This prospective study was performed between June 2017 and September 2018 at the Veterinary Teaching Hospital, Bursa Uludag University (BUU), Bursa / Turkey.
Dogs and groups
This study consisted of a total of 20 client-owned dogs of different breed, age, body weight, and both sexes. The dogs were divided in two groups: a control group integrated by healthy dogs (n=10) and the group of dogs with acute CHF (n=10). A complete physical, laboratory, thoracic radiography, electrocardiography (ECG) and echocardiographic examination were made in order to include each dog in the group of healthy and acute CHF dogs.
Inclusion criteria
This study included only dogs with stage C acute CHF due to MMVD. CHF was staged according to the American College of Veterinary Internal Medicine (ACVIM) consensus guidelines for the diagnosis and treatment of MMVD in dogs (3). For this study, dogs were selected, showing both criteria at the same time: 1) presence of clinical signs of CHF (coughing, exercise intolerance, and tachypnea, etc.) along with the presence of physical examination findings such as systolic heart murmur (at least grade 4/6 over the mitral puncta maxima) and precordial thrill, and 2) radiographic (vertebral heart score [VHS] > 11.0) and echocardiographic evidence (left atrial to aortic root ratio [LA/Ao] > 1.6 and/or left ventricular end diastolic diameter normalized for body weight [LVIDDn] > 1.7) of left-sided cardiac remodeling due to MMVD (3, 72).
MMVD was diagnosed by trans-thoracic echocardiography (CarisPlus®, color Doppler, Italy), based on the combination of following criteria: the presence of mitral valve prolapse (MVP) and/or thickening of the mitral valve leaflets by 2-D echocardiography on right parasternal long-axis (RPLA) and apical 4-chamber views, and identification of mitral valve regurgitation on left apical 4-chamber view by color Doppler examination. Mitral regurgitation was also confirmed by color M-mode examination at right parasternal short axis view – mitral valve level (73, 74).
The cases of acute CHF due to MMVD were selected, and acute CHF was characterized by the current clinical (increased respiratory rate or difficulty breathing) and radiographic findings of pulmonary edema. For this purpose, thoracic radiographic score was used to describe the probability of CHF. Briefly, this score was estimated based on the absence or presence (and its severity) of left atrial enlargement, pulmonary venous congestion, pulmonary infiltrates, and pleural effusion (75). The dogs with score >4 were forwarded to echocardiographic examination for final diagnosis. Radiographic and clinical findings were interpreted in a combination with the echocardiography, providing a final diagnosis of acute CHF due to MMVD (73-75).
In addition, these dogs had at least one cardiac rhythm abnormalities (i.e., sinus tachycardia and atrial fibrillation) identified by ECG and higher serum cardiac troponin I (cTnI) level than the reference ranges (<0.03 - 0.07 ng/mL) at the time of first admission to the clinic (I-STAT®, Abaxis). Cardiologic examination was performed by PL (first author) and then MMVD with Stage C CHF was confirmed by specialists, MK and ZY.
Control dogs were obtained from staff and students of BUU Veterinary Teaching Hospital. These control dogs according to the owners did not have any history of chronic disease and in the last two months did have any disease and not receive any drug, preventive treatment or vaccination. These dogs did not show any alteration at physical and cardiological examination (including ECG and echocardiography) and showed values of hemogram and biochemistry analysis (including serum cTnI) within the reference values of our laboratory.
Exclusion criteria
Cases with a history of any disease different to MMVD and CHF for the past 2 months and that had any chronic disease or were under medication were excluded of the study. Dogs were excluded if they had a congenital heart disease or another acquired cardiac disorder such as dilated cardiomyopathy (DCM). Asymptomatic dogs with MMVD with (stage B2) or without cardiac remodeling (stage B1) and symptomatic dogs with refractory CHF (stage D) were also excluded. The dogs who had a lower than or equal to 4 of radiographic composite score were not included in this study. Besides, routine hematological and biochemical data were used to identify and exclude patients who had anemia, renal failure, endocrine diseases (diabetes mellitus and hypothyroidism), or hepatobiliary diseases accompanying primary problem. Ehrlichia, Lyme disease, Dirofilaria, Leishmania and/or Anaplasma positive cases were excluded by rapid screening tests (Anigen CaniV and LeishVet, Bionate). If the dogs received the medication such as steroids, non-steroids, antibiotics, inotropes or diuretics before inclusion, they were not included to the study, because of their potential effects on platelet protein expressions.
The dogs included in the study were treated by conventional medical approaches (diuretics, inotropic, and angiotensin converting enzyme inhibitors, etc.) for heart failure, and then re-examined as needed. Thus, dogs studied continued their lives under the responsibility of the patient owners. We have a permission to collect the animals’ samples from the owners.
Sample collection and measurements
Clinical examinations
In this study, cardiological examination was performed following the well-known procedures including physical examination, electrocardiography (ECG), thoracic radiography and echocardiography on admission day in all dogs. Thoracic radiographs were taken in different imaging planes (right and left lateral, and ventrodorsal positions) to evaluate the heart, vertebral heart score, lung and thoracic vessels, and then each of which was used to estimate radiographic composite score (75).
ECG was performed using 3 bipolar standard limb leads in un-sedated dogs. Cardiac rhythm analyses and measurements were performed with a standard calibration (10 mm/mV and 50 mm/sec), as reported in our previous studies (76, 77).
Echocardiographic examinations were performed using conventional methods (2‐D, M-mode, and color Doppler) and imaging techniques (right parasternal short – RPSA, and long axis - RPLA, left apical 4-5 chamber and subcostal views) with three different choices (2.5-5, 5-7.5, and 7.5-10 MHz) of phased-array cardiac transducers in all dogs (Caris Plus Esaote, Italy) (76-78). Left ventricular (LV) - related parameters such as interventricular septum (IVS) and post-wall (LVPW) thickness, LV internal dimension at diastole and systole (LVDd and LVDs), and E-point to septal separation (EPSS) were measured at RPLA 4 chamber view by 2D and M-mode echocardiography. LV systolic function was evaluated by ejection fraction (EF %) and fractional-shortening (FS %), and both of them were calculated automatically with M-mode measurement (Teicholz method). The LA/Ao was obtained from the 2‐D RPSA view – aortic level. LVDDn was calculated according to Cornell's method of allometric scaling: LVDDn = LVDd (cm) /body weight (kg)0.294 (72). For PV/PA estimation, PV and PA diameters were measured by M-Mode echo at RPSLA 4‐chamber view as previously described (79).
LV diastolic function was evaluated by trans-mitral inflow profile including early LV filling (E), late atrial contraction (A) and their ratio (E/A) by Pulsed-wave Doppler echo at apical 4-chamber view. At the same image, the presence of mitral regurgitation was determined by color Doppler mode, and its severity was estimated by the measurements of regurgitant jet velocity with continuous Doppler mode (80). As a standard procedure, other measurements such as main pulmonary artery (MPA), and Ao Doppler flow velocities were determined as suggested (81).
Laboratory examinations
Blood samples were collected, at the same day of cardiological and radiographic examinations, before starting to treatment, from the cephalic veins into EDTA containing tubes for complete blood counting, and acid citrate dextrose (ACD) containing tubes for platelet isolation (15), as well as into anticoagulant-free tubes for routine serum biochemistry analysis.
For platelet isolation, a volume of 20 ml of venous blood was collected into the tubes with ACD containing trisodium citrate (22.0g/L), citric acid (8.0g/L) and dextrose (24.5g/L) (BD Vacutainer), and platelets were then isolated according to the method modified from Cevik et al. (15). Briefly, platelet isolation steps included:
i- Blood was drawn into ACD tubes for platelet isolation.
ii- It was centrifuged (150 g / 15 min, at room temperature) to obtain platelet-rich plasma (PRP).
iii- PRP was transferred to dry tubes. HEPES buffer solution (137 mM NaCl, 3.8 mM HEPES, 5.6 mM Glucose, 2.7 mM KCl, 1 mM Magnesium sulfate, pH 7.4; Sigma) was added over the PRP samples. This was suspended with prostaglandin E1 (1 mM/L, Sigma) to inhibit platelet activation during high-speed centrifugation (82). This mix was centrifuged at 800 g for 15 min to concentrate the platelets.
iv- Platelet pellets were re-suspended in ammonium bicarbonate solution (50 mM, Sigma) and then centrifuged at 800 g for 15 min to obtain final platelet pellets. Platelet extractions with WBCs and RBCs contaminations less than 0.5 and 0.1 % by Diff-Quick staining, respectively, were considered sufficient to perform platelet proteomic study.
Serum samples and pure platelet pellets were stored in cryo tubes at -80 degrees. After the sample collections in both groups were completed, all platelet pellets were analysed at the same time.
Proteomic analysis
Label-free differential proteomics analysis method was used to detect altered protein expressions in this study. The method provides changes in protein amount across the sample set rather than quantifying absolute amounts of each protein in the samples. Protein quantitation is based on the ion intensities of its non-conflicting peptide features. Unique peptides identified for the protein is used for calculating the protein intensity. Differential expression changes of the proteins reported here are relative ratios showing the up-regulation or down-regulation of a specific protein.
LC-MS based label free proteomics analysis was done for 10 control samples and 10 CHF samples being each sample individually analysed. Every injected sample is a separate biological replicate. Steps of platelet proteomic analysis were performed in a total of 20 different biological samples, as described in a previous paper (15). Before starting the analysis, the detector and calibration settings were made by the MassLynx program (V4.1-Waters) which is specific to Xevo G2-XS Q TOF (Waters) device where the analysis were performed. The method was switched to SONAR and sensitivity mode and the tryptic peptides generated were subjected to 132 min reverse phase chromatography at 300 nL / min flow rate in a HSS T3 (Waters-186008818) nano column. Separation of the peptides from the column was achieved by increasing in the range of 5-35% acetonitrile according to their hydrophobicity and analyzed by mass spectrometry. During the analysis, data were collected for peptides that could be identified in the m / z range 50-1950. MS analysis was performed for 0.7 s and information was collected about the entire peptide. Then, MS / MS analysis was performed for 0.7 sec and the peptide fragmentation and sequence information were obtained.
Throughout this analyses, [Glu1]-fibrinopeptide B human peptide standard was infused to the mass spectrometer every 60 sec in a 50:50 ACN:Water mixture to be used as a mass calibrant. Peptides were mass re-calibrated based on the standard peptide mass value. In a label free data independent acquisition method an intensity calibrant was not used. Our results are relative ratios and not absolute quantities. All data pertaining to protein identifications with peptide sequences, mass errors and modifications are given as a supplementary file (additional file-3).
Statistical analysis
In this study, the student t test was applied for the clinical, laboratory (hematologic and biochemical) and cardiological examination results in two groups. Results were given as mean ± standard deviation, and P < 0.05 was considered statistically significant (SigmaStat 12.0, GmBH). Proteins obtained in three separate sets were paired with previously described proteins for Canis lupus familiaris in the gene bank. Protein identification and statistical analysis was performed using Progenesis QIP software (Waters-2018). In the method, samples from different patients were compared as a group and the proteins separating the groups were identified. Various statistical calculations were used in order to test reliability of the results obtained, to compare hundreds of proteins at the same time and to reach significant information by software package programs. Platelet proteins with at least P < 0.05 and more than 1.2-fold change were accepted as significant in dogs with acute CHF due to MMVD compared to control group.
Bioinformatic analysis was performed to show the protein – protein interaction (STRING; www.string-db.org), and the roles of proteins in molecular, cellular, and biological process, and pathway analysis was made to evaluate the possible association between proteins and biological functions (REACTOME; www.reactome.org and www.pantherdb.org).