Introduction

Isoproterenol (ISO), a synthetic non-selective β-adrenergic receptor agonist, is widely utilized to induce experimental myocardial dysfunction in rodents. High-dose ISO administration produces acute cardiac injury that closely resembles catecholamine-induced cardiomyopathy and shares key pathophysiological features with Takotsubo syndrome (TTS)1. Following an initial surge in heart rate and myocardial contractility, high-dose ISO exposure leads to transient and largely reversible left ventricular dysfunction, mimicking the TTS-like cardiac alterations observed in preclinical models2.

The myocardial injury induced by ISO is primarily attributed to excessive catecholaminergic stimulation and heightened generation of reactive oxygen species (ROS) resulting from catecholamine autoxidation. This oxidative burden promotes membrane lipid peroxidation, inflammatory cascades, and contractile dysfunction3. Clinically, TTS is an acute, stress-induced cardiomyopathy characterized by reversible left ventricular wall motion abnormalities in the absence of obstructive coronary artery disease, typically precipitated by severe emotional or physical stress accompanied by supraphysiological catecholamine levels4. The syndrome classically manifests as acute transient heart failure with distinct regional wall motion abnormalities, most notably apical akinesis or ballooning alongside basal hyperkinesis5.

Notably, TTS predominantly affects postmenopausal women, pointing to a critical role for estrogen deficiency in disease susceptibility. Estrogens confer multifaceted cardioprotection, in part by attenuating sympathetic nervous system overactivation and limiting catecholamine-mediated myocardial toxicity6. Consistent with this paradigm, ISO-induced cardiomyopathy in rodent myocardium is characterized by pronounced oxidative stress and activation of apoptotic signaling pathways1,7. These biochemical alterations include the upregulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, elevated malondialdehyde (MDA) concentrations reflecting lipid peroxidation, and depletion of reduced glutathione (GSH), indicative of compromised endogenous antioxidant defenses8.

Mitochondria represent a primary intracellular source of ROS, and excessive mitochondria-derived oxidative stress is heavily implicated in TTS-like myocardial injury. Preclinical investigations have demonstrated that targeted antioxidant interventions can mitigate mitochondrial ROS generation in stress cardiomyopathy models8. Elevated mitochondrial oxygen free radical production has likewise been documented in ISO-treated rats9. Furthermore, ultrastructural and molecular analyses reveal substantial mitochondrial swelling, diminished adenosine triphosphate (ATP) availability, and downregulation of key proteins governing mitochondrial biogenesis, fusion, and fission during the acute phase of experimental TTS-like cardiomyopathy10.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) serves as a master transcriptional coactivator of mitochondrial biogenesis and cellular energy metabolism, playing an essential role in defending the myocardium against oxidative damage11. Downregulated or dysregulated PGC-1α expression is closely linked to the progression of heart failure, whereas preservation of PGC-1α activity sustains mitochondrial integrity and myocardial contractility under pathological stress conditions12.

Sirtuin-1 (SIRT1), a nicotinamide adenine dinucleotide (NAD⁺)-dependent class III histone deacetylase, is another pivotal regulator of mitochondrial homeostasis and cellular stress resistance. SIRT1 exerts potent cardioprotective effects by mitigating oxidative stress and suppressing pro-apoptotic signaling pathways13. Crucially, SIRT1 acts directly upstream of PGC-1α, catalyzing its deacetylation and activation to enhance mitochondrial biogenesis and bolster cellular antioxidant capacity11.

Glucagon-like peptide-1 (GLP-1) is an endogenous incretin hormone integral to glucose homeostasis, and its long-acting receptor agonist liraglutide (LIR) is widely prescribed for the management of type 2 diabetes mellitus and obesity14. Beyond its established glucoregulatory actions, GLP-1 receptor signaling directly modulates cardiovascular physiology, supported by the expression of functional GLP-1 receptors on cardiomyocytes, vascular smooth muscle cells, and endothelial cells15. Experimental evidence indicates that LIR confers direct cardioprotection through antioxidant, anti-inflammatory, and anti-apoptotic mechanisms that operate independently of glycemic control14,16. Although LIR has demonstrated beneficial effects in experimental models of cardiac injury and TTS3,8,16,17,18, its specific impact on myocardial oxidative and nitrosative stress alongside the SIRT1/PGC-1α signaling axis in estrogen-deficient, catecholamine-induced cardiomyopathy remains unexplored. Therefore, the present study aimed to investigate the effects of LIR on myocardial redox status, SIRT1/PGC-1α protein expression, and left ventricular function in an ovariectomized rat model of ISO-induced TTS-like cardiomyopathy.

Materials and Methods

Experimental Groups and Protocol

Six weeks after ovariectomy, rats were randomly allocated into three experimental groups (n = 8 per group):

1. Ovariectomized control group (OVX): OVX rats receiving vehicle without ISO or LIR administration.

2. ISO-induced TTS-like cardiomyopathy group (ISO): OVX rats receiving a single high dose of ISO to induce acute catecholamine-mediated cardiac dysfunction.

3. ISO plus liraglutide treatment group (ISO+LIR): OVX rats receiving prophylactic and post-insult LIR treatment alongside ISO administration.

Acute TTS-like cardiac dysfunction was induced by a single intraperitoneal injection of isoproterenol hydrochloride (Cat. No. I6504; Sigma-Aldrich, St. Louis, MO, USA) at a dose of 150 mg/kg, as previously established5. Rats assigned to the ISO+LIR group received subcutaneous (s.c.) injections of liraglutide (Saxenda®; Novo Nordisk, Bagsværd, Denmark) at a dose of 150 µg/kg/day for five consecutive days prior to ISO administration, with treatment continuing for two additional days following the ISO challenge (total treatment duration: 7 days)19. This prophylactic regimen was selected based on prior experimental evidence demonstrating that LIR pretreatment effectively attenuates myocardial oxidative injury and preserves cardiac function via GLP-1 receptor–dependent signaling3,16. On day 6 of the protocol, ISO (150 mg/kg, i.p.) was administered to animals in the ISO and ISO+LIR groups. Forty-eight hours after ISO administration (end of day 7), all surviving rats were deeply anesthetized with ketamine (90 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and euthanized by exsanguination. The hearts and uteri were rapidly excised, weighed, and snap-frozen at −80 °C for subsequent biochemical and molecular analyses (Figure 1).

Transthoracic Echocardiography

Serial transthoracic echocardiographic assessments were performed at 90 minutes, 24 hours, and 48 hours after ISO injection by two experienced investigators blinded to group allocation. Imaging was conducted using a Philips Affiniti 30 ultrasound system equipped with an S8-3 phased-array transducer, accompanied by continuous surface electrocardiogram (ECG) monitoring20. All echocardiographic examinations were performed under ketamine/xylazine anesthesia (70 mg/kg and 10 mg/kg, i.p., respectively). Although ketamine/xylazine anesthesia exerts known negative chronotropic and inotropic effects, identical anesthetic dosing and timing were applied uniformly across all groups to minimize intergroup confounding. Two-dimensional guided M-mode echocardiographic tracings were acquired from the parasternal long-axis view and analyzed offline using the integrated system software. Interventricular septal thickness in diastole and systole (IVSd and IVSs), left ventricular posterior wall thickness in diastole and systole (LVPWd and LVPWs), left ventricular ejection fraction (LVEF), and heart rate (HR) were recorded at each time point (Figure 2).

Results

During the seven-day experimental protocol prior to ISO challenge, no overt clinical abnormalities were observed. Following high-dose ISO administration, no mortality occurred in the OVX control group (final n = 8), whereas two rats died in the untreated ISO group (final n = 6) and one rat died in the ISO+LIR group (final n = 7). Animals that succumbed during the post-ISO observation period were included in the echocardiographic analyses up to their last completed assessment time point.

Myocardial SIRT1 and PGC-1α Protein Expression

Myocardial protein levels of SIRT1 and PGC-1α quantified by ELISA are illustrated in Figure 3. High-dose ISO administration caused a profound reduction in both myocardial SIRT1 and PGC-1α levels compared with the OVX control group (p < 0.05; Figure 3A, B). Conversely, LIR treatment significantly restored myocardial SIRT1 and PGC-1α protein expression in the ISO+LIR group compared with the untreated ISO group (p < 0.05; Figure 3A, B). While SIRT1 expression in the ISO+LIR group recovered toward OVX control levels, PGC-1α expression in the ISO+LIR group remained statistically lower than that of the OVX control group (p < 0.05; Figure 3B).

Discussion

In the present study, an ovariectomized rat model of ISO-induced catecholamine-mediated, TTS-like cardiomyopathy was utilized to evaluate the cardioprotective potential of the GLP-1 receptor agonist LIR. Our principal findings demonstrate that a seven-day treatment regimen with LIR (150 µg/kg/day) significantly attenuated myocardial lipid peroxidation and nitrosative stress—as evidenced by decreased myocardial MDA and nitrate concentrations—while upregulating myocardial SIRT1 and PGC-1α protein expression. Crucially, these molecular and biochemical improvements were accompanied by a significant recovery of left ventricular systolic function (LVEF) at 48 hours post-ISO, underscoring a functional benefit of LIR in catecholamine-induced myocardial dysfunction.

Takotsubo syndrome is characterized by acute, transient left ventricular systolic dysfunction typically precipitated by intense emotional or physical stress. Although its precise pathogenesis remains incompletely delineated, sympathetic nervous system hyperactivation and catecholamine surge are recognized as central drivers, supported by clinical observations of markedly higher circulating catecholamine levels in patients with TTS than in those with acute myocardial infarction25. Consequently, ISO—a synthetic non-selective β₁/β₂-adrenergic receptor agonist structurally analogous to epinephrine—is widely employed in preclinical models to recapitulate catecholamine-induced myocardial injury and TTS-like cardiac dysfunction2,3. High-dose ISO provokes myocardial damage primarily through excessive β-adrenergic stimulation, intracellular calcium overload, heightened myocardial oxygen demand, and severe oxidative stress1.

Numerous experimental investigations have confirmed that high-dose ISO administration reproduces hallmark functional, electrocardiographic, and biochemical features of TTS in rodents1,2,4,5. However, gross morphological apical ballooning is inconsistently captured in small-animal models. In the current study, a single intraperitoneal injection of ISO (150 mg/kg) induced profound reductions in LVEF (falling to 27.4% at 90 minutes and 21.2% at 24 hours) alongside marked ST-segment elevation, consistent with severe catecholamine-mediated myocardial stunning. Although discrete apical ballooning was not visualized on transthoracic echocardiography, the absence of this localized geometric feature does not preclude substantial TTS-like myocardial injury. Indeed, multiple studies utilizing comparable ISO dosing protocols have documented severe global systolic dysfunction and TTS-like molecular pathology without overt apical ballooning1, reflecting recognized inter-individual and inter-strain heterogeneity in rodent ventricular remodeling. Moreover, previous investigations have validated the 150 mg/kg ISO dose as highly effective for inducing reproducible TTS-like cardiomyopathy in adult female rats26,27.

The striking epidemiological predominance of TTS in postmenopausal women implicates estrogen deprivation as a key predisposing factor. Under physiological conditions, estrogens preserve coronary endothelial function, modulate autonomic tone, and blunt excessive β-adrenergic signaling. Bilateral ovariectomy is therefore the gold-standard experimental paradigm for modeling postmenopausal hypoestrogenism in rodents. Because a sham-operated (gonadally intact) control group was not included in the present study design, the independent contribution of ovariectomy cannot be isolated from the effects of ISO or LIR; thus, conclusions regarding menopausal susceptibility specifically should be interpreted with caution. Nevertheless, seminal work by Ueyama et al. demonstrated that OVX rats exhibit heightened vulnerability to stress-induced left ventricular dysfunction compared with estradiol-replaced counterparts28. Furthermore, while acute neuroendocrine fluctuations partially stabilize within two weeks after ovariectomy, sustained depletion of circulating estradiol and progesterone is firmly established between three and six weeks post-surgery29. Consequently, the six-week post-ovariectomy interval employed in our protocol represents a pathophysiologically robust timeframe for modeling chronic postmenopausal estrogen deficiency.

Oxidative stress is a cornerstone mechanism underlying ISO-induced cardiotoxicity1. Supraphysiological catecholamine exposure accelerates auto-oxidation into adrenochromes, generates excessive ROS, disrupts redox-sensitive signaling cascades, and triggers membrane lipid peroxidation and contractile impairment. In our study, LIR treatment significantly lowered myocardial MDA concentrations compared with untreated ISO rats and promoted a significant recovery of LVEF at 48 hours, aligning with prior reports documenting the potent antioxidant and cardioprotective properties of LIR30. In contrast, myocardial GSH concentrations remained unaltered across groups.

Alongside oxidative damage, nitrosative stress has been increasingly implicated in the pathogenesis of TTS30,31. Excessive β₂-adrenergic receptor stimulation augments nitric oxide (NO) synthesis in cardiomyocytes; in the presence of abundant superoxide anions, NO rapidly reacts to form peroxynitrite (ONOO⁻), a potent reactive nitrogen species that induces protein tyrosine nitration, cellular inflammation, and myocardial energetic failure31,32. Under basal conditions, physiological myocardial NO production is predominantly regulated by endothelial nitric oxide synthase (eNOS), whereas pathological NO overproduction is driven by inducible nitric oxide synthase (iNOS). Notably, long-term ovariectomy shifts this enzymatic equilibrium toward heightened iNOS expression and diminished eNOS activity, lowering myocardial tolerance to stress33. In the present study, myocardial nitrate concentrations—an indirect indicator of tissue NO generation—were elevated following ISO administration and significantly suppressed by LIR treatment. Because direct measurements of myocardial peroxynitrite, 3-nitrotyrosine accumulation, and eNOS/iNOS isoform expression were not performed, our mechanistic inferences regarding nitrosative signaling remain indirect and grounded in established literature31,33.

Extensive preclinical and clinical literature supports the cardiovascular benefits of LIR across diverse disease states. In experimental diabetic cardiomyopathy and doxorubicin-induced cardiotoxicity, LIR enhances myocardial insulin sensitivity, activates adenosine monophosphate–activated protein kinase (AMPK) signaling, limits lipid peroxidation, reduces circulating and tissue MDA levels, and preserves ventricular contractility34,35. At the clinical level, landmark cardiovascular outcome trials such as the LEADER trial have established that GLP-1 receptor agonists significantly reduce major adverse cardiovascular events and cardiovascular mortality in patients with type 2 diabetes, mediated in part through favorable modulation of myocardial bioenergetics and vascular inflammation36. In our experimental design, LIR was administered beginning five days prior to ISO injection and continued for 48 hours post-insult, consistent with established cardioprotective pretreatment protocols against acute oxidative injury16. Although prophylactic administration does not mirror the unpredictable acute clinical onset of TTS, this regimen enables a rigorous evaluation of the preventive and molecular mechanisms by which GLP-1 receptor agonism counteracts catecholamine-induced cardiotoxicity. Future translational studies incorporating therapeutic (post-ISO) administration of LIR are warranted to evaluate its efficacy as an acute rescue therapy.

At the molecular level, SIRT1 is a critical NAD⁺-dependent deacetylase that orchestrates cellular stress resistance, mitochondrial quality control, and cardiomyocyte survival11. The AMPK/SIRT1/PGC-1α signaling axis serves as a central rheostat of mitochondrial homeostasis and myocardial redox balance37. Specifically, PGC-1α requires both AMPK-mediated phosphorylation and SIRT1-catalyzed deacetylation to achieve full transcriptional activity11. High-dose ISO exposure disrupts mitochondrial ultrastructure and suppresses mitochondrial biogenesis pathways during acute TTS-like injury38. In the present investigation, ISO administration markedly depleted myocardial SIRT1 and PGC-1α protein levels, whereas LIR treatment significantly restored the expression of both proteins (Figure 3). Because enzymatic deacetylase activity, downstream nuclear respiratory factor targets, and mitochondrial respiration were not directly assayed, these findings reflect preservation of protein abundance rather than definitive functional activation of the signaling cascade.

Nevertheless, our observations align closely with prior studies evaluating SIRT1 and PGC-1α protein expression in models of organ protection30,39,40. For instance, LIR has been shown to preserve mitochondrial architecture and mitigate oxidative stress via the SIRT1/SIRT3–FOXO3a pathway in non-alcoholic fatty liver disease41. Similarly, Zhang et al. demonstrated that LIR attenuates myocardial damage and pyroptosis in diabetic rats via activation of the SIRT1/AMPK pathway42. Furthermore, LIR pretreatment upregulates PGC-1α expression and promotes mitochondrial biogenesis to protect against gentamicin-induced acute kidney injury43. Collectively, these convergent lines of evidence support the hypothesis that preservation of the SIRT1/PGC-1α axis contributes to the cardioprotective actions of LIR during catecholamine overload.

Interestingly, although certain studies have reported LIR-mediated increases in tissue GSH concentrations30,35, we observed no significant differences in myocardial GSH levels among our experimental groups. This divergence may be attributable to dosing differences, as studies reporting GSH elevation frequently employed higher doses or longer durations of LIR treatment than the low, euglycemic dose (150 µg/kg/day for seven days) used here. Importantly, this 150 µg/kg/day regimen was sufficient to suppress lipid peroxidation (MDA), reduce nitrosative stress (nitrate), upregulate SIRT1/PGC-1α expression, and improve LVEF without inducing hypoglycemia. Mechanistically, PGC-1α coregulates endogenous antioxidant gene expression partly via nuclear factor erythroid 2–related factor 2 (Nrf2) signaling, and LIR has been shown to promote Nrf2 nuclear translocation to attenuate MDA accumulation30. Future investigations incorporating functional mitochondrial respirometry and downstream Nrf2/antioxidant enzyme assays are needed to fully validate these pathways.

Several limitations of the present study should be acknowledged. First, classical apical ballooning was not detected, likely reflecting the spatial and temporal resolution constraints of standard M-mode small-animal echocardiography under ketamine/xylazine anesthesia. Second, myocardial histopathological examination, direct mitochondrial functional assays, and apoptotic marker analyses were not performed. Third, our evaluation of oxidative and nitrosative stress was restricted to tissue MDA, GSH, and nitrate levels. Fourth, LIR was administered using a prophylactic pretreatment schedule rather than a post-injury therapeutic protocol. Finally, because only ovariectomized female rats were studied without a sham-operated non-ovariectomized control group, the specific interaction between estrogen deficiency and GLP-1 receptor agonism could not be independently dissected.

Conclusion

In an ovariectomized rat model of ISO-induced TTS-like cardiomyopathy, LIR treatment significantly attenuated myocardial lipid peroxidation and nitrosative stress—as evidenced by reduced tissue MDA and nitrate levels—while restoring myocardial SIRT1 and PGC-1α protein expression and improving left ventricular systolic function at 48 hours post-injury. These findings indicate that the cardioprotective effects of LIR against catecholamine-induced myocardial dysfunction are associated with modulation of redox balance and preservation of the SIRT1/PGC-1α signaling pathway. Future studies incorporating post-injury therapeutic dosing, sham-operated controls, and direct functional assessments of mitochondrial bioenergetics and apoptosis are warranted to further establish the translational potential of GLP-1 receptor agonists in stress-induced cardiomyopathy.

Declarations