Simvastatin s Mitochondrial Defenses against Angiotensin II-Induced Heart Failure

Research Article

Austin J Pharmacol Ther. 2023; 11(2): 1176.

“Simvastatin’s Mitochondrial Defenses against Angiotensin II-Induced Heart Failure”

Hari Prasad Sonwani*

Apollo College of Pharmacy, Anjora, Durg (C.G), India

*Corresponding author: Hari Prasad Sonwani Apollo College of Pharmacy, Anjora, Durg (C.G), India. Email: [email protected]

Received: October 03, 2023 Accepted: November 09, 2023 Published: November 16, 2023


Background and Aims: Heart failure is one cardiovascular condition that might progress due to mitochondrial dysfunction. In chronic heart failure, 3hydroxy-3-methylglutaryl CoA reductase inhibitors (statins), which prevent the production of ROS, have cardioprotective benefits. However, it is still unknown how statins can protect the mitochondria in heart failure Experimental Strategy: Angiotensin II (1.5 mg/kg/day) or co-administered simvastatin (oral, 10 mg/kg/day) were given to rats for 14 days, after which the treatment was withdrawn. Wheat germ agglutinin staining and echocardiography were used to analyze the structure and function of the heart. Transmission electron microscopy was used to analyze the shape of the mitochondria as well as the numbers of lipid droplets, lysosomes, autophagosomes, and mitophagosomes. After stimulating human cardiomyocytes, flow cytometry was used to assess changes in intracellular ROS and mitochondrial membrane potential (m). and, respectively, JC1 staining. By using immunohistochemistry and western blotting, apoptotic proteins that are associated to autophagy, mitophagy, and mitochondrial regulation were identified. Key outcomes Simvastatin mitigated the disruption of m and dramatically decreased ROS generation. Simvastatin stimulated autophagy and mitophagy, caused lipid droplets to accumulate, and provided energy for maintaining mitochondrial function and impeded apoptosis that was mediated by mitochondria. According to these results, simvastatin-mediated mitochondrial protection prevents heart failure by modifying antioxidant status and enhancing energy sources for autophagy and mitophagy, which prevent mitochondrial damage and cardiomyocyte apoptosis. Final Thoughts and Implications Mitochondria are crucial in controlling the course of heart failure. Simvastatin reduced angiotensin II-induced heart failure through mitochondrial preservation and may offer a new treatment for heart failure prevention.

Keywords: Cardiovascular; Simvastatin; Echocardiography; Cardiomyocytes


Heart Failure (HF) is a condition that affects the entire world and is brought on by the aging global population. According to Bhatt and Butler (2018), HF is a frequent reason for individuals over 65 to be admitted to the hospital and will have a significant financial impact on healthcare globally. The production of ROS, mitochondrial dysfunction, and reduced cardiac contractility are typical pathological findings in HF [40]. One of the frequent reasons why HF progresses is pressure overload caused by angiotensin II (Ang II). According to studies, mito-chondrial dysfunction is a major factor in the development of HF [59]. However, there are currently no clinically proven ways to stop or even stop the progression of HF. The renin-angiotensin system in the body generates the peptide Ang II. Regulates ROS generation, mitochondrial dysfunction, pro-inflammatory cytokine expression, autophagy, apoptosis, and pathophysiology of the cardiovascular system, including hypertension and Heart Failure (HF) [51]. There are a lot of mitochondria in heart cells. Cardiomyocytes employ efficient preventative strategies to preserve mitochondrial homeostasis through mitochondrial quality management, which involves the regulation of mitochondrial dynamics and mitochondrial autophagy (also known as mitophagy) [16]. Recently, cardiovascular illnesses such cardiomyopathy and HF have been linked to mitochondrial quality, morphology, and function [25]. Mitophagy is the process by which damaged mitochondria are broken down. During this process, damaged mitochondria are consumed by autophagosomes and then broken down in lysosomes [50]. Additionally, it was recently shown that Ang II causes cardiomyocytes to engage in autophagy, with possible effects on HF linked to Ang II [28]. The stability of cardiomyocyte ATP production affects the progression of cardiac illness and mito chondrial respiratory function in addition to maintaining the cellular energy state [47]. One of the primary energy sources in cardiomyocytes is lipid. Cardiomyocytes increase their fatty acid content when heart injury is caused by hypoxia, ischaemia, or stress overload. According to Minami et al. (2017), acid (FA) intake results in intracellular Lipid Droplets (LDs), which are then hydrolyzed for usage by lysosomes or peroxisomes. Recent research has shown that the lysosome route is essential for the conversion of lipids to FAs [1,41]. Additionally, Dupont et al. (2014) showed that LD accumulation could encourage autophagy because LDs provide lipid precursors for the developing autophagosome membrane. According to Lee, Zhang, Choi, and Kim (2013), LD formation is a global stress response that is triggered by cardiac mitochondrial dys-function. In addition, Yokoyama et al. (2007) showed that taking a statin caused the cytosolic LDs to build up, giving the cell extra energy. Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, also known as statins, are a common class of medications used to treat Overproduction of cholesterol [38]. In addition to their effects on lipid levels, statins have been shown to have anti-inflammatory, anti-oxidative, and anti-cancer properties [28,52]; they also play a role in endothelial regulation and have an impact on cell autophagy and apoptosis [42,46]. However, there are still many issues that need to be researched further in relation to simvastatin's function in the heart. To determine whether simvastatin's control of We used an HF animal model and cultured Human Cardiomyocytes (HCMs) to investigate the preventive effects of simvastatin in Ang II-induced mitochondrial damage. Mitophagy participates in mitochondrial protection in HF. By controlling LD accumulation, lysosomal activation to destroy LDs, inhibiting ROS production, and preventing alteration of mitochondrial membrane potential (m), simvastatin reduces Ang II-mediated HF, according to our findings. As a result, there is an increase in mitophagy, which prevents the activation of apoptosis by the mitochondria.


Treatment of Animals

The animal treatments were carried out in a facility that was approved by the Association for Assessment and Accreditation of Laboratory Animal Care International and strictly adhered to Taiwanese law. Eight-week-old male Sprague-Dawley rats were kept in a specified pathogen-free environment with a 12/12-hour light/dark cycle.having free access to regular food and water, as well as a temperature-controlled room. Throughout the trial, all rats were weighed once a week, and three experimental groups (each with six rats) were randomly selected. After isoflurane anaes- thesia (1.5%), each group received a subcutaneously implanted Alzet® osmotic pump (Durect Corporation, Cupertino, CA, USA; infusion rate of 0.5 μl·hr-1) with (a) PBS; control group, (b) Ang II (1.5 mg·kg-1·day-1; , Ang II group or (c) Ang II infusion and co-administration of simvastatin (oral, 10 mg·kg-1·day-1, Merck), AngII+SIM group. After being administered for 14 days, the infusions were ceased for the next 14. The rats were put to death (with CO2) on day 28, and the hearts were immediately taken. Upon completion of the experiment weight of the entire heart, the coronal heart portion (from apex to apex), the lung, and the gastrocnemius muscle were also recorded throughout this time. The authors claim that every attempt was made to reduce both the quantity and severity of the suffering experienced by the animals.


Animals were given isoflurane (Panion & BF Biotech Inc., Taiwan) anesthesia before having M-mode transthoracic echocardiography performed utilizing an iE33TM imaging equipment and an S124 (12-4 MHz) pediatric probe. large sampling frequency (150 mm/s). At the level of the left atrium, two-dimensional targeted M-mode echocardiographic pictures were taken. to measure the left ventricular mass (LVM), LVM contractility, left ventricular ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter in diastole, and left ventricular internal diameter in systole. For each assessment, three cycles were measured, and the average values were computed. The formula LVM (g) = 0.8 [1.04 (IVS + PP + LVEDD)3 LVEDD3] + 0.6 was used to calculate LVM (g), while Teichholz's formula was used to determine EF.

Oil Red O staining, germ agglutinin staining, immunohistochemistry, TUNEL labeling, and histology

As previously described [48], portions of the heart were fixed with 4% paraformaldehyde, wax embedded, and sectioned (5 m thick) for H&E staining (Invitrogen, Carlsbad, CA, USA). The size of cardiomyocytes, and Wheat germ agglutinin (WGA) staining and ImageJ analysis (RRID: SCR_003070, NIH, Bethesda, Rockville, MD, USA) were used to estimate length. Oil Red O staining was quantified to determine the lipid content (Bio Vision, Mountain View, CA, USA; Catalog #K58024). The samples were stained with haematoxylin, washed with distilled water, and then were washed three more times with 60% isopropanol for five minutes each while being gently rocked. They were then replaced in 24 well plates for measurement after being removed by Oil Red O stain with 100% isopropanol for 5 min with gentle shaking. After subtracting the background signal using 100% isopropanol as a background control, the absorbance was measured at 495 nm. The methods in this investigation that were based on antibodies abide by the British Journal of Pharmacology's recommendations. Measurements of autophagic or apoptotic protein expression were made using immunohistochemical staining and western blotting. Tissue sections were first incubated in blocking buffer (0.5% BSA, 0.05% Tween 20, and PBS) for 1 hour at room temperature, then specific primary antibodies against Bcl2 (1:100, sc7382, RRID: AB_626736, Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c The substance According to manufacturer's protocol, staining was created using a 3,3'-diaminobenzidine detection system (Catalog #760124, Ventana Medical Systems, Tucson, AZ, USA) and counterstained with hematoxylin. According to the manufacturer's instructions, TUNEL assays were carried out using a TUNEL assay kit (In Situ Cell Death Detection Kit, Catalog 11684795910, Roche, Mannheim, Germany). A fluorescent microscope (Leica, Wetzlar, Germany) was used to view the cell nuclei after they had been counterstained with DAPI, cleaned, mounted with VECTASHIELD® mounting media (Vector Laboratories, Burlingame, CA, USA), and washed.

Electron Microscope that Uses Field-Emission Tomography

According to previous descriptions [45], field-emission trans mission electron microscopy (FETEM) was implemented. Briefly stated, tissue samples were preserved for two hours at 4°C with 2.5% glutaraldehyde. Following a thorough cleaning, the samples were post-fixed for two hours in 1% osmium tetroxide, dehydrated in graded acetone, infiltrated, and finally embedded in epoxy resin. Using a Leica, ultrathin 70 nm slices were cut. At an accelerating voltage of 80 kV, samples were cut with a microtome (Leica RM2165, Japan) and analyzed using a FE-TEM (HITACHI HT-7700, Japan).

Measuring the level of Mitochondrial Damage

A mitochondrion is made up of five different components and has a double-membrane structure made of proteins and phospholipid bilayers: the mitochondrial outer membrane, (b the inner mitochondrial membrane, (c) the cristae space (produced by infoldings of the inner membrane), (d) the cristae space (the space within the inner membrane), and (e) the matrix (the space within the inner membrane). The shape of the mitochondrial cristae directly reflects the health of the mitochondria, and FE-TEM images demonstrate various degrees of mitochondrial damage: Score 1: cristae are joined to the intermembrane space by well-defined, many crista junctions and cristae, indicating healthy mitochondria (well-defined, undamaged, ordered membranes); Score 2: early stages of enlarged mitochondria (sometimes enlarged cristae, rather erratic); Megamitochondria receive a 3 (severe deformities, significant swelling and disarray of the cristae, and discontinuous membrane and cristae); Score 4: incredibly large, enlarged matrix mitochondria (membranes and cristae separated into particles to Score 5: vacuolization (delamination of the inner and outer mitochondrial membranes, absence of cristae, vacuolization); and Score 4: widespread mitochondrial ghosts.

Culture of Cells

The cells were grown in accordance with earlier instructions [45]. HCMs were cultured in cardiac myocyte medium (ScienCell Research Laboratories; Catalog #6200; Carlsbad, CA, USA) with 5% FBS (Life Technologies; Ref. 10437-028; Lot 1700200), 1% cardiac myocyte growth supplement (ScienCell Research Laboratories), and 1% penicillin/streptomycin solution (Life Technologies; Ref. 15140-122; Lot 1881449) added. The culture media was changed every 4 to 5 days while the cells were incubated at 37°C in a 5% CO2 environment. The cells were utilized exclusively between Pas- sages 3 and 9.

Measurement in Millimeters

A JC-1 m detection kit (ThermoFisher Scientific, Waltham, MA, USA; Catalog M31152) was used to assess the Ang II-induced changes in m. in the past [4]. HCMs (1 103) were seeded on the cover slip for 48 hours, after which they were exposed to 2 gml of JC1 at 37°C for 20 min. They were subsequently treated with Ang II or Ang II + simvastatin for 2 hours. A BD LSR II flow cytometer (BD Bioscience, Singapore) or an Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan) was used to analyze and take pictures of the cells. A rough estimate of JC1's excitation peak is 488 nm. For monomeric and J-aggregate forms, the estimated emission peaks are 529 and 590 nm, respectively.

ROS Measurement

Simvastatin (0.5 M, Sigma Aldrich, St. Louis, MO, USA) was pretreated with or without the addition of mitoSOXTM (5 M, ThermoFisher Scientific) for 2 hours in HCMs. ThermoFisher Scientific; Catalog 6827) for 30 min at 37°C and then treated with Ang II (10 M) for 1.5 hrs. 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA; 20 M, ThermoFisher Scientific; Catalog 6827) for 10 min at 37°C. Using flow cytometry, the mitochondrial and intracellular ROS levels were assessed. The fluorescence emission and excitation at 510 and 580 nm (MitoSOXTM) and 490 and 520 nm (H2DCFDA), respectively, were measured using the BD LSR II instrument (BD Bioscience).

Western Blot Examination

Western blot analyses were carried out as previously explained (Kuo et al., 2016). The Lowry assay was used to assess the protein levels in cell or tissue lysates. then 30g of protein samples

Depending on the molecular weight of the proteins of interest, they were separated using 7.5%, 10%, or 12.5% SDS-PAGE, and then electroblotted onto a nitrocellulose membrane. The membranes were then incubated with specific primary antibodies against Bcl2 (1:500, sc-7382, Santa Cruz Biotechnology), cytochrome c (1:1,000, ADI-AAM-175, Enzo Biochem), caspase 3 (1:1,000, #9662, Cell Signaling Technology), and LC3I/II (1:500, sc-7382, Santa Cruz Biotechnology).

Parkin, p62 (1:500, GTX100685, Gene Tex), GTX127375, Gene Tex GAPDH (1:2,000, sc137179, Santa Cruz Biotechnology), PINK1 (1:1,000, sc32282, Novus Biotechnology), and GAPDH were diluted before being detected using secondary antibodies that were HRP-conjugated. Fluorography and an improved detection kit (ECL, GE Healthcare Life Sciences, Buckinghamshire Amersham Pharmacia International) were used to visualize the signals.

Information and Analysis

The data and statistical analysis adhere to the British Journal of Pharmacology's guidelines for experimental design and pharmacology analysis. The results were analyzed using ANOVA, followed by Dunnett's post hoc tests, and are shown as the means SEM of each group. SigmaStat version 3.5 (RRID: SCR_010285, Systat Software Inc., Chicago, IL, USA) was used to generate all statistics, and P .05 was regarded as statistically significant.

Target and Ligand Nomenclature

Key protein targets and ligands are permanently archived in the Concise Guide to Pharmacology 2017/18 (Alexander, Cidlowski et al., 2017; Alexander, Fabbro, 2018) and are hyperlinked to corresponding entries in, the common portal for data from the IUPHAR/BPS Guide to Pharmacology (Harding et al., 2018).


LVM contractility (Figure 2d) and (Figure 2c). The left ventricular internal diameter, on the other hand, demonstrated that Ang II did not directly alter cardiac dilation. Simvastatin enhances cardiac performance in Ang II-induced heart failure The body weight of the study's experimental animals was measured at both the beginning (8 weeks) and the end (12 weeks), and it was determined to be 358.5 10.9 g 434.8 34.4 g in the control group, 354.6 11.8 g 399.6 13.7 g in the Ang II group, and 354.6 11.8 g 399.6 13.7 g in the Ang II group. In the Ang II + SIM group, the weights were 355.5 13.4 g 422.4 33.6 g. The most prevalent anatomical abnormality known as cardiac cachexia is severe HF, left ventricular hypertrophy, and skeletal muscle wasting (Delafontaine & Akao, 2004). Figure 1 displays the data that were gathered after the experiment. Body weight and gastrocnemius muscle weight are presented in Figures 1a,b, whereas Figure 1c depicts whole-heart imaging and H&E staining to identify histological changes. WGA staining and quantification were used to demonstrate that the treatment with Ang II did enhance cardiomyocyte size when compared to the control or simvastatin-treated groups (Figure 1d). The preventive benefits of simvastatin were subsequently investigated using echocardiography, which clarified the structure and function of the heart. Simvastatin and Ang II were administered to rats for 14 days, followed by 14 days of recovery. The rats' EF and FS were lowered at day 28 (Figure 2a and Figure 2b). LVM (Figure 2 eter in diastole (Figure 2e) and left ventricular internal diameter in systole (Figure 2f) values also rose (Figure 2b). In the last stages of HF, significant consequences such cardiac hypertrophy and pulmonary oedema frequently manifest [7,33]. Rat heart/body weight ratios (Figure 2g), lung/body weight ratios (Figure 2h), and gastrocnemius muscle weight/body weight ratio (Figure 2i) were calculated at the conclusion of the experiment, as in previous studies [22,25], in order to track the progression of Ang II-induced HF. Our findings demonstrated that Ang II significantly increased pulmonary oedema and cachexia in rats, which weren't seen in the SIM group plus Ang II. According to these findings, simvastatin supplementation reduced Ang II-induced ventricular hypertrophy, EF/FS decline, and pulmonary oedema. Simvastatin prevents in vivo myocardial mitochondrial damage caused by Ang II.