Animals used

Male Sprague Dawley rats weighing 180-200 g (12-14 months old) were used in this study. The animals had unlimited access to water and a standard laboratory diet. They were housed in the central animal facility, which maintained a 12-hour light: 12- hour dark cycle. This experimental protocol was approved by the institutional animal ethics committee (IAEC approval number: IAEC/05/2025). The IAEC guidelines were followed during the trials.

Experimental design

The experimental design consisted of seven groups of eight rats each.

Group I: Animals served as naive controls and did not receive any drug administration.

Animals served as a negative control in Group II. For seven consecutive days, this group of animals received intraperitoneal (i.p.) treatment of ISO (5 mg/kg/day) to induce heart failure (HF). Approximately 0.05% ascorbic acid in 0.9% sodium chloride (NaCl) was used to prepare the ISO solution.

Groups III and IV: Animals served as test subjects for the drug treatments. These groups of animals were administered AST (25 and 50 mg/kg) orally for 21 consecutive days after the last dose of ISO.

Group V-VII: Animals were used as reference drug treatments. These groups of animals received oral administration of specific drugs: a direct renin inhibitor, aliskiren (30 mg/kg); an angiotensin-converting enzyme (ACE) inhibitor, ramipril (4 mg/kg); and an angiotensin II receptor antagonist, telmisartan (8 mg/kg), for 21 consecutive days following the last dose of ISO administration.

At 0, 7, 14, 21, and 28 days, HF-related changes in heart rate (HR), diastolic blood pressure (DAP), and systolic blood pressure (SAP) were recorded. Blood samples were collected on the twenty-eighth day for biochemical analysis of plasma renin activity (PRA), ACE activity, and serum creatine kinase isoenzyme-MB (CK-MB). Afterwards, all animals were weighed and euthanized with diethyl ether. To assess the heart-to-body weight ratio, the heart was removed and weighed. Immediately, the heart was perfused prior to tissue homogenization, and the cardiac chambers were opened to remove residual blood. Then, cardiac tissue was homogenized in ice-cold phosphate buffer (pH 7.4). Centrifugation at 769 x g was performed to obtain a clear supernatant, which was used to measure lactate dehydrogenase (LDH) activity and hydroxyproline content, both of which are tissue biomarkers. Additionally, ISO-induced histopathological changes were examined using eosinhematoxylin staining.

Assessment of Isoproterenol-induced functional changes in the heart

The ISO induced the HF with alteration of functional changes of the heart, such as blood pressure and heart rate, using the non-invasive tail-cuff method described by Wang et al. ²². Briefly, the animal was placed in appropriate holders based on its body size (CODA-HT8, CODA high-throughput noninvasive blood pressure system, Kent Scientific Corporation, Torrington, United States). The tail was positioned into the rear side of the tail port with the screw of the rear hatch without pinching it. The animals were given five minutes to acclimate to the holder. Frequent contact with the animals was avoided to reduce stress and irritation. Without applying any force, the tail occlusion cuff was placed at the base of the tail. The distance between the tail occlusion cuff and the volume pressure recording (VPR) sensor cuff was 2 mm. Body temperature was measured with an infrared thermometer, and it was maintained between 34 and 35°C using a rodent heating pad. Data were recorded as averages of multiple measurements using CODA Data Acquisition Software.

Assessment of Isoproterenol-induced changes in serum and plasma biomarkers

Serum biomarkers, including CK-MB, ACE activity, and PRA, were measured in rat samples. Serum CK-MB was assessed using the rat CK-MB ELISA kit (NBP2-75313, Novus Biologicals, Kuala Lumpur, Malaysia) according to the manufacturer’s protocol. Briefly, 100 μL of serum and 100 μL of biotinylated detection antibody working solution were combined in a well plate and incubated at 37 °C for 90 minutes; the plate was then washed with 350 μL of wash buffer. Subsequently, 100 μL of the horseradish peroxidase conjugate working solution was added, and the mixture was incubated at 37 °C for 30 minutes. Importantly, 90 μL of substrate reagent was added after the plate had been washed. After 15 minutes of incubation at 37 °C, 50 μL of stop solution was added to halt the reaction. A microplate reader (BioTek Microplate Instruments, Penang, Malaysia), operating at 450 nanometers, was used to measure the chromogen changes. The reference standard curve was prepared using standards from 31.25 to 2000 pg/mL for CK-MB activity.

Serum ACE levels were measured using the rat ACE ELISA kit (CSB-E04490r; Cusabio Technology LLC, Houston, United States) following the manufacturer’s instructions. Briefly, microplate wells were filled with 100 μL of serum and incubated at 37°C for two hours. Afterward, 100 μL of biotinylated detection antibody working solution was added, and the plate was incubated again at 37ºC for 60 minutes. The plate was washed with 200 μL of wash buffer solution. Next, 100 μL of horseradish peroxidase conjugate working solution was added, followed by incubation at 37°C for 60 minutes. After washing, 90 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate reagent was added. The reaction was stopped after 15 to 30 minutes of incubation at 37°C in the dark by adding 50 μL of stop solution. The change in chromogen was measured at 450 nm using a microplate reader (Bio-Tek Microplate Instruments, Penang, Malaysia). A standard curve was prepared with standard concentrations ranging from 31.25 to 2000 ng/mL of ACE activity.

Plasma renin activity (PRA) was measured using the rat PRA ELISA kit (IB59131; Immuno-Biological Laboratories Inc., Minneapolis, United States) following the manufacturer’s instructions. Briefly, 500 μL of plasma was added to the microplate wells and incubated for 2 hours at 37°C. Then, 5 μL of phenylmethylsulfonyl fluoride (PMSF) solution was added and vortexed, followed by the addition of 50 μL of the generation buffer. Next, 60 μL of the subtract solution was combined with 250 μL of the sample and transferred to another well plate. After incubating the plate at 37°C for 15 to 30 minutes, 50 μL of stop solution was added to halt the reaction. Changes in chromogen were measured using a microplate reader (Bio-Tek Microplate Instruments, Penang, Malaysia) at 450 nm. The reference standard curve was prepared with standards ranging from 0.2 to 60 ng/mL of PRA activity.

Assessment of Isoproterenol-induced changes in tissue biomarkers

Cardiac tissue biomarkers, such as hydroxyproline content and LDH, were measured. Hydroxyproline, an amino acid, results from the hydrolysis of connective tissue proteins like collagen; therefore, it plays a key role in stabilizing collagen. The tissue hydroxyproline content was determined following the method of Stegemann and Stalder²³, with slight modifications from Cissell et al.²⁴. Briefly, about 30 μL of tissue sample was diluted to 100 μL with papain solution to extract maximum collagen, then centrifuged at 1968 G for 10 minutes. Next, 30 μL of this aliquot was mixed with 100 μL of 4 N sodium hydroxide (NaOH), and the mixture was incubated at 120°C for 15 minutes. After cooling to room temperature, 100 μL of 4 N hydrochloric acid (HCl) was added to neutralize the alkaline solution. A mixture containing 0.625 mL of chloramine-T (0.05 M in 74% v/v distilled water), 2-propanolol (26% v/v), NaOH (0.629 M), citric acid (0.140 M), sodium acetate (0.453 M), and glacial acetic acid (0.112 M) was used to oxidize hydroxyproline into pyrrole-2-carboxylate. This mixture was incubated at room temperature for 20 minutes. Each sample was then vortexed immediately after adding 0.625 mL of p-dimethylaminobenzaldehyde (DMAB; 1 M, 15% w/v in 2-propanol plus concentrated acid; Ehrlich’s solution). After another 20-minute incubation at 65ºC, the samples were cooled to room temperature. The resulting red chromophore was measured with a spectrophotometer (DU 640B, Beckman Coulter Inc., Brea, CA, USA) at 558 nm. A standard curve was prepared using 0.5, 0.75, 1, and 1.5 μg of pure hydroxyproline per milliliter. The hydroxyproline concentration was multiplied by a factor of 8.2 to estimate collagen content. Results were expressed as micrograms of hydroxyproline per milligram of tissue protein.

Lactate dehydrogenase (LDH, EC 1.1.1.27) is a major oxidoreductase enzyme involved in the anaerobic metabolism pathway. When β-nicotinamide adenine dinucleotide (NAD+) is reduced to nicotinamideadenine dinucleotide hydrogen (NADH), lactate reversibly converts to pyruvate. The tissue LDH content was assessed as described in the method of Singh et al. ²⁵, with a slight modification from Dewi et al. ²⁶. Briefly, about 50 μL of tris-(hydroxymethyl)-aminomethane hydrochloride (200 mM, pH 8), 50 μL of lithium lactate (50 mM; 49 mg lithium lactate in 2.5 mL water), and 50 μL of phenazine methosulfate (PMS), iodonitrotetrazolium chloride (INT), and NAD solutions (PIN) mixture were combined with 50 μL of sample (adding all reagents first, then the aliquot). The PIN mixture was prepared by mixing 100 μL of PMS (0.9 mg in 100 μL of water), 100 μL of INT (3.3 mg in 100 μL of DMSO), and 2.3 mL of NAD (8.6 mg NAD in 2.3 mL of water). The microplate was incubated at room temperature for 5 minutes. Changes in chromogen were measured using a microplate reader (Bio-Tek Microplate Instruments, Penang, Malaysia) at 490 nm via the endpoint assay method. The standard curve was prepared using 50 μL of standards at 0, 2.5, 5, 7.5, 10, and 12.5 nmoles of NADH. LDH activity was calculated using the following equation:

In this case, B represents the amount of NADH produced (nmole). The sample volume (mL) injected into the well is represented by V, the sample dilution factor by DF, and absorbance changes by ΔA. The amount of LDH enzyme that catalyzes the conversion of lactate to pyruvate, producing 1.0 μmole of NADH per minute at 37°C, is defined as 1 unit of LDH activity. The LDH activity results are expressed as units per gram of protein (U/g protein).

Estimation of tissue total proteins

The total tissue proteins were estimated using the method described by Lowry et al.²⁷. The results were expressed as milligrams of total protein per gram of tissue.

Evaluation of histopathological changes

The ISO-induced cardiac histopathological changes were evaluated using eosinhematoxylin techniques, as described by Grimm ⁷, with minor modifications by Nikam et al. ²⁸. Briefly, tissue was fixed in 10% formalin and sectioned into 4 μm transverse slices. Cardiac tissue alterations were observed, and images were captured using an Olympus EP50 microscope camera (Olympus Corporation, Tokyo, Japan). Microscopic analyses were performed with a 400x light microscope, including a 35 μm scale bar.

Statistical analysis

All of the data were represented as mean ± standard deviations (SD). Data on systolic and diastolic blood pressure and heart rate were examined using a two-way analysis of variance (ANOVA) with a Bonferroni post hoc test. Data for CK-MB, ACE & PRA activities, hydroxyproline content, and LDH activity levels were examined using one- way ANOVA and Tukey’s Multiple Range tests in GraphPad Prism version 5.0 software. A probability (p) value < 0.05 was judged statistically significant.

Effect of astaxanthin on isoproterenol-induced cardiac functional changes

Administration of ISO (5 mg/kg/day) for seven consecutive days in rats caused a significant (p<0.05) decrease in SAP and DAP and a significant increase in HR compared to the normal group. This suggests that ISO may induce potential HF through strong, nonselective β-adrenergic (β1) receptor agonist activity, which enhances inotropic and lusitropic effects in cardiac muscle. Oral administration of AST (25 and 50 mg/kg for 21 consecutive days) alleviates ISO-induced cardiac functional changes in a dose-dependent manner compared to the ISO group. This effect is similar to that seen with aliskiren (30 mg/kg), ramipril (4 mg/ kg), and telmisartan (8 mg/kg) in the 21- day treatment groups. The impact of AST on ISO-induced changes in cardiac function, such as SAP, DAP, and HR, is shown in Fig. 1.

Fig. 1 Effect of AST on ISO-induced cardiac functional changes. Fig. 1A shows changes in SAP; Fig. 1B shows changes in DAP; and Fig. 1C shows changes in heart rate. Values in parentheses indicate the dose in mg/kg. Results are presented as mean ± standard deviation (SD), with n = 8 rats per group, analyzed using a two-way ANOVA with Bonferroni post hoc test. *p<0.05 vs. control group; #p<0.05 vs. ISO group. Abbreviations: AST, astaxanthin; DAP, diastolic blood pressure; HR, heart rate; ISO, isoproterenol; mmHg, millimeter of mercury (blood pressure units); and SAP, systolic blood pressure. 

Effect of astaxanthin on isoproterenol-induced changes in serum and plasma biomarkers

Administering ISO (5 mg/kg/day) to rats for seven days caused significant (p<0.05) increases in CK-MB, ACE, and PRA levels compared to the control group. ISO may induce heart failure by promoting cardiac muscle injury and degeneration, along with elevated circulating RAS mediators. Oral administration of AST (25 and 50 mg/kg) for 21 days decreased ISO-induced changes in serum and plasma biomarkers in a dose-dependent fashion, compared to the ISO group. This effect was comparable to results seen in groups treated for 21 days with aliskiren (30 mg/kg), ramipril (4 mg/ kg), and telmisartan (8 mg/kg). Table 1 illustrates how AST influences ISO-induced changes in serum and plasma biomarkers, including CK-MB, ACE, and PRA levels.

Effect of astaxanthin on isoproterenol-induced changes in heart and body weight ratio

Administration of ISO (5 mg/kg/day) for 7 days in rats resulted in a significant (p<0.05) increase in the heart-to-body weight ratio compared to the control group. This indicates that ISO may induce potential heart failure (HF) through degeneration of cardiac muscle. Oral administration of AST (25 and 50 mg/kg) for 21 days reduced the ISO-induced changes in the heart-to-body weight ratio in a dose-dependent manner compared to the ISO group. The effect was similar to that observed in the 21-day treatment groups with aliskiren (30 mg/kg), ramipril (4 mg/kg), and telmisartan (8 mg/ kg). The impact of AST on the ISO-induced heart and body weight ratio is summarized in Fig. 2.

Fig. 2 Effect of AST on ISO-induced changes in the heart and body weight ratio. The values in parentheses indicate a dose in mg/ kg. Results are shown as mean ± standard deviation (SD), with n = 8 rats per group, analyzed using a one-way ANOVA and Tukey’s Multiple Comparisons test. *p<0.05 compared to the control group; #p<0.05 compared to the ISO group. Abbreviations: AST, astaxanthin; ISO, isoproterenol. 

Effect of astaxanthin on isoproterenol-induced changes in tissue biomarkers

Administering ISO (5 mg/kg/day) for 7 days to rats caused significant (p<0.05) increases in hydroxyproline and LDH activity levels compared to the control group. This suggests that ISO may induce potential HF by affecting diastolic function, myocardial structure, muscle mass, and energy metabolism. Oral treatment with AST (25 and 50 mg/kg for 21 days) reduces ISO-induced changes in tissue biomarkers in a dose-dependent way, similar to the effects seen in the groups treated for 21 days with aliskiren (30 mg/kg), ramipril (4 mg/kg), and telmisartan (8 mg/kg). The impact of AST on ISO-induced alterations in tissue biomarkers, such as hydroxyproline and LDH levels, is illustrated in Table 2.

Effect of astaxanthin on isoproterenol-induced histopathological changes

The histological changes in rat cardiac tissue in naive control animals showed no alterations in rat cardiomyocytes, i.e., branched cardiomyocytes with a central oval vesicular nucleus and mild separation of the connective tissue. In contrast, ISO (5 mg/kg/day; for 7 days) caused potential microscopic changes in cardiac tissue, such as cell destruction, pyknotic and karyolytic nuclei, hemorrhage, edema, and myocyte degeneration. Oral administration of AST (25 and 50 mg/kg) for 21 days alleviated ISO-induced histological alterations in cardiac tissue. These findings were comparable to those seen with reference RAS modulators, i.e., aliskiren (30 mg/kg), ramipril (4 mg/ kg), and telmisartan (8 mg/kg) treatment groups. This indicates that AST exhibits cardioprotective effects against ISO-induced myocardial damage and dysfunction (Fig. 3). The changes were observed at 400× magnification (scale bar: 35 μm).

Fig. 3 Effect of AST on ISO-induced histopathological changes in rat cardiac tissue. In each group, two rats were used to evaluate cardiac tissue histopathology. Tissue sections were stained with eosin and hematoxylin. Figures 3a to 3g display histological changes in cardiac tissue from naïve control, ISO (5 mg/kg/day for 7 days), AST (25 mg/kg for 21 days), AST (50 mg/kg for 21 days), aliskiren (30 mg/kg for 21 days), ramipril (4 mg/kg for 21 days), and telmisartan (8 mg/kg for 21 days) groups, respectively. Fig. 3a shows normal cardiac tissue structure. Fig. 3b depicts ISO-associated myocardial damage. Fig. 3c and 3g demonstrate that AST and RAS modulators have cardioprotective effects against ISO toxicity. In this figure, the black arrow points to a normal pyknotic and karyolytic nucleus; the black arrowhead indicates normal cardiac cell structure; the white arrowhead shows cell destruction; the red arrow highlights abnormalities in myocardial fiber arrangement; the red arrowhead signifies hemorrhage and edema; and the yellow star indicates myocyte degeneration. Microscopic examinations were performed at 400× magnification; scale bar, 35 μm.