted in the improvement of cell viability in primary porcine cardiomyocytes against simulated ischemic reperfusion induction. The findings from cardiac surgery demonstrated that mean arterial pressure and heart rate are constantly stable in cardioplegic solution combined with the GBR group, while the trend of potassium and lactase concentration was decreased in the animals receiving GBR group. Consistently, all parameters from arterial blood gas showed better outcomes in animals receiving GBR; however, there were no statistically significant differences between groups, except hepatic enzymes. CONCLUSION: Therefore, GBR might exert cardio-protective effects against ischemic reperfusion injury in the porcine cardiac surgery model due to anti-inflammatory response. These protective actions of GBR may explain the benefits gained from applying GBR products as a possible therapeutic supplement on cardiac diseases.