Invest Clin 67(1): 125 - 138, 2026 https://doi.org/10.54817/IC.v67n1a09

Corresponding author. Furong Qiao. Department of Cardiology, Xi’an Daxing Hospital, Xi’an, Shaanxi Province,

710003, China. Telephone: +86 17719654188. Email: furong644759573@sina.com

Ameliorative potential of astaxanthin

in isoproterenol-induced heart failure

in rats via the regulation of the

renin-angiotensin system.

Liang Chang and Furong Qiao

Department of Cardiology, Xi’an Daxing Hospital, Xi’an, Shaanxi Province, China.

Keywords: Angiotensin-Converting Enzyme;Arterial Pressure; Endomyocardial Fibrosis;

Lactate Dehydrogenase; Renin

Abstract. Heart failure (HF) is a condition in which the heart cannot pump

blood effectively to the body. Isoproterenol (ISO) induces HF in rodents by

affecting the renin-angiotensin system (RAS). Astaxanthin (AST) is known to

have protective effects on the cardiovascular system. However, clear evidence

showing that AST improves HF through RAS regulation has not yet been report-

ed. This study aimed to investigate the role of AST in ISO-induced HF in rats.

HF was induced by intraperitoneal (i.p.) injection of ISO (5 mg/kg/day) for

seven consecutive days. AST (25 and 50 mg/kg), aliskiren (30 mg/kg), ramipril

(4 mg/kg), and telmisartan (8 mg/kg) were administered orally for 21 days,

starting from the last dose of ISO (day 8). Changes in systolic and diastolic

blood pressure and heart rate associated with HF were measured on days 0,

7, 14, 21, and 28. Additionally, changes in heart-to-body weight ratio, serum

creatine kinase-MB (CK-MB), serum angiotensin-converting enzyme (ACE) ac-

tivity, plasma renin activity (PRA), tissue hydroxyproline, and lactate dehydro-

genase (LDH) activity, along with histopathological alterations, were evaluated.

The administration of AST and RAS-modulating agents reduced ISO-induced

changes in cardiac function and biochemical markers. It also demonstrated car-

dioprotective effects. Therefore, AST may be useful for treating cardiotoxic HF

due to its RAS-regulatory actions. However, further studies are needed to con-

firm this therapeutic potential across different HF models and animal species.

126 Chang and Qiao

Investigación Clínica 67(1): 2026

Potencial mejorador de la astaxantina en la insuficiencia

cardíaca inducida por isoproterenol en ratas a través

de la regulación del sistema renina-angiotensina.

Invest Clin 2026; 67 (1): 125 – 138

Palabras clave: Enzima Convertidora de Angiotensina; Presión Arterial; Fibrosis

Endomiocárdica; Lactato Deshidrogenasa; Renina.

Resumen. La insuficiencia cardíaca (IC) es una alteración de la capaci-

dad funcional del corazón para bombear sangre al organismo. El isoproterenol

(ISO) induce IC en roedores mediante la alteración del sistema renina-angio-

tensina (SRA). Se sabe que la astaxantina (AST) ejerce una acción reguladora

sobre el sistema cardiovascular. Sin embargo, aún no se ha descrito evidencia

clara de que la AST mejore la IC mediante la regulación del SRA. El presente

estudio fue diseñado para investigar el papel de la AST en la IC inducida por ISO

en ratas. La IC se indujo mediante administración intraperitoneal (i.p.) de ISO

(5 mg/kg/día) durante siete días consecutivos. La AST (25 y 50 mg/kg), aliski-

ren (30 mg/kg), ramipril (4 mg/kg) y telmisartán (8 mg/kg) se administraron

por vía oral durante 21 días consecutivos, desde la última dosis de ISO (día 8).

Los cambios asociados a la IC en la presión arterial sistólica y diastólica y en la

frecuencia cardíaca se evaluaron en diferentes puntos temporales, es decir, los

días 0, 7, 14, 21 y 28. También se evaluaron los cambios en la relación entre el

peso corporal y el corazón, la creatinquinasa sérica-MB (CK-MB), la actividad

sérica de la enzima convertidora de angiotensina (ECA) y la actividad plasmá-

tica de la renina (PRA); y la actividad tisular de la hidroxiprolina y la lactato

deshidrogenasa (LDH) junto con los cambios histopatológicos. La administra-

ción de agentes moduladores de AST y SRA atenúa los parámetros bioquímicos

y funcionales cardíacos inducidos por ISO. También demuestra efectos cardio-

protectores. Por lo tanto, la AST puede utilizarse para la IC asociada a cardio-

toxinas debido a sus acciones reguladoras del SRA. Sin embargo, se requieren

estudios más exhaustivos para demostrar su eficacia terapéutica en diferentes

IC y especies animales.

Received: 14-10-2025 Accepted: 21-12-2025

INTRODUCTION

Heart failure (HF) is also known as con-

gestive heart failure (CHF), which indicates

impairment of the pumping of blood by

the heart. The symptoms of HF are fatigue,

shortness of breath, and leg swelling 1. The

global prevalence rate of HF is rising to 24%

and affects one person out of four in their

lifetime. Furthermore, it is expected to in-

crease by 8.5% among persons aged 65 to

70 years 2. Isoproterenol (ISO) is widely used

to treat bradycardia in patients with arrhyth-

mias. Structurally, it is similar to epineph-

rine and an agonist of non-selective beta-

adrenergic receptors 3. Experimentally, ISO

causes myocardial fibrosis, raises diaphrag-

matic contractility, and mimics the patho-

genesis of cardiomyopathy and human heart

failure 4,5 and the mechanism of the effects,

Astaxanthin ameliorates isoproterenol-induced heart failure in rats 127

Vol. 67(1): 125 - 138, 2026

of isoproterenol on diaphragmatic contrac-

tility and fatigue in septic peritonitis in vi-

tro. Furthermore, the administration of ISO

is known to induce HF by modulating the

renin-angiotensin system (RAS) 6–8 changes

in circulating and tissue renin-angiotensin

system (RAS). Moreover, ISO is known to in-

crease heart rate and systemic arterial blood

pressure 9,10. Even a single dose of ISO also

induces type 2 diabetes mellitus-associated

myocardial infarctions 11. Besides, the mul-

tiple doses of ISO cause chronic heart failure

in mice 12. A one-week administration of ISO

at 5 mg/kg induces changes in hemodynamic

parameters in rats by increasing angiotensin

II levels and angiotensin-converting enzyme

2 (ACE2) activity 6,13 5 mg/kg/day, intraperi-

toneally. The activation of ACE2 may be due

to the fact that raising cardiac angiotensin-

II peptide concentrations is associated with

increased ACE2 expression in normal rats 14.

However, these effects are absent in other

conditions and other species 15. In most cas-

es, ISO does not affect ACE2 expression. An

experimental study also demonstrated that

ISO increases the cardiac renin-angiotensin

system function in a rat model of cardiac hy-

pertrophy 6,16.

Astaxanthin (AST) is a type of keto-ca-

rotenoid belonging to the terpene compound

group. AST is a metabolite of canthaxanthin

and zeaxanthin. It contains hydroxyl and ketone

groups, which have key antioxidant properties

and act as free radical scavengers and chelators

of metal ions 17. It improves cardiac function

and exercise tolerance in HF patients18. Addi-

tionally, AST provides myocardial protection

against steroid-induced cardio-renal dysfunc-

tion and related hypertensive complications 19.

Moreover, AST also has modulating effects on

the RAS system and enhances vascular func-

tion against cardiotoxins 20,21. However, further

research is necessary to fully understand the

role of AST in ISO-induced HF and RAS regula-

tion under these conditions. This study exam-

ined the effects of specific RAS modulators and

AST on cardiovascular dysfunction caused by

ISO in rats. Therefore, the impact of AST was

evaluated in this research regarding its effects

on ISO-induced HF and its possible modulation

of the RAS.

METHODS

Animals used

Male Sprague Dawley rats weighing

180–200 g (12–14 months old) were used

in this study. The animals had unlimited ac-

cess to water and a standard laboratory diet.

They were housed in the central animal fa-

cility, 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 con-

trols and did not receive any drug adminis-

tration.

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 refer-

ence drug treatments. These groups of ani-

mals 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, telmisar-

tan (8 mg/kg), for 21 consecutive days fol-

lowing the last dose of ISO administration.

At 0, 7, 14, 21, and 28 days, HF-related

changes in heart rate (HR), diastolic blood

128 Chang and Qiao

Investigación Clínica 67(1): 2026

pressure (DAP), and systolic blood pres-

sure (SAP) were recorded. Blood samples

were collected on the twenty-eighth day for

biochemical analysis of plasma renin activ-

ity (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 buf-

fer (pH 7.4). Centrifugation at 769 x g was

performed to obtain a clear supernatant,

which was used to measure lactate dehydro-

genase (LDH) activity and hydroxyproline

content, both of which are tissue biomark-

ers. Additionally, ISO-induced histopatho-

logical changes were examined using eosin-

hematoxylin staining.

Assessment of Isoproterenol-induced

functional changes in the heart

The ISO induced the HF with altera-

tion 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 nonin-

vasive 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. Fre-

quent contact with the animals was avoided

to reduce stress and irritation. Without ap-

plying any force, the tail occlusion cuff was

placed at the base of the tail. The distance

between the tail occlusion cuff and the vol-

ume 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 us-

ing 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 bioti-

nylated 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. Subse-

quently, 100 μL of the horseradish peroxi-

dase 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 (Bio-

Tek Microplate Instruments, Penang, Malay-

sia), 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 us-

ing the rat ACE ELISA kit (CSB-E04490r;

Cusabio Technology LLC, Houston, United

States) following the manufacturer’s in-

structions. Briefly, microplate wells were

filled with 100 μL of serum and incubated at

37°C for two hours. Afterward, 100 μL of bi-

otinylated detection antibody working solu-

tion 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 con-

jugate working solution was added, followed

by incubation at 37°C for 60 minutes. After

washing, 90 μL of 3,3′,5,5′-tetramethylben-

zidine (TMB) substrate reagent was added.

The reaction was stopped after 15 to 30 min-

utes of incubation at 37°C in the dark by

Astaxanthin ameliorates isoproterenol-induced heart failure in rats 129

Vol. 67(1): 125 - 138, 2026

adding 50 μL of stop solution. The change

in chromogen was measured at 450 nm us-

ing a microplate reader (Bio-Tek Microplate

Instruments, Penang, Malaysia). A standard

curve was prepared with standard concentra-

tions ranging from 31.25 to 2000 ng/mL of

ACE activity.

Plasma renin activity (PRA) was mea-

sured using the rat PRA ELISA kit (IB59131;

Immuno-Biological Laboratories Inc., Min-

neapolis, United States) following the man-

ufacturer’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 deter-

mined 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 col-

lagen, then centrifuged at 1968 G for 10

minutes. Next, 30 μL of this aliquot was

mixed with 100 μL of 4 N sodium hydrox-

ide (NaOH), and the mixture was incubated

at 120°C for 15 minutes. After cooling to

room temperature, 100 μL of 4 N hydro-

chloric 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), so-

dium acetate (0.453 M), and glacial acetic

acid (0.112 M) was used to oxidize hydroxy-

proline into pyrrole-2-carboxylate. This mix-

ture was incubated at room temperature

for 20 minutes. Each sample was then vor-

texed immediately after adding 0.625 mL

of p-dimethylaminobenzaldehyde (DMAB; 1

M, 15% w/v in 2-propanol plus concentrat-

ed acid; Ehrlich’s solution). After another

20-minute incubation at 65ºC, the samples

were cooled to room temperature. The re-

sulting red chromophore was measured

with a spectrophotometer (DU 640B, Beck-

man 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 hydroxypro-

line per milliliter. The hydroxyproline con-

centration 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 path-

way. When β-nicotinamide adenine dinucle-

otide (NAD+) is reduced to nicotinamide-

adenine dinucleotide hydrogen (NADH),

lactate reversibly converts to pyruvate. The

tissue LDH content was assessed as de-

scribed 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), iodo-

nitrotetrazolium 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

130 Chang and Qiao

Investigación Clínica 67(1): 2026

mg in 100 μL of DMSO), and 2.3 mL of NAD

(8.6 mg NAD in 2.3 mL of water). The mi-

croplate was incubated at room tempera-

ture for 5 minutes. Changes in chromogen

were measured using a microplate reader

(Bio-Tek Microplate Instruments, Penang,

Malaysia) at 490 nm via the endpoint as-

say method. The standard curve was pre-

pared 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 repre-

sented 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 estimat-

ed using the method described by Lowry

et al.27. The results were expressed as mil-

ligrams of total protein per gram of tissue.

Evaluation of histopathological changes

The ISO-induced cardiac histopatho-

logical changes were evaluated using eosin-

hematoxylin techniques, as described by

Grimm 7, with minor modifications by Ni-

kam et al. 28. Briefly, tissue was fixed in 10%

formalin and sectioned into 4 μm transverse

slices. Cardiac tissue alterations were ob-

served, and images were captured using an

Olympus EP50 microscope camera (Olym-

pus 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 Bonfer-

roni 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 sta-

tistically significant.

RESULTS

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 com-

pared to the normal group. This suggests

that ISO may induce potential HF through

strong, nonselective β-adrenergic (β1) re-

ceptor 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.

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,

Astaxanthin ameliorates isoproterenol-induced heart failure in rats 131

Vol. 67(1): 125 - 138, 2026

along with elevated circulating RAS media-

tors. 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 il-

lustrates 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 treat-

ment 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.

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 metabo-

lism. Oral treatment with AST (25 and 50

mg/kg for 21 days) reduces ISO-induced

changes in tissue biomarkers in a dose-de-

pendent way, similar to the effects seen in

the groups treated for 21 days with aliskiren

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 Bon-

ferroni post hoc test. *p<0.05 vs. control

group; #p<0.05 vs. ISO group. Abbrevia-

tions: AST, astaxanthin; DAP, diastolic blood

pressure; HR, heart rate; ISO, isoproterenol;

mmHg, millimeter of mercury (blood pres-

sure units); and SAP, systolic blood pressure.

132 Chang and Qiao

Investigación Clínica 67(1): 2026

(30 mg/kg), ramipril (4 mg/kg), and telmis-

artan (8 mg/kg). The impact of AST on ISO-

induced alterations in tissue biomarkers,

such as hydroxyproline and LDH levels, is il-

lustrated in Table 2.

Effect of astaxanthin on isoproterenol

-induced histopathological changes

The histological changes in rat car-

diac 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 de-

generation. 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 car-

dioprotective effects against ISO-induced

myocardial damage and dysfunction (Fig. 3).

The changes were observed at 400× magni-

fication (scale bar: 35 μm).

Table 1. Effect of astaxanthin on isoproterenol - induced changes in serum and plasma biomarkers.

Groups CK-MB

(pg/mL)

ACE

(ng/mL)

PRA

(ng/mL)

Naive control 212 ± 13.5 3.22 ± 1.52 10.3 ± 0.9

ISO (5) 456 ± 14.9* 14.72 ± 1.14* 43.4 ± 1.3*

ISO + AST (25) 293 ± 12.7# 7.48 ± 0.93# 17.5 ± 0.8#

ISO + AST (50) 264 ± 8.6# 5.61 ± 1.04# 11.9 ± 1.2#

ISO + Aliskiren (30) 241 ± 13.1# 4.35 ± 1.47# 9.7 ± 0.4#

ISO + Ramipril (4) 227 ± 11.2# 4.39 ± 0.95# 15.4 ± 0.7#

ISO + Telmisartan (8) 217 ± 9.7# 3.45 ± 1.13# 13.7 ± 0.8#

The values in parentheses represent a dose in mg/kg. The results are presented as mean ± SD, with eight rats in

each group. *p<0.05 vs. control group; #p<0.05 vs. ISO group. ACE, angiotensin-converting enzyme; AST, asta-

xanthin; CK-MB, creatine kinase isoenzyme-MB; ISO, isoproterenol; PRA, plasma renin activity. Groups were analy-

zed using a one-way ANOVA and Tukey’s Multiple Comparisons test.

Fig. 2. Effect of AST on ISO-induced

changes in the heart and body

weight ratio. The values in pa-

rentheses 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 Mul-

tiple Comparisons test. *p<0.05

compared to the control group;

#p<0.05 compared to the ISO

group. Abbreviations: AST, astax-

anthin; ISO, isoproterenol.

Heart and Body Weight Ratio (mg/g)

Astaxanthin ameliorates isoproterenol-induced heart failure in rats 133

Vol. 67(1): 125 - 138, 2026

DISCUSSION

The i.p. administration of ISO (5 mg/

kg/day for seven days) in rats demonstrated

a significant (p<0.05) development of car-

diac dysfunction through alterations in he-

modynamic parameters, such as reductions

in SAP and DAP, along with increases in HR

levels and elevated serum CK-MB, ACE, and

PRA levels. Furthermore, there was an in-

crease in the heart-to-body weight ratio and

in tissue hydroxyproline and LDH activity lev-

Table 2. Effect of astaxanthin on isoproterenol - induced changes in tissue biomarkers.

Groups Hydroxyproline

(μg/mg of tissue protein)

LDH activity

(U/g protein)

Naive control 1.67 ± 0.92 1176.81 ± 12.73

ISO (5) 6.91 ± 1.02* 1492.18 ± 17.62*

ISO + AST (25) 2.29 ± 0.43#1232.25 ± 9.04#

ISO + AST (50) 2.13 ± 0.67# 1213.94 ± 11.14#

ISO + Aliskiren (30) 2.03 ± 1.05# 1199.07 ± 10.46#

ISO + Ramipril (4) 1.82 ± 0.87# 1204.63 ± 11.06#

ISO + Telmisartan (8) 1.94 ± 1.03#1191.04 ± 9.83#

The values in parentheses indicate a dose in mg/kg. The results are shown as mean ± SD, with eight

rats in each group. *p<0.05 versus control group; #p<0.05 versus ISO group. AST, astaxanthin; ISO,

isoproterenol; and LDH, lactate dehydrogenase.

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 he-

matoxylin. 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, respecti-

vely. 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 he-

morrhage and edema; and the yellow star indicates myocyte degeneration. Microscopic examinations

were performed at 400× magnification; scale bar, 35 μm.

134 Chang and Qiao

Investigación Clínica 67(1): 2026

els. ISO was also shown to destroy myocytes,

resulting in pyknotic cells, karyolytic nuclei,

hemorrhage, edema, and myocyte degenera-

tion. These findings indicate that ISO causes

potential HF through multiple pathway ab-

normalities, including free radical-associat-

ed cardiac damage, altered energy demands,

and RAS pathway disruptions 29,30. Similar

results were observed in ISO-induced HF

in rats, which showed changes in the same

hemodynamic parameters and myocardial

function. These outcomes are consistent

with those reported in other studies 31. Oral

AST therapy at 25 and 50 mg/kg for 21 days

significantly reduced ISO-related cardiac

dysfunctions, biomarker alterations, and

histological abnormalities. This effect was

comparable to that seen in the RAS modu-

lator treatment groups (aliskiren, ramipril,

and telmisartan).

Circulatory and local ACE activity, plas-

ma renin content, and the renin-angiotensin

system in the heart and aorta contribute to

the development of cardiovascular issues, in-

cluding myocardial infarction and heart fail-

ure 32,33. Proteomic analysis showed that ISO

increases myocardial tissue weight by 55% in

hypertrophied rat hearts 34. Additionally, ISO

raises left ventricular tissue weight by 33%

and decreases systolic and diastolic blood

pressure by 13%. Administering ISO (1, 10,

100, and 500 μg/kg) increases plasma re-

nin activity in a dose-dependent manner 33.

Inhibitors of the renin-angiotensin system

are known to improve heart failure in experi-

mental animals 35,36. RAS modulators, such

as spironolactone and captopril, reduce ISO-

induced cardiac remodeling in rats37. Aliski-

ren has been shown to lower serum cardiac

enzymes like LDH and CK-MB 38, decrease

cardiac biomass 39, and also reduce oxidative

stress and apoptosis 38,40. In this study, the

AST treatment group exhibited results simi-

lar to those of the control group. However,

the ACE inhibitor ramipril worsens ISO-in-

duced myocardial dysfunctions by regulating

hydroxyproline content in rats41,42. The cur-

rent research demonstrates that AST allevi-

ates ISO-induced cardiac damage by enhanc-

ing mitochondrial function and scavenging

free radicals in rats 43. AST is also known to

decrease ACE protein expression, mitigate

hypertensive conditions 21, prevent ISO-

induced loss of cardiac muscle mass 43, and

reduce hydroxyproline content 44.

Normally, ISO does not influence ACE2

expression. However, some studies have re-

ported elevated ACE2 levels in Sprague-

Dawley rats 6. Another study indicated that

increased cardiac angiotensin-II peptide

levels are linked to higher ACE2 expression

in normal rats 14. Nonetheless, these effects

are not observed in other conditions or spe-

cies15. Therefore, the impact of ISO on the

renin-angiotensin system is complex and

varies depending on the specific tissue and

animal model. Experimentally, ISO is known

to raise plasma angiotensin-II peptide levels,

which can lead to left ventricular hypertro-

phy and worsen heart failure progression16.

Additionally, angiotensin II directly influ-

ences cardiac tissue via the angiotensin II

type 2 receptor, resulting in myocardial

damage 45. Furthermore, administering an

angiotensin II receptor antagonist, such as

telmisartan, can reduce ISO-induced car-

diac dysfunction46. Similar findings are ob-

served in this study: telmisartan (8 mg/kg)

and AST administration normalize elevated

blood pressure and modify biomarker levels,

including serum CK-MB, ACE, PRA, hydroxy-

proline, and LDH activity. Moreover, AST

has been shown to lower blood pressure in

spontaneously hypertensive rats 47 by inhib-

iting ACE activity and regulating the RAS 48.

Additionally, AST supplementation has dem-

onstrated the ability to decrease blood pres-

sure in humans by suppressing the RAS 49.

Our study also indicates that AST improves

ISO-induced cardiac dysfunction and blood

pressure control through RAS regulatory

mechanisms.

The current study revealed that oral

treatment with AST reduces ISO-induced

cardiac dysfunction by regulating SAP and

DAP, decreasing elevated HR, CK-MB, ACE,

Astaxanthin ameliorates isoproterenol-induced heart failure in rats 135

Vol. 67(1): 125 - 138, 2026

and PRA levels, and lowering the heart-to-

body weight ratio, tissue hydroxyproline,

and LDH activity levels. Therefore, AST pro-

vides cardioprotection against ISO-induced

toxicity by regulating the RAS system. It can

potentially be used for various heart failure

conditions. However, further research using

diverse preclinical animal models and hu-

man subjects is necessary.

Acknowledgments

The authors acknowledge the Depart-

ment of Cardiology at Xi’an Daxing Hospital,

Xi’an, Shaanxi Province, 710003, China, for

its unconditional support.

Funding

The authors received no financial sup-

port for this research.

Conflict of interest

The authors declare no conflicts of in-

terest regarding the present study.

ORCID number of authors

• Furong Qiao (FQ):

0009-0008-7478-1109

• Liang Chang (LC):

0009-0003-2808-3522

Contribution of the authors

LC: performed the experiments, col-

lected data, conceived the study, and drafted

the manuscript. FQ: supervised the project,

secured funding, and critically revised the

intellectual content.

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