Protective Effect of Geraniol on Oxidative, Inflammatory and Apoptotic Alterations in Isoproterenol-Induced Cardiotoxicity: Role of the Keap1/Nrf2/HO-1 and PI3K/Akt/mTOR Pathways

Background: Myocardial infarction (MI) is still a major contributor to mortality worldwide, and therefore, searching for new drugs is an urgent priority. Natural products are a renewable source for medicinally and pharmacologically active molecules. The objective of this study was to explore the potential of geraniol, a monoterpene alcohol, to protect against MI. Methods: Five groups of Wister rats were used: a control group; a group treated only with geraniol; a group treated only with isoproterenol, to induce MI; and two groups pretreated with geraniol (100 or 200 mg/kg, respectively) for 14 days and challenged with isoproterenol on the 13th and 14th days. Several parameters were measured including electrocardiogram (ECG), cardiac markers, the expression of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and other downstream antioxidant enzymes, as well as the expression of phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and other downstream apoptotic and inflammatory mediators. Results: Geraniol treatment reduced the size of the infarct region, attenuated the levels of cardiac indicators, and diminished myocardial necrosis and immune cell infiltration. Geraniol treatment also activated the Keap1/Nrf2/heme oxygenase-1 (HO-1) pathway, increased antioxidant enzyme activities, modulated the PI3K/Akt/mTOR pathway, and ameliorated myocardial autophagy, inflammation, and apoptosis. Conclusion: Geraniol may possess a protective effect against MI through moderating MI-induced myocardial oxidative stress (glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), and Keap1/Nrf2 pathway), inflammation (IL-1β, IL-6, TNF-α, and Nuclear factor-κB (NF-κB)), apoptosis (caspase-3, caspase-9, Bcl2, and Bax), and autophagy (PI3K/Akt/mTOR pathway).


Introduction
Cardiovascular diseases (CVDs) are the main cause of mortality globally, contributing to nearly 33% of the total worldwide deaths, with myocardial infarction (MI, heart attack) as a major attributor [1,2].
The tissue damage associated with myocardial infraction has been ascribed to reactive oxygen species (ROS) and numerous related pathways such as tumor protein p53 (p53), AMP-activated protein

Infarct Size Determination
The dissected hearts were cut into four to five transverse sections and incubated in 10% triphenyl tetrazolium chloride (TTC, solubilized in phosphate buffer with pH 7.4, for 30 min in room temperature in the dark). Transverse heart sections were fixed with 10% formalin [5] and then used to calculate the infract area using Image J ® (National Institutes of Health, University of Wisconsin, Madison, WI, USA).

Estimation of Antioxidant Markers, ATP, and Ca 2+ in Heart Mitochondrial Fraction
Mitochondria were isolated from the experimental animals' heart tissues as previously described [23]. In brief, heart tissues were homogenized in ice-cold 50 mM Tris-Hcl containing 0.25 M sucrose (pH 7.4), and then centrifuged for 20 min at 700× g. The supernatant was centrifuged again for 15 min at 9000× g, and then the resulting pellets were washed and then suspended in 10 mM of homogenization buffer and stored at −20 • C.
Antioxidant markers (SOD, CAT, GPx, and GST) and the levels of GSH, mitochondrial ATP, and Ca 2+ were quantified in the previously prepared mitochondria using corresponding assay kits according to the manufactures' protocols.

Gene Expression Analysis
Real-time PCR was performed as previously described [22]. Quantification analyses were performed in an Opticon-2 Real-time PCR reactor (MJ Research, Reno, NV, USA).
Step PE Applied Biosystems (Perkin Elmer, Waltham, MA, USA) software was used to analyze real time-PCR results. The expression of each target gene was measured and adjusted to the expression of β-actin as the reference gene. The primer sequences used in this study are listed in Table 1.

Assessment of Inflammatory and Apoptotic Markers
The extracted heart tissues were homogenized in 10% phosphate buffer to be used for the measurement of the apoptotic markers: caspase-3 and caspase-9 and inflammatory mediators: IL-1β, IL-6, TNF-α and NF-κB. ELISA kits were consumed to measure these markers according to the manufacturers' protocols and using a microplate reader SpectraMax i3x (Molecular Devices).

Histopathological and Immunohistochemical Assays
Heart sections (5 µm-thick) were cut, deparaffinized, dehydrated, and stained with hematoxylin and eosin (H&E) for the evaluation of pathological variations in the heart tissues under light microscopy (Leica DM 300 LED binocular, New York, NY, USA).
Separate heart sections were used for immunohistochemical staining. 3% hydrogen peroxide (H 2 O 2 ) in methanol was used to block the endogenous peroxidase enzyme in the obtained sections at 21-25 • C for 30 min, followed by rinsing three times in phosphate-buffered saline (PBS). Afterward, the sections were incubated with NF-κB antibodies (1:100, Thermo Fisher Scientific, Oxford, UK), stored overnight at 4 • C in a humidified chamber, and then goat anti-rabbit-horseradish peroxidase (HRP)-conjugated IgG antibody (1:1000; cat. no. ab6721; Abcam) was added for 1 h at 37 • C. Finally, the sections were developed with 1% diaminobenzidine for 5 min, counterstained with 1% hematoxylin for 2 min at 21-25 • C, and mounted with neutral gum. Heart slices were imagined using a microscope fitted with a digital camera (Nikon Instruments Inc., Tokyo, Japan). NIS-Elements software was used for the quantitative analysis of NFκB. First, the area of the immunohistochemical reaction in the picture was selected. Then, the average optical density in the selected area of each picture was measured. Positive cells were counted under 400× magnification observing 10 consecutive non-overlapping fields per animal in a blinded manner.

Statistical Analysis
Data are presented as mean ± SD. For multiple comparisons, one-way ANOVA followed by Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used as the statistical significance level. The ISO group was compared with the control group, and significance was indicated by the symbol "€" in all the relevant figures and tables in the manuscript. The two ISO + geraniol groups were

Geraniol Effect on Infarct Area Size and Heart-to-Body Ratio
The control group and the geraniol group demonstrated minor infarct areas and normal heartto-body ratios. In contrast, isoproterenol (ISO)-treated animals disclosed significantly increased heart-to-body ratios together with the presence of infarct areas. Geraniol pretreatment (100 or 200 mg/kg) caused a significant reduction in the infarct areas and heart-to-body ratio compared to the ISO group. There was no significant difference between the two doses of geraniol in infarct area size and heart-to-body ratio as shown in Figure 3a,b, respectively. ", respectively, in all the relevant figures and tables in the manuscript. All statistical analyses were performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, USA) software, version 5.

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST segment, the P wave, the QT, P-R, and R-R intervals, and the QRS complex were electronically recorded. The control group and the geraniol group both displayed normal ECG traces, whereas the ISO-induced MI group showed numerous ECG alterations including a wider ST segment and QT interval, and a shorter P wave, QRS complex, and P-R and R-R intervals compared to controls. Geraniol pretreatment (100 or 200 mg/kg) reversed most of the ECG alterations, as detailed in Table 2. Data are presented as mean ± SD. For multiple comparis Tukey-Kramer as a post-hoc test was performed. p < 0.05 was use The ISO group was compared with the control group, and signi "€" in all the relevant figures and tables in the manuscript. T compared to each other and to the ISO group, and significance w "҂", respectively, in all the relevant figures and tables in the man performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, U

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST se R-R intervals, and the QRS complex were electronically recorded group both displayed normal ECG traces, whereas the ISO-in ECG alterations including a wider ST segment and QT interval, and P-R and R-R intervals compared to controls. Geraniol pretr most of the ECG alterations, as detailed in Table 2.

Geraniol Effect on Infarct Area Size and Heart-to-Body Ratio
The control group and the geraniol group demonstrated m to-body ratios. In contrast, isoproterenol (ISO)-treated anima heart-to-body ratios together with the presence of infarct areas mg/kg) caused a significant reduction in the infarct areas and ISO group. There was no significant difference between the two and heart-to-body ratio as shown in Figure 3a,b, respectively. 0.0810 ± 0.0066 Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive consecutive non-overlapping field

Statistical Analysis
Data are presented as mean Tukey-Kramer as a post-hoc test w The ISO group was compared with "€" in all the relevant figures and compared to each other and to the "҂", respectively, in all the relevan performed using Prism (GraphPad

Geraniol Reversed Electrocardiog
Electrocardiographic (ECG) co R-R intervals, and the QRS comple group both displayed normal ECG ECG alterations including a wider and P-R and R-R intervals compar most of the ECG alterations, as det

Geraniol Effect on Infarct Area Si
The control group and the ge to-body ratios. In contrast, isopr heart-to-body ratios together with mg/kg) caused a significant reduc ISO group. There was no significan and heart-to-body ratio as shown i Data are presented as mean ± SD. For multiple comparis Tukey-Kramer as a post-hoc test was performed. p < 0.05 was use The ISO group was compared with the control group, and signi "€" in all the relevant figures and tables in the manuscript. T compared to each other and to the ISO group, and significance w "҂", respectively, in all the relevant figures and tables in the man performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, U

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST se R-R intervals, and the QRS complex were electronically recorded group both displayed normal ECG traces, whereas the ISO-in ECG alterations including a wider ST segment and QT interval, and P-R and R-R intervals compared to controls. Geraniol pretr most of the ECG alterations, as detailed in Table 2.

Geraniol Effect on Infarct Area Size and Heart-to-Body Ratio
The control group and the geraniol group demonstrated m to-body ratios. In contrast, isoproterenol (ISO)-treated anima heart-to-body ratios together with the presence of infarct areas mg/kg) caused a significant reduction in the infarct areas and ISO group. There was no significant difference between the two and heart-to-body ratio as shown in Figure 3a,b, respectively. 0.0643 ± 0.0028 Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive consecutive non-overlapping field

Statistical Analysis
Data are presented as mean Tukey-Kramer as a post-hoc test w The ISO group was compared with "€" in all the relevant figures and compared to each other and to the "҂", respectively, in all the relevan performed using Prism (GraphPad

Geraniol Reversed Electrocardiog
Electrocardiographic (ECG) co R-R intervals, and the QRS comple group both displayed normal ECG ECG alterations including a wider and P-R and R-R intervals compar most of the ECG alterations, as det

Geraniol Effect on Infarct Area Si
The control group and the ge to-body ratios. In contrast, isopr heart-to-body ratios together with mg/kg) caused a significant reduc ISO group. There was no significan and heart-to-body ratio as shown i * P Wave (sec) 0.0192 ± 0.0030 0.0190 ± 0.0019 0.0047 ± 0.0005 € 0.0133 ± 0.0008 Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive cells were counted under consecutive non-overlapping fields per animal in a blinded man

Statistical Analysis
Data are presented as mean ± SD. For multiple comparis Tukey-Kramer as a post-hoc test was performed. p < 0.05 was use The ISO group was compared with the control group, and signi "€" in all the relevant figures and tables in the manuscript. T compared to each other and to the ISO group, and significance w "҂", respectively, in all the relevant figures and tables in the man performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, U

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST se R-R intervals, and the QRS complex were electronically recorded group both displayed normal ECG traces, whereas the ISO-in ECG alterations including a wider ST segment and QT interval, and P-R and R-R intervals compared to controls. Geraniol pretr most of the ECG alterations, as detailed in Table 2.

Geraniol Effect on Infarct Area Size and Heart-to-Body Ratio
The control group and the geraniol group demonstrated m to-body ratios. In contrast, isoproterenol (ISO)-treated anima heart-to-body ratios together with the presence of infarct areas mg/kg) caused a significant reduction in the infarct areas and ISO group. There was no significant difference between the two and heart-to-body ratio as shown in Figure 3a,b, respectively. 0.0163 ± 0.0008 Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive consecutive non-overlapping field

Statistical Analysis
Data are presented as mean Tukey-Kramer as a post-hoc test w The ISO group was compared with "€" in all the relevant figures and compared to each other and to the "҂", respectively, in all the relevan performed using Prism (GraphPad

Geraniol Reversed Electrocardiog
Electrocardiographic (ECG) co R-R intervals, and the QRS comple group both displayed normal ECG ECG alterations including a wider and P-R and R-R intervals compar most of the ECG alterations, as det

Geraniol Effect on Infarct Area Si
The control group and the ge to-body ratios. In contrast, isopr heart-to-body ratios together with mg/kg) caused a significant reduc ISO group. There was no significan and heart-to-body ratio as shown i * QRS complex (sec) 0.0416 ± 0.0008 0.0417 ± 0.0005 0.0231 ± 0.0009 € 0.0305 ± 0.0009 Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive cells were counted under consecutive non-overlapping fields per animal in a blinded man

Statistical Analysis
Data are presented as mean ± SD. For multiple comparis Tukey-Kramer as a post-hoc test was performed. p < 0.05 was use The ISO group was compared with the control group, and signi "€" in all the relevant figures and tables in the manuscript. T compared to each other and to the ISO group, and significance w "҂", respectively, in all the relevant figures and tables in the man performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, U

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST se R-R intervals, and the QRS complex were electronically recorded group both displayed normal ECG traces, whereas the ISO-in ECG alterations including a wider ST segment and QT interval, and P-R and R-R intervals compared to controls. Geraniol pretr most of the ECG alterations, as detailed in Table 2. 0.1453 ± 0.0008 € All values were stated as mean ± SD (n = 6). €, p < 0.05 compare compared to the ISO-induced MI group; *, p < 0.05 compared to th Comparisons done using one-way ANOVA followed by Tukey-K

Geraniol Effect on Infarct Area Size and Heart-to-Body Ratio
The control group and the geraniol group demonstrated m to-body ratios. In contrast, isoproterenol (ISO)-treated anima heart-to-body ratios together with the presence of infarct areas mg/kg) caused a significant reduction in the infarct areas and ISO group. There was no significant difference between the two and heart-to-body ratio as shown in Figure 3a,b, respectively. 0.0338 ± 0.0009 Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive consecutive non-overlapping field

Statistical Analysis
Data are presented as mean Tukey-Kramer as a post-hoc test w The ISO group was compared with "€" in all the relevant figures and compared to each other and to the "҂", respectively, in all the relevan performed using Prism (GraphPad

Geraniol Reversed Electrocardiog
Electrocardiographic (ECG) co R-R intervals, and the QRS comple group both displayed normal ECG ECG alterations including a wider and P-R and R-R intervals compar most of the ECG alterations, as det

Geraniol Effect on Infarct Area Si
The control group and the ge to-body ratios. In contrast, isopr heart-to-body ratios together with mg/kg) caused a significant reduc ISO group. There was no significan and heart-to-body ratio as shown i Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive cells were counted under consecutive non-overlapping fields per animal in a blinded man

Statistical Analysis
Data are presented as mean ± SD. For multiple comparis Tukey-Kramer as a post-hoc test was performed. p < 0.05 was use The ISO group was compared with the control group, and signi "€" in all the relevant figures and tables in the manuscript. T compared to each other and to the ISO group, and significance w "҂", respectively, in all the relevant figures and tables in the man performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, U

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST se R-R intervals, and the QRS complex were electronically recorded group both displayed normal ECG traces, whereas the ISO-in ECG alterations including a wider ST segment and QT interval, and P-R and R-R intervals compared to controls. Geraniol pretr most of the ECG alterations, as detailed in Table 2. 0.1453 ± 0.0008 € All values were stated as mean ± SD (n = 6). €, p < 0.05 compare compared to the ISO-induced MI group; *, p < 0.05 compared to th Comparisons done using one-way ANOVA followed by Tukey-K

Geraniol Effect on Infarct Area Size and Heart-to-Body Ratio
The control group and the geraniol group demonstrated m to-body ratios. In contrast, isoproterenol (ISO)-treated anima heart-to-body ratios together with the presence of infarct areas mg/kg) caused a significant reduction in the infarct areas and ISO group. There was no significant difference between the two and heart-to-body ratio as shown in Figure 3a,b, respectively. 0.2117 ± 0.0194 Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive consecutive non-overlapping field

Statistical Analysis
Data are presented as mean Tukey-Kramer as a post-hoc test w The ISO group was compared with "€" in all the relevant figures and compared to each other and to the "҂", respectively, in all the relevan performed using Prism (GraphPad

Geraniol Reversed Electrocardiog
Electrocardiographic (ECG) co R-R intervals, and the QRS comple group both displayed normal ECG ECG alterations including a wider and P-R and R-R intervals compar most of the ECG alterations, as det

Geraniol Effect on Infarct Area Si
The control group and the ge to-body ratios. In contrast, isopr heart-to-body ratios together with mg/kg) caused a significant reduc ISO group. There was no significan and heart-to-body ratio as shown i Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive cells were counted under consecutive non-overlapping fields per animal in a blinded man

Statistical Analysis
Data are presented as mean ± SD. For multiple comparis Tukey-Kramer as a post-hoc test was performed. p < 0.05 was use The ISO group was compared with the control group, and signi "€" in all the relevant figures and tables in the manuscript. T compared to each other and to the ISO group, and significance w "҂", respectively, in all the relevant figures and tables in the man performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, U

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST se R-R intervals, and the QRS complex were electronically recorded group both displayed normal ECG traces, whereas the ISO-in ECG alterations including a wider ST segment and QT interval, and P-R and R-R intervals compared to controls. Geraniol pretr most of the ECG alterations, as detailed in Table 2.

Geraniol Effect on Infarct Area Size and Heart-to-Body Ratio
The control group and the geraniol group demonstrated m to-body ratios. In contrast, isoproterenol (ISO)-treated anima heart-to-body ratios together with the presence of infarct areas mg/kg) caused a significant reduction in the infarct areas and ISO group. There was no significant difference between the two and heart-to-body ratio as shown in Figure 3a,b, respectively. 0.1983 ± 0.0023 Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive consecutive non-overlapping field

Statistical Analysis
Data are presented as mean Tukey-Kramer as a post-hoc test w The ISO group was compared with "€" in all the relevant figures and compared to each other and to the "҂", respectively, in all the relevan performed using Prism (GraphPad

Geraniol Reversed Electrocardiog
Electrocardiographic (ECG) co R-R intervals, and the QRS comple group both displayed normal ECG ECG alterations including a wider and P-R and R-R intervals compar most of the ECG alterations, as det

Geraniol Effect on Infarct Area Si
The control group and the ge to-body ratios. In contrast, isopr heart-to-body ratios together with mg/kg) caused a significant reduc ISO group. There was no significan and heart-to-body ratio as shown i * All values were stated as mean ± SD (n = 6). €, p < 0.05 compared to the normal group; Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive cells wer consecutive non-overlapping fields per anim

Statistical Analysis
Data are presented as mean ± SD. For Tukey-Kramer as a post-hoc test was perform The ISO group was compared with the contr "€" in all the relevant figures and tables in compared to each other and to the ISO group "҂", respectively, in all the relevant figures an performed using Prism (GraphPad Sotfware

Geraniol Reversed Electrocardiographic Alter
Electrocardiographic (ECG) constituents R-R intervals, and the QRS complex were elec group both displayed normal ECG traces, w ECG alterations including a wider ST segmen and P-R and R-R intervals compared to contr most of the ECG alterations, as detailed in Ta All values were stated as mean ± SD (n = 6 compared to the ISO-induced MI group; *, p < Comparisons done using one-way ANOVA , p < 0.05 compared to the ISO-induced MI group; *, p < 0.05 compared to the ISO + geraniol (100 mg/kg) group. Comparisons done using one-way ANOVA followed by Tukey-Kramer post-hoc test.

Geraniol Effect on Infarct Area Size and Heart-to-Body Ratio
The control group and the geraniol group demonstrated minor infarct areas and normal heart-to-body ratios. In contrast, isoproterenol (ISO)-treated animals disclosed significantly increased heart-to-body ratios together with the presence of infarct areas. Geraniol pretreatment (100 or 200 mg/kg) caused a significant reduction in the infarct areas and heart-to-body ratio compared to the ISO group. There was no significant difference between the two doses of geraniol in infarct area size and heart-to-body ratio as shown in Figure 3a,b, respectively.

Geraniol Effect on Cardiac Enzymes
ISO control animals showed a substantial intensification in numerous cardiac enzymes including Creatine Phosphokinase (CPK), Creatine Kinase Myocardial Bound (CK-MB), Cardiac Tropinine T (cTnT), and Cardiac Tropinine I (cTnI) compared to the control groups, whereas pretreatment with geraniol in both doses-100 and 200 mg/kg-considerably declined these cardiac enzymes, reaching a percentage reduction of 30.2% and 42% for CPK, 32.2% and 41.8% for CK-MB, 21.31% and 51% for cTnT, and 29.4% and 45% for cTnI, as illustrated in Figure 3c-f, respectively.

Geraniol Effect on Mitochondrial Antioxidant Activities
To appraise the role of geraniol on the mitochondrial antioxidant status, the levels of the non-enzymatic antioxidant GSH and the activities of antioxidant enzymes SOD, CAT, GPx, and GST were measured in the various treatment groups ( Table 3). The GSH level as well as the activities of mitochondrial antioxidant enzymes were significantly decreased in ISO-challenged animals when compared with controls. As shown in Table 3, geraniol administration (100 or 200 mg/day) increased the GSH levels and the antioxidant enzyme activities. Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive cells were counted under 400× magn consecutive non-overlapping fields per animal in a blinded manner.

Statistical Analysis
Data are presented as mean ± SD. For multiple comparisons, one-way Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used as the stati The ISO group was compared with the control group, and significance was in "€" in all the relevant figures and tables in the manuscript. The two ISO + compared to each other and to the ISO group, and significance was indicated "҂", respectively, in all the relevant figures and tables in the manuscript. All s performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, USA) software

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST segment, the P R-R intervals, and the QRS complex were electronically recorded. The control group both displayed normal ECG traces, whereas the ISO-induced MI gro ECG alterations including a wider ST segment and QT interval, and a shorter and P-R and R-R intervals compared to controls. Geraniol pretreatment (100 most of the ECG alterations, as detailed in Table 2. The control group and the geraniol group demonstrated minor infarct a to-body ratios. In contrast, isoproterenol (ISO)-treated animals disclosed , p < 0.05 compared to the ISO-induced MI group; *, p < 0.05 compared to the ISO + geraniol (100 mg/kg) group. Comparisons done using one-way ANOVA followed by the Tukey-Kramer post-hoc test.

Statistical Analysis
Data are presented as mean ± SD. For multiple comparison Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used The ISO group was compared with the control group, and signific "€" in all the relevant figures and tables in the manuscript. The compared to each other and to the ISO group, and significance wa "҂", respectively, in all the relevant figures and tables in the manu performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, US

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST seg R-R intervals, and the QRS complex were electronically recorded. T group both displayed normal ECG traces, whereas the ISO-indu ECG alterations including a wider ST segment and QT interval, an and P-R and R-R intervals compared to controls. Geraniol pretrea most of the ECG alterations, as detailed in Table 2.

Geraniol Effect on Infarct Area Size and Heart-to-Body Ratio
The control group and the geraniol group demonstrated min to-body ratios. In contrast, isoproterenol (ISO)-treated animals heart-to-body ratios together with the presence of infarct areas. mg/kg) caused a significant reduction in the infarct areas and he ISO group. There was no significant difference between the two d and heart-to-body ratio as shown in Figure 3a,b, respectively.
6.24 ± 0.26 Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive c consecutive non-overlapping fields p

Statistical Analysis
Data are presented as mean ± Tukey-Kramer as a post-hoc test was The ISO group was compared with t "€" in all the relevant figures and t compared to each other and to the IS "҂", respectively, in all the relevant f performed using Prism (GraphPad S

Geraniol Reversed Electrocardiogra
Electrocardiographic (ECG) con R-R intervals, and the QRS complex w group both displayed normal ECG ECG alterations including a wider ST and P-R and R-R intervals compared most of the ECG alterations, as detai

Geraniol Effect on Infarct Area Size
The control group and the gera to-body ratios. In contrast, isoprot heart-to-body ratios together with t mg/kg) caused a significant reductio ISO group. There was no significant and heart-to-body ratio as shown in

Statistical Analysis
Data are presented as mean ± SD. For multiple comparison Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used The ISO group was compared with the control group, and signifi "€" in all the relevant figures and tables in the manuscript. The compared to each other and to the ISO group, and significance wa "҂", respectively, in all the relevant figures and tables in the manu performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, US

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST seg R-R intervals, and the QRS complex were electronically recorded. group both displayed normal ECG traces, whereas the ISO-indu ECG alterations including a wider ST segment and QT interval, an and P-R and R-R intervals compared to controls. Geraniol pretrea most of the ECG alterations, as detailed in Table 2.

Geraniol Effect on Infarct Area Size and Heart-to-Body Ratio
The control group and the geraniol group demonstrated min to-body ratios. In contrast, isoproterenol (ISO)-treated animals heart-to-body ratios together with the presence of infarct areas. mg/kg) caused a significant reduction in the infarct areas and he 95.19 ± 6.10 Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive c consecutive non-overlapping fields

Statistical Analysis
Data are presented as mean ± Tukey-Kramer as a post-hoc test was The ISO group was compared with "€" in all the relevant figures and compared to each other and to the IS "҂", respectively, in all the relevant f performed using Prism (GraphPad S

Geraniol Reversed Electrocardiogra
Electrocardiographic (ECG) con R-R intervals, and the QRS complex group both displayed normal ECG ECG alterations including a wider S and P-R and R-R intervals compared most of the ECG alterations, as deta

Geraniol Effect on Infarct Area Siz
The control group and the gera to-body ratios. In contrast, isoprot heart-to-body ratios together with t mg/kg) caused a significant reducti

Statistical Analysis
Data are presented as mean ± SD. For multiple comparison Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used The ISO group was compared with the control group, and signific "€" in all the relevant figures and tables in the manuscript. The compared to each other and to the ISO group, and significance wa "҂", respectively, in all the relevant figures and tables in the manu performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, US

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST seg R-R intervals, and the QRS complex were electronically recorded. T group both displayed normal ECG traces, whereas the ISO-indu ECG alterations including a wider ST segment and QT interval, an and P-R and R-R intervals compared to controls. Geraniol pretrea most of the ECG alterations, as detailed in Table 2.

Geraniol Effect on Infarct Area Size and Heart-to-Body Ratio
The control group and the geraniol group demonstrated min to-body ratios. In contrast, isoproterenol (ISO)-treated animals 1.17 ± 0.03 Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive c consecutive non-overlapping fields

Statistical Analysis
Data are presented as mean ± Tukey-Kramer as a post-hoc test was The ISO group was compared with "€" in all the relevant figures and compared to each other and to the IS "҂", respectively, in all the relevant f performed using Prism (GraphPad S

Geraniol Reversed Electrocardiogra
Electrocardiographic (ECG) con R-R intervals, and the QRS complex group both displayed normal ECG ECG alterations including a wider S and P-R and R-R intervals compared most of the ECG alterations, as detai

Statistical Analysis
Data are presented as mean ± SD. For multiple comparison Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used The ISO group was compared with the control group, and signifi "€" in all the relevant figures and tables in the manuscript. The compared to each other and to the ISO group, and significance wa "҂", respectively, in all the relevant figures and tables in the manu performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, US

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST seg R-R intervals, and the QRS complex were electronically recorded. group both displayed normal ECG traces, whereas the ISO-indu ECG alterations including a wider ST segment and QT interval, an and P-R and R-R intervals compared to controls. Geraniol pretrea most of the ECG alterations, as detailed in Table 2.

Statistical Analysis
Data are presented as mean ± Tukey-Kramer as a post-hoc test was The ISO group was compared with "€" in all the relevant figures and compared to each other and to the IS "҂", respectively, in all the relevant f performed using Prism (GraphPad S

Geraniol Reversed Electrocardiogra
Electrocardiographic (ECG) con R-R intervals, and the QRS complex group both displayed normal ECG ECG alterations including a wider S and P-R and R-R intervals compared most of the ECG alterations, as deta

Statistical Analysis
Data are presented as mean ± SD. For multiple compariso Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used The ISO group was compared with the control group, and signif "€" in all the relevant figures and tables in the manuscript. Th compared to each other and to the ISO group, and significance w "҂", respectively, in all the relevant figures and tables in the manu performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, U

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST seg R-R intervals, and the QRS complex were electronically recorded. group both displayed normal ECG traces, whereas the ISO-ind ECG alterations including a wider ST segment and QT interval, a and P-R and R-R intervals compared to controls. Geraniol pretre most of the ECG alterations, as detailed in Table 2.

Statistical Analysis
Data are presented as mean ± Tukey-Kramer as a post-hoc test was The ISO group was compared with t "€" in all the relevant figures and t compared to each other and to the IS "҂", respectively, in all the relevant f performed using Prism (GraphPad S

Geraniol Reversed Electrocardiogra
Electrocardiographic (ECG) con R-R intervals, and the QRS complex group both displayed normal ECG ECG alterations including a wider ST and P-R and R-R intervals compared most of the ECG alterations, as detai GPx, glutathione peroxidase; GST: glutathione-S-transferase; All values were stated as mean ± SD (n = 6). €, p < 0.05 compared to the normal group; Antioxidants 2020, 9, x FOR PEER REVIEW 6 of 16 picture was measured. Positive cells were counted under 400× magnification observing 10 consecutive non-overlapping fields per animal in a blinded manner.

Statistical Analysis
Data are presented as mean ± SD. For multiple comparisons, one-way ANOVA followed by Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used as the statistical significance level. The ISO group was compared with the control group, and significance was indicated by the symbol "€" in all the relevant figures and tables in the manuscript. The two ISO + geraniol groups were compared to each other and to the ISO group, and significance was indicated by the symbols "*" and "҂", respectively, in all the relevant figures and tables in the manuscript. All statistical analyses were performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, USA) software, version 5.

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST segment, the P wave, the QT, P-R, and R-R intervals, and the QRS complex were electronically recorded. The control group and the geraniol group both displayed normal ECG traces, whereas the ISO-induced MI group showed numerous ECG alterations including a wider ST segment and QT interval, and a shorter P wave, QRS complex, and P-R and R-R intervals compared to controls. Geraniol pretreatment (100 or 200 mg/kg) reversed most of the ECG alterations, as detailed in Table 2.

Effects of Geraniol on the Keap1/Nrf2/HO-1 Pathway
To estimate geraniol's capability to activate Nrf2 signaling in myocardial tissues, both mRNA and protein expression levels of Keap1, Nrf2, and HO-1 were assessed; the results are shown in Figure 4. Challenge with ISO increased the mRNA and protein expression levels of Keap1, Nrf2, and HO-1. Geraniol pretreatment significantly increased the nuclear accumulation of Nrf2 and the mRNA and protein expression levels of HO-1, and it decreased both the mRNA and protein expression levels of Keap1.

Effects of Geraniol on the PI3k/Akt/mTOR Pathway
The mRNA and protein expression levels of PI3K, Akt, and mTOR were evaluated, and the results are shown in Figure 5. In the ISO-induced MI group, the protein expression levels of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt) and phosphorylated mTOR (pmTOR) were significantly decreased. On the other hand, geraniol pretreatment significantly increased the protein expression levels of p-PI3K, p-Akt, and pmTOR in a dose-dependent manner. These findings suggest that geraniol might exert its heart protective function via the activation of PI3k/Akt/mTOR signaling, thereby promoting myocardium autophagy in the ISO-induced MI model. Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positiv consecutive non-overlapping field

Statistical Analysis
Data are presented as mean Tukey-Kramer as a post-hoc test w The ISO group was compared wi "€" in all the relevant figures an compared to each other and to the "҂", respectively, in all the relevan performed using Prism (GraphPa

Geraniol Reversed Electrocardio
Electrocardiographic (ECG) c R-R intervals, and the QRS compl group both displayed normal EC ECG alterations including a wide and P-R and R-R intervals compa most of the ECG alterations, as de All values were stated as mean compared to the ISO-induced M Comparisons done using one-w , p < 0.05 compared to the ISO-induced MI group; *, p < 0.05 compared to the ISO + geraniol (100 mg/kg) group. Comparisons done using one-way ANOVA followed by Tukey-Kramer post-hoc test.
5 Figure 5. Geraniol (100 or 200 mg/kg) increased the gene and protein expression levels of the PI3k/Akt/mTOR pathway in myocardial infraction rat models. The mRNA expression levels of (a) PI3K, (b) Akt, and (c) mTOR, and the protein expression levels of (d) phosphorylated PI3K (pPI3k), (e) phosphorylated Akt (pAkt), and (f) phosphorylated mTOR (pmTOR) were increased in response to geraniol pretreatment. ISO: isoproterenol. All values were stated as mean ± SD (n = 6). €, p < 0.05 compared to the normal group; Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive cells were counted under 400× magnification consecutive non-overlapping fields per animal in a blinded manner.

Statistical Analysis
Data are presented as mean ± SD. For multiple comparisons, one-way ANOVA Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used as the statistical sign The ISO group was compared with the control group, and significance was indicated "€" in all the relevant figures and tables in the manuscript. The two ISO + geraniol compared to each other and to the ISO group, and significance was indicated by the sy "҂", respectively, in all the relevant figures and tables in the manuscript. All statistical performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, USA) software, version

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST segment, the P wave, the R-R intervals, and the QRS complex were electronically recorded. The control group an group both displayed normal ECG traces, whereas the ISO-induced MI group show ECG alterations including a wider ST segment and QT interval, and a shorter P wave, , p < 0.05 compared to the ISO-induced MI group; *, p < 0.05 compared to the ISO + geraniol (100 mg/kg) group. Comparisons done using one-way ANOVA followed by Tukey-Kramer post-hoc test.

Geraniol Pretreatement Inhibited Inflammatory Markers
The control group and the geraniol group showed no difference in the levels of inflammatory markers (IL-1β, IL-6, TNF-α, and NF-κB), whereas ISO-induced MI caused a significant elevation of these markers. Pretreatment with geraniol (100 or 200 mg/kg) ameliorated the MI-induced elevation in inflammatory mediators, with respective reductions of 32.57% and 53.64% for IL-1β, 29.04% and 47.73% for IL-6, 28.57% and 46.68% for TNF-α, and 24.67% and 45.74% for NF-κB ( Figure 6). Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive cells were counted under 400× magnification observi consecutive non-overlapping fields per animal in a blinded manner.

Statistical Analysis
Data are presented as mean ± SD. For multiple comparisons, one-way ANOVA follow Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used as the statistical significance The ISO group was compared with the control group, and significance was indicated by the s "€" in all the relevant figures and tables in the manuscript. The two ISO + geraniol groups compared to each other and to the ISO group, and significance was indicated by the symbols " "҂", respectively, in all the relevant figures and tables in the manuscript. All statistical analyse performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, USA) software, version 5.

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST segment, the P wave, the QT, P-R-R intervals, and the QRS complex were electronically recorded. The control group and the ge group both displayed normal ECG traces, whereas the ISO-induced MI group showed num ECG alterations including a wider ST segment and QT interval, and a shorter P wave, QRS com and P-R and R-R intervals compared to controls. Geraniol pretreatment (100 or 200 mg/kg) rev most of the ECG alterations, as detailed in Table 2. , p < 0.05 compared to the ISO-induced MI group; *, p < 0.05 compared to the ISO + geraniol (100 mg/kg) group. Comparisons done using one-way ANOVA followed by Tukey-Kramer post-hoc test.

Effects of Geraniol on the Apoptotic Status
The apoptotic markers Bax, Bcl2, caspase-3, and caspase-9 showed no differences between the control group and the geraniol group. In the ISO-treated group, the mRNA expression levels of Bax and the protein levels of cleaved caspase-3 and caspase-9 were significantly increased, whereas Bcl-2 mRNA expression levels were decreased, suggesting an apoptotic status within the myocardium of ISO-induced MI animals ( Figure 7). However, animals pretreated with geraniol showed ameliorated mRNA expression levels of Bax and protein levels of cleaved caspase-3 and caspase-9, and higher mRNA expression levels of Bcl-2, suggesting that geraniol might limit MI-induced myocardial apoptosis (Figure 7). and the protein levels of cleaved caspase-3 and caspase-9 were significantly increased, whereas Bcl-2 mRNA expression levels were decreased, suggesting an apoptotic status within the myocardium of ISO-induced MI animals ( Figure 7). However, animals pretreated with geraniol showed ameliorated mRNA expression levels of Bax and protein levels of cleaved caspase-3 and caspase-9, and higher mRNA expression levels of Bcl-2, suggesting that geraniol might limit MI-induced myocardial apoptosis (Figure 7). . All values were stated as mean ± SD (n = 6). €, p < 0.05 compared to the normal group; ҂, p < 0.05 compared to the ISO-induced MI group; *, p < 0.05 compared to the ISO + geraniol (100 mg/kg) group. Comparisons done using one-way ANOVA followed by Tukey-Kramer post-hoc test.

Histopathological and Immunohistochemical Assays
In the control and geraniol-treated groups, H&E staining of myocardial tissue sections revealed a regular and clear myocardial structure, with no edema or other signs of inflammation (Figure 8a). On the other hand, animals in the ISO-treated group showed moderate-to-severe myocardial necrosis, edema, and extensive infiltration by immune cells. Animals pretreated with geraniol Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive cells were counted under 400× magnification observi consecutive non-overlapping fields per animal in a blinded manner.

Statistical Analysis
Data are presented as mean ± SD. For multiple comparisons, one-way ANOVA follow Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used as the statistical significance The ISO group was compared with the control group, and significance was indicated by the s "€" in all the relevant figures and tables in the manuscript. The two ISO + geraniol groups compared to each other and to the ISO group, and significance was indicated by the symbols " "҂", respectively, in all the relevant figures and tables in the manuscript. All statistical analyse performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, USA) software, version 5.

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST segment, the P wave, the QT, P-R-R intervals, and the QRS complex were electronically recorded. The control group and the ge group both displayed normal ECG traces, whereas the ISO-induced MI group showed num ECG alterations including a wider ST segment and QT interval, and a shorter P wave, QRS com and P-R and R-R intervals compared to controls. Geraniol pretreatment (100 or 200 mg/kg) rev most of the ECG alterations, as detailed in Table 2. , p < 0.05 compared to the ISO-induced MI group; *, p < 0.05 compared to the ISO + geraniol (100 mg/kg) group. Comparisons done using one-way ANOVA followed by Tukey-Kramer post-hoc test.

Histopathological and Immunohistochemical Assays
In the control and geraniol-treated groups, H&E staining of myocardial tissue sections revealed a regular and clear myocardial structure, with no edema or other signs of inflammation (Figure 8a). On the other hand, animals in the ISO-treated group showed moderate-to-severe myocardial necrosis, edema, and extensive infiltration by immune cells. Animals pretreated with geraniol showed significantly diminished myocardial necrosis as well as reduced edema and immune cells infiltration compared to animals in the ISO-treated group.
Immunohistochemical staining of myocardium samples with an antibody recognizing NF-κB showed no substantial immunoreactivity in the control group and the geraniol group. NF-κB-immunoreactive cells were widely present in the group of ISO-induced MI, whereas animals in the groups pretreated with geraniol showed fewer immunoreactive cells (Figure 8b).

Effects of Geraniol on Heart Mitochondrial Ca 2+ and ATP Content
The effects of geraniol on Ca 2+ and ATP content were evaluated in cardiac cells' mitochondria. In the group of ISO-induced MI animals, Ca 2+ levels in cardiac cells' mitochondrial were significantly increased compared to the control group, whereas mitochondrial ATP levels were decreased. Pretreatment with geraniol ameliorated the Ca 2+ content and the mitochondrial ATP levels compared with the ISO-treated group (Figure 9).
showed significantly diminished myocardial necrosis as well as reduced edema and immune cells infiltration compared to animals in the ISO-treated group.
Immunohistochemical staining of myocardium samples with an antibody recognizing NF-κB showed no substantial immunoreactivity in the control group and the geraniol group. NF-κBimmunoreactive cells were widely present in the group of ISO-induced MI, whereas animals in the groups pretreated with geraniol showed fewer immunoreactive cells (Figure 8b). Figure 8. Histopathological investigation (a), immunohistochemical assay (b,c) immunohistochemical scoring of geraniol (100 or 200 mg/kg)-pretreated rats subjected to isoproterenol (ISO)-induced myocardial infraction (MI). Histopathological analysis was performed using hematoxylin and eosin (H&E) staining of the myocardial tissue sections; immunohistochemical staining of cardiac tissues was performed with antibody recognizing NF-κB. All values were stated as mean ± SD (n = 6). €, p < 0.05 compared to the normal group; ҂, p < 0.05 compared to the ISO-induced MI group; *, p < 0.05 compared to the ISO + geraniol (100 mg/kg) group. Comparisons done using one-way ANOVA followed by Tukey-Kramer post-hoc test.

Effects of Geraniol on Heart Mitochondrial Ca 2+ and ATP Content
The effects of geraniol on Ca 2+ and ATP content were evaluated in cardiac cells' mitochondria. In the group of ISO-induced MI animals, Ca 2+ levels in cardiac cells' mitochondrial were significantly increased compared to the control group, whereas mitochondrial ATP levels were decreased. Pretreatment with geraniol ameliorated the Ca 2+ content and the mitochondrial ATP levels compared with the ISO-treated group (Figure 9). Figure 8. Histopathological investigation (a), immunohistochemical assay (b,c) immunohistochemical scoring of geraniol (100 or 200 mg/kg)-pretreated rats subjected to isoproterenol (ISO)-induced myocardial infraction (MI). Histopathological analysis was performed using hematoxylin and eosin (H&E) staining of the myocardial tissue sections; immunohistochemical staining of cardiac tissues was performed with antibody recognizing NF-κB. All values were stated as mean ± SD (n = 6). €, p < 0.05 compared to the normal group; Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive cells were counted under 400× magnification consecutive non-overlapping fields per animal in a blinded manner.

Statistical Analysis
Data are presented as mean ± SD. For multiple comparisons, one-way ANOVA Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used as the statistical sign The ISO group was compared with the control group, and significance was indicated "€" in all the relevant figures and tables in the manuscript. The two ISO + geraniol compared to each other and to the ISO group, and significance was indicated by the sy "҂", respectively, in all the relevant figures and tables in the manuscript. All statistical performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, USA) software, version

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST segment, the P wave, the R-R intervals, and the QRS complex were electronically recorded. The control group an group both displayed normal ECG traces, whereas the ISO-induced MI group show ECG alterations including a wider ST segment and QT interval, and a shorter P wave, and P-R and R-R intervals compared to controls. Geraniol pretreatment (100 or 200 mg most of the ECG alterations, as detailed in Table 2.  Figure 9. Levels of mitochondrial Ca 2+ (a) and ATP (b) in isoproterenol (ISO)-induced myocardial infraction (MI) and effects of geraniol pretreatment. All values were stated as mean ± SD (n = 6). €, p < 0.05 compared to the normal group; ҂, p < 0.05 compared to the ISO-induced MI group; *, p < 0.05 compared to the ISO + geraniol (100 mg/kg) group. Comparisons done using one-way ANOVA followed by Tukey-Kramer post-hoc test.

Discussion
A deeper understanding of the pathological and biochemical developments in MI has inspired the search for new preventive or therapeutic drugs that could effectively control or cure the myocardial impairment. Essential oils are one of the precious sources for pharmacologically and medicinally active natural compounds. Some essential oils, such as those derived from Lavandula Angustifolia, have been used as treatment or protection against many cardiovascular diseases, including ischemic heart disease [24]; however, these oils suffer from qualitative and quantitative alterations in the main components due to environmental and climate factors. The isolation and investigation of the medicinal and pharmacological effects of essential oil components is more useful, Figure 9. Levels of mitochondrial Ca 2+ (a) and ATP (b) in isoproterenol (ISO)-induced myocardial infraction (MI) and effects of geraniol pretreatment. All values were stated as mean ± SD (n = 6). €, p < 0.05 compared to the normal group; Antioxidants 2020, 9, x FOR PEER REVIEW picture was measured. Positive cells were counted under 400× magn consecutive non-overlapping fields per animal in a blinded manner.

Statistical Analysis
Data are presented as mean ± SD. For multiple comparisons, one-way Tukey-Kramer as a post-hoc test was performed. p < 0.05 was used as the stati The ISO group was compared with the control group, and significance was in "€" in all the relevant figures and tables in the manuscript. The two ISO + compared to each other and to the ISO group, and significance was indicated "҂", respectively, in all the relevant figures and tables in the manuscript. All s performed using Prism (GraphPad Sotfware Inc., La Jolla, CA, USA) software

Geraniol Reversed Electrocardiographic Alterations
Electrocardiographic (ECG) constituents including the ST segment, the P R-R intervals, and the QRS complex were electronically recorded. The control group both displayed normal ECG traces, whereas the ISO-induced MI gro ECG alterations including a wider ST segment and QT interval, and a shorter and P-R and R-R intervals compared to controls. Geraniol pretreatment (100 most of the ECG alterations, as detailed in Table 2. , p < 0.05 compared to the ISO-induced MI group; *, p < 0.05 compared to the ISO + geraniol (100 mg/kg) group. Comparisons done using one-way ANOVA followed by Tukey-Kramer post-hoc test.

Discussion
A deeper understanding of the pathological and biochemical developments in MI has inspired the search for new preventive or therapeutic drugs that could effectively control or cure the myocardial impairment. Essential oils are one of the precious sources for pharmacologically and medicinally active natural compounds. Some essential oils, such as those derived from Lavandula Angustifolia, have been used as treatment or protection against many cardiovascular diseases, including ischemic heart disease [24]; however, these oils suffer from qualitative and quantitative alterations in the main components due to environmental and climate factors. The isolation and investigation of the medicinal and pharmacological effects of essential oil components is more useful, and it is current common practice. The present study aimed to characterize the anti-MI properties of geraniol, an essential oil component, and to shed light into its potential mechanisms of action, especially those related to the oxidative stress and apoptosis.

Cardioprotective Effects of Geraniol
The ISO-induced MI group showed numerous ECG alterations, presence of an infarct area, increase in the levels of cardiac enzymes, moderate-to-severe myocardial necrosis, edema, and extensive immune cells infiltration. It has been previously reported that ISO causes detrimental cardiac effects, including necrosis, apoptosis, mitochondrial modifications, oxidative injury, and inflammatory cell infiltration, comparable to what is observed in the ischemic human heart [8,23,25,26]. Compared to ISO-challenged animals, geraniol pretreatment led to a size reduction of the infarct region, attenuation of cardiac indicator enzymes, as well as diminished myocardial necrosis, edema, and immune cells infiltration. All these activities indicated the potential cardioprotective effect of geraniol, prompting investigations of the potential underlying mechanism(s) of action.
Geraniol pretreatment decreased the Ca 2+ content and increased the ATP content. Earlier reports on the cardiac actions of geraniol by El-Bassossy et al. [27] showed that geraniol could improve the weakened vascular reactivity in diabetes and metabolic syndrome through blocking both voltage-dependent and receptor-operated calcium channels. Furthermore, geraniol proved to exert negative inotropic and chronotropic actions via decreasing both L-type Ca 2+ and voltage-gated K + currents, acting as a promising anti-arrhythmic candidate [12].
Two main events, which are associated with the MI insult, could contribute to its pathogenesis and the functional deterioration of the cardiac tissue: the increase in ROS levels (oxidative stress) and the induction of apoptosis. Ischemic cells contribute to oxidative stress status through the generation of ROS; meanwhile, the affected cells induce apoptosis in themselves and nearby healthy cells. Therefore, in the current study, pathways related to both events were investigated.

Geraniol and Oxidative Stress: The Keap1/Nrf2 Pathway
The potential antioxidant properties of geraniol to counteract the toxic effects of MI-induced ROS were studied. Specifically, the antioxidant potential of geraniol was investigated through its effect on the Keap1/Nrf2/HO-1 as a redox signaling pathway. Activation of Nrf2 is known to defend against reactive oxidants, while decreased Nrf2 activity has the opposite action [5]. In the current study, challenge with ISO caused the depletion of GSH, reduction of mitochondrial antioxidant enzymes activities, and increase of Keap1 levels, and it decreased Nrf2 and HO-1 mRNA and protein expressions levels, which is indicative of oxidative stress. Former studies demonstrated that ISO administration reduces the activity of mitochondrial antioxidant enzymes leading to cardiac contractile dysfunction and cardiotoxicity, finally resulting in myocardial necrosis [23]. In contrast, geraniol reduced Keap1 expression and increased the nuclear accumulation of Nrf2, which is associated with higher expression levels of HO-1, GSH, and mitochondrial antioxidant enzymes. These results are in accordance with earlier reports suggesting geraniol as an anti-oxidative stress agent. A previous study proved that geraniol (5-200 µM) diminished the endogenous production of ROS in normoxic and hypoxic neonatal rat ventricular cardiomyocytes [1]. Another report proposed that geraniol reduced the levels of the oxidants malondialdehyde and 3-nitrotyrosine and decreased the expression levels of iNOS in animals suffering spinal cord injury [20]. The present study characterized geraniol as a cardioprotective agent in MI, which is possibly associated with the modulation of the Keap1/Nrf2 pathway, enhancing the cytochrome C and caspase-9, thereby improving the neurodegeneration state and its functional effects on movement [20].

Conclusions
The present study highlighted geraniol as a potential cardioprotective agent against ISO-induced MI. Geraniol protected the heart against the MI insult as reflected in the ECG, infarct size, cardiac Ca 2+ , ATP, and marker enzymes (CPK, CK-MB, cTnT, and cTnI). The protective effect of geraniol might be linked to its activity against oxidative stress via the Keap1/Nrf2 pathway modulation and the associated overexpression of the downstream antioxidant enzymes such as HO-1, SOD, CAT, GPx, and GST. Another mechanism contributing to geraniol's cardioprotective activity might be its regulatory effect of the PI3K/Akt/mTOR signaling cascade, which could control autophagy and apoptosis. Therefore, this study may contribute to the potential future utilization of geraniol as a cardioprotective agent against MI, expanding the medicinal value of this natural monoterpene alcohol.