Introduction

Mitral regurgitation is the most prevalent valvular disease in the aging population1, and the most common heart disease found in small-breed, middle-old aged dogs2. The renin–angiotensin–aldosterone system (RAAS) and natriuretic peptide system (NPs) are essential mechanisms that are accelerated as compensatory mechanisms to preserve cardiac output and stabilize haemodynamic status when mitral regurgitation occurs along with its consequences, including left ventricular (LV) volume overload, LV systolic dysfunction, and LV remodeling3,4. However, if MR progression continuously develops with chronic activation of these systems, they will become a vicious cycle, culminating in subsequent heart failure (HF)5. HF is a necessary consequence of MR, resulting in a high and increasing mortality rate worldwide6.

Recently, studies have mentioned several cellular mechanisms contributing to the disease progression through HF. Volume overload and RAAS activation are critical factors that initiate reactive oxygen species (ROS) production, inducing mitochondrial damage and dysfunction and leading to cardiac injury, remodeling, and HF7. During this period, autophagy upregulation is responsible for dysfunctional protein turnover and cell biogenesis preservation8,9. Microtubule-associated protein 1A/1B-light chain 3 (LC3), an indicator of autophagy, is upregulated in MR10 and was significantly associated with the severity of the myolysis in MR11. According to this point of view, novel pharmacological interventions for HF patients are continuously being developed that focus deeper on mechanisms involved with disease pathophysiology12. The drugs that can positively affect these mechanisms will be effective treatments for cardiac patients in the long run.

Sacubitril/valsartan (SAC/VAL) combines an angiotensin II type 1 receptor (AT1R) blocker and a neprilysin inhibitor, ARNi, was approved by the U.S. Food and Drug Administration for heart failure (HF) with reduced ejection fraction treatment in 201513. The dual mechanisms of SAC/VAL are to inhibit the over-activation of pathological RAAS through an AT1R blocker and enhance the salutary effects of NPs by degrading the neprilysin enzyme and having potentially synergistic effects14. Previous studies revealed that SAC/VAL improved cardiac function and limited cardiac remodeling, inflammation, and fibrosis in experimental and clinical studies of cardiovascular diseases14,15,16,49. After the successful creation of MR, the thoracic wall and skin were anatomically closed with suitable suture materials. A similar surgical procedure was performed without the LV and mitral leaflet puncture for the sham operation. The recovering animals were moved to an individual recovery cage where they were provided a warm and dry environment and monitored until they presented completely normal behavior. They were administered diluted enrofloxacin (10 mg/kg, SC) and tramadol (12.5 mg/kg, SID, IP) daily for 5 days after surgery.

The experimental procedures/pharmacological therapy

Four weeks after surgery, transthoracic echocardiography was done to assess the severity of the MR-volume overload model focusing on the presence of MR (> 30% of LA area) or LA enlargement (left atrium to the aortic root ratio; LA: Ao > 1.6) in all rats and used as baseline data. The MR rats (n = 16 rats) were randomly assigned to two groups: the MR group (drinking water; n = 8) and the sacubitril/valsartan (SAC/VAL) group (68 mg/kg PO daily; n = 8)14. All rats received the treatments daily via oral gavage for 8 weeks using the appropriate doses in the previous study14. For the sham group, the rats were given the vehicle daily (drinking water) in the same volume as other treatment groups.

Echocardiography

Transthoracic echocardiography was done 4 weeks after surgery (baseline) and at the end of week 8 after the drug administration to evaluate drug effects on cardiac geometry and cardiac function in all groups. The Mindray M9 echocardiographic machine (Mindray, Shenzhen, China) equipped with a P10-4E (4–10 MHz) phase array transducer probe was used to acquire conventional echocardiographic parameters. The rats were mildly sedated with isoflurane, and the fur around the thoracic area was clipped. Then, the rats were placed in the right, lateral recumbent position and electrocardiographic (ECG) electrodes were attached to both forelimbs and the left hindlimb and connected to the machine.

LV conventional echocardiography consists of the interventricular septum and LV posterior wall thickness during systole and diastole (IVSd, IVSs, LVPWd, and LVPWs) and the left ventricular internal dimension end-diastole and end-systole (LVIDd, LVIDs) derived from the M-mode of the right parasternal short-axis view at the head of the papillary muscle level. From this view, the LV FS, LV EF, LV EDV, and LV ESV were calculated using the Teichholz method50,51. A standard two-dimensional (2D) right parasternal short-axis view at the base of the heart was performed to acquire the left atrium to aortic root ratio (LA: Ao). The right parasternal long-axis view with color map** was performed to assess the MR jet area (%), which indicated the severity of MR22. In addition, the aortic flow velocity (AV Vmax) was obtained. All parameters were obtained from an average of six consecutive cardiac cycles.

Tissue collection and fibrosis assessment

At the end of 8 weeks after drug administration, all rats were euthanized with overdose of isoflurane in an induction chamber connected to scavenging system. The heart was instantly harvested and perfused with ice-cold phosphate-buffered saline (PBS). Heart weight was obtained for the HW to BW ratio calculation. The LV-free wall was separated into three segments, similar regions, for further analysis. For the first part, approximately 30 mg of tissue was preserved in RNAlater and kept at − 20 °C for further PCR analysis. In another part, approximately 300 mg of the LV-free wall was immediately used as fresh LV tissue for mitochondrial isolation and mitochondrial quality analyses. The remaining LV section was preserved in 10% buffered formalin and embedded in paraffin. Then, myocardium-embedded blocks were sectioned and stained with hematoxylin and eosin stain (H&E) for histomorphology analyses. In addition, the blocks were sectioned into 5-µm-thick slices and stained with Masson’s trichrome stain to evaluate myocardial collagen and fibrosis. The fibrotic area was quantitatively evaluated from scanning of the whole LV section using NIS-element BR analysis software (Nikon Corporation, Tokyo, Japan): the collagen-stained area/the entire sectioned area × 100 and expressed as a percentage of the fibrotic area52. The left ventricular myocyte cross-sectional area was measured using NIS-element BR analysis software as previously described31. Briefly, each myocyte was identified, and its border was traced and the pixels within the border were calibrated against a scale to measure the area. The CSA data were averaged from 30 different cells in each image.

Evaluation of mitochondrial quality

All processes associated with mitochondrial quality analyses were modified from previous studies53.

Mitochondrial isolation

Fresh LV tissue (approximately 300 mg) was excised and washed in ice-cold PBS solution. Then, the tissue was homogenized in ice-cold isolated buffer (300 mM sucrose, 0.2 mM EGTA, 5 mM TES, pH 7.2: 1 mL per tissue 100 mg). The homogenized tissue solution was centrifuged at 800 × g, 4 °C for 10 min. The supernatant (S1) was transferred to a precooled Eppendorf tube and re-centrifuged at 8000 × g, 4 °C for 10 min. Then, the supernatant (S2) was discarded, and the mitochondrial pellet (P2) was washed by resuspending in an ice-cold isolation buffer again and re-centrifuged at 8000 × g, 4 °C for 10 min. Finally, the isolated cardiac mitochondrial protein was resuspended with respiration buffer (100 mM KCl, 50 mM sucrose, 10 mM HEPES, and 5 mM KH2PO4, pH 7.2), and the mitochondrial protein concentration was determined using a Bradford assay (HiMedia Laboratories, Maharashtra, India) at λ 595 nm. Bovine serum albumin was used as a protein standard. The final concentration of the isolated cardiac mitochondrial stock was diluted to 0.4 mg/mL.

Determination of mitochondrial swelling

Mitochondrial swelling is the condition used to determine the sensitivity to membrane permeability transition (mPT)54. Five μL of respiration buffer and 20 mM CaCl2 were separately added into 2 wells of a 96-well plate, labeling them with and without calcium conditions. The isolated cardiac mitochondrial stock (200 μL) was pipetted into these two wells. The plate was measured to determine the rapid loss of the absorbance at λ 540 nm via the spectrophotometric method at 10- and 30-min time points at room temperature. The maximal mitochondrial (∆OD540 nm) swelling amplitude was calculated from the optical density at 10 min (OD1)–30 min (OD2)55. An increase in the ∆OD540 nm indicated mitochondrial swelling.

Determination of mitochondrial ROS production

Non-fluorescent 2′7′-dichlorofluorescein-diacetate (DCFH-DA), which was hydrolyzed by ROS in mitochondria and changed the form to fluorescent 2′,7′-dichlorohydrofluorescein (DCF), was used to evaluate mitochondrial ROS production56. The isolated cardiac mitochondrial stock was incubated with 2 μM DCFH-DA (prepared from 5 mL of respiratory buffer + 10 μL stock of 1 mM DCFH-DA) at 37 °C in the dark for 30 min. A fluorescence microplate reader with the excitation of λ 485 nm and emission of λ 530 nm was used to determine the fluorescence intensity of DCF, which indicated the increase in ROS production.

Determination of mitochondrial membrane potential changes

The JC-1 dye or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolecarbocyanine iodide (5 μM) was used in this study for an indirect estimate of the mitochondrial membrane potential change (∆ψm). Five μM of JC-1 (prepared from 5 mL of respiratory buffer + 10 μL stock of 2.5 mM JC-1) was incubated with isolated cardiac mitochondrial stock at 37 °C in the dark for 30 min. When membrane depolarization occurs (∆ψm augmentation), the expression of the red and green fluorescence is altered, leading to a decrease in the red: green ratio54. The red and green fluorescence intensity was measured via a fluorescence microplate reader with the excitation at λ 485 nm and emission at λ 530 nm for red and λ 590 nm for green fluorescein. The results revealed the red: green fluorescence intensity ratio.

Real-time polymerase chain reaction (real-time PCR) analysis

The expression levels of autophagy-related genes (LC3-phosphatidylethanolamine conjugate and p62), mitophagy-related genes (PINK and Parkin), and natriuretic peptides (ANP and BNP) were determined using real-time PCR analysis. The 18S rRNA were selected and used as an internal control in this study. The used primer sequences of these genes were obtained from a previous study in rat cardiomyocytes and are displayed in Table 157,58,59. Total RNA was extracted from the LV tissue using an AccuPure tissue RNA mini kit (AccuBioMed®, New Taipei City, Taiwan) in accordance with the manufacturer’s instructions and reverse transcribed to the first-strand complementary DNA (cDNA) using the iScript™ reverse transcription supermix (BioRad, Hercules, USA). The expression level of the target genes and housekee** genes was detected by real-time PCR analysis using PowerUp™ SYBR™ Green Master Mix in conjunction with QuantStudio™ 5 (Thermo Fisher Scientific, Massachusetts, USA), performing duplicate per reactions. The standard reaction conditions were 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 1 min with a total of 40 cycles. The threshold cycle (CT) of 18S rRNA was used to normalize the CT of the target genes (∆CT). Thereafter, the fold change mRNA expression of the target genes to the control group and the fold change mRNA expression of the target genes to the MR group was calculated using formula 2−∆∆CT60.

Table 1 Target genes and internal control primer sequences.
Full size table

Statistical analysis

Statistical analysis was performed using IBM® SPSS® statistics commercial software (SPSS Inc., IL, U.S.A.). Continuous variables are demonstrated as mean ± standard error of mean (SEM). Student t-test was performed to differentiate the difference between the Sham and MR groups at baseline of the study. One-way ANOVA test with Tukey’s correction for multiple comparisons was used to evaluate the pharmacological effects of the drugs among groups. P value < 05 was considered statistically significance.