Background Long‐term use of doxorubicin (DOX) is limited by cumulative dose‐dependent cardiotoxicity. Objectives Identify plasma extracellular vesicle (EV)‐associated microRNAs (miRNAs) as a biomarker for cardiotoxicity in dogs by correlating changes with cardiac troponin I (cTnI) concentrations and, echocardiographic and histologic findings. Animals Prospective study of 9 client‐owned dogs diagnosed with sarcoma and receiving DOX single‐agent chemotherapy (total of 5 DOX treatments). Dogs with clinically relevant metastatic disease, preexisting heart disease, or breeds predisposed to cardiomyopathy were excluded. Methods Serum concentration of cTnI was monitored before each treatment and 1 month after the treatment completion. Echocardiography was performed before treatments 1, 3, 5, and 1 month after completion. The EV‐miRNA was isolated and sequenced before treatments 1 and 3, and 1 month after completion. Results Linear mixed model analysis for repeated measurements was used to evaluate the effect of DOX. The miR‐107 (P = .03) and miR‐146a (P = .02) were significantly downregulated whereas miR‐502 (P = .02) was upregulated. Changes in miR‐502 were significant before administration of the third chemotherapeutic dose. When stratifying miRNA expression for change in left ventricular ejection fraction, upregulation of miR‐181d was noted (P = .01). Serum concentration of cTnI changed significantly but only 1 month after treatment completion, and concentrations correlated with left ventricular ejection fraction and left ventricular internal dimension in diastole. Conclusion and Clinical Significance Downregulation of miR‐502 was detected before significant changes in cTnI concentrations or echocardiographic parameters. Further validation using a larger sample size will be required.

Canine myxomatous mitral valve disease (MMVD) resembles the early stages of myxomatous pathology seen in human non-syndromic mitral valve prolapse, a common valvular heart disease in the adult human population. Canine MMVD is seen in older subjects, suggesting age-related epigenetic dysregulation leading to derangements in valvular cell populations and matrix synthesis or degradation. We hypothesized that valvular interstitial cells (VICs) undergo disease-relevant changes in miRNA expression. In primary VIC lines from diseased and control valves, miRNA expression was profiled using RT-qPCR and next generation sequencing. VICs from diseased valves showed phenotypic changes consistent with myofibroblastic differentiation (vimentin^low+, α-SMA^high+), increases in senescence markers (p21, SA-β-gαl), and decreased cell viability and proliferation potential. RT-qPCR and miRNA sequencing analyses both showed significant (p<0.05) downregulation of let-7c, miR-17, miR-20a, and miR-30d in VICs from diseased valves compared to controls. Decreased let-7c, miR-17, and miR-20a may contribute to myofibroblastic differentiation in addition to cell senescence, and decreased miR-30d may disinhibit cell apoptosis. These data support the hypothesis that epigenetic dysregulation plays an important role in age-related canine MMVD.

OBJECTIVE: To evaluate whether mesenchymal stem cells (MSCs) can be safely administered IV to dogs with congestive heart failure (CHF) secondary to myxomatous mitral valve disease (MMVD) to improve cardiac function and prolong survival time. ANIMALS: 10 client-owned dogs with CHF secondary to MMVD. PROCEDURES: Dogs with an initial episode of CHF secondary to MMVD were enrolled in a double-blind, placebo-controlled clinical trial. Five dogs in the MSC group received allogeneic Wharton jelly–derived MSCs (2 X 10⁶ cells/kg, IV), and 5 dogs in the placebo group received a 1% solution of autologous serum (IV) for 3 injections 3 weeks apart. Cell-release criteria included trilineage differentiation, expression of CD44 and CD90 and not CD34 and major histocompatability complex class II, normal karyotype, and absence of contamination by pathogenic microorganisms. Patients were followed for 6 months or until death or euthanasia. Echocardiographic data, ECG findings, serum cardiac biomarker concentrations, CBC, and serum biochemical analysis results were obtained prior to and 4 hours after the first injection and every 3 months after the final injection. RESULTS: Lymphocyte and eosinophil counts decreased significantly 4 hours after injection, and monocytes decreased significantly only in dogs that received an MSC injection. No significant differences were seen in the echocardiographic variables, ECG results, serum cardiac biomarker concentrations, survival time, and time to first diuretic drug dosage escalation between the 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: This study showed that MSCs can be easily collected from canine Wharton jelly as an allogeneic source of MSCs and can be safely delivered IV to dogs with CHF secondary to MMVD.

Chemotherapy-induced impairment of autophagy is implicated in cardiac toxicity induced by anti-cancer drugs. Imperfect translation from rodent models and lack of in vitro models of toxicity has limited investigation of autophagic flux dysregulation, preventing design of novel cardioprotective strategies based on autophagy control. Development of an adult heart tissue culture technique from a translational model will improve investigation of cardiac toxicity. We aimed to optimize a canine cardiac slice culture system for exploration of cancer therapy impact on intact cardiac tissue, creating a translatable model that maintains autophagy in culture and is amenable to autophagy modulation. Canine cardiac tissue slices (350 μm) were generated from left ventricular free wall collected from euthanized client-owned dogs (n = 7) free of cardiovascular disease at the Foster Hospital for Small Animals at Tufts University. Cell viability and apoptosis were quantified with MTT assay and TUNEL staining. Cardiac slices were challenged with doxorubicin and an autophagy activator (rapamycin) or inhibitor (chloroquine). Autophagic flux components (LC3, p62) were quantified by western blot. Cardiac slices retained high cell viability for >7 days in culture and basal levels of autophagic markers remained unchanged. Doxorubicin treatment resulted in perturbation of the autophagic flux and cell death, while rapamycin co-treatment restored normal autophagic flux and maintained cell survival. We developed an adult canine cardiac slice culture system appropriate for studying the effects of autophagic flux that may be applicable to drug toxicity evaluations.