Association between high expression macrophage migration inhibitory factor (MIF) alleles and West Nile virus encephalitis.

Infection with mosquito-borne West Nile virus (WNV) is usually asymptomatic but can lead to severe WNV encephalitis. The innate cytokine, macrophage migration inhibitory factor (MIF), is elevated in patients with WNV encephalitis and promotes viral neuroinvasion and mortality in animal models. In a case-control study, we examined functional polymorphisms in the MIF locus in a cohort of 454 North American patients with neuroinvasive WNV disease and found patients homozygous for high-expression MIF alleles to be >20-fold (p=0.008) more likely to have WNV encephalitis. These data indicate that MIF is an important determinant of severity of WNV neuropathogenesis and may be a therapeutic target.

Urine sodium concentration after intravenous furosemide in dogs with acute congestive heart failure and correlation with treatment efficacy.

BACKGROUND: Poor natriuresis is a potential marker of diuretic resistance in dogs with acute congestive heart failure (CHF) but little is known about the relationship between urine sodium concentration (uNa) and frequency of successful decongestion. Supplemental O2 is a common treatment in dogs with severe CHF. The time from start to discontinuation of supplemental O2 therapy (DCSO2 ) typically reflects the time course and ease of decongestion. HYPOTHESIS/OBJECTIVES: Urine Na concentration after IV administration of furosemide will be correlated with duration of treatment with supplemental O2 (timeO2 ) and the cumulative frequency of successful DCSO2 during hospitalization. ANIMALS: Fifty-one dogs with acute CHF. METHODS: Retrospective observational single center study. RESULTS: Dogs with low uNa had significantly longer mean timeO2 than dogs with high uNa (uNa <87 mmol/L, 24.2 ± 2.6 hours vs uNa ≥87 mmol/L, 16.6 ± 1.7 hours; P = .02). Low uNa was correlated with lower cumulative frequency of DCSO2 (12 hour, 28%; 24 hour, 42%; 36 hour, 73%) compared to high uNa (12 hour, 28%; 24 hour, 88%; 36 hour, 96%; P = .005). History of PO loop diuretics, low serum chloride concentration (sCl), and high PCV were associated with low uNa. Urine Na concentration outperformed other metrics of diuretic responsiveness including weight loss. CONCLUSIONS AND CLINICAL IMPORTANCE: Urine Na concentration after IV furosemide predicted timeO2 and cumulative frequency of DCSO2 in dogs with acute CHF, which likely reflects important aspects of diuretic responsiveness. Urine Na can assess diuretic responsiveness and treatment efficacy in dogs with CHF.

Utility of focused cardiac ultrasonography training in veterinary students to differentiate stages of subclinical myxomatous mitral valve disease in dogs.

BACKGROUND: Differentiation of the subclinical phases of myxomatous mitral valve disease (MMVD) in dogs relies heavily on echocardiography. Focused cardiac ultrasonography (FCU) is a point-of-care technique that can assess heart size. HYPOTHESIS/OBJECTIVES: Veterinary students trained in FCU can differentiate dogs with subclinical MMVD based on left ventricular (LV) and left atrial (LA) dimensions. ANIMALS: Forty-eight dogs with subclinical MMVD. METHODS: Veterinary students were trained to measure LV dimension and LA-to-aortic root dimension ratio (LA : Ao) using FCU. Dogs were categorized into 2 cohorts based on whether or not the LV normalized internal diastolic dimension was ≥1.7 and LA : Ao was ≥1.6. Agreement between FCU and echocardiographic studies performed by cardiologists was evaluated. RESULTS: One-hundred and forty-six FCU examinations were performed by 58 veterinary students on 48 dogs. Overall agreement between students and cardiologists was moderate (Fleiss' kappa, 0.54; 95% confidence interval [CI], 0.39-0.69; P < .001). Percentage accuracy in observations with heart dimensions less than the cutoffs (86/89, 97%) was significantly higher than in observations in with larger hearts (31/57, 54%; P < .001). Agreement increased from moderate to good as heart sizes became more extreme. Degree of confidence by students in performing FCU was significantly higher at the end vs start of the study. CONCLUSIONS AND CLINICAL IMPORTANCE: Categorization of dogs with subclinical MMVD by veterinary students using FCU was associated with moderate to good agreement with echocardiography. Focused cardiac ultrasonography is a point-of-care method that can help assess clinical stage in dogs with subclinical MMVD.

Focused Cardiac Ultrasonography in Cats.

Heart disease is a common cause of morbidity and mortality in cats. Focused cardiac ultrasonography (FCU) is a useful diagnostic tool for identifying heart disease in symptomatic and asymptomatic cats when performed by trained veterinarians. When used in conjunction with other diagnostics such as physical examination, blood biomarkers, electrocardiography, Global FAST, and other point-of-care ultrasonographic examinations, FCU may improve clinical decision making and help clinicians prioritize which cats would benefit from referral for complete echocardiography and cardiac consultation. This article reviews the definition, advantages, clinical indications, limitations, training recommendations, and a protocol for FCU in cats.

Effect of angiotensin receptor blockers and angiotensin converting enzyme 2 on plasma equilibrium angiotensin peptide concentrations in dogs with heart disease.

BACKGROUND: The pathophysiology of heart failure involves maladaptive angiotensin peptides (APs) and enzymes, including angiotensin 2 (AT2) and angiotensin converting enzyme (ACE), as well as recently described alternative components, such as angiotensin 1-7 (Ang1-7) and angiotensin converting enzyme 2 (ACE2). The relative effects of different neurohormonal-targeting drugs on balance of APs in dogs with heart disease are unknown. HYPOTHESIS/OBJECTIVES: Plasma AP concentrations differ in dogs receiving angiotensin converting enzyme inhibitors (ACEIs) vs angiotensin receptor blockers (ARBs) and recombinant human ACE2 (rhACE2) will further increase these differences. ANIMALS: Eight dogs with degenerative mitral valve disease (DMVD). METHODS: Prospective open-label trial. Equilibrium concentrations of APs from plasma during PO ACEI treatment and then after 14 days of PO ARB treatment using telmisartan were measured using liquid chromatography-tandem mass spectroscopy before and after in vitro incubation with rhACE2. RESULTS: Concentration of Ang1-7 was increased during ARB treatment (Ang1-7: 443 pg/mL; 95% confidence interval [CI] = 247-794 pg/mL) vs ACEI (Ang1-7: 182 pg/mL; 95% CI = 66.2-503 pg/mL; P = .01). Incubation with rhACE2 decreased traditional APs while increasing beneficial alternative APs, and Ang1-7 was significantly higher in the ARB + rhACE2 (880 pg/mL; 95% CI = 560-1383 pg/mL) vs ACEI + rhACE2 (455 pg/mL; 95% CI = 188-1104 pg/mL; P = .03) group. The most favorable theoretical AP profile was achieved in the ARB + rhACE2 group. CONCLUSIONS AND CLINICAL IMPORTANCE: The AP profile during telmisartan treatment is associated with higher plasma Ang1-7 as compared with during ACEI. This favorable shift is potentiated in vitro by combination of ARB + rhACE2. These data support potential AP-targeting strategies and drugs in dogs with DMVD.

Effect of angiotensin receptor blockers and angiotensin-converting enzyme 2 on plasma equilibrium angiotensin peptide concentrations in cats with heart disease.

BACKGROUND: Little is known about the effect of renin angiotensin aldosterone system-inhibiting (RAASi) drugs on alternative angiotensin peptides (APs) such as angiotensin 1-7 (Ang1-7), which are mediated by angiotensin-converting enzyme 2 (ACE2). HYPOTHESIS/OBJECTIVES: Angiotensin receptor blockers (ARBs) would alter balance of APs and differences would be magnified in vitro by incubation of plasma samples with recombinant human ACE2 (rhACE2). ANIMALS: Six cats with cardiomyopathy (CM), 8 healthy cats. METHODS: Prospective open label trial. Plasma equilibrium concentrations of APs were measured in healthy cats as well as in CM cats that first received no RAASi drugs (CMnoRAASi ) and then after 14 days of PO telmisartan (CMARB ). Plasma APs also were measured after in vitro incubation with rhACE2. RESULTS: No significant differences were found between healthy and CMnoRAASi groups. Concentrations of several APs, including angiotensin I (AT1) and angiotensin II (AT2) were significantly different between CMnoRAASi and CMARB groups. Incubation with rhACE2 decreased AT1 and AT2 in both groups. The geometric mean concentration of Ang1-7 was significantly higher in CMARB (4.9 pg/mL; 95% confidence interval [CI], 3.7-6.4 pg/mL) vs CMnoRAASi (3.2 pg/mL; 95% CI, 2.2-4.7 pg/mL; P = .01) and in CMARB + ACE2 (5.0 pg/mL; 95% CI, 3.9-6.4 pg/mL) vs CMnoRAASi + ACE2 (3.0 pg/mL; 95% CI, 1.7-5.5 pg/mL; P = .01). The most favorable theoretical AP profile that maximized Ang1-7 and other alternative APs was CMARB + ACE2. CONCLUSIONS AND CLINICAL IMPORTANCE: Balance between traditional and alternative APs can be favorably shifted using ARBs and in vitro incubation with rhACE2. These data shed light on new AP-targeting strategies in cats with CM.

Congenital dyserythropoiesis and polymyopathy without cardiac disease in male Labrador retriever littermates.

BACKGROUND: Two Labrador retriever littermates were identified based on incidentally noted marked microcytosis and inappropriate metarubricytosis. Muscle atrophy was noted and associated with distinctive pathological findings in biopsy samples from 1 dog studied. The disorder represents a rare clinical entity of suspected congenital dyserythropoiesis and polymyopathy. Clinicopathologic changes were similar to a previously reported syndrome of congenital dyserythropoiesis, congenital polymyopathy, and cardiac disease in 3 related English Springer Spaniel (ESS) dogs, but the dogs reported here did not have apparent cardiac disease. INTERVENTIONS: Bone marrow aspiration, electromyography, muscle biopsies, and an echocardiogram were performed on dog 1. Results supported dyserythropoiesis and congenital polymyopathy similar to reports in ESS dogs, but did not identify obvious cardiac disease. CONCLUSION: The clinicopathologic changes of dyserythropoiesis and polymyopathy provide an easily recognizable phenotype for what appears to be a low morbidity syndrome. Early recognition may decrease unnecessary testing or euthanasia.

Metabolomics Analysis Reveals Deranged Energy Metabolism and Amino Acid Metabolic Reprogramming in Dogs With Myxomatous Mitral Valve Disease.

Background Myxomatous mitral valve disease (MMVD), a naturally occurring heart disease, affects 10% to 15% of the canine population. Canine MMVD shares many similarities with human MMVD. Untargeted metabolomics was performed to identify changes in metabolic pathways and biomarkers with potential clinical utilities. Methods and Results Serum samples from 27 healthy, 22 stage B1, 18 stage B2 preclinical MMVD dogs, and 17 MMVD dogs with a history of congestive heart failure (CHF) were analyzed. Linear regression analysis identified 173 known metabolites whose concentrations were different among the 4 groups (adjusted P<0.05), of which 40% belonged to amino acid super pathways, while 30% were lipids. More than 50% of significant metabolites were correlated with left atrial diameter but not left ventricular dimension. Acylcarnitines, tricarboxylic acid cycle intermediates, and creatine accumulated in proportion to MMVD severity. α-Ketobutyrate and ketone bodies were increased as MMVD advanced. Nicotinamide, a key substrate of the main nicotinamide adenine dinucleotide (NAD+) salvage pathway, was decreased, while quinolinate of the de novo NAD+ biosynthesis was increased in CHF dogs versus healthy dogs. 3-Methylhistidine, marker for myofibrillar protein degradation, was higher in CHF dogs than non-CHF dogs. Trimethylamine N-oxide (TMAO) and TMAO-producing precursors, including carnitine, phosphatidylcholine, betaine, and trimethyllysine, were increased in CHF dogs versus non-CHF dogs. Elevated levels of uremic toxins, including guanidino compounds, TMAO, and urea, were observed in CHF dogs. Pathway analysis highlighted the importance of bioenergetics and amino acid metabolism in canine MMVD. Conclusions Our study revealed altered energy metabolism, amino acid metabolic programming, and reduced renal function in the development of MMVD and CHF. Complex interplays along the heart-kidney-gut axis were implicated.

Gut Dysbiosis and Its Associations with Gut Microbiota-Derived Metabolites in Dogs with Myxomatous Mitral Valve Disease.

Gut dysbiosis and gut microbiota-derived metabolites, including bile acid (BA), short-chain fatty acid, and trimethylamine N-oxide (TMAO), are associated with cardiovascular disease. Canine myxomatous mitral valve disease (MMVD) is a model for human MMVD. The aim of the study is to evaluate gut microbial dysbiosis and its relationship with gut-produced metabolites in dogs with MMVD. Fecal samples from 92 privately owned dogs, including 17 healthy, 23 and 27 asymptomatic MMVD dogs without (stage B1) and with (stage B2) secondary cardiac enlargement, respectively, and 25 MMVD dogs with history of congestive heart failure (stage C or D), were analyzed by 16S rRNA sequencing. Alpha and beta diversities were different between healthy and MMVD dogs (adjusted P < 0.05). The average dysbiosis indexes were -1.48, -0.6, 0.01, and 1.47 for healthy, B1, B2, and C/D dogs, respectively (P = 0.07). Dysbiosis index was negatively correlated with Clostridium hiranonis (P < 0.0001, r = -0.79). Escherichia coli, capable of trimethylamine production in the gut, had an increased abundance (adjusted P < 0.05) and may be responsible for the increased circulating TMAO levels in stage B2 and C/D MMVD dogs. Primary and secondary BAs showed opposite associations with C. hiranonis, a key BA converter (P < 0.0001 for both, r = -0.94 and 0.95, respectively). Secondary BAs appeared to promote the growth of Fusobacterium and Faecalibacterium but inhibit that of E. coli Multivariate analysis revealed significant but weak associations between gut microbiota and several circulating metabolites, including short-chain acylcarnitines and TMAO.IMPORTANCE Our study expands the current "gut hypothesis" to include gut dysbiosis at the preclinical stage, prior to the onset of heart failure. Gut dysbiosis index increases in proportion to the severity of myxomatous mitral valve disease (MMVD) and is inversely associated with Clostridium hiranonis, a key bile acid (BA) converter in the gut. Secondary BAs appear to promote the growth of beneficial bacteria but inhibit that of harmful ones. An intricate interplay between gut microbiota, gut microbiota-produced metabolites, and MMVD pathophysiological progression is implicated.

Prediction and measurement of diuretic responsiveness after oral administration of furosemide to healthy dogs and dogs with congestive heart failure.

BACKGROUND: In human patients, cumulative urine volume (uVol) and urine sodium (uNa) can be predicted using spot urine samples and these quantitative measures help detect low diuretic responsiveness (LDR). HYPOTHESIS/OBJECTIVES: Formulas using spot urine samples predict cumulative uVol and uNa output after oral administration of furosemide to dogs. ANIMALS: Eight healthy dogs, 6 dogs with congestive heart failure (CHF). METHODS: Prospective interventional study. Spot urine samples at 180 and 270 minutes after furosemide (3 mg/kg PO) were used to predict cumulative uVol and uNa output over 7 hours. Differentiation of dogs fulfilling predefined criteria for LDR was examined using receiver operating characteristic (ROC) curves. RESULTS: Predicted uNa output at 180 minutes (rs = 0.763, [95% confidence interval [CI], 0.375-0.923], P = .002) and 270 minutes (r = 0.816, [95% CI, 0.503-0.940], P < .001) was highly correlated to 7-hour uNa output. Predicted uVol at 180 minutes (r = 0.598, [95% CI, 0.098-0.857], P = .02) and 270 minutes (r = 0.791, [95% CI, 0.450-0.931], P < .001) was moderately correlated to 7-hour uVol. Predicted uNa using 180-minute (area under the curve [AUC], 0.933 [95% CI, 0.804-1.000]) and 270-minute (AUC, 0.911 [95% CI, 0.756-1.000]) samples identified dogs with LDR (n = 5) with high accuracy. CONCLUSIONS AND CLINICAL IMPORTANCE: Urinary Na excretion and uVol are complementary but distinct aspects of diuretic responsiveness in dogs. Quantification of diuretic responsiveness in the clinical setting opens new diagnostic, treatment, and monitoring strategies.

Comparative pathology of human and canine myxomatous mitral valve degeneration: 5HT and TGF-ß mechanisms.

Myxomatous mitral valve degeneration (MMVD) is a leading cause of valve repair or replacement secondary to the production of mitral regurgitation, cardiac enlargement, systolic dysfunction, and heart failure. The pathophysiology of myxomatous mitral valve degeneration is complex and incompletely understood, but key features include activation and transformation of mitral valve (MV) valvular interstitial cells (VICs) into an active phenotype leading to remodeling of the extracellular matrix and compromise of the structural components of the mitral valve leaflets. Uncovering the mechanisms behind these events offers the potential for therapies to prevent, delay, or reverse myxomatous mitral valve degeneration. One such mechanism involves the neurotransmitter serotonin (5HT), which has been linked to development of valvulopathy in a variety of settings, including valvulopathy induced by serotonergic drugs, Serotonin-producing carcinoid tumors, and development of valvulopathy in laboratory animals exposed to high levels of serotonin. Similar to humans, the domestic dog also experiences naturally occurring myxomatous mitral valve degeneration, and in some breeds of dogs, the lifetime prevalence of myxomatous mitral valve degeneration reaches 100%. In dogs, myxomatous mitral valve degeneration has been associated with high serum serotonin, increased expression of serotonin-receptors, autocrine production of serotonin within the mitral valve leaflets, and downregulation of serotonin clearance mechanisms. One pathway closely associated with serotonin involves transforming growth factor beta (TGF-ß) and the two pathways share a common ability to activate mitral valve valvular interstitial cells in both humans and dogs. Understanding the role of serotonin and transforming growth factor beta in myxomatous mitral valve degeneration gives rise to potential therapies, such as 5HT receptor (5HT-R) antagonists. The main purposes of this review are to highlight the commonalities between myxomatous mitral valve degeneration in humans and dogs, with specific regards to serotonin and transforming growth factor beta, and to champion the dog as a relevant and particularly valuable model of human disease that can accelerate development of novel therapies.

Echocardiographic indices and severity of mitral regurgitation in dogs with preclinical degenerative mitral valve disease.

BACKGROUND: Describing severity of mitral regurgitation (MR) in dogs with degenerative mitral valve disease (DMVD) is challenging. HYPOTHESIS/OBJECTIVES: Mitral regurgitant fraction (RF), effective regurgitant orifice area (EROA), and the ratio of mitral regurgitant to aortic flow (QMR :QAo ) can be calculated from routine echocardiographic measurements and provide additional information regarding MR severity. ANIMALS: Fifty-seven dogs with preclinical DMVD including 36 without and 21 with cardiomegaly. METHODS: Prospective observational study. The expected relationships among RF, EROA, and QMR :QAo and 1-dimensional measurements including left atrium to aortic root diameter ratio (LA:Ao) and normalized left ventricular internal dimension at end-diastole (LVIDdN) were mathematically derived and calculated using echocardiographic data from the study population. Nonlinear goodness of fit was determined by calculation of the root mean standard error. The correlations between 1-dimensional and multidimensional indices were analyzed using receiver operating characteristic curves. RESULTS: The relationships among RF, EROA, QMR :QAo , and both LA:Ao and LVIDdN were curvilinear, and the multidimensional indices differentiated MR of variable severity. By contrast, 1-dimensional measurements were insensitive to MR severity until RF equaled or exceeded 50%. Regurgitant fraction ≥50%, EROA to body surface area ≥0.347 and QMR :QAo ≥0.79 were strongly associated with LA:Ao ≥1.6 and LVIDdN ≥1.7. CONCLUSIONS AND CLINICAL IMPORTANCE: Regurgitant fraction, EROA, and QMR :QAo quantify MR severity in dogs with preclinical DMVD in a manner that 1-dimensional measurements do not.

The use of focused cardiac ultrasound to screen for occult heart disease in asymptomatic cats.

BACKGROUND: Focused cardiac ultrasound (FCU) helps detect occult heart disease in human patients. HYPOTHESIS: Focused cardiac ultrasound by a nonspecialist practitioner (NSP) will increase the detection of occult heart disease in asymptomatic cats compared with physical examination and ECG. ANIMALS: Three hundred forty-three client-owned cats: 54 excluded and 289 analyzed. METHODS: Multicenter prospective cohort study. Twenty-two NSPs were trained to perform FCU. Cats without clinical signs of heart disease were recruited, and NSPs performed the following in sequential order: physical examination, ECG, FCU, and point-of-care N-terminal pro-B-type natriuretic peptide assay (POC-BNP). After each step, NSPs indicated yes, no, or equivocal as to whether they believed heart disease was present. The level of agreement between the NSP diagnosis and a blinded cardiologist's diagnosis after echocardiogram was evaluated using Cohen's kappa test. RESULTS: Cardiologist diagnoses included 148 normal cats, 102 with heart disease, and 39 equivocal ones. Agreement between NSP and cardiologist was slight after physical examination (kappa 0.253 [95% CI, 0.172-0.340]), did not increase after ECG (0.256 [0.161-0.345]; P = .96), increased after FCU (0.468 [0.376-0.558]; P = .002), and the level of agreement was similar after POC-BNP (0.498 [0.419-0.580]; P = .67). In cats with mild, moderate, and marked occult heart disease, the proportion of cats having a NSP diagnosis of heart disease after FCU was 45.6%, 93.1%, and 100%, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Focused cardiac ultrasound performed by NSPs increased the detection of occult heart disease, especially in cats with moderate to marked disease. Focused cardiac ultrasound appears to be a feasible and useful tool to assist NSPs in the detection of heart disease in cats.

Plasma and tissue angiotensin-converting enzyme 2 activity and plasma equilibrium concentrations of angiotensin peptides in dogs with heart disease.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) is a homologue of angiotensin-converting enzyme (ACE) and produces angiotensin peptides (APs), such as angiotensin 1-9 and 1-7 that are vasodilatory and natriuretic, and act to counterbalance angiotensin II. HYPOTHESIS: Evidence of ACE2 can be found in tissues and plasma of dogs. Equilibrium concentrations of renin angiotensin aldosterone system (RAAS) APs differ in dogs with heart disease compared to healthy dogs and recombinant human ACE2 (rhACE2) alters relative concentrations of APs. ANIMALS: Forty-nine dogs with and 34 dogs without heart disease. METHODS: Immunohistochemistry and assays for tissue and plasma ACE2 activity and equilibrium concentrations of plasma RAAS APs were performed. RESULTS: Immunolabeling for ACE2 was present in kidney and myocardial tissue. Median plasma ACE2 activity was significantly increased in dogs with congestive heart failure (CHF; 6.9 mU/mg; interquartile range [IQR], 5.1-12.1) as compared to control (2.2 mU/mg; IQR, 1.8-3.0; P = .0003). Plasma equilibrium analysis of RAAS APs identified significant increases in the median concentrations of beneficial APs, such as angiotensin 1-7, in dogs with CHF (486.7 pg/mL; IQR, 214.2-1168) as compared to those with preclinical disease (41.0 pg/mL; IQR, 27.4-45.1; P < .0001) or control (11.4 pg/mL; IQR, 7.1-25.3; P = .01). Incubation of plasma samples from dogs with CHF with rhACE2 increased beneficial APs, such as angiotensin 1-9 (preincubation, 10.3 pg/mL; IQR, 4.4-37.2; postincubation, 2431 pg/mL; IQR, 1355-3037; P = .02), while simultaneously decreasing maladaptive APs, such as angiotensin II (preincubation, 53.4 pg/mL; IQR, 28.6-226.4; postincubation, 2.4 pg/mL; IQR, 0.50-5.8; P = .02). CONCLUSIONS AND CLINICAL IMPORTANCE: Recognition of the ACE2 system expands the conventional view of the RAAS in the dog and represents an important potential therapeutic target.

Circulating exosome microRNA associated with heart failure secondary to myxomatous mitral valve disease in a naturally occurring canine model.

Myxomatous mitral valve disease (MMVD) is functionally and histologically identical to mitral valve prolapse (MVP) in humans. Currently, there are no medical treatments that can delay the progression of this valvular disease or associated cardiac remodelling. Therefore, there is a need to understand the molecular pathology associated with MMVD and MVP better, and thus identify potential therapeutic targets. Circulating exosomes contain small RNA, including miRNA, which reflect cell physiology and pathology. This study explored the association between circulating exosomal miRNA (ex-miRNA) content and MMVD, heart failure due to MMVD (MMVD-CHF) and ageing, which is strongly associated with MMVD. Ex-miRNA was isolated from old normal/healthy dogs (n = 6), young normal dogs (n = 7), dogs with MMVD (n = 7) and dogs with MMVD-CHF (n = 7). Separately, total plasma miRNA was isolated from normal dogs (n = 8), dogs with MMVD (n = 8) and dogs with MMVD-CHF (n = 11). Using reverse transcription quantitative polymerase chain reaction, exosomal miR-181c (p = 0.003) and miR-495 (p = 0.0001) significantly increased in dogs with MMVD-CHF compared to the other three groups. Exosomal miR-9 (p = 0.002) increased in dogs with MMVD and MMVD-CHF compared to age-matched (old) normal dogs. Exosomal miR-599 (p = 0.002) decreased in dogs with MMVD compared to old normal dogs. In total plasma, 58 miRNA were deemed significantly different (p < 0.04) between normal dogs, dogs with MMVD and dogs with MMVD-CHF. However, in contrast to ex-miRNA, none of the miRNA in total plasma remained statistically significant if the false discovery rate was <15%. Changes in ex-miRNA are observed in dogs as they age (miR-9, miR-495 and miR-599), develop MMVD (miR-9 and miR-599) and progress from MMVD to CHF (miR-181c and miR-495). Ex-miRNA expression-level changes appear to be more specific to disease states than total plasma miRNA. RESPONSIBLE EDITOR Elena Aikawa, Harvard Medical School, USA.