The Protective Effect of Carvacrol Against Isoproterenol-Induced Cardiotoxicity in Rats

Abstract

Objective

Isoproterenol (ISO)-induced myocardial injury involves oxidative stress (OS) and inflammation. Given carvacrol (CAR)’s antioxidant and cardioprotective properties, we aimed to investigate its protection against ISO-induced myocardial injury in rats.

Methods

Thirty male Wistar albino rats were assigned to three groups: Control; ISO (100 mg/kg, subcutaneous, for 2 days); and CAR+ISO (CAR 50 mg/kg/day, orally at 9:00, for 7 days + ISO at 16:00 on days 6-7). CAR and ISO doses were chosen based on prior evidence of CAR’s antioxidant effects and ISO-induced oxidative cardiac stress. Diastolic and mean arterial pressures, corrected QT interval (QTc), T-wave changes, and serum troponin I levels were assessed. Cardiac OS biomarkers [malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT)] and histopathology were evaluated.

Results

ISO markedly increased troponin I from 43.9 to 508.9 ng/mL and slightly reduced GSH from 1502.4 to 1330 nmol/g wet tissue and SOD from 1080.25 to 1010.34 U/g protein. ISO markedly reduced MDA (95.30 to 71.78 nmol/g wet tissue at 535 nm; 77.49 to 61.24 nmol/g wet tissue at 520 nm), likely due to depletion of lipid peroxidation substrates caused by excessive reactive oxygen species production, while slightly increasing CAT (132.66 to 162.04 K/g protein), possibly as a compensatory response. QTc and histopathological scores increased significantly; histopathological components were cardiomyocyte degeneration (CD) (range, 0.0 to 2.0), interstitial edema (IE) (range, 0.0 to 2.0), and granulation tissue (GT) (range, 0.0 to 1.0). CAR attenuated electrocardiographic abnormalities, significantly reduced troponin I (from 508.9 to 38.05 ng/mL), and restored GSH (from 1330 to 1396.8), SOD (from 1010.34 to 1094.42), and CAT (from 162.04 to 135.58). CAR significantly ameliorated histopathological scores: CD (2.0 to 1.0), IE (2.0 to 1.0), and GT (1.0 to 0.0).

Conclusions

CAR exerts protective effects against ISO-induced myocardial injury via antioxidant, membrane-stabilizing, and possible anti-arrhythmic properties. These findings support the therapeutic potential of CAR in OS-mediated cardiac pathologies and warrant further investigation in chronic and molecularly targeted models.

INTRODUCTION

Cardiovascular diseases (CVDs) represent a major global health burden, accounting for approximately 19.8 million deaths annually, according to the World Health Organization¹. Among the various pathological conditions encompassed under CVDs, myocardial infarction (MI) remains one of the most life-threatening events, often resulting from prolonged ischemia (I), oxidative stress (OS), and inflammatory cascades that culminate in cardiomyocyte death and impaired cardiac function². To better understand the pathophysiology of MI and evaluate potential therapeutic interventions, experimental models that reliably mimic human cardiac injury are indispensable³. In this context, isoproterenol (ISO), a synthetic non-selective β-adrenergic receptor agonist, has been extensively employed to induce cardiotoxicity in rodents. High-dose ISO administration triggers excessive catecholamine stimulation, leading to myocardial necrosis, mitochondrial dysfunction, calcium overload, and a surge in reactive oxygen species (ROS), thereby replicating key features of human ischemic heart disease⁴.

In recent decades, there has been growing scientific and clinical interest in phytochemicals as adjunctive or alternative cardioprotective agents due to their multi-targeted mechanisms and favorable safety profiles⁵. Among these natural compounds, carvacrol (CAR), a monoterpenoid phenol predominantly found in the essential oils of Origanum vulgare, Thymus vulgaris, and other plants of the Lamiaceae family, has attracted considerable attention for its broad-spectrum biological activities⁶. CAR exhibits well-documented antioxidant, anti-inflammatory, anti-apoptotic, and antimicrobial properties⁷. Importantly, accumulating preclinical evidence suggests that CAR may exert significant protective effects against various forms of organ toxicity, including hepatotoxicity, nephrotoxicity, and notably, cardiotoxicity^{8, 9}

The cardioprotective potential of CAR appears to be mediated primarily through its ability to scavenge free radicals, enhance endogenous antioxidant defenses, and modulate key signaling pathways involved in inflammation and cell survival^{10, 11, 12}. For instance, CAR has been shown to suppress the activation of nuclear factor-kappa B (NF-κB), a master regulator of pro-inflammatory cytokine production, and to upregulate the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway, which plays a pivotal role in cellular defense against OS¹³.

Despite these promising findings, a comprehensive evaluation of CAR’s efficacy—particularly with respect to dose-response relationships, long-term functional outcomes, and molecular mechanisms beyond OS—is warranted. Moreover, comparative analyses with standard cardioprotective agents and investigations into potential synergistic effects with conventional therapies could further validate its translational relevance. Therefore, the present study was designed to systematically investigate the protective effects of CAR in a well-established rat model of ISO-induced cardiotoxicity. We assessed a range of parameters, including hemodynamic function, biochemical markers of myocardial damage, OS indices, and histopathological changes in cardiac tissue. The primary endpoint of our study was myocardial malondialdehyde (MDA) level, selected as the principal marker of ISO-induced oxidative injury¹². The results of our investigation are intended to provide robust preclinical evidence supporting the potential of CAR as a natural adjuvant therapy for the management of ischemic heart disease and other OS-mediated cardiac disorders.

MATERIALS and METHODS

Ethics Committee Information

All procedures were approved by the Inonu University Institutional Animal Care and Use Committee (IACUC; approval no. 2021/22-6, date: 10.11.2021). Experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and complied with the ARRIVE reporting guidelines¹⁴.

Study Design

Thirty male Wistar albino rats (12 weeks old; ~350 g) were obtained from the Inonu University Laboratory Animals Research Center. Animals were housed under controlled conditions (21±2 °C; relative humidity 60±5%) on a 12:12 h light-dark cycle. Rats had unrestricted access to standard pelleted chow and tap water.

A computer-generated random number list was used for unbiased group allocation of 30 rats into three experimental groups.

Control Group (n=10): sunflower oil (1 mL/day, per os=p.o.) was administered at 9:00 AM for 7 days. Saline (0.5 mL/day, subcutaneous=s.c.) was administered at 4:00 PM on days 6-7.

ISO Group (n=10): Sunflower oil (1 mL/day, p.o.) was administered at 9:00 AM for 7 days. ISO (150 mg/kg/day, s.c.) was administered at 4:00 PM on days 6-7.

CAR+ISO Group (n=10): CAR (80 mg/kg/day, p.o.) was given at 9:00 AM for 7 days; ISO (150 mg/kg/day, s.c.) was given at 4:00 PM on days 6-7.

The dose and duration of ISO administration (Isoprenaline hydrochloride, CAS number: 51-30-9, Sigma-Aldrich, MO, USA) were determined based on previous studies demonstrating that ISO induces acute myocardial injury via OS. The chosen dose and treatment length for CAR (CAS number: 499-75-2, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were based on earlier research demonstrating its antioxidant and cardioprotective properties^{15, 16}. Rats were weighed at baseline and again at the end of the study. Twenty-four hours after the final ISO dose, all animals were anesthetized with urethane (ethyl carbamate; 1.2 g/kg, i.p.; Acros Organics, Geel, Belgium). Under anesthesia, systolic, diastolic, and mean arterial blood pressure (BP) were measured invasively via carotid artery cannulation, and heart rate (HR) and standard electrocardiography (ECG) were recorded. HR, arterial BP, and ECG were recorded with a Biopac MP100 data acquisition system (BIOPAC Systems, Inc., Santa Barbara, CA, USA). ECG signals were acquired for ≥3 minutes at 500 Hz using disposable surface electrodes placed on the thorax, as in our previous study¹⁷. After recording, tracings were visually reviewed by two experienced investigators to determine HR and to identify major ECG abnormalities (e.g., arrhythmias, ST-segment elevation or depression, inverted T-waves, and conduction disturbances) according to the Lambeth Conventions diagnostic criteria¹⁸. In addition, the analysis examined the high-precision measurements of the durations and variability of PR, QRS, and corrected QT interval (QTc) intervals between the groups. QTc rather than QT was used in our study because the QT interval varies with HR, and correction is recommended in both experimental and clinical practice. QTc was calculated from the measured QT interval values ​​using the Hodge formula [QTc=QT+0.00175x(HR-60)], which was suggested to be the most appropriate formula for Wistar rats¹⁹. Intracardiac blood was collected from rats while they were still under deep anesthesia. The collected blood samples were centrifuged at 2500 rpm for 7 minutes, and the separated serum was stored at -80°C until biochemical analyses were performed. Animals were euthanized by surgical exsanguination and the hearts were rapidly excised. Whole-heart weight was measured using an analytical balance. Each heart was bisected along the longitudinal axis: one half was snap-frozen and stored at -80 °C for biochemical assays of antioxidant enzymes and OS markers, and the other half was fixed in 10% neutral-buffered formalin for histopathology. Histological evaluation of cardiac tissue was performed by light microscopy.

Heart Tissue Biochemical Analysis

The cardiac tissue was homogenized in 10% ice-cold 150 mM KCl (pH 7.5) after thawing. A portion of the homogenized sample was subjected to centrifugation at 3000 rpm at +4°C. The supernatant was then used to assess reduced glutathione (GSH) content and the activities of superoxide dismutase (SOD) and catalase (CAT). MDA concentrations were analyzed directly in the homogenate. Each measurement was performed in duplicate.

Measurement of MDA Concentrations

MDA concentrations in the homogenates were quantified spectrophotometrically based on thiobarbituric acid-reactive substances. Briefly, 0.5 mL of homogenate or MDA standard was added to 10-mL glass tubes containing 1 mL of 0.6% (w/v) thiobarbituric acid and 3 mL of 1% H₃PO₄. The mixtures were incubated in a water bath at 95-100°C for 45 minutes. Following incubation, the samples were allowed to cool, after which 4 mL of n-butanol was added. The mixtures were vigorously shaken for 5 minutes to extract the reaction products. Phase separation was achieved by centrifugation at 1500 × g for 10 minutes, and the upper butanol layer was collected for analysis. Absorbance readings were obtained at 535 nm and 520 nm using a UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan). The absorbance difference between the two wavelengths was used to calculate the MDA concentration. Quantification was performed using a standard curve generated from 1,1,3,3-tetramethoxypropane. Results were expressed in nmol per gram of wet tissue (nmol/g wet tissue). The coefficients of variation within and between assays were 4.5% and 9%, respectively.

Measurement of GSH Levels

GSH levels were analyzed using Ellman’s method²¹. To prepare the samples for GSH determination, we added trichloroacetic acid to the collected supernatant, mixed thoroughly, and performed a second centrifugation. The resulting clear solution was transferred into test tubes and thoroughly vortexed. After allowing the reaction to proceed for 5 minutes, the absorbance of the developed color was measured spectrophotometrically at 410 nm, and the GSH concentrations were evaluated from the GSH standard graph and given as nmol/gram wet tissue.

Determination of SOD Activity

SOD activity was determined according to the method of Sun et al.²². To measure SOD enzyme activity, the nitro blue tetrazolium (NBT)-reduction method based on superoxide radicals was used, with the xanthine-xanthine oxidase system serving as the superoxide source. The xanthine-xanthine oxidase system produces superoxide radicals. Superoxide radicals reduce NBT, producing a colored formazan. This reduction exhibits a maximum absorbance at 560 nm, forming a blue formazan. In the absence of enzymes, this reduction is maximal, producing a deep blue color. In the presence of SOD, the enzyme converts the superoxide anion into hydrogen peroxide, thereby decreasing NBT reduction and lightening the color. When SOD is low, reduction of NBT by O₂- and the absorbance of the resulting formazan dye at 560 nm are increased. If SOD is high, NBT reduction will be decreased, resulting in a lower absorbance of the formazan dye at 560 nm. Thus, SOD activity is calculated from the absorbance of the formazan formed at 560 nm. One unit of SOD was defined as the amount of enzyme that caused 50% inhibition of the rate of NBT reduction. SOD activity was given as units per gram protein (U/g protein). The composition of reagents and sample volumes used in the SOD activity assay are shown in (Table 1).

Table 1. Composition of reagents and sample volumes used in the SOD activity assay.

Reagents	Sample	Control
Assay reagent (0.3 mM/L xantine, 0.6 mM/L Na2EDTA, 150 μmol/L NBT, 400 mmol/L Na2CO3, 1 g/L BSA)	2.45 mL	2.45 mL
Supernatant	0.5 mL	-
Distiled water	0.5 mL	-
Xantine oxidase (167 U/L)	0.05 mL	0.05 mL

SOD: Superoxide dismutase

Determination of CAT Activity

CAT activity was measured using Aebi’s method by tracking the initial rate of H₂O₂ (10 mM) disappearance at 240 nm in a spectrophotometer²³. Results were expressed as a constant rate per gram of protein (K/g protein). The composition of reagents and sample volumes used in the CAT activity assay are shown in (Table 2).

Table 2. Composition of reagents and sample volumes used in the CAT activity assay.

Reagents	Sample	Control
Phosphate buffer (50 mM, ph: 7.0)	-	2.99 mL
Phosphate buffer with H2O2	2.99 mL	0.01 mL
Supernatant	0.01 mL	-

CAT: Catalase

Equation.

Protein Measurement

Protein amount was measured according to the method of Lowry et al.²⁴.

Serum Troponin Analysis

Serum troponin I levels were measured using a human-specific ELISA kit via contracted laboratory services at the Inonu University Turgut Ozal Medical Center (Malatya, Türkiye). All procedures were conducted in accordance with the manufacturer’s protocol, and results were expressed in nanograms per milliliter (ng/mL).

Histological Analyses

Myocardial tissues were preserved using 10% formaldehyde solution. Paraffin-embedded tissue blocks, following standard processing, were sectioned at 4-5 µm and stained with hematoxylin and eosin for histopathological analysis. The samples were examined for signs of cardiomyocyte damage, including myofibril loss, cellular swelling, vacuole formation, intensely eosinophilic cytoplasm, and pyknotic nuclei, as well as for interstitial edema and the development of granulation tissue. Histopathological alterations were assessed in ten randomly selected fields and graded on a scale of 0-3: 0= none, 1= mild, 2= moderate, and 3= severe. Examinations were conducted with a Leica DFC-280 microscope and the images were analyzed with Leica Q Win software (Leica Microsystems, Cambridge, UK).

Statistical Analysis

A power analysis was performed to determine the necessary sample size for this study. Based on a Type I error rate (alpha) of 0.05, a power (1-beta) of 0.8, and an effect size of 0.62, a sample size calculation indicated that at least 10 animals per group were required, for a total of 30 animals. Values for myocardial MDA level and their variability reported in the literature for ISO models were used to estimate the expected effect size^{25, 26}.

SPSS software (IBM SPSS Statistics version 25) was used for statistical analysis of the obtained data. The normality of quantitative data was assessed using the Kolmogorov-Smirnov test. For indicators that met the normality assumption, between-group differences in quantitative variables were analyzed using one-way ANOVA after confirming homogeneity of variance with Levene’s test; multiple comparisons were performed using Tukey HSD. When the assumption of normality was not met, between-group differences in quantitative variables were assessed using the Kruskal-Wallis H test. Multiple comparisons were performed using the Mann-Whitney U test with Bonferroni correction. Outliers were not explicitly tested in this study. Quantitative data in the study were presented as mean ± standard deviation for parametric tests and as median (minimum-maximum) for non-parametric tests. Values of p<0.05 were considered statistically significant.

RESULTS

Rat Body and Heart Weights

Baseline body weights were significantly higher in both the ISO and CAR+ISO groups than in the control group (p<0.05). However, there was no statistically significant difference in post-treatment body weights among the groups (p=0.080). ISO administration led to a marked increase in absolute heart weight compared to both the control and CAR+ISO groups (p<0.05). Furthermore, the heart-to-body weight ratio was significantly elevated in the ISO group relative to the control group (p<0.05), indicating ISO-induced cardiac hypertrophy. Notably, co-administration of CAR appeared to attenuate this effect. Evaluation of the body- and heart-weight parameters is presented in Table 3.

Table 3. Rat body and heart weights.

Parameters	Control group	ISO group	CAR+ISO group	p-value
Pre-experiment rat weight (g)	226 (194-284)	277.5a (256-305)	269.5a (248-301)	0.001
Post-experiment rat weight (g)	247.5 (205-309)	285 (247-315)	267 (237-302)	0.080
Heart weight (g)	0.86 (0.69-1.46)	1.24ab (1.06-1.31)	1 (0.87-1.23)	0.001
Heart/rat weight ratio	0.003 (0.002-0.006)	0.004a (0.004-0.005)	0.004 (0.003-0.004)	0.035

Data are expressed as “median (min-max)”.

^a: Significantly different from the Control group, p<0.05.

^b: Significantly different from the CAR+ISO group, p<0.05.

CAR: Carvacrol, ISO: Isoproterenol

Hemodynamic Parameters

Evaluation of the hemodynamic parameters, as presented in Figure 1 and Table 4, demonstrated that ISO administration significantly affected BP and cardiac electrical activity. Diastolic arterial pressure (DAP) and mean arterial pressure (MAP) were significantly lower in the ISO group compared with the control group (p=0.03 for both), indicating impaired hemodynamic function following ISO-induced myocardial injury. Furthermore, the QTc interval was significantly prolonged in the ISO group relative to the control group (p=0.04), suggesting a delay in ventricular repolarization. These alterations were less pronounced in the CAR+ISO group, although the differences did not reach statistical significance compared to the control group. Other parameters such as HR, systolic arterial pressure, and PR interval did not show statistically significant differences among the groups (p>0.05).

Figure 1.

Comparative bar graphs showing A), Diastolic arterial pressure (DAP), B), Mean arterial pressure (MAP), and C), QTc interval values across the Control, ISO, and CAR+ISO groups. Data are presented as mean ± standard deviation. ISO administration significantly reduced DAP and MAP, and prolonged the QTc interval compared to the Control group (p<0.05). CAR co-treatment partially ameliorated these changes.

ISO: Isoproterenol, CAR: Carvacrol, QTc: Corrected QT interval, DAP: Diastolic arterial pressure

Table 4. Findings related to the hemodynamics of rats.

Variables	Groups			p-value
	Control	ISO	CAR+ISO
Heart rate (beat per minute)	358.57±54.77	333±43.82	318.11±51.78	0.290
SAP (mmHg)	86.81±10.23	67.28±19.09	75.77±12.92	0.052
DAP (mmHg)	81.97±10.27	60.79a±18.69	69.39±12.54	0.30
MAP (mmHg)	83.59±10.22	62.95a±18.6	71.52±12.59	0.30
PR interval (ms)	46.29±1.8	47.78±2.11	48.89±4.81	0.310
QRS interval (ms)	40 (4)	44 (10)	44 (12)	0.060
QTc interval (ms)	77.24±12.68	98.26a±12.19	92.01±19.32	0.040

Data are expressed as “median (interquartile range)” or “arithmetic mean ± standard deviation”.

^a: Significantly different from the Control group, p<0.05

SBP: Systolic arterial pressure, DAP: Diastolic arterial pressure, MAP: Mean arterial pressure, CAR: Carvacrol, ISO: Isoproterenol, QTc: Corrected QT interval

Electrocardiographic Analysis

The distribution of electrocardiographic abnormalities among the groups is presented in Table 5. While no ECG abnormalities were observed in the control group, various pathological changes were detected in rats exposed to ISO. In the ISO group, T-wave negativity was observed in 3 rats (33.33%) and biphasic T-waves were observed in 1 rat (11.11%), indicating myocardial electrical instability. In the CAR+ISO group, T-wave negativity and biphasic T-waves were observed in 2 rats (22.22%) and 1 rat (11.11%), respectively. Additionally, a bundle branch block was detected in 1 rat (11.11%). Although no formal statistical test was applied due to categorical data and limited sample size, these qualitative observations suggest that CAR co-administration may partially reduce ISO-induced ECG abnormalities.

Table 5. Distribution of ECG abnormalities among groups.

Groups	Arrhythmia	ST depression	ST elevation	T negativity	Biphasic T	Bundle branch block
Control (n=7)	0	0	0	0	0	0
ISO (n=9)	0	0	0	3 (33.33%)	1 (11.11%)	0
CAR+ISO (n=9)	0	0	0	2 (22.22%)	1 (11.11%)	1 (11.11%)

Data are presented as the number of animals (n) and the percentage (%).

ECG: Electrocardiography, CAR: Carvacrol, ISO: Isoproterenol

Biochemical Analysis

The comparison of OS and antioxidant parameters among groups is summarized in Figure 2 and Table 6. MDA levels, measured at both 532 nm and 520 nm, were significantly reduced in the ISO group compared to the control group (p<0.001), indicating an unexpected decline in lipid peroxidation following ISO administration. However, MDA levels were significantly higher in the CAR+ISO group than in the ISO group (p<0.001), approaching values seen in the control group, suggesting a modulatory effect of CAR on oxidative processes.

Figure 2.

Bar graphs representing malondialdehyde (MDA) levels in cardiac tissue measured at A), 520 nm and B), 535 nm in Control, ISO, and CAR+ISO groups. Data are shown as mean ± standard deviation. ISO administration significantly reduced MDA levels compared to the Control group (p<0.001), while co-treatment with CAR significantly restored MDA levels toward normal (p<0.001 vs. ISO group).

ISO: Isoproterenol, CAR: Carvacrol

Table 6. Comparison of antioxidant and lipid peroxidation parameters among groups.

Parameters	Groups			p-value
	Control	ISO	CAR+ISO
MDA (nmol/g wet tissue 535 nm)	95.30±8.11	71.78a±16.47	100.81b±15.78	<0.001
MDA (nmol/g wet tissue 520 nm)	77.49±4.41	61.24a±10.16	75.85b±9.1	<0.001
GSH (nmol/g wet tissue)	1502.4±144.2	1330±180.9	1396.8±112.6	0.051
SOD (U/g protein)	1080.25±105.11	1010.34±107.09	1094.42±83.41	0.160
CAT (K/g protein)	132.66±50.5	162.04±53.72	135.58±39.49	0.360

Data are expressed as “arithmetic mean ± standard deviation”.

^a: Significantly different from the Control group, p<0.05

^b: Significantly different from the ISO group, p<0.05

MDA: Malondialdehyde, GSH: Glutathione, SOD: Superoxide dismutase, CAT: Catalase, CAR: Carvacrol, ISO: Isoproterenol

Although GSH levels and SOD activity tended to be lower in the ISO group and higher in the CAR+ISO group, these differences did not reach statistical significance (p=0.051, p=0.16, respectively). CAT levels tended to be higher in the ISO group and lower in the CAR+ISO group, these differences did not reach statistical significance (p=0.36).

Troponin Levels

Serum troponin levels among the experimental groups are presented in Figure 3 and Table 7. Troponin levels were markedly elevated in the ISO group compared to the control group (p<0.05), confirming significant myocardial injury following ISO administration. In contrast, rats in the CAR+ISO group exhibited significantly lower troponin levels than the ISO group (p<0.05), suggesting that CAR treatment effectively attenuated ISO-induced cardiac damage. Notably, troponin levels in the CAR+ISO group were comparable to those of the control group, further supporting the cardioprotective role of CAR.

Figure 3.

Bar graph showing serum troponin levels (median ± interquartile range) in Control, ISO, and CAR+ISO groups. ISO administration significantly elevated troponin concentrations compared to the Control group (p<0.05), indicating myocardial injury. CAR co-treatment significantly attenuated this elevation (p<0.05 vs. ISO group).

ISO: Isoproterenol, CAR: Carvacrol

Table 7. Comparison of troponin values among groups.

Parameters	Groups			p-value
	Control	ISO	CAR+ISO
Troponin (ng/mL)	43.9 (154.45)	508.9a (2069.1)	38.05b (69.55)	0.30

Data are expressed as “median (interquartile range)”.

^a: Significantly different from the Control group, p<0.05

^b: Significantly different from the ISO group, p<0.05

CAR: Carvacrol, ISO: Isoproterenol

Histopathological Analysis

In the control group, the myocardium exhibited a normal histological appearance, except for mild interstitial edema observed in some areas and the presence of a small number of degenerated cardiomyocytes (Figure 4a). In the myocardium of the ISO group, a marked increase in the density of degenerated cardiomyocytes and interstitial edema was observed. Another notable change observed in the ISO group was the presence of granulation tissue. In many specimens of this group, granulation tissue replaced myocardial tissue in some areas (Figure 4b). The difference between the control and ISO groups with respect to these changes was statistically significant (p<0.0001). In the CAR+ISO group, histopathological changes were mild (Figure 4c) and were significantly reduced compared with those in the ISO group (p<0.0001). The histopathological evaluation results for the groups are presented in Table 8.

Figure 4.

Control group a), normal histological appearance of the myocardium. ISO group b), dense granulation tissue (star), degenerated cardiomyocytes (arrow), and interstitial edema (arrow head) are observed. CAR+ISO group c), a marked reduction in histopathological changes is noted. H&E x20.

H&E: Hematoxylin and eosin, ISO: Isoproterenol, CAR: Carvacrol

Table 8. Myocardial histopathology assessment.

Experimental groups	Myocyte degeneration score	Interstitial edema score	Granulation tissue formation
Control	0.0 (0.0-2.0)	0.0 (0.0-0.0)	0.0 (0.0-0.0)
ISO	2.0 (0.0-3.0)a	2.0 (0.0-3.0)a	1.0 (0.0-3.0)a
CAR+ISO	1.0 (0.0-2.0)b	1.0 (0.0-3.0)b	0.0 (0.0-2.0)b

Values are presented as median (minimum-maximum).

^a: Indicates a statistically significant elevation versus the control (p<0.0001).

^b: Indicates a statistically significant reduction versus the ISO group (p<0.0001).

CAR: Carvacrol, ISO: Isoproterenol

DISCUSSION

CAR, a phenolic monoterpenoid, has attracted attention because of its protective role in several models of cardiac injury, including myocardial I/reperfusion (R) and sepsis-related myocardial dysfunction. Findings from experimental studies show that treatment with CAR is associated with smaller infarct areas and improved cardiac function. These improvements accompany reductions in OS, inflammatory activity, and programmed cell death. Rather than acting through a single mechanism, CAR appears to influence multiple signaling processes that regulate cardiomyocyte survival. Evidence suggests that it interferes with mitogen-activated protein kinase (MAPK)-associated signaling and regulated kinase (ERK) activation, while simultaneously promoting Akt-mediated stimulation of endothelial nitric oxide synthase (eNOS), thereby supporting nitric oxide production and vascular homeostasis. Moreover, CAR dampens inflammatory signaling by limiting activation of the Toll-like receptor 4 (TLR4)-myeloid differentiation primary response 88 (MyD88) axis and the subsequent transcriptional activity of NF-κB. Through the combined regulation of oxidative and inflammatory pathways, CAR contributes to preservation of myocardial structure and function^{10, 27, 28, 29, 30}. These molecular actions are particularly relevant to ISO-induced cardiotoxicity, characterized by pronounced OS and inflammation. Consistent with this interpretation, previous investigations have demonstrated cardioprotective effects of CAR in both I/R injury and diabetic cardiomyopathy models^{27, 28, 29}.

This study provides comprehensive evidence that CAR protects against ISO-induced myocardial injury through its antioxidant, cardioprotective, and histology-preserving effects. ISO-induced myocardial injury is a well-established experimental model characterized by OS, inflammation, electrical instability, and histological degeneration of cardiomyocytes⁴. Our findings support and expand upon these pathological hallmarks, showing that CAR attenuates the biochemical, functional, and structural damage induced by ISO.

Hemodynamic and ECG data revealed that ISO led to significant reductions in DAP and MAP, as well as prolonged QTc intervals, which are indicative of impaired ventricular repolarization and heightened arrhythmogenic risk. These findings are consistent with previous observations. ISO, a non-selective β-adrenergic agonist, consistently causes significant reductions in DAP and MAP across animal and human studies. This hypotensive effect is attributed to its potent vasodilatory action and increased cardiac output, which lower peripheral vascular resistance and arterial pressures. In both healthy and diseased models, ISO administration leads to marked decreases in DAP and MAP, often accompanied by tachycardia and increased myocardial oxygen demand^{31, 32}. ISO is well-documented to prolong the QT interval, reflecting impaired ventricular repolarization and heightened risk for arrhythmias. In animal models, ISO-induced myocardial injury is associated with significant QT prolongation and increased incidence of early afterdepolarizations, which are key triggers for ventricular arrhythmias^{33, 34, 35}. In patients with congenital long QT syndrome, ISO further prolongs the QT interval and increases the risk of arrhythmogenic events such as torsade de pointes^{34, 36}. Mechanistically, ISO enhances calcium influx and disrupts potassium currents, contributing to repolarization abnormalities and arrhythmogenesis^{33, 35, 37}.

Co-administration of CAR mitigated these disturbances, indicating partial restoration of cardiac function and electrical stability. CAR has been shown to modulate cardiac electrical activity and improve cardiac function in multiple preclinical models. At higher concentrations, CAR alters action potential parameters, including reducing upstroke velocity and shortening action potential duration, and slows conduction through the atrioventricular node, likely via inhibition of sodium and calcium channels. These effects are reversible and do not significantly impact resting membrane potential, suggesting a modulatory rather than a toxic effect on cardiac electrophysiology^{38, 39}. CAR also reduces ventricular cell contractility and induces a negative chronotropic effect, potentially through modulation of transient receptor potential cation channel subfamily M member 4 (TRPM4) and TRPM7 channels⁴⁰.

Importantly, ECG abnormalities, such as T-wave inversion, biphasic T-waves, and bundle branch blocks, were observed predominantly in the ISO group, whereas the CAR+ISO group showed fewer such abnormalities. These results align with Xu et al.³⁰, who reported that CAR alleviates lipopolysaccharide (LPS)-induced myocardial dysfunction by inhibiting the TLR4/MyD88/NF-κB pathway, thereby modulating arrhythmogenic signaling. CAR achieves this by downregulating the TLR4/MyD88/NF-κB signaling pathway, which is central to the inflammatory response triggered by LPS. This leads to reduced expression of pro-inflammatory cytokines, decreased OS, and reduced cardiomyocyte apoptosis. CAR also suppresses nucleotide-binding domain, leucine-rich-containing family, NOD-like receptor pyrin domain-containing-3 (NLRP3) inflammasome signaling, resulting in reduced inflammatory activity and pyroptosis in cardiac cells³⁰.

CAR consistently demonstrates antioxidant and cardioprotective effects in models of myocardial injury and of OS. CAR administration significantly lowers MDA levels, a key marker of lipid peroxidation and OS, in myocardial tissue following injury or stressors such as ISO or ketamine. CAR increases the activity of endogenous antioxidant enzymes, including SOD, CAT, and GSH peroxidase, and elevates GSH levels, restoring redox balance. The cardioprotective and antioxidant effects of CAR are linked to the activation of MAPK/ERK and Akt/eNOS signaling pathways, as well as the Nrf2 transcription factor/HO-1 axis, which are central to cellular antioxidant responses^{27, 28, 41, 42}. Our biochemical analysis demonstrated a significant decline in myocardial MDA levels following ISO treatment, a finding that appears paradoxical. However, similar observations have been reported by Küçükler et al.¹², who suggested that excessive ROS production may lead to the consumption of measurable lipid peroxidation substrates over time. CAR restored MDA levels toward normal, potentially reflecting the normalization of oxidative status via scavenging activity or activation of the Nrf2/HO-1 axis¹³. Consistent with our findings, Khatua et al.⁴³ also reported decreased cardiac MDA levels during ISO-induced OS, despite observing reduced cardiac GSH levels and lower activities of GSH peroxidase and CAT. They proposed that this decline in MDA could be due to heightened activity of the antioxidant system. Similarly, our previous study showed that cardiac MDA levels were lower in the ISO group than in the control group. These findings suggest that MDA may decrease due to the overactivation of the antioxidant system during the early stage of ISO treatment⁴⁴. Also, Maleki Dizaji et al.⁴⁵ reported that ISO slightly reduces MDA levels in the heart, even though histopathological evidence shows cardiac damage, including cardiomyocyte necrosis and disintegration. Alternatively, MDA may not be the only product of lipid peroxidation; other compounds, such as propanal, hexanal, and 4-hydroxynonenal, are also generated. Analyzing these additional lipid peroxidation products could provide clearer insights into our observations⁴⁶. Csonka et al.⁴⁷ reported elevated CAT activity in spontaneously hypertensive rat hearts exposed to OS induced by H₂0₂ infusion. Similarly, we determined that CAT activity tended to decrease in the CAR+ISO group, while increasing in the ISO group. This suggests that the antioxidant defense system is activated in response to oxidative damage caused by ISO. Also, SOD activity and GSH levels tended to increase in the CAR+ISO group. This aligns with results from Yu et al.⁴¹, who demonstrated increased GSH level and SOD activity in CAR-treated rats in an acute MI model. These findings suggest that CAR may selectively boost endogenous antioxidant capacity, depending on the extent and timing of the oxidative insult.

Elevated troponin levels are a hallmark of cardiomyocyte necrosis and myocardial injury. Recent research demonstrates that CAR co-treatment markedly reduces troponin levels in animal models of ISO-induced MI, indicating a strong cardioprotective effect. Troponin levels, which are sensitive markers of cardiomyocyte necrosis, were markedly elevated in the ISO group but were significantly reduced with CAR co-treatment. This confirms the membrane-stabilizing and anti-necrotic effects of CAR, consistent with prior work by Koçak et al.⁹, who showed that CAR preserves myocardial integrity in ISO-induced infarction models. In a controlled rat study, ISO administration significantly increased troponin T levels, reflecting cardiac injury. CAR co-treatment (50 mg/kg for six weeks) led to a significant reduction in troponin T compared to the ISO-only group, suggesting mitigation of cardiomyocyte necrosis⁹.

Histologically, ISO exposure caused widespread cardiomyocyte degeneration, interstitial edema, and the formation of granulation tissue—typical features of acute myocardial injury. ISO administration in animal models reliably produces myocardial injury that closely mimics human acute MI. Histological examination consistently reveals, widespread cardiomyocyte degeneration and necrosis, interstitial edema, inflammatory cell infiltration, granulation tissue formation, fibrosis and myocyte disarray in chronic or severe cases^{48, 49, 50, 51, 52, 53}. These changes were substantially attenuated in the CAR+ISO group, as evidenced by reduced damage scores. These findings echo those of Bağcı et al.⁵⁴, who demonstrated histological preservation in ISO-exposed rats treated with CAR in brain and heart tissues. Mechanistically, the cardioprotective effects of CAR are likely mediated via multiple complementary pathways. As shown by Yan et al.¹⁰ and Hou et al.²⁹, CAR activates ERK1/2 and Akt/eNOS pathways, suppresses the NLRP3 inflammasome, and regulates oxidative-inflammatory signaling cascades, including Nrf2, NF-κB, and peroxisome proliferator-activated receptor gamma-dependent autophagic responses^{10, 29}. Multiple studies show that CAR administration reduces cardiomyocyte degeneration and interstitial edema in models of myocardial I/R, drug-induced cardiotoxicity, and metabolic stress. Histopathological analyses reveal that CAR-treated groups have less myocyte degeneration, reduced inflammatory infiltration, and milder edema compared to untreated controls^{29, 30, 42, 55, 56, 57}. Our findings are consistent with these mechanisms and suggest that CAR’s pharmacological effects extend beyond simple radical scavenging.

Moreover, the observed reduction in ISO-induced hypertrophy, as reflected by heart weight and heart/body weight ratio, with CAR treatment may be attributed to its modulation of anti-hypertrophic signaling pathways. The reduction in heart weight and heart-to-body weight ratio observed with CAR treatment in hypertrophy models is supported by research demonstrating that CAR exerts anti-hypertrophic effects by modulating key signaling pathways and OS. CAR treatment significantly reduces heart weight and the heart-to-body weight ratio in animal models of cardiac hypertrophy, indicating a direct anti-hypertrophic effect. CAR lowers the expression of atrial natriuretic peptide mRNA, a marker of cardiac hypertrophy, in both in vivo and in vitro models. CAR increases antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl radical scavenging) and decreases MDA levels, suggesting that its anti-hypertrophic effects are partly due to reduced oxidative damage. CAR decreases the number of apoptotic cells and may reduce fibrosis, further protecting cardiac tissue from hypertrophic remodeling⁵⁸.

While both the current study and the previously published work by Bağcı et al.⁵⁴ evaluated the protective effects of CAR against ISO-induced myocardial injury, several key methodological and analytical differences distinguish this study⁴⁵. Notably, the present investigation includes hemodynamic measurements (DAP, MAP, QTc interval) that were not assessed in the previous study, allowing for a more comprehensive functional evaluation of cardiac performance. Additionally, the electrocardiographic evaluation in this study provides new insights into the anti-arrhythmic potential of CAR, which was not addressed in the earlier work. Another important distinction is the use of serum troponin levels as a sensitive biomarker of myocardial injury. While the prior study focused on cardiac injury markers such as creatine kinase- muscle-brain, the current study incorporates troponin as a clinically relevant and more specific indicator of cardiomyocyte damage. Furthermore, OS was assessed using a broader panel of biochemical markers, including GSH, SOD, CAT, and MDA, accompanied by a more detailed statistical interpretation. Finally, the histopathological scoring system and the graphical representation of the results were expanded, offering clearer visualization and quantification of myocardial protection. These methodological enhancements contribute to a more robust and translationally relevant dataset, thus reinforcing and extending the evidence for the cardioprotective role of CAR in acute ISO-induced cardiac injury.

Although the cardioprotective properties of CAR against ISO-induced myocardial injury have been previously explored, the present study differs substantially from earlier work in both scope and methodology⁹. In contrast to the recent study by Koçak et al.⁹, which primarily focused on OS markers and pro-inflammatory cytokines in heart and kidney tissues, the current investigation emphasizes hemodynamic parameters, electrocardiographic findings, and serum troponin levels-markers more directly associated with clinical cardiac function and damage. Additionally, this study uniquely incorporates a detailed ECG analysis, including QTc interval assessment and classification of specific conduction abnormalities (e.g., T-wave inversion, biphasic T-waves, and bundle branch block), providing novel insights into the potential anti-arrhythmic effects of CAR. Such analyses were not conducted in the previous report. Another key difference lies in the histopathological evaluation: the current study implements a refined scoring system and provides more comprehensive documentation of myocardial degeneration, edema, and granulation tissue formation. Furthermore, while both studies assessed OS, the current work introduces a broader panel of antioxidant defense markers (e.g., GSH, SOD, CAT) and interprets these findings with respect to functional cardiac outcomes, offering a more integrated view of redox biology and cardiac physiology. Together, these distinctions highlight the originality of the present study and expand the mechanistic understanding of CAR’s cardioprotective role by combining biochemical, electrophysiological, and histological perspectives in a unified experimental model.

Compared to the recent investigation by Küçükler et al.¹², which primarily assessed the biochemical and histopathological effects of CAR in ISO-induced myocardial injury, the present study provides a more comprehensive functional and electrophysiological evaluation. Notably, we included hemodynamic measurements (DAP, MAP) and electrocardiographic parameters (QTc interval, T-wave abnormalities, conduction disturbances), thereby offering a more translational perspective on cardiac performance and arrhythmogenic risk. These functional endpoints were not examined in the aforementioned study. While Küçükler et al.¹² focused on OS markers such as MDA and antioxidant enzymes, our study expands this biochemical profile by incorporating serum troponin levels, thus enhancing the clinical relevance of the findings. Furthermore, the current work uses quantitative histological scoring to assess tissue injury, providing a more detailed and standardized evaluation of structural damage. These differences underscore the novelty of our approach, which integrates biochemical, functional, and histological parameters to better elucidate the cardioprotective mechanisms of CAR in the context of ISO-induced myocardial injury. By combining these multidimensional assessments, our findings offer a broader understanding of CAR’s therapeutic potential with greater translational applicability.

Taken together, our data reinforce the concept that CAR exerts a broad spectrum of protective actions in the setting of acute cardiac stress, including suppression of oxidative injury, maintenance of membrane integrity, stabilization of hemodynamic function, and attenuation of structural damage. Given its multi-targeted effects and favorable safety profile⁶, CAR represents a promising candidate for adjunctive therapy in ischemic heart disease, especially in conditions driven by OS and inflammation.

Study Limitations

This study has some limitations that should be acknowledged. First, the duration of the study was limited to acute exposure; thus, long-term outcomes of CAR treatment in chronic cardiac injury models remain unexplored. Second, although several OS markers and histological evaluations were performed, the study did not include molecular-level assessments, such as analyses of gene or protein expression (e.g., Nrf2, NF-κB, or apoptotic markers), which could provide deeper mechanistic insights. Third, not using a rat-specific troponin I ELISA kit is a limitation because it could have improved the reliability of the results. Fourth, invasive cardiac measurements were performed under anesthesia because of methodological and ethical constraints. Employing noninvasive methods can eliminate the potential effects of anesthesia on cardiac physiology. Fifth, the lack of pre- and post-intervention measurements prevents within-subject longitudinal comparisons and limits the assessment of individual temporal responses. Finally, the findings are based on a rat model and may not be directly generalizable to human physiology without further translational studies.

CONCLUSIONS

In conclusion, the present study demonstrates that CAR provides significant protection against ISO-induced myocardial injury by improving hemodynamic function, reducing electrocardiographic abnormalities, lowering serum troponin levels, and attenuating histopathological damage. These cardioprotective effects are likely mediated by antioxidant mechanisms and membrane stabilization. The findings support the potential use of CAR as an adjunctive therapeutic agent in the management of acute cardiac injury associated with OS. Further investigations involving chronic models, dose-response analyses, and molecular pathway evaluations are warranted to confirm and expand upon these results.

Ethics

Ethics Committee Approval: All procedures were approved by the Inonu University Institutional Animal Care and Use Committee (IACUC; approval no. 2021/22-6, date: 10.11.2021).

Informed Consent: This is a study conducted on animals.