Effects of 5-methoxytryptophan on cardiac structure and functions in a rat model of diabetic cardiomyopathy

Abstract

BACKGROUND

Diabetic cardiomyopathy (DCM) is characterized by ventricular remodeling and cardiac dysfunction, with limited therapies targeting the underlying myocardial remodeling process.

AIM

To investigate the effects of 5-methoxytryptophan (5-MTP) on cardiac structure and function in a rat model of DCM.

METHODS

Forty-eight Sprague-Dawley rats with DCM induced by a high-fat, high-fructose diet and streptozotocin (25 mg/kg) were randomized to a control group or 5-MTP treatment groups (25 mg/kg, 50 mg/kg, or 100 mg/kg). Animals were evaluated after 8 days, 16 days, or 32 days of treatment. Cardiac structure and systolic and diastolic functions were assessed by echocardiography, and serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels were measured and compared among groups.

RESULTS

5-MTP significantly reduced NT-proBNP levels after 16 days and 32 days of treatment (vs controls; P = 0.029 and P < 0.05, respectively) but not after 8 days. At day 8, 5-MTP significantly reduced left ventricular end-diastolic diameter (LVEDD; P = 0.026) and left ventricular end-systolic diameter (LVESD; P = 0.044). Significant reductions in LVEDD and LVESD from baseline were observed in the 25 mg/kg and 50 mg/kg treatment groups. Preservation of interventricular septal thickness in diastole and left ventricular posterior wall thickness in diastole was observed during the early treatment phase. However, these structural effects were not maintained after 16 days or 32 days of treatment. No significant improvements in systolic or diastolic function were observed in any treatment group.

CONCLUSION

5-MTP reduced NT-proBNP levels and attenuated early structural remodeling in this rat model of DCM, although these structural effects were not sustained nor accompanied by improvements in cardiac function. Additional studies are needed to determine whether alternative dosing strategies or longer treatment durations can produce durable structural and functional benefit.

Key Words: Diabetic cardiomyopathy; 5-Methoxytryptophan; Cardiac remodeling; Echocardiography; N-terminal pro-B-type natriuretic peptide; Experimental model

Core Tip: Diabetic cardiomyopathy (DCM) is characterized by progressive myocardial remodeling and a lack of therapies that specifically target the underlying inflammatory and fibrotic processes. This study evaluated the effects of 5-methoxytryptophan (5-MTP) in a rat model of DCM. Treatment with 5-MTP significantly reduced N-terminal pro-B-type natriuretic peptide levels and attenuated early structural remodeling but did not improve systolic or diastolic cardiac function. These findings suggest that 5-MTP may influence early myocardial remodeling; however, additional studies are needed to determine whether optimized dosing, longer treatment durations, or other treatment strategies can produce sustained structural and functional benefits.

INTRODUCTION

Diabetes mellitus (DM) remains a major global health burden and is strongly associated with cardiovascular morbidity and mortality. Epidemiological studies suggest that the increasing prevalence of DM is driven by rising rates of obesity, westernized lifestyles, and global economic development[1]. Beyond atherosclerotic complications, diabetes directly affects myocardial structure and function, contributing to the development of diabetic cardiomyopathy (DCM), a distinct myocardial disorder characterized by ventricular remodeling and systolic and diastolic dysfunction in the absence of coronary artery disease, hypertension, or valvular heart disease[2].

DCM is an increasingly recognized cardiovascular complication of DM and an important contributor to the development of heart failure. Patients with diabetes have a two- to four-fold higher risk of developing heart failure than individuals without diabetes. Furthermore, echocardiographic studies have demonstrated a high prevalence of subclinical cardiac abnormalities among patients with type 2 DM, with left ventricular diastolic dysfunction reported in approximately 35%-48% of patients. As the global burden of diabetes continues to increase, DCM is expected to become an increasingly important cause of heart failure and cardiovascular morbidity[3,4].

The pathogenesis of DCM is multifactorial and involves chronic hyperglycemia-induced inflammation, oxidative stress, apoptosis, and profibrotic signaling. A hallmark of DCM is interstitial fibrosis, characterized by excessive deposition of extracellular matrix proteins, predominantly collagen, ultimately leading to systolic and diastolic dysfunction[5].

Current therapies for DCM include renin-angiotensin-aldosterone system inhibitors and sodium-glucose cotransporter 2 inhibitors. However, no therapies specifically target the inflammatory and fibrotic myocardial remodeling that underlies the pathophysiology of DCM[6].

5-Methoxytryptophan (5-MTP), an endogenous tryptophan metabolite generated through the tryptophan hydroxylase pathway, has recently emerged as a potential antifibrotic and anti-inflammatory mediator. Experimental studies suggest that 5-MTP suppresses macrophage migration, inhibits fibroblast-to-myofibroblast differentiation, reduces collagen deposition, and modulates TGF-β-related profibrotic signaling. Notably, previous animal studies have demonstrated that 5-MTP attenuates fibrosis by inhibiting the TGF-β/SMAD3 and PI3K/AKT signaling pathways, both of which are implicated in the pathogenesis of DCM. These findings support the hypothesis that 5-MTP may mitigate adverse cardiac remodeling and preserve ventricular function in DCM[7,8].

However, the effects of 5-MTP on structural and functional cardiac alterations in DCM remain incompletely characterized. Although previous studies have demonstrated the anti-inflammatory and antifibrotic properties of 5-MTP in various experimental disease models, its effects on cardiac remodeling and ventricular function in DCM have not been fully elucidated. To our knowledge, no previous study has systematically evaluated the effects of 5-MTP on echocardiographic measures of cardiac remodeling, systolic and diastolic functions, and N-terminal pro-B-type natriuretic peptide (NT-proBNP) as a marker of myocardial stress in an experimental model of DCM.

Therefore, this study aimed to investigate the effects of 5-MTP on cardiac structure and function in a rat model of DCM by evaluating cardiac structural changes, systolic and diastolic functions using echocardiography, and BNP expression as an indicator of myocardial stress.

MATERIALS AND METHODS

Study design and sample size

This in vivo experimental study used a randomized parallel-group design in a rat model of DCM. Rats with DCM were randomly assigned to one of four groups: A control group or one of three treatment groups receiving 5-MTP at doses of 25 mg/kg, 50 mg/kg, or 100 mg/kg. Each group was further stratified by treatment duration into 8-day, 16-day, and 32-day follow-up subgroups. Each treatment group comprised 12 rats, with 3 rats assigned to each follow-up subgroup, yielding a total of 48 rats.

Sample size was determined using Federer’s formula for experimental animal studies, which indicated a minimum of three animals per subgroup. Baseline assessments of cardiac structure and function, along with NT-proBNP measurement, were performed at study initiation, followed by repeat evaluations at the designated follow-up time points (Figure 1).

Figure 1 Study design of effects of different dose 5-methoxytryptophan on rat model of diabetic cardiomyopathy on different treatment duration. 5-MTP: 5-methoxytryptophan; DCM: Diabetic cardiomyopathy.

Establishing rat model of DCM

Male Sprague-Dawley (Rattus norvegicus) rats aged 7-8 weeks, weighing approximately 200-250 g, were used in this study. Before induction of DCM, the rats underwent a 7-day acclimatization period during which they were fed a standard chow diet. A rat model of DCM was established by administering a high-fat, high-fructose (HFHF) diet ad libitum for 12 weeks, followed by a single intraperitoneal injection of streptozotocin (STZ; 25 mg/kg), adapted from previously published HFHF/HFD-STZ models of DCM[9]. Successful induction of diabetes was confirmed by fasting blood glucose levels > 200 mg/dL 3 days after STZ injection. Detailed information regarding the HFHF diet composition, STZ preparation and administration, and animal husbandry procedures is provided in Supplementary material.

Before initiation of the present intervention study, the HFHF/STZ protocol had been characterized in a separate pilot study using the same dietary and STZ induction protocol. The corresponding model characterization data are provided in Supplementary Tables 1 and 2; Supplementary Figure 1. After completion of the designated follow-up period, rats were euthanized by ketamine administration (15 mg/kg).

A previous study by Wen et al[9] had successfully established a rat model of DCM using this protocol. Three months after STZ injection, diabetic rats exhibited persistent hyperglycemia, insulin resistance, and characteristic histopathological abnormalities, including cardiac hypertrophy, fibrosis, and diastolic dysfunction[9]. The HFHF diet combined with low-dose STZ is widely recognized as a model of type 2 DM because the HFHF diet induces insulin resistance, whereas low-dose STZ causes partial pancreatic β-cell dysfunction rather than complete β-cell destruction. Consequently, this model more closely recapitulates the metabolic characteristics of type 2 DM[7,9].

5-MTP

Treatment with 5-MTP was initiated at week 12, following successful establishment of the DCM model. Rats in the treatment groups received 5-MTP at doses of 25 mg/kg, 50 mg/kg, or 100 mg/kg via intraperitoneal injection every other day for treatment durations of 8 days, 16 days, or 32 days.

The selected doses were based on previously reported in vivo studies demonstrating the biological activity of 5-MTP within a comparable dose range. Doses of approximately 23.4-25 mg/kg have been shown to exert anti-inflammatory and antifibrotic effects, whereas higher doses of up to 100 mg/kg have also been used in experimental models[7,10]. Accordingly, the 25 mg/kg and 100 mg/kg doses were selected based on previously reported effective doses, whereas the 50 mg/kg dose was included as an intermediate dose to evaluate a potential dose–response relationship. Treatment durations of 8 days, 16 days, and 32 days were selected to evaluate the time-dependent effects of 5-MTP.

Before intraperitoneal administration, 5-MTP was weighed according to the assigned dose and each rat’s body weight and then dissolved in sterile phosphate-buffered saline (pH 7.4) before injection.

Evaluation of cardiac structure and function in the rat model of DCM

Cardiac structure and function were evaluated noninvasively using transthoracic echocardiography performed before treatment initiation (prior to 5-MTP injection) and repeated during the final week of the treatment period before euthanasia. Hair over the thoracic region was removed using a body trimmer. The transducer was positioned in a stationary holder perpendicular to the rat, with manual adjustment performed as needed to obtain optimal images.

Doppler echocardiography was performed using a Versana Active ultrasound system (GE Healthcare, Chicago, IL, United States) equipped with a 15-MHz linear array transducer, following previously described techniques[11]. Echocardiographic measures of cardiac structure included left ventricular end-diastolic diameter (LVEDD, in mm), left ventricular end-systolic diameter (LVESD, in mm), interventricular septal thickness at end-diastole (IVSd, in mm), and left ventricular posterior wall thickness at end-diastole (LVPWd, in mm). Systolic function was evaluated by left ventricular ejection fraction (LVEF, %) and fractional shortening (FS, %). Diastolic function was assessed using peak early transmitral flow velocity (E, mm/s), early diastolic mitral annular tissue velocity (e′, mm/s), peak late transmitral flow velocity (A, mm/s), the ratio of E to early diastolic mitral annular tissue velocity (E/e′), and the ratio of early to late transmitral flow velocity (E/A).

NT-proBNP evaluation

At the end of the follow-up period, blood samples were collected into tubes containing a clot activator and centrifuged at 2500 rpm for 15 minutes to obtain serum. The separated serum was stored at -80 °C until analysis. Serum NT-proBNP levels were measured using a commercially available rat NT-proBNP enzyme-linked immunosorbent assay kit (E-EL-R3023; Elabscience^®, Wuhan, China) according to the manufacturer’s instructions. Detailed assay procedures are provided in Supplementary material.

Statistical analysis

Baseline characteristics are presented as the mean ± SD for normally distributed variables and as the median (interquartile range) for non-normally distributed variables, as appropriate. Normality was assessed using the Shapiro-Wilk test, with P > 0.05 indicating a normal distribution. Comparisons among groups were performed using one-way ANOVA for normally distributed data and the Kruskal-Wallis test for non-normally distributed data. Statistical significance was defined as P < 0.05.

When significant treatment effects were identified, multivariate analysis of variance (MANOVA) was performed to evaluate the simultaneous effect of treatment across multiple correlated outcomes. Overall multivariate differences among groups were assessed using Wilks’ lambda. When significant, follow-up univariate analyses (tests of between-subjects effects) were performed to identify the individual outcomes contributing to group differences, followed by Bonferroni-adjusted pairwise comparisons, as appropriate.

RESULTS

Characteristics of the rat model of DCM

A total of 48 rat models with DCM were included in the study. Mean body weight increased from 226.0 ± 18.87 g at baseline to 260.0 ± 49.91 g following HFHF diet and STZ induction. Mean body length similarly increased from 19.49 ± 1.21 cm at baseline to 20.88 ± 0.91 cm after DCM induction. Fasting blood glucose levels increased from 95.73 ± 14.92 mg/dL at baseline to 109.7 ± 15.22 mg/dL after HFHF diet exposure and further increased to 243.5 ± 88.74 mg/dL following STZ induction, confirming successful establishment of diabetes.

Baseline characteristics were generally comparable among treatment groups, with no significant between-group differences except for body weight in the 32-day treatment group. In this subgroup, rats in the control and 100 mg/kg 5-MTP group had lower body weights (209.5 g and 239.5 g, respectively) than those in the 25 mg/kg and 50 mg/kg 5-MTP groups (266.5 g and 275.5 g, respectively, P = 0.026). Detailed baseline characteristics for each treatment group are provided in Supplementary Table 3.

Effects of 5-MTP on the cardiac biomarker NT-proBNP

In the 8-day treatment group, 5-MTP treatment at any dose did not significantly affect NT-proBNP levels (P = 0.56). However, in the 16-day and 32-day treatment groups, 5-MTP significantly reduced NT-proBNP levels compared with the control group (P < 0.01; Figure 2).

Figure 2 Changes in N-terminal pro-B-type natriuretic peptide levels following treatment with different doses of 5-methoxytryptophan. A: 8-day treatment group; B: 16-day treatment group; C: 32-day treatment group. Normally distributed data are presented as mean ± SD and were analyzed using one-way analysis of variance, whereas non-normally distributed numerical data are presented as the median (minimum-maximum) and were analyzed using the Kruskal-Wallis test. Comparisons between the control and treatment groups were performed using Fisher’s least significant difference test for normally distributed data and Bonferroni-adjusted pairwise comparisons for non-normally distributed data. ^aP < 0.05, ^bP < 0.01 indicated for the indicated between-group comparisons. 5-MTP: 5-Methoxytryptophan; DCM: Diabetic cardiomyopathy.

Effects of 5-MTP on cardiac structure

In the 8-day treatment group, rats treated with 5-MTP exhibited a significant reduction in LVEDD, regardless of dose, compared with the control group (P = 0.026). In addition, the 25 mg/kg and 50 mg/kg 5-MTP groups showed significant reductions in LVEDD from baseline (ΔLVEDD -0.07 ± 0.13 and -0.10 ± 0.12, respectively; P < 0.05). However, no significant differences in LVEDD were observed among treatment groups or relative to baseline in the 16-day and 32-day treatment groups. Similar findings were observed for LVESD, which was also significantly reduced in the 5-MTP treatment group compared with the control group (P = 0.044) (Table 1).

Table 1 Changes in cardiac structural parameters after 8 days, 16 days, and 32 days of 5-methoxytryptophan treatment in a rat model of diabetic cardiomyopathy.

Treatment group	Day 8	P value	Day 16	P value	Day 32	P value
ΔLVEDD
DCM (control)	0.19 ± 0.14		-0.01 ± 0.15		0.001 ± 0.06
DCM + 5-MTP 25 mg/kg	-0.07 ± 0.13	0.013a	-0.07 ± 0.10	0.529	0.02 ± 0.14	0.799
DCM + 5-MTP 50 mg/kg	-0.10 ± 0.12	0.006a	-0.02 ± 0.15	0.969	-0.12 ± 0.07	0.077
DCM + 5-MTP 100 mg/kg	0.03 ± 0.09	0.094	0.01 ± 0.07	0.835	0.04 ± 0.05	0.587
P for treatment vs control		0.026		0.843		0.106
ΔLVESD
DCM (control)	0.19 ± 0.06		0.02 ± 0.09		-0.02 ± 0.05
DCM + 5-MTP 25 mg/kg	-0.02 ± 0.11	0.013a	-0.07 ± 0.08	0.232	0.01 ± 0.09	0.593
DCM + 5-MTP 50 mg/kg	-0.01 ± 0.14	0.021a	-0.05 ± 0.12	0.390	-0.05 ± 0.08	0.482
DCM + 5-MTP 100 mg/kg	0.01 ± 0.10	0.028	-0.02 ± 0.10	0.644	0.07 ± 0.08	0.155
P for treatment vs control		0.044		0.634		0.203

^aP < 0.05.

Normally distributed data are presented as mean ± SD and analyzed using one-way analysis of variance; non-normally distributed data are presented as the median (minimum-maximum) and analyzed using the Kruskal-Wallis test. Comparisons between the control group and treatment groups were performed using post-hoc least significant difference for normally distributed data and pairwise Bonferroni testing for non-normally distributed data. 5-MTP: 5-Methoxytryptophan; DCM: Diabetic cardiomyopathy; LVEDD: Left ventricular end-diastolic diameter; LVESD: Left ventricular end-systolic diameter.

For IVSd, significant increases from baseline were observed in the 25 mg/kg and 50 mg/kg 5-MTP groups in the 8-day treatment group (ΔIVSd 0.03 ± 0.02 and 0.02 ± 0.03, respectively; P < 0.05), whereas no significant differences were observed in the other groups. Similar findings were observed for LVPWd, with significant increases from baseline in the 50 mg/kg and 100 mg/kg 5-MTP groups (ΔLVPWd 0.01 ± 0.05 and 0.02 ± 0.06, respectively; P < 0.05).

Effects of 5-MTP on cardiac systolic function

There were no significant differences in changes in LVEF (ΔLVEF) between any 5-MTP treatment group and the control group. Likewise, no significant changes in LVEF from baseline to follow-up were observed within any treatment duration group. In the 16-day treatment group, mean ΔLVEF values were numerically greater than baseline in the 25 mg/kg, 50 mg/kg, and 100 mg/kg 5-MTP groups (6.94%, 5.95%, and 3.71%, respectively); however, these increases did not reach statistical significance (P = 0.21, 0.27, and 0.45, respectively) (Table 2).

Table 2 Changes in cardiac systolic function after 8 days, 16 days, and 32 days of 5-methoxytryptophan treatment in a rat model of diabetic cardiomyopathy.

Treatment group	Day 8	P value	Day 16	P value	Day 32	P value
ΔLVEF
DCM (control)	-13.83 ± 3.87		-1.07 ± 8.51		2.43 ± 8.17
DCM + 5-MTP 25 mg/kg	-2.12 ± 5.20	0.135	6.94 ± 7.38	0.212	-3.07 ± 14.37	0.511
DCM + 5-MTP 50 mg/kg	-5.40 ± 17.47	0.271	5.95 ± 6.39	0.271	-3.10 ± 13.91	0.509
DCM + 5-MTP 100 mg/kg	1.32 ± 8.86	0.060	3.71 ± 11.29	0.447	-6.44 ± 7.80	0.296
P for treatment vs control		0.245		0.576		0.749
ΔFS
DCM (control)	-14.18 ± 7.05		-0.73 ± 6.90		3.61 ± 8.73
DCM + 5-MTP 25 mg/kg	-2.72 ± 4.99	0.106	6.56 ± 7.28	0.259	-1.06 ± 9.51	0.543
DCM + 5-MTP 50 mg/kg	-4.24 ± 14.01	0.155	5.93 ± 6.20	0.300	-0.94 ± 13.73	0.553
DCM + 5-MTP 100 mg/kg	1.67 ± 8.50	0.032a	5.69 ± 12.78	0.317	-6.97 ± 9.45	0.181
P for treatment vs control		0.155		0.617		0.583

^aP < 0.05.

Normally distributed data are presented as mean ± SD and analyzed using one-way analysis of variance; non-normally distributed numerical data are presented as median (minimum-maximum) and analyzed using the Kruskal-Wallis test. Comparisons between the control group and treatment groups were performed using post hoc least significant difference for normally distributed data and pairwise Bonferroni testing for non-normally distributed data. 5-MTP: 5-Methoxytryptophan; DCM: Diabetic cardiomyopathy; LVEF: Left ventricular ejection fraction; FS: Fractional shortening.

Similar findings were observed for FS (ΔFS). In the 16-day treatment group, mean ∆FS values increased by 6.56%, 5.93%, and 5.69% in the 25 mg/kg, 50 mg/kg, and 100 mg/kg 5-MTP groups, respectively, although these changes were not statistically significant (Table 2).

Effects of 5-MTP on cardiac diastolic function

There were no significant differences in changes in diastolic function parameters (E, e′, A, E/e′, and E/A) between any 5-MTP treatment group and the control group. Likewise, no significant changes from baseline to follow-up were observed within any treatment duration group, except in the 32-day treatment duration group receiving 25 mg/kg 5-MTP group, which showed a significant increase in the E/A ratio compared with baseline (ΔE/A 0.66 ± 0.29, P = 0.003) (Table 3).

Table 3 Changes in cardiac diastolic function after 8 days, 16 days, and 32 days of 5-methoxytryptophan treatment in a rat model of diabetic cardiomyopathy.

Treatment group	Day 8	P value	Day 16	P value	Day 32	P value
ΔE/e'
DCM (control)	-4.63 ± 12.06		-6.83 ± 9.98		-10.46 ± 16.38
DCM + 5-MTP 25 mg/kg	1.72 ± 4.96	0.269	-2.27 ± 13.41	0.527	-1.82 ± 11.76	0.299
DCM + 5-MTP 50 mg/kg	-0.16 ± 6.32	0.431	-3.46 ± 9.48	0.638	4.73 ± 5.45	0.081
DCM + 5-MTP 100 mg/kg	1.53 ± 5.47	0.283	-1.13 ± 4.60	0.430	4.18 ± 8.48	0.091
P for treatment vs control		0.639		0.861		0.248
ΔE/A
DCM (control)	0.09 ± 0.92		-0.09 ± 0.83		-0.31 ± 0.11
DCM + 5-MTP 25 mg/kg	-0.40 ± 0.35	0.296	-0.30 ± 1.87	0.801	0.66 ± 0.29	0.003a
DCM + 5-MTP 50 mg/kg	-0.07 ± 0.25	0.730	0.34 ± 0.54	0.590	0.01 ± 0.29	0.264
DCM + 5-MTP 100 mg/kg	0.05 ± 0.62	0.936	0.25 ± 0.60	0.669	-0.06 ± 0.62	0.376
P for treatment vs control		0.693		0.834		0.020a

^aP < 0.05.

Normally distributed data are presented as mean ± SD and analyzed using one-way analysis of variance; non-normally distributed numerical data are presented as median (minimum-maximum) and analyzed using the Kruskal-Wallis test. Comparisons between the control group and treatment groups were performed using post hoc least significant difference for normally distributed data and pairwise Bonferroni testing for non-normally distributed data. 5-MTP: 5-Methoxytryptophan; DCM: Diabetic cardiomyopathy.

For the ΔE/e’ ratio, the 25 mg/kg, 50 mg/kg, and 100 mg/kg 5-MTP groups exhibited reductions from baseline in the 16-day treatment duration group (ΔE/e’ -2.27, -3.46, and -1.13, respectively); however, these changes did not reach statistical significance (Table 3).

MANOVA analysis

In the 8-day treatment group, MANOVA did not identify a significant multivariate effect across the evaluated outcomes (P = 0.26). In contrast, a significant multivariate effect was observed in the 32-day treatment group (Wilks’ lambda = 0.277, P = 0.018). Follow-up univariate analyses identified a significant difference in ΔE/A among treatment groups (P = 0.020), whereas the difference in ΔNT-proBNP among treatment groups did not reach statistical significance (P = 0.054).

DISCUSSION

The main findings of this in vivo experimental study were as follows: (1) Treatment with 5-MTP at all tested doses significantly reduced NT-proBNP levels compared with the control group in the 16-day and 32-day treatment groups but not in the 8-day treatment group; (2) Assessment of cardiac structure, as reflected by LVEDD, LVESD, IVSd, and LVPWd, demonstrated early improvement with 5-MTP treatment, particularly in the 8-day treatment group; and (3) No consistent improvements in systolic or diastolic function were observed.

In this study, we successfully established a rat model of DCM using a HFHF diet combined with low-dose STZ (25 mg/kg), as evidenced by elevated fasting blood glucose levels and abnormalities in cardiac structure and function. This HFHF/low-dose STZ protocol has been widely used to model type 2 DM because the HFHF diet induces insulin resistance, whereas low-dose STZ causes partial pancreatic β-cell dysfunction rather than complete β-cell destruction[9,12,13]. Previous work by Wen et al[9] demonstrated that this model produces persistent hyperglycemia, insulin resistance, cardiac hypertrophy, fibrosis, and diastolic dysfunction. Additional mechanisms contributing to cardiomyopathy development include increased reactive oxygen species production, activation of pro-inflammatory pathways, and subsequent myocardial remodeling and fibrosis[14-16]. In addition to the diabetic phenotype, elevated NT-proBNP levels were observed, likely reflecting increased myocardial wall stress, hemodynamic burden, and structural remodeling, further supporting successful establishment of DCM in this model.

Our findings suggest a potential cardioprotective effect of 5-MTP in this rat model of DCM. 5-MTP is an endogenous tryptophan metabolite with emerging anti-inflammatory, anti-apoptotic, and anti-fibrotic properties. These pleiotropic effects are particularly relevant to DCM, in which inflammation, apoptosis, fibrosis, and adverse myocardial remodeling contribute to progressive ventricular dysfunction and increased wall stress[17]. NT-proBNP is a well-established biomarker for the diagnosis and prognosis of heart failure, and longitudinal changes in NT-proBNP have been associated with disease progression and clinical outcomes, including in DCM[18]. In our study, treatment with 5-MTP significantly reduced NT-proBNP levels after 16 days and 32 days of treatment but not after 8 days. This delayed reduction suggest that the biological effects of 5-MTP may require sustained treatment before translating into measurable attenuation of ventricular stress. Experimental studies have shown that 5-MTP suppresses oxidative stress and inflammatory signaling while improving cardiac structure and function, including reductions in left ventricular dilatation and apoptosis and improved systolic performance in a rat model of post-infarction cardiac injury[19]. Collectively, these findings provide biological plausibility that 5-MTP may attenuate ventricular wall stress, as reflected by reduced NT-proBNP levels.

Additionally, we observed an early beneficial effect of 5-MTP on cardiac remodeling, as reflected by reductions in LVEDD and LVESD in the 8-day treatment group. LVEDD and LVESD are echocardiographic measures of left ventricular chamber size, and increases in these parameters are indicative of adverse ventricular remodeling that may ultimately impair cardiac function[20]. We also observed preservation of IVSd and LVPWd, suggesting attenuation of progressive myocardial wall thinning and providing further evidence of an early structural benefit. However, these effects were not sustained at longer treatment durations. This may reflect the chronic and dynamic nature of remodeling in DCM, in which early structural changes may occur rapidly but require prolonged treatment to achieve sustained reverse remodeling.

This interpretation is supported by previous experimental studies. Xiao et al[21] demonstrated that luteolin therapy reduced LVEDD and ventricular wall abnormalities in diabetic rats within approximately 1 week, likely through attenuation of inflammation and oxidative stress, suggesting that structural responses to cardioprotective interventions may occur early. In contrast, Kim et al[22] reported that more sustained reverse remodeling, including reductions in the LVEDD index and left ventricular mass index, required 10 weeks of thalidomide treatment, whereas Zhao et al[23] observed partial reversal of ventricular dilatation only after 16 weeks of liraglutide therapy, likely through antifibrotic mechanisms involving suppression of collagen synthesis. Collectively, these studies suggest that although early changes in ventricular dimensions may occur rapidly, sustained reverse remodeling, particularly fibrosis-related remodeling, often requires prolonged therapeutic exposure.

The anti-inflammatory properties of 5-MTP may also contribute to maintenance of LVPWd through suppression of NF-κB signaling, reduction of pro-inflammatory cytokines, including interleukin-1β and interleukin-6, inhibition of the NLRP3 inflammasome, and attenuation of macrophage infiltration[17,19,24]. Together, these mechanisms may limit extracellular matrix deposition, ventricular stiffening, and adverse myocardial remodeling, thereby helping to preserve LVPWd.

In our study, we did not observe a significant benefit of 5-MTP on cardiac systolic or diastolic function. Despite evidence of favorable structural effects and reduced NT-proBNP levels, these changes did not translate into measurable functional improvement. One possible explanation is that the follow-up duration was insufficient for early cellular and structural reverse remodeling to manifest as functional recovery, which may occur later in the disease-modification process. Previous experimental studies support this interpretation. Chan et al[25] reported functional improvement with metformin only after approximately 6 weeks of treatment, whereas Zhao et al[23] demonstrated improved systolic function with liraglutide after 16 weeks of therapy, suggesting that restoration of cardiac function may require more prolonged treatment than early structural remodeling. Differences among studies may also reflect variation in animal models, therapeutic agents, treatment duration, and the timing of intervention relative to the stage of myocardial remodeling. Accordingly, these findings should be interpreted cautiously, as the absence of significant functional improvement precludes definitive conclusions regarding the effects of 5-MTP on cardiac function.

Although MANOVA demonstrated a significant overall multivariate effect in the 32-day treatment group, subsequent univariate analyses identified a significant difference only for ΔE/A, whereas the remaining individual outcomes did not reach statistical significance. These findings suggest that the overall treatment effect may reflect modest combined changes across multiple correlated cardiac parameters rather than substantial effects on individual variables.

Several factors may explain the inconsistent functional findings observed in this study. First, the limited sample size may have reduced the statistical power to detect modest treatment effects. Second, the relatively short follow-up period may have been insufficient to capture delayed functional benefits. Third, dose optimization may be necessary to enhance treatment response and sustain the early structural improvements observed in this study. In addition, the absence of a non-diabetic control group and a standard treatment comparator limits assessment of disease progression and comparison with established therapies. Finally, molecular and histopathological analyses would provide additional mechanistic insight into the observed treatment effects. Collectively, these findings suggest that 5-MTP may exert an early modulatory effect on cardiac remodeling, whereas demonstrable functional recovery may require longer treatment duration, optimized dosing, and extended follow-up.

CONCLUSION

5-MTP may attenuate myocardial wall stress and early structural remodeling in an experimental rat model of DCM, as evidenced by significant reductions in NT-proBNP levels. However, no significant improvements in systolic or diastolic cardiac function were observed. Further studies with longer treatment durations, optimized dosing strategies, and comprehensive mechanistic evaluation are warranted to better define the therapeutic potential of 5-MTP.

ACKNOWLEDGEMENTS

The authors would like to express their sincere gratitude to the staff of the Animal Research Facility, IMERI, Jakarta, Indonesia, and the Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia, for their invaluable support and assistance during this study.