Background

Degenerative mitral valve disease (DMVD) is the most common heart disease in dogs responsible for approximately 75% of heart disease cases visiting veterinary practitioners worldwide, especially in North America [1]. In DMVD, the mitral valve does not close properly, causing blood to flow backward into the left atrium from the left ventricle as it contracts. This disease initiates volume overload, leading to several subsequent compensatory mechanisms [2]. Ultimately, these compensatory adjustments weaken the heart and lead to decompensated mitral regurgitation (MR) (i.e., heart failure). In recent prospective studies from randomized clinical trials in the field of veterinary cardiology, pimobendan administration delays the onset of congestive heart failure (CHF) by a median of 15 months in dogs with DMVD stage B2 [3]. Moreover, adding pimobendan to heart failure therapy in dogs with furosemide and angiotensin-converting enzyme inhibitors delayed the onset of refractory signs of heart failure, improved clinical conditions, and increased survival time [4,5,6]. Although pimobendan inhibits phosphodiesterase-III (PDE-III) causing an increase in cyclic adenosine monophosphate (cAMP) and sensitizes the cardiac contractile apparatus (i.e., troponin C) to intracellular calcium, the definite mechanism of pimobendan underlying the delay in the onset of CHF or refractory signs of CHF is still unclear.

Cardiac mitochondrial dysfunction has been demonstrated to play a crucial role in HF and is believed to contribute to the progression of HF through both diminished high-energy phosphate production and amplified production of reactive oxygen species (ROS), resulting in high levels of oxidative stress [7, 8]. The intracellular ROS generated by damaged or malfunctional mitochondria also causes mitochondrial dysfunction, which initiates cardiac remodeling and damages sarcomeric proteins [9]. Furthermore, stretched myocytes produce ROS, leading to cardiomyocyte degeneration, and are associated with contractile dysfunctions in the pathophysiology of HF [20]. Briefly, the incision was made in the fifth intercostal space, and the pericardial sac was cut open. The left auricle was lifted, and the mitral valve was punctured with a 20-G needle through the left ventricular free wall just below the valve location. MR was confirmed by a transthoracic echocardiography machine equipped with a 4–10-MHz phased array probe (M9, Mindray, Shenzhen, China). The MR jet area of 45–60% of the total left atrial area was considered successful and was included in the study (Fig. 1). After that, the thoracic cavity, muscular layer, and skin were closed with an absorbable suture. A similar surgical procedure was performed without puncturing the left ventricle (LV) and mitral leaflet for the sham operation. Enrofloxacin and tramadol were given daily for 7 days. All rats were allowed to develop LA and LV anatomic and molecular remodeling and altered systolic function for 8 weeks before initiating the treatment intervention.

Experimental procedure

Eight weeks after surgery, echocardiography was performed to confirm and document the presence of MR in the MR group, whereas no MR was visible in the sham group. The rats in the MR group were randomly divided into two groups: MR rats (n = 10) receiving drinking water 5 mL/kg, PO, q12h, and MR rats receiving pimobendan (MR + PIMO; n = 10) 0.3 mg/kg, PO, divided twice daily. The dose of pimobendan used in this study was selected based on a previous publication [43] in which chronic administration of pimobendan resulted in an increased EF in catecholamine-induced myocardial injury. Echocardiography was performed to ensure that there were no differences in echocardiographic parameters observed between the two groups at the baseline. The sham group was given drinking water (5 mL/kg, PO, q12h) as a placebo. Echocardiography was conducted again 4 weeks after therapy was initiated (Fig. 9). Four weeks after the initiation of treatment, the rats were sacrificed with an overdose of isoflurane in an anesthetic chamber to harvest their heart. Thoracotomy and vital organ removal (i.e., heart) were used as a physical confirmation of death in the current study. The heart was perfused with ice-cold phosphate-buffered saline (PBS) solution and weighed. A small portion of the LV apex (approximately 300 mg) was collected for mitochondrial isolation, whereas the trabeculae muscle of the left ventricle was obtained for TEM. The remaining part of the heart was placed in 10% formalin for 24 h, and histopathological processes were performed for hematoxylin and eosin staining.

Fig. 9
figure 9

Study design to assess the effects of pimobendan (PIMO) on cardiac function, mitochondrial quality, and cardiac ultrastructure and mitochondrial morphology in rats with mitral regurgitation (MR). Eight weeks after surgical induction of MR, pimobendan (0.15 mg/kg) was given orally to MR + PIMO rats twice daily for 4 weeks, whereas MR rats received drinking water orally (5 mL/kg). Echocardiography images were obtained at baseline (8 weeks after the operation) and at the end of the study (12 weeks after treatment). At the end of the experiments, all hearts were harvested to evaluate their weight per bodyweight ratio. In addition, a heart section was processed for transmission electron microscopy to quantify mitochondrial morphology and sarcomere length. Mitochondrial quality was inferred from three mitochondrial parameters: swelling, membrane potential changes, and reactive oxygen production

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Echocardiography

Echocardiography was performed in all rats between 1.5 and 3.0 h post-pimobendan or placebo dosing. Rats were lightly sedated with isoflurane 2–3% mixed with oxygen through the nose cone. Images were acquired with a 4–10-MHz ultrasonic phased array probe connected to a Mindray M9 echocardiography machine, and electrocardiographic (ECG) electrodes were attached to both forelimbs and the left hindlimb and connected to the machine. Color Doppler map** of MR jets was used to semiquantitatively assess the severity of MR. MR jet areas were assessed in the right parasternal long-axis view. LV septal and posterior wall thickness (i.e., interventricular septal at end-diastole, interventricular septal at end-systole, LV posterior wall at end-diastole, and left ventricular posterior wall at end-systole) were measured by M-mode echocardiography at the level of the head of the papillary muscle following previously published guidelines [44]. From this view, fractional shortening, EF, EDV, and ESV were calculated using the Teichholz method [45, 46]. At the base of the heart, a right parasternal short-axis view was obtained to evaluate the left atrium-to-aortic root ratio (LA/Ao). The right parasternal long-axis view with color map** was also obtained to measure the MR jet area (%). The MR jet area (%) was quantified by the area of mosaic color observed during systole inside the left atrium divided by the total area of the left atrium and multiply by 100 which was described previously [47]. All parameters were evaluated on an average of six consecutive cardiac cycles.

Determination of mitochondrial quality: mitochondrial ROS production, membrane potential changes, and swelling

The isolation of cardiac mitochondria was described previously [48]. Briefly, a piece of the LV apex was homogenized with an isolation buffer (300 mM sucrose, 0.2 mM EGTA, 5 mM TES; pH 7.2) in cold conditions using a Dounce homogenizer for the evaluation of mitochondrial function. A differential centrifuge was used to isolate the mitochondria. The mitochondrial pellet was re-suspended with 500 µL of ice-cold respiration buffer (100 Mm, KCl; 50 mM, sucrose; 10 mM, HEPES; 5mM, K2HPO4; pH, 7.2), and the mitochondrial protein concentration was immediately measured using the Bradford protein assay. Bovine serum albumin was used for creating a standard curve and quantifying the concentration equation.

The ROS production by the mitochondria was determined using dichloro-dihydro-fluorescein diacetate (DCFDA) in a fluorescent microplate reader [49]. The mixture of isolated cardiac mitochondria and DCFDA was prepared and added into a black microplate to obtain a final concentration of mitochondria protein of 0.4 mg/mL in a total volume of 200 µL and incubated at 37 °C for 30 min. DCFDA passed into the mitochondria and oxidized in the presence of ROS to form DCF, a fluorescent form. Fluorescence intensity was measured at excitation and emission wavelengths of λ 485 nm and λ 530 nm, respectively.

Cardiac mitochondrial membrane potential changes were determined using 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) [49]. The diluted JC-1 solution (5 µM) was added into a black microplate, followed by a mitochondrial solution, to obtain 0.4 mg/mL in a total volume of 200 µL and incubated at 37 °C for 30 min in the dark. Fluorescence intensities of green (JC-1 monomer) and red (JC-1 aggregate) were measured at excitation and emission wavelengths of λ 485 and λ 530 nm, and λ 485 and 590 nm, respectively. The red/green intensity ratio was calculated and represented to indicate the quality of mitochondria [50]. A reduction in the red/green fluorescence intensity indicates mitochondrial depolarization.

Mitochondrial swelling was determined by measuring the change in the optical density value at λ540 nm in the respiration buffer (mitochondrial protein concentration of 0.4 mg/mL in a total volume of 200 µL) using a microplate reader [49]. Absorbance was measured immediately and every 5 min after mitochondria were added to the buffer. A rapid loss of the absorbance measured at 30 min indicates mitochondrial swelling.

Visualization of the myocyte ultrastructure by TEM

An LV trabeculae muscle (n = 2/group) was cut and fixed with 3% glutaraldehyde in 0.1 M phosphate buffer and stored at 4 °C for the TEM study. Then, the tissues were postfixed with 2% osmium tetroxide in 0.1 M phosphate buffer, dehydrated with an alcohol series, and embedded in epon (Epon 812; Electron Microscopy Sciences, Fort Washington, PA, USA) and polymerized at 70 °C overnight. Lead citrate and uranyl acetate were used to stain the ultrathin Sects. (65–70 nm). Mitochondrial morphology, sarcomere length, and vacuole size were visualized under a transmission electron microscope (JEM-1400 Plus; JEOL, Tokyo, Japan). Images were captured at low magnification, i.e., 2,500× for sarcomere length assessment and 10,000× for mitochondrial morphology assessment. Sarcomere lengths were measured using NIS-Elements Advance Research (Nikon, USA).

In vitro study of the effect of pimobendan on H2 O2 -induced intracellular ROS production in H9c2 cells.

Cell culture and treatment

The rat ventricular myoblast cell line H9c2 (ATCC number CRL-1446) was purchased from the American Type Culture Collection (ATCC®, Manassas, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (D5030, Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS-G1-12 A, Biocom Biotech) and 100 U/mL penicillin and 100 µg/mL streptomycin. The cells were incubated in a humidified incubator at 37 °C, under 95% air and 5% CO2. The medium was refreshed every 2 days. Subculture was performed when the cell density reached 80–90% confluence.

The first protocol was designed to obtain the optimum dosage of pimobendan for a subsequent study. The H9c2 cells were seeded at a density of 3 × 103 cells/well in a 96-well plate until reaching 80–90% confluence. Pimobendan (Vetmedin injection, 0.75 mg/mL, MW 334.37) was freshly diluted with fresh media to several concentrations at 0.1, 1, 10, and 100 µM. These ranges of concentration were chosen as they effectively cause augmented glucose-induced insulin release in a dose-dependent manner by sensitizing the intracellular calcium in rats [51]. The cell viability assay was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) cell viability assay, which was evaluated under a microscope after incubating pimobendan for 1, 2, 6, 12, and 24 h. The dosage that did not cause 10% of the individuals to die, or lethal dose 10% (LD10), was chosen for the next protocol. In the second protocol, the effects of pimobendan on the alteration of the mitochondrial membrane potential and intracellular ROS production were determined.

Mitochondrial membrane potential

H9c2 cells were seeded at a density of 3 × 103 cells in 200 µL to a black 96-well plate. Then, 5 µM of JC-1 dye was added and incubated at 37 °C for 30 min in the dark. Fluorescence intensity was determined using a microplate reader. The fluorescence intensity for monomeric green fluorescein was determined by a fluorescence microplate reader with the excitation and emission wavelengths at 485 and 530 nm, respectively, while the aggregate red fluorescein was determined by a fluorescence microplate reader with excitation and emission wavelengths at 485 and 590 nm, respectively.

Intracellular ROS production

H9c2 cells were seeded at a concentration of 5 × 103 cells/mL in 200 µL. Cells were allowed to attach for at least 24 h. After pimobendan treatment, the media was discarded and incubated with 100 µL of complete media containing 5 µM DCFDA. The plate was incubated at 37 °C for 60 min in the dark. To determine ROS production, the fluorescence intensity was determined by a fluorescence microplate ready with excitation and emission wavelengths at 485 and 530 nm, respectively.

Cell viability

H9c2 cells were seeded at a density of 3 × 103 cells in 200 µL until reaching 80% confluence. Then, cells were incubated with a complete medium in the presence and absence of pimobendan and incubated at 37 °C. Cell viability was assessed by the MTT cell viability assay by incubation with 0.5 mg/mL of MTT dye at 37 °C for 2 h. After incubation, the MTT reagent was discarded and dimethylsulfoxide (DMSO) was added for solubilizing the formazan dye. The optical density was determined by a spectrophotometer at λ 490 nm using DMSO as a blank. The relative percentage of cell viability was compared with the control group [52].

Determination of intracellular ROS production by H2O2 challenging

H9c2 cells were seeded at a density of 105 cells in 200 µL until reaching 80% confluence. Pimobendan was freshly prepared by diluting with the fresh media to obtain a concentration of 0, 0.1, 1, 10, and 100 µM. Two protocols were established as an ROS formation preventer and an ROS scavenger.

In the first protocol, to determine the pretreatment effect of pimobendan on intracellular ROS generation, cells were incubated with 25 µM DCFDA in a medium at 37 °C 30 min before incubation with several concentrations of pimobendan. Then, the culture medium was discarded, and cells were treated with PBS or 250 µM H2O2 in PBS for 30 min. The fluorescence signal of DCF was measured at excitation and emission wavelengths of 485 and 530 nm, respectively.

In the second protocol, for determining the ROS-scavenging properties of pimobendan, different concentrations of pimobendan were incubated with PBS (control) or H2O2 to obtain 250 µM H2O2 at 37 °C for 30 min. Before treatment, the cells were incubated with 25 µM DCFDA in a medium for 30 min. Then, the culture medium was replaced by pimobendan–PBS or pimobendan–H2O2 mixtures and incubated at 37 °C for 30 min. The fluorescence signal of DCF was measured at excitation and emission wavelengths of 485 and 530 nm, respectively. The mean fluorescence intensities are expressed as percentages of the untreated control.

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). Heart-to-bodyweight ratios were calculated for each rat using the data at the end of the study. Statistical analysis was performed using IBM® SPSS® Statistics software (IBM Corp., Armonk, NY, USA). The normal distribution of variables was assessed using the Shapiro–Wilk test. A one-way analysis of variance (ANOVA) test with Tukey’s correction for multiple comparisons was used to evaluate the differences among groups. If the values failed to display normality, ANOVA on ranks was used to evaluate the differences among groups. P-value < 0.05 was considered statistically significant.