Artículo de investigación

La N-lactoil-fenilalanina mejora la función cardíaca en un modelo de ratón de miocardiopatía diabética mediante la regulación del metabolismo lipídico

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DOI:

10.3791/72633

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1 de septiembre de 2026

 ,  ,  ,  ,  ,  , 
1Nanjing Drum Tower Hospital Clinical College, Nanjing University of Chinese Medicine, 2Affiliated Hospital of Medical School, Nanjing University, 3Institute of Advanced Synthesis, School of Chemistry and Molecular Engineering, Nanjing Tech University

En este artículo

Resumen

Este protocolo describe enfoques in vivo e in vitro para investigar cómo el N-lactoil-fenilalanina afecta la cardiomiopatía diabética, el metabolismo lipídico cardíaco y la síntesis de colesterol, con especial atención a la vía de la proteína quinasa activada por AMP alfa 1/reductasa de la hidroximetilglutaril-coenzima A.

Resumen

La cardiomiopatía diabética (DCM) es una de las complicaciones más graves de la diabetes. La N-lactoil-fenilalanina (Lac-Phe), un metabolito endógeno, puede promover la pérdida de peso y mejorar la homeostasis glucídica. Este estudio tuvo como objetivo investigar el efecto terapéutico de Lac-Phe sobre la DCM y explorar además el mecanismo subyacente. Se administró Lac-Phe a ratones con diabetes tipo 2 que presentaban DCM. Lac-Phe mejoró la función contráctil cardíaca, redujo el remodelado patológico y disminuyó la acumulación de lípidos en el corazón. In vitro, se estudiaron los efectos de Lac-Phe en células H9c2 estimuladas con glucosa alta y ácido palmítico. Lac-Phe redujo la acumulación de lípidos al disminuir la síntesis de colesterol en las células H9c2. Mecanísticamente, Lac-Phe promovió la fosforilación y activación de la proteína quinasa activada por monofosfato de adenosina alfa 1 (AMPKα1). Posteriormente, la AMPKα1 activada fosforiló e inhibió su diana descendente, la 3-hidroxi-3-metilglutárico-coenzima A reductasa (HMGCR), lo que condujo a una reducción en la síntesis de colesterol y, en última instancia, a la mejora del desequilibrio del metabolismo lipídico en las células H9c2. Estos hallazgos indican que Lac-Phe mejora el metabolismo lipídico en células H9c2 en el contexto de la DCM a través de la vía AMPKα1/HMGCR, mejorando así la función contráctil cardíaca y reduciendo el remodelado patológico. Lac-Phe podría representar un enfoque terapéutico potencial para la cardiomiopatía diabética.

Introducción

La diabetes es una enfermedad metabólica caracterizada por hiperglucemia persistente1. En las últimas dos décadas, la diabetes se ha convertido en una de las diez principales causas de muerte en todo el mundo, con más de seis millones de personas fallecidas cada año debido a la diabetes y sus complicaciones.2. La miocardiopatía diabética (DCM), una afección cardíaca patológica resultante de la diabetes, representa una de las complicaciones más graves asociadas con la diabetes3,4. Actúa como un factor de riesgo independiente para la insuficiencia cardíaca y se caracteriza por alteraciones estructurales y funcionales del miocardio5,6, incluyendo fibrosis cardíaca e hipertrofia de los miocitos cardíacos, que finalmente conducen a disfunción diastólica y sistólica. Aunque varias terapias cardiovasculares convencionales, como β-bloqueantes, inhibidores de la enzima convertidora de angiotensina e inhibidores del receptor de tipo 1 de angiotensina II, se utilizan actualmente en el manejo de la miocardiopatía dilatada7,8, su eficacia sigue siendo subóptima en entornos clínicos. Dado que los mecanismos subyacentes a la miocardiopatía diabética no se han estudiado exhaustivamente9, actualmente no existe una estrategia de tratamiento clara dirigida a esta enfermedad10,11Las manifestaciones clínicas de la miocardiopatía dilatada progresan desde una disfunción diastólica asintomática en la etapa inicial hasta una disfunción sistólica e insuficiencia cardíaca clínica en la etapa avanzada, e incluso la muerte12,13Por lo tanto, existe una necesidad urgente de explorar y desarrollar opciones de tratamiento eficaces para esta afección.

El metabolismo lipídico desequilibrado es una característica clave de la miocardiopatía diabética (DCM)14. En la DCM, los niveles elevados de ácidos grasos libres (AGL) en sangre pueden provocar una captación excesiva de ácidos grasos por los cardiomiocitos15,16. El metabolismo de ácidos grasos en los cardiomiocitos depende principalmente de la oxidación de ácidos grasos. Con el tiempo, la oxidación de ácidos grasos en los cardiomiocitos es insuficiente para metabolizar completamente todos los ácidos grasos ingeridos, y se acumulan excesos de ácidos grasos y sus metabolitos intermedios en los cardiomiocitos17. Esta sobrecarga lipídica excesiva induce lipotoxicidad, altera el suministro energético miocárdico y promueve el estrés oxidativo cardíaco y las respuestas inflamatorias, causando finalmente un daño severo a los cardiomiocitos18,19. Por lo tanto, regular activamente el metabolismo lipídico cardíaco podría representar un objetivo terapéutico potencial para la DCM20.

La N-lactoil-fenilalanina (Lac-Phe) es un metabolito endógeno cuya concentración aumenta rápidamente después del ejercicio21. Mecánicamente, la Lac-Phe es sintetizada por la enzima citosólica carnosina dipeptidasa 2 (CNDP2) mediante la condensación de lactato y fenilalanina. Esta vía biosintética está ampliamente distribuida en diversos tipos celulares, incluidos macrófagos, células epiteliales y células madre mesenquimales, que secretan Lac-Phe a la circulación tras la actividad física22. Se ha demostrado que la administración crónica de Lac-Phe reduce eficazmente la ingesta de alimentos, disminuye la obesidad y el peso corporal, y mejora la homeostasis glucosa21. Un estudio reciente ha mostrado que la Lac-Phe puede mejorar la capacidad del metabolismo lipídico de las microglías/macrófagos tras una lesión de la médula espinal23. Curiosamente, estudios indican que tanto la administración aguda como crónica de metformina, un fármaco de primera línea para la diabetes tipo 2, puede elevar los niveles de Lac-Phe y que esta última media los efectos antiobesidad de la metformina22. Además, Sha et al. recientemente demostraron que la Lac-Phe mejora la muerte celular miocárdica aliviar la disfunción mitocondrial, mejorando así la insuficiencia cardíaca inducida por la constricción aórtica transversal (TAC)24. La hiperglucemia prolongada y la obesidad son factores importantes que contribuyen a la desregulación del metabolismo lipídico en la miocardiopatía diabética (DCM)25. En conjunto, esta evidencia sugiere que la Lac-Phe podría tener un efecto terapéutico potencial sobre la DCM. Sin embargo, hasta la fecha, no se ha investigado el papel específico de la Lac-Phe en la DCM.

En este estudio, descubrimos que la administración exógena de Lac-Phe a ratones con cardiomiopatía diabética mejoró eficazmente la función contráctil del corazón y atenuó la remodelación patológica cardíaca. Encontramos que Lac-Phe mejoró la función cardíaca en ratones con MCD mediante la regulación del desequilibrio del metabolismo lipídico. Lac-Phe redujo la acumulación de gotas lipídicas (GL) en cardiomiocitos de ratones con MCD y disminuyó el contenido de colesterol en el corazón. Mecánicamente, Lac-Phe promovió la fosforilación y activación de la proteína quinasa activada por adenosina monofosfato alfa 1 (AMPKα1), promoviendo así la fosforilación de la 3-hidroxi-3-metilglutaril-coenzima A reductasa (HMGCR). La fosforilación de HMGCR reduce su actividad, lo que resulta en una menor producción de colesterol y, por ende, mejora el desequilibrio del metabolismo lipídico en los cardiomiocitos. Estos resultados proporcionan una base teórica para el posible tratamiento de la MCD con Lac-Phe.

Protocolo

The animal study protocol was approved by the Institutional Ethics Committee of Nanjing Drum Tower Hospital (Approval Number: 2025AE1065, 26 September 2025) and was conducted in accordance with the guidelines outlined in the Guidelines for the Care and Use of Laboratory Animals (8th Edition), published by the National Institutes of Health of the United States.

Animals

Thirty 5-week-old male C57BL/6J mice were used in this study. After 1 week of adaptive feeding, all mice were housed with access to food and water under an alternating 12 h day-night cycle at a temperature of 22°C ± 2°C and humidity of 40%–60%. An HFD combined with low-dose STZ was used to establish the DCM mouse model (Figure 1A). Four weeks after initiation of high-fat diet (HFD; 60% fat, 20% protein, and 20% carbohydrate) feeding, mice were fasted overnight and subsequently administered streptozotocin (STZ; 8 mg/mL dissolved in citrate buffer, pH 4.5) via intraperitoneal injection at a dosage of 35 mg/kg/day for three consecutive days. Control group mice were fed a control diet (10% fat, 20% protein, and 70% carbohydrate) and received equivalent volumes of citrate buffer. Following 15 additional weeks of high-fat diet maintenance, mice meeting both criteria, random blood glucose of ≥13.3 mmol/L and left ventricular ejection fraction (LVEF) of <55%, were defined as successfully established DCM models. These validated DCM mice were numbered and randomly assigned to the DCM and DCM + Lac-Phe groups using a random number table. Approximately half of the mice failed to develop the model; these animals were euthanized in accordance with animal ethics requirements. In brief, mice were divided into three groups: Control, DCM, and DCM + Lac-Phe (n = 10 for each group). All 10 mice per group underwent echocardiography, body weight measurement, and OGTT. Following the 28-day treatment period, hearts were collected for subsequent analyses. There were 10 mice in each group. Of these, three mice per group were used for transcriptome sequencing, three were used for transmission electron microscopy and histological staining, and three were used for protein expression analysis. The remaining mouse in each group was reserved as a backup. After successful establishment of the DCM model, Lac-Phe was dissolved in normal saline to a final concentration of 10 mg/mL. Mice in the DCM + Lac-Phe group received intraperitoneal Lac-Phe at 50 mg/kg/day in an injection volume of 5 mL/kg, once daily for 28 consecutive days. Mice in the Control and DCM groups were administered an equivalent volume of normal saline as vehicle control.

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Figure 1. Lac-Phe improves cardiac function and pathological remodeling in mice with diabetic cardiomyopathy. (A) Schematic of the HFD/STZ-induced DCM mouse model and Lac-Phe treatment protocol. (B) Body weights of mice in the Control, DCM, and DCM + Lac-Phe groups during the 4-week treatment period; n = 10 per group. (C) Oral glucose tolerance test (OGTT) in the indicated groups; n = 10 per group. (D) Representative echocardiographic images at 0, 2, and 4 weeks of treatment. (E,F) Quantification of left ventricular ejection fraction (LVEF) (E) and fractional shortening (FS) (F); n = 10 per group. (G) Representative hematoxylin and eosin (H&E)-stained heart sections. Scale bar = 1 mm. (H,I) Representative Masson’s trichrome staining of heart sections (H) and quantification of fibrotic area (I); n = 3 per group. Scale bar = 50 μm. (J,K) Representative wheat germ agglutinin (WGA) staining of heart sections (J) and quantification of relative cardiomyocyte area (K); n = 3 per group. Scale bar = 20 μm. Data are presented as the mean ± standard deviation. P < 0.05, **P < 0.001. Please click here to view a larger version of this figure.

Cardiac function

Following anesthesia with isoflurane inhalation, mice were secured in a supine position on a temperature-controlled platform (37°C). Left ventricular dimensions and mitral valve blood flow velocity during diastole and systole were measured using an echocardiography machine equipped with a 40 MHz transducer, from which LVEF and shortening fraction (FS) were calculated. Three consecutive cardiac cycles were collected from each mouse.

Cell treatment

H9c2 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Cells were passaged every 2–3 days upon reaching 70%–80% confluence. For experiments, cells were seeded in 10 cm dishes and then divided into three groups: Control, high glucose/palmitic acid (HG/PA), and HG/PA + Lac-Phe. Cells in the HG/PA and HG/PA + Lac-Phe groups were first exposed to 0.25 mM palmitic acid and 50 mM glucose for 24 h. Thereafter, cells in the HG/PA + Lac-Phe group were treated with 10 μg/mL Lac-Phe for an additional 24 h while HG/PA exposure was maintained; cells in the HG/PA group received an equivalent volume of PBS under the same HG/PA conditions. In the p-AMPK inhibition experiments, 10 μM AMPK-IN-3 (dissolved in DMSO) was added simultaneously with Lac-Phe for 24 h, and the corresponding control groups received an equal volume of DMSO. For the atorvastatin experiments, H9c2 cells under HG/PA conditions were treated with 10 μM atorvastatin for 24 h, either alone or in combination with 10 μg/mL Lac-Phe.

Histological staining

After 28 days of Lac-Phe administration, mouse hearts were harvested and immersion-fixed in 4% paraformaldehyde (pH 7.4) for 24 h. Fixed cardiac tissues were paraffin-embedded and sectioned into 6-μm-thick slices. Tissue sections were stained with hematoxylin and eosin, wheat germ agglutinin, and Masson’s trichrome. All staining procedures were performed strictly following the manufacturers’ protocols. Whole-slide imaging was performed at 20× magnification using a high-resolution brightfield microscope equipped with a digital camera system.

Western blot analysis

Protein concentration was determined using the Bradford assay. Tissue lysates were resolved by SDS-PAGE on 10% (w/v) acrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes. Nonspecific binding sites were blocked with 5% bovine serum albumin for 2 h at room temperature. Membranes were then incubated overnight at 4°C with primary antibodies (diluted 1:1,000), followed by six washes with TBST (5 min each) on a shaker. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1:5,000) for 2 h at room temperature, followed by another six washes with TBST (5 min each). Protein bands were visualized using an enhanced chemiluminescence reagent kit. Quantification of western blot results was performed by densitometric analysis using ImageJ software. All antibodies used in this study are listed in the Table of Materials.

Transcriptomics

Total RNA was isolated from cardiac tissue of C57BL/6J mice using TRIzol Reagent and RNeasy mini kits. RNA quality was evaluated using a 2100 bioanalyzer, demonstrating high RNA purity and integrity. RNA-seq libraries were prepared according to the VAHTS Universal V10 RNA-seq Library Prep Kit and sequenced using an Illumina NovaSeq 6000 System (single-read, 150 bp). Differentially expressed genes were identified using DESeq2, with an adjusted P value < 0.05 and an absolute log₂ fold change > 1 considered statistically significant. Gene Ontology (GO) enrichment analysis was conducted on differentially expressed genes, and the significance of enrichment of differentially expressed genes in GO terms was calculated using the hypergeometric distribution algorithm.

Oil Red O staining and LD540 lipid staining

Cardiac tissue was embedded in OCT compound and then cut into 10-μm-thick sections. The frozen sections were equilibrated at room temperature for 30 min and fixed with 4% paraformaldehyde for 10 min. After washing with PBS, the sections were placed in 60% isopropyl alcohol for 15 s and covered with Oil Red O working solution for 30 min. The sections were then treated with 60% isopropyl alcohol for 15 s and washed three times with PBS. For LD540 lipid staining, H9c2 cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were then incubated with 10 μM LD540 for 30 min at room temperature.

Transmission electron microscopy (TEM)

Fresh mouse heart tissues were cut into 1–2 mm3 pieces, fixed in electron microscope fixative at 4°C for 4 h, then washed with double-distilled water and immersed in ethanol solutions of progressively increasing concentrations. The samples were embedded, sectioned, and then observed under a transmission electron microscope.

Total cholesterol (TC) content

Total cholesterol (TC) levels in serum and H9c2 cell samples were measured using the Amplex Red Cholesterol and Cholesteryl Ester Assay Kit according to the manufacturer’s instructions.

Oral Glucose Tolerance Test (OGTT)

Mice were fasted for 14 h (from 19:00 to 9:00) and gavaged with glucose (20% stock, 2 g/kg). Blood glucose levels were determined using blood collected from the tail vein at 0, 15, 30, 60, and 120 min after gavage.

Cell counting kit-8 (CCK-8) measurement

CCK-8 was used to measure cell viability. H9c2 cells were seeded into 96-well plates at an initial density of 3 × 104 cells/well. After treatment with different concentrations of Lac-Phe under HG/PA conditions for 24 h, 10 μL of the kit reagent was added to 100 μL of culture medium per well, followed by incubation at 37°C for 4 h. The absorbance was measured at 450 nm using a multimode microplate reader to obtain the optical density (OD) value.

Molecular docking

The crystal structures of Lac-Phe and AMPKα1 were downloaded from PubChem26. The molecular docking process was carried out using CB-DOCK227.

Statistical analysis

All echocardiographic measurements, histological staining, and image quantifications were performed by investigators blinded to group assignment. All data analyses were performed using Prism and SPSS 20.0. Each experiment was repeated at least three times, and the data were presented as the mean ± standard deviation. Homogeneity of variance was assessed using Levene’s test. Normality was confirmed by graphical inspection of residuals. Comparisons among multiple groups were conducted using one-way analysis of variance, followed by Tukey's post hoc test for pairwise comparisons. For OGTT data, two-way repeated-measures ANOVA was used, with group as the between-subjects factor and time as the within-subjects factor, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.

Resultados

Lac-Phe improves cardiac function in diabetic cardiomyopathy mice

We induced DCM in C57BL/6J mice using an HFD combined with STZ injections (Figure 1A) and randomly assigned the successfully established DCM mice to an untreated group (DCM group) and a Lac-Phe treatment group, with control C57BL/6J mice serving as the negative control. According to the study by Li et al.21, we selected 50 mg/kg as the therapeutic dose of Lac-Phe. After successful model establishment, the Lac-Phe treatment group received four weeks of exogenous Lac-Phe injections, while the untreated group received injections of an equivalent volume of saline. Compared with the DCM group, mice with diabetic cardiomyopathy treated with Lac-Phe exhibited a significant reduction in body weight after four weeks (Figure 1B). OGTT revealed that, compared with the negative control group, mice in the DCM group had significantly elevated blood glucose levels at all measured time points, indicating impaired glucose tolerance. In contrast, DCM mice treated with Lac-Phe showed improved glucose tolerance (Figure 1C). Echocardiographic analysis revealed that, compared with negative control mice, mice with DCM exhibited significantly reduced LVEF and FS (Figure 1D–F). Treatment with Lac-Phe increased LVEF and FS in DCM mice. Histopathological analysis using H&E staining showed pathological hypertrophy in the cardiac tissue of DCM mice compared with control mice (Figure 1G). Lac-Phe treatment markedly alleviated myocardial hypertrophy. Masson’s trichrome staining further demonstrated that Lac-Phe significantly alleviated myocardial fibrosis in DCM hearts (Figure 1H,I). WGA staining indicated that Lac-Phe significantly reduced the cross-sectional area of cardiomyocytes in DCM hearts (Figure 1J,K). These experiments demonstrate that exogenous administration of Lac-Phe effectively improves cardiac function and ameliorates pathological remodeling in mice with DCM.

Lac-Phe ameliorates dysregulated lipid metabolism in DCM

To further elucidate the mechanism of Lac-Phe in DCM, we performed transcriptomic analysis of mouse cardiac tissues. Following data acquisition, GO and KEGG pathway analyses were conducted. GSEA of the fatty acid metabolic process and KEGG pathway enrichment analysis indicated alterations in pathways related to lipid metabolism (Figure 2A,B). To further investigate how Lac-Phe functions in the hearts of DCM mice, we conducted TEM analysis (Figure 2C,D) to examine whether Lac-Phe treatment altered the ultrastructural morphology of the hearts of DCM mice. TEM imaging revealed a significant increase in LD density within the hearts of DCM mice compared with control mice, whereas Lac-Phe administration effectively reduced this LD density. Given the established significance of lipid metabolism dysregulation in DCM, we further assessed cardiac lipid accumulation using Oil Red O staining (Figure 2E,F). Histological analysis demonstrated markedly increased lipid accumulation in the hearts of DCM mice, whereas Lac-Phe treatment reduced this lipid accumulation. Lac-Phe treatment also reduced the TC level in the serum of DCM mice (Figure 2G). To select an appropriate dose of Lac-Phe for the in vitro experiments, we employed the CCK-8 assay to measure the viability of H9c2 cells treated with various concentrations of Lac-Phe under HG/PA conditions (Figure 2H). H9c2 cells treated with Lac-Phe at a concentration of 10 μg/mL under HG/PA conditions exhibited the highest survival rate among the tested concentrations. Thus, we selected 10 μg/mL Lac-Phe for the subsequent experiments. H9c2 cells exposed to HG/PA showed significant lipid accumulation, as demonstrated by LD540 staining and fluorescence quantification (Figure 2I,J), and TC content was also increased (Figure 2K). Lac-Phe treatment reversed these changes, significantly reducing both lipid accumulation and TC levels in H9c2 cells. Collectively, these experimental findings demonstrate that Lac-Phe effectively attenuates cardiac lipid accumulation and ameliorates lipid metabolism dysregulation in DCM mice and reduces lipid accumulation and TC levels in HG/PA-treated H9c2 cells.

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Figure 2. Lac-Phe ameliorates dysregulated lipid metabolism in diabetic cardiomyopathy. (A) Gene set enrichment analysis (GSEA) of the fatty acid metabolic process in the Control and DCM groups. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis comparing the DCM and DCM + Lac-Phe groups. (C) Representative transmission electron microscopy (TEM) images of cardiac tissues, with red arrows indicating lipid droplets (LDs). Scale bar = 2 μm. (D) Quantification of the mean number of LDs per 100 μm2; n = 3 per group. (E,F) Representative Oil Red O staining of cardiac tissues (E) and quantification of the relative Oil Red O-positive area (F); n = 3 per group. Scale bar = 20 μm. (G) Relative total cholesterol (TC) levels in serum from the indicated groups; n = 3 per group. (H) Cell viability measured by the CCK-8 assay in H9c2 cells treated with 0, 1, 5, 10, or 20 μg/mL Lac-Phe under HG/PA conditions. (I,J) Representative LD540 staining of H9c2 cells (I) and quantification of mean LD540 fluorescence intensity (J); n = 3 per group. Scale bars = 200 μm. (K) TC levels in H9c2 cells from the indicated groups; n = 3 per group. Data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Please click here to view a larger version of this figure.

Lac-Phe increases HMGCR phosphorylation in the hearts of DCM mice and H9c2 cells

To identify potential targets through which Lac-Phe improves lipid metabolism, we screened DCM-associated proteins using GeneCards28 and predicted proteins potentially interacting with Lac-Phe using SwissTargetPrediction29 (Figure 3A). By comparing the results, we identified six potential target genes (Figure 3B). Analysis of these six genes revealed that HMGCR is a core regulator of cholesterol metabolism and also plays a central role in DCM (Figure 3C)30. To determine whether Lac-Phe regulates lipid metabolism via HMGCR, we first examined the p-HMGCR/HMGCR ratio in mouse hearts. Western blot analysis revealed that the p-HMGCR/HMGCR ratio was decreased in the hearts of DCM mice, whereas Lac-Phe treatment increased this ratio (Figure 3D,E). These findings are consistent with increased HMGCR phosphorylation and reduced HMGCR activity following Lac-Phe treatment. In vitro experiments showed that, under HG/PA conditions, the p-HMGCR/HMGCR ratio was decreased in H9c2 cells, whereas Lac-Phe treatment increased this ratio (Figure 3F,G). These results demonstrate that Lac-Phe increases HMGCR phosphorylation in the hearts of DCM mice and in HG/PA-treated H9c2 cells, supporting the involvement of HMGCR in the effects of Lac-Phe on lipid metabolism.

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Figure 3. Lac-Phe increases HMGCR phosphorylation in the hearts of mice with diabetic cardiomyopathy and in H9c2 cells. (A) Venn diagram showing the overlap between DCM-associated proteins and predicted Lac-Phe-associated proteins. (B) Six overlapping proteins associated with DCM and Lac-Phe. (C) Protein-protein interaction network showing HMGCR within the network of DCM-associated proteins. (D,E) Representative western blot images of p-HMGCR and HMGCR in cardiac tissues from the indicated groups (D) and quantification of the p-HMGCR/HMGCR ratio (E); n = 3 per group. (F,G) Representative western blot images of p-HMGCR and HMGCR in H9c2 cells from the indicated groups (F) and quantification of the p-HMGCR/HMGCR ratio (G); n = 3 per group. β-Tubulin was used as the loading control. Data are presented as the mean ± standard deviation. *P < 0.05. Please click here to view a larger version of this figure.

Lac-Phe combined with atorvastatin improves lipid metabolism in H9c2 cells

To further evaluate the effect of Lac-Phe on lipid metabolism via HMGCR, we used atorvastatin as a control. In vitro experiments showed that, under HG/PA conditions, combined treatment with Lac-Phe and atorvastatin produced a greater reduction in lipid accumulation than atorvastatin treatment alone. LD540 staining and fluorescence quantification demonstrated that combined treatment with Lac-Phe and atorvastatin reduced LD accumulation in H9c2 cells (Figure 4A,B). TC content measurement showed a similar effect (Figure 4C). Western blot analysis showed that combined treatment with Lac-Phe and atorvastatin significantly increased the p-HMGCR/HMGCR ratio (Figure 4D,E). These results indicate that combined Lac-Phe and atorvastatin treatment is associated with reduced lipid accumulation and TC levels and increased HMGCR phosphorylation in H9c2 cells.

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Figure 4. Lac-Phe combined with atorvastatin improves lipid metabolism in H9c2 cells. (A,B) Representative LD540 staining of H9c2 cells from the indicated treatment groups (A) and quantification of mean LD540 fluorescence intensity (B); n = 3 per group. Scale bar = 200 μm. (C) Total cholesterol (TC) levels in H9c2 cells from the indicated treatment groups; n = 3 per group. (D,E) Representative western blot images of p-HMGCR and HMGCR in H9c2 cells from the indicated treatment groups (D) and quantification of the p-HMGCR/HMGCR ratio (E); n = 3 per group. β-Tubulin was used as the loading control. Data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, ****P < 0.0001. Please click here to view a larger version of this figure.

Lac-Phe increases AMPKα1 phosphorylation

To further explore the molecular mechanism by which Lac-Phe regulates HMGCR, we reanalyzed the mouse cardiac transcriptome data. Through KEGG analysis of the transcriptomic data, we identified 160 differentially enriched pathways between the DCM and Control groups and 89 differentially enriched pathways between the DCM and DCM + Lac-Phe groups (Figure 5A). Sixty pathways overlapped between these two comparisons. From these 60 overlapping pathways, we selected the AMPK pathway. AMPK plays a critical role in regulating lipid metabolism in DCM31. Furthermore, AMPK is upstream of HMGCR (Figure 5B). We hypothesized that Lac-Phe might regulate HMGCR by modulating AMPK, ultimately improving lipid metabolism. To test this hypothesis, we first performed molecular docking of AMPKα1 and Lac-Phe using CB-DOCK2. The molecular docking results showed that the best-ranked predicted binding conformation between Lac-Phe and AMPKα1 had an affinity of −7.6 kcal/mol (Figure 5C). These results suggest a potential interaction between Lac-Phe and AMPKα1 but do not establish direct intracellular binding. To determine whether Lac-Phe affects the AMPK pathway in DCM, we examined AMPKα1 phosphorylation in the hearts of DCM mice. Western blot analysis revealed that the p-AMPKα1/AMPKα1 ratio was decreased in the hearts of DCM mice, whereas Lac-Phe treatment increased this ratio (Figure 5D,E). In vitro experiments showed that, under HG/PA conditions, the p-AMPKα1/AMPKα1 ratio was decreased in H9c2 cells, whereas Lac-Phe treatment increased this ratio (Figure 5F,G). These findings indicate that Lac-Phe increases AMPKα1 phosphorylation in DCM mouse hearts and HG/PA-treated H9c2 cells.

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Figure 5. Lac-Phe increases AMPKα1 phosphorylation in the hearts of mice with diabetic cardiomyopathy and in H9c2 cells. (A) Venn diagram showing the overlap between pathway enrichment results for the DCM/Control and DCM + Lac-Phe/DCM comparisons. (B) Schematic illustrating AMPK and HMGCR phosphorylation and the associated inhibition of cholesterol synthesis. (C) Predicted three-dimensional molecular docking interaction between Lac-Phe and AMPKα1; binding affinity = −7.6 kcal/mol. (D,E) Representative western blot images of p-AMPKα1 and AMPKα1 in cardiac tissues from the indicated groups (D) and quantification of the p-AMPKα1/AMPKα1 ratio (E); n = 3 per group. (F,G) Representative western blot images of p-AMPKα1 and AMPKα1 in H9c2 cells from the indicated groups (F) and quantification of the p-AMPKα1/AMPKα1 ratio (G); n = 3 per group. GAPDH was used as the loading control. Data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01. Please click here to view a larger version of this figure.

AMPK inhibition attenuates Lac-Phe-mediated HMGCR phosphorylation and improvement of lipid metabolism in H9c2 cells

We then added a p-AMPK inhibitor to H9c2 cells receiving Lac-Phe treatment. The p-AMPK inhibitor decreased the p-AMPKα1/AMPKα1 ratio (Figure 6A,B) and reduced the p-HMGCR/HMGCR ratio (Figure 6C,D). These findings indicate that inhibition of AMPK phosphorylation is accompanied by reduced HMGCR phosphorylation during Lac-Phe treatment. The p-AMPK inhibitor also diminished the effect of Lac-Phe on lipid metabolism. LD540 staining and fluorescence quantification showed increased lipid accumulation in H9c2 cells treated with the p-AMPK inhibitor compared with cells receiving Lac-Phe without AMPK inhibition (Figure 6E,F). TC levels in H9c2 cells also increased following AMPK inhibition (Figure 6G). These results collectively support the involvement of AMPKα1/HMGCR signaling in the effects of Lac-Phe on lipid metabolism. Lac-Phe increased AMPKα1 phosphorylation, which was associated with increased HMGCR phosphorylation and reduced lipid accumulation and TC levels. Inhibition of AMPK attenuated these effects in H9c2 cells, supporting the hypothesis that Lac-Phe improves lipid metabolism through regulation of the AMPKα1/HMGCR pathway.

figure-results-5
Figure 6. AMPK inhibition attenuates the effects of Lac-Phe on HMGCR phosphorylation and lipid accumulation in H9c2 cells. (A,B) Representative western blot images of p-AMPKα1 and AMPKα1 in H9c2 cells treated with HG/PA, Lac-Phe, and/or AMPK-IN-3 as indicated (A) and quantification of the p-AMPKα1/AMPKα1 ratio (B); n = 3 per group. (C,D) Representative western blot images of p-HMGCR and HMGCR in H9c2 cells from the indicated treatment groups (C) and quantification of the p-HMGCR/HMGCR ratio (D); n = 3 per group. GAPDH and β-tubulin were used as loading controls in (A) and (C), respectively. (E,F) Representative LD540 staining of H9c2 cells from the indicated treatment groups (E) and quantification of mean LD540 fluorescence intensity (F); n = 3 per group. Scale bar = 200 μm. (G) Total cholesterol (TC) levels in H9c2 cells from the indicated treatment groups; n = 3 per group. Data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. Please click here to view a larger version of this figure.

Collectively, the findings from the in vivo and in vitro experiments support a proposed mechanism whereby Lac-Phe increases AMPKα1 and HMGCR phosphorylation, which is associated with reduced cholesterol levels and lipid accumulation and improved cardiac function and pathological remodeling in DCM. The proposed AMPKα1/HMGCR pathway underlying the effects of Lac-Phe is summarized schematically in Figure 7.

figure-results-6
Figure 7. Proposed mechanism by which Lac-Phe improves lipid metabolism in diabetic cardiomyopathy through the AMPK/HMGCR pathway. Schematic illustrating the proposed mechanism whereby Lac-Phe promotes AMPKα1 phosphorylation, followed by HMGCR phosphorylation, which is associated with reduced lipid droplet accumulation and lipotoxicity in diabetic cardiomyopathy. AMPK, AMP-activated protein kinase; HMGCR, HMG-CoA reductase; LD, lipid droplet. Please click here to view a larger version of this figure.

Data Availability:

The raw data supporting the findings of this study have been deposited in Zenodo and are publicly available at https://zenodo.org/records/21991371 

Discusión

En este estudio, presentamos evidencia que respalda un efecto terapéutico de Lac-Phe en la miocardiopatía dilatada (DCM). Lac-Phe mejoró el remodelado cardíaco y la disfunción cardíaca en la DCM, al mismo tiempo que redujo la producción de colesterol en los cardiomiocitos y mejoró el desequilibrio del metabolismo lipídico. Mecánicamente, Lac-Phe incrementó la fosforilación de AMPKα1. Posteriormente, el aumento en la fosforilación de AMPKα1 se asoció con un incremento en la fosforilación y la inhibición de su diana descendente HMGCR, lo que condujo a una reducción en la síntesis de colesterol y, finalmente, a la mejora del desequilibrio del metabolismo lipídico en los cardiomiocitos. En resumen, este estudio demuestra que Lac-Phe mejora el metabolismo lipídico cardíaco en la DCM a través de la vía de señalización AMPK/HMGCR. Se presenta un resumen esquemático de los hallazgos principales y del mecanismo propuesto en la Figura 7.

El desequilibrio del metabolismo de lípidos es uno de los factores clave que contribuyen al daño miocárdico en la MCD15. La acumulación excesiva de lípidos en los cardiomiocitos conduce a lipotoxicidad, lo que resulta en disfunción de los cardiomiocitos y remodelación cardíaca patológica16. La hiperglucemia prolongada y la obesidad son factores importantes que conducen a trastornos del metabolismo de lípidos25. Se ha demostrado que Lac-Phe es una sustancia endógena que puede reducir eficazmente la ingesta de alimentos, disminuir la obesidad y el peso corporal, y mejorar la homeostasis glucídica21. Informes recientes también han mostrado que Lac-Phe puede regular el metabolismo de lípidos en microglía/macrófagos durante la lesión de la médula espinal23, lo que demuestra que Lac-Phe puede regular el metabolismo de lípidos en otro modelo de enfermedad. Como metabolito endógeno, el papel de Lac-Phe en las enfermedades metabólicas ha despertado un gran interés. Sin embargo, el papel de Lac-Phe en la MCD aún no ha sido investigado completamente. Nuestro estudio reveló un papel de Lac-Phe en el metabolismo de lípidos en cardiomiocitos en la MCD. Mediante análisis transcriptómico, encontramos que el tratamiento con Lac-Phe se asoció con alteraciones en vías relacionadas con el metabolismo de lípidos en los corazones de ratones con MCD. Las tinciones con rojo de aceite y las observaciones mediante microscopía electrónica de transmisión confirmaron una reducción en la acumulación de lípidos en los corazones de ratones con MCD tratados con Lac-Phe. Estos hallazgos sugieren que Lac-Phe ejerce efectos cardioprotectores en la MCD al reducir la acumulación de lípidos en el corazón y aliviar la remodelación patológica. Experimentos in vitro también demostraron que la administración de Lac-Phe alivió la acumulación de lípidos en células H9c2 expuestas a condiciones de HG/PA.

HMGCR es una enzima dependiente de NADPH y limitante de la velocidad en la vía del mevalonato30. La HMGCR cataliza la conversión de HMG-CoA en mevalonato, un paso crítico en la biosíntesis del colesterol30,31,32. HMGCR representa un objetivo terapéutico relevante para la enfermedad cardiovascular y la miocardiopatía dilatada33Clínicamente, la inhibición de HMGCR es un enfoque establecido para reducir el riesgo cardiovascular34Nuestros datos experimentales demuestran que Lac-Phe aumenta la fosforilación de HMGCR en los corazones de ratones con diabetes tipo 2, lo cual es consistente con la inhibición de la actividad de HMGCR y la reducción de la síntesis de colesterol. Estudios in vitro mostraron además que la administración de Lac-Phe incrementó la fosforilación de HMGCR en células H9c2 expuestas a condiciones de glucosa alta/ácido palmítico (HG/PA), acompañado de una reducción en los niveles de colesterol. La combinación de Lac-Phe con el fármaco hipolipemiante atorvastatina produjo efectos mayores que la atorvastatina sola en las condiciones experimentales analizadas. En células H9c2 tratadas con atorvastatina, la adición de Lac-Phe aumentó la fosforilación de HMGCR y disminuyó la acumulación de lípidos. Estos hallazgos respaldan la viabilidad de modular la fosforilación de HMGCR mediante Lac-Phe para reducir la producción de colesterol y restablecer el equilibrio del metabolismo lipídico en la miocardiopatía diabética (DCM). Sin embargo, se requiere investigación adicional para determinar si Lac-Phe podría constituir una estrategia terapéutica segura y eficaz en pacientes con DCM.

AMPK, una enzima clave en el metabolismo energético mitocondrial, es fundamental para el estudio de enfermedades metabólicas como la diabetes35,36. A través de la regulación de la fosforilación y desfosforilación, AMPK desempeña un papel crucial en el metabolismo cardíaco de lípidos. AMPK fosforilada representa su forma activa37. p-AMPK promueve la entrada de ácidos grasos en las mitocondrias para la β-oxidación de ácidos grasos (FAO) mediante la regulación de CPT-138. En consonancia con estudios previos39, nuestros resultados mostraron que el aumento de la fosforilación de AMPK se asoció con un incremento en la fosforilación de HMGCR y una reducción en la síntesis de colesterol. En ratones con miocardiopatía diabética (DCM) inducida por STZ y una dieta rica en grasas (HFD), la hiperglucemia persistente puede provocar cambios en los niveles cardíacos de p-AMPK40. En la etapa inicial de la diabetes, cuando la función cardíaca aún no ha cambiado, los niveles cardíacos de p-AMPK pueden permanecer sin alteraciones10,37,41. En la diabetes avanzada, con hiperglucemia persistente durante más de 8 semanas, comienza el remodelado cardíaco patológico y los niveles de p-AMPK pueden disminuir10. Se ha propuesto que mantener la activación de AMPK constituye una estrategia eficaz para prevenir la DCM10,42. La metformina, un fármaco ampliamente utilizado para tratar la diabetes tipo 2, ha demostrado activar la vía de AMPK, aunque se ha informado que tiene un efecto neutro sobre el corazón43,44. Curiosamente, tanto la administración aguda como crónica de metformina puede aumentar los niveles de Lac-Phe22. Estas observaciones plantean la posibilidad de que Lac-Phe pueda contribuir a la señalización metabólica asociada con la activación de AMPK; sin embargo, el presente estudio no establece que Lac-Phe exógeno produzca un efecto más específico o seguro que la metformina.

En nuestro estudio, la fosforilación de AMPKα1 se redujo notablemente en el modelo de miocardiopatía dilatada en etapa tardía, como se muestra en la Figura 5D,E, un hallazgo consistente con investigaciones previas42. El análisis transcriptómico reveló cambios relacionados con la vía de AMPK en los corazones de ratones diabéticos tipo 2 tratados con Lac-Phe, y el análisis por inmunotransferencia mostró un aumento en la fosforilación de AMPKα1 tras el tratamiento con Lac-Phe. El acoplamiento molecular sugirió una posible interacción entre Lac-Phe y AMPKα1, lo cual es coherente con un estudio reciente23. Es importante destacar que el acoplamiento molecular por sí solo no establece una unión intracelular directa entre Lac-Phe y AMPKα1. Experimentos in vitro mostraron que el tratamiento con el inhibidor de AMPK suprimió la fosforilación de AMPKα1 y se asoció con una reducción en la fosforilación de HMGCR. Tras la adición del inhibidor de AMPK, los efectos de Lac-Phe se atenuaron, coincidiendo con un aumento en la acumulación de lípidos y un contenido elevado de colesterol en células H9c2. En conjunto, estos resultados respaldan la participación de la señalización AMPKα1/HMGCR en los efectos de Lac-Phe sobre el metabolismo lipídico. Lac-Phe incrementó la fosforilación de AMPKα1, lo que se asoció con un aumento en la fosforilación de HMGCR, niveles reducidos de colesterol y disminución en la acumulación de lípidos; la inhibición de AMPK atenuó estos efectos.

Este estudio tiene varias limitaciones. En primer lugar, todos los experimentos con animales se realizaron en ratones macho. Aunque demostramos el efecto terapéutico del Lac-Phe en la miocardiopatía dilatada (DCM) en ratones C57BL/6J macho, el sexo es un factor importante que influye en las enfermedades cardiovasculares45. Por lo tanto, se desconoce si el Lac-Phe tiene los mismos efectos en ratones hembra. En segundo lugar, la secuenciación del transcriptoma se realizó en muestras de corazón completo, lo que podría haber ocultado cambios específicos de tipo celular en el corazón tras el tratamiento con Lac-Phe. La secuenciación a nivel de una sola célula podría ofrecer una visión más profunda sobre la heterogeneidad celular y molecular subyacente a los efectos observados46,47 y representa un enfoque alternativo para investigar más a fondo esta hipótesis. Por último, debido a las limitaciones del equipo de ultrasonido utilizado en este estudio, no fue posible evaluar la función diastólica cardíaca en ratones. Por consiguiente, se necesitan estudios adicionales para aclarar los efectos terapéuticos del Lac-Phe en la DCM, evaluar posibles efectos dependientes del sexo, investigar las respuestas específicas por tipo celular, evaluar la función diastólica y profundizar en los mecanismos subyacentes a los efectos observados. En resumen, este estudio aporta evidencia que respalda al Lac-Phe como un posible metabolito endógeno de interés para el tratamiento de la DCM. Además, nuestros hallazgos respaldan un mecanismo propuesto mediante el cual el Lac-Phe reduce la acumulación de lípidos en los cardiomiocitos durante la DCM a través de la vía AMPKα1/HMGCR. Específicamente, el Lac-Phe incrementó la fosforilación de AMPKα1, lo que se asoció con un aumento en la fosforilación de HMGCR, una reducción en la síntesis de colesterol y en la acumulación de lípidos, y una mejoría en el remodelado patológico. Se requiere una investigación adicional para establecer la interacción molecular entre el Lac-Phe y AMPKα1 y para determinar las posibles aplicaciones traslacionales de estos hallazgos.

Divulgaciones

Conflicto de intereses:

Los autores no tienen nada que declarar.

Agradecimientos

Queremos expresar nuestro sincero agradecimiento a los investigadores y al personal del Hospital Clínico Colegio Nanjing Drum Tower y del Hospital Afiliado de la Facultad de Medicina de la Universidad de Nanjing por su apoyo técnico. Esta investigación fue financiada por subvenciones de la Fundación Nacional de Ciencias Naturales de China (números de subvención 81870291 y 82300384), la Fundación China para la Ciencia Postdoctoral (número de subvención 2023M731625) y la Fundación de Ciencias Naturales de la Provincia de Jiangsu (número de subvención BK20220175). También agradecemos los recursos y el apoyo colaborativo proporcionados por el Instituto de Síntesis Avanzada de la Universidad de Nanjing Tech.

Materiales

Lista de materiales utilizados en este artículo
NombreEmpresaNúmero de catálogoComentarios
AMPK-IN-3 (inhibidor de p-AMPK)MedChemExpress (MCE)HY-132921Utilizado en 10 μM durante experimentos de inhibición de AMPK para inhibir la señalización de AMPK en células tratadas con Lac-Phe.
Kit de ensayo Amplex Red para colesterol y esterol de colesterolBeyotimeS0211SUtilizado para determinar los niveles totales de colesterol en suero y en muestras de células H9c2.
Anti-AMPKα1Cell Signaling Technology2532; RRID: AB_330331Anticuerpo primario utilizado para el análisis por inmunotransferencia de AMPKα1.
Anti-GAPDHCell Signaling Technology5174; RRID: AB_10622025Anticuerpo primario utilizado para el análisis por inmunotransferencia de GAPDH.
Anti-HMGCRAbcamab174830; RRID: AB_2749818Anticuerpo primario utilizado para el análisis por inmunotransferencia de HMGCR.
Anti-p-AMPKα1 (Thr172)Cell Signaling Technology2535; RRID: AB_331250Anticuerpo primario utilizado para el análisis por inmunotransferencia de AMPK fosforiladaα1.
Anti-p-HMGCR (Ser872)Abcamab124350Anticuerpo primario utilizado para el análisis por inmunotransferencia de HMGCR fosforilada.
anticuerpo secundario anti-IgG de conejo, conjugado con HRPCell Signaling Technology7074; RRID: AB_2099233Utilizado a una dilución 1:5.000 para la detección en inmunotransferencia western.
Anti-β-tubulinaCell Signaling Technology2128; RRID: AB_823664Anticuerpo primario utilizado para el análisis por inmunotransferencia de β-tubulina.
AtorvastatinaMedChemExpress (MCE)HY-17379Utilizado solo o en combinación con Lac-Phe para evaluar los efectos sobre la acumulación de lípidos, los niveles totales de colesterol y la fosforilación de HMGCR en células H9c2 tratadas con HG/PA.
Albúmina sérica bovinaSigma-AldrichA7906Utilizado al 5 % para bloquear la unión inespecífica durante el análisis de western blot.
reactivo/kit de ensayo de BradfordThermo Fisher Scientific23200Utilizado para determinar la concentración de proteína antes del análisis mediante western blot.
C57BL/6J ratones (machos, de 5 semanas de edad)Centro de Investigación de Animales Modelo de la Universidad de NanjingN/AUtilizado para establecer el modelo murino de cardiomiopatía diabética inducida por dieta rica en grasas (HFD) y estreptozotocina (STZ).
Kit de conteo celular-8 (CCK-8)SharebioSB-CCK8SUtilizado para medir la viabilidad de células H9c2 tras el tratamiento.
Tampón de citratoPreparado internamenteN/APreparado a pH 4,5 y utilizado para disolver la STZ y como control vehicular correspondiente.
Dieta control (10 % grasa, 20 % proteína, 70 % carbohidrato)Research Diets, Inc.D12450JUtilizado como dieta para los ratones del grupo control.
D-(+)-GlucosaSigma-AldrichN/AUtilizado para el tratamiento de células HG/PA y para la prueba de tolerancia oral a la glucosa.
DMSO (dimetilsulfóxido)Sigma-AldrichD2650Utilizado como disolvente para AMPK-IN-3 y como control vehicular correspondiente.
Dulbecco’s modificado de Eagle’medio sGibco, Thermo Fisher Scientific11965092Utilizado como medio de cultivo basal para células H9c2.
Equipo de ecocardiografía con transductor de 40 MHzFUJIFILM VisualSonicsVevo 3100 LT; transductor de 40 MHzUtilizado para medir las dimensiones del ventrículo izquierdo y la velocidad del flujo sanguíneo en la válvula mitral, y para derivar la FEVI y la FS.
Fijador para microscopía electrónicaServiceBioG1102Utilizado para fijar tejido cardíaco fresco de ratón para microscopía electrónica de transmisión.
EtanolSigma-AldrichE7023Utilizado en concentraciones progresivamente crecientes para la deshidratación durante la preparación de muestras para microscopía electrónica de transmisión.
Suero bovino fetalGibco, Thermo Fisher Scientific10099141Utilizado al 10% para suplementar el medio de cultivo celular H9c2.
Línea celular H9c2SunncellRRID: CVCL_0286Utilizado como modelo celular cardíaco in vitro para experimentos de tratamiento con HG/PA y Lac-Phe.
Tinción con hematoxilina y eosinaServiceBioG1005-1/2Utilizado para la evaluación histológica del tejido cardíaco.
Dieta alta en grasas (60 % kcal de grasas)Research Diets, Inc.D12492Utilizado con STZ de baja dosis para establecer el modelo de ratón de cardiomiopatía diabética.
Microscopio de campo claro de alta resoluciónLeicaDM2500Utilizado para la obtención de imágenes completas de cortes de tejido cardíaco teñidos a 20× aumento
ImageJInstitutos Nacionales de la SaludN/AUtilizado para la cuantificación densitométrica de los resultados de inmunotransferencia en Western blot.
IsofluranoSigma-Aldrich792632Utilizado para anestesia durante la ecocardiografía en ratones.
Alcohol isopropílicoSigma-AldrichI9516Utilizado al 60 % durante la tinción con Oil Red O de secciones cardíacas congeladas.
tinción lipídica LD540BeyotimeC2050S-1Utilizado en 10 μM para teñir lípidos intracelulares.
Masson’tinción con tricrómico sServiceBioG1006Utilizado para la tinción histológica de tejido cardíaco.
N-lactoil-fenilalanina (Lac-Phe)MedChemExpress (MCE)HY-146098Administrado a ratones con miocardiopatía dilatada y utilizado para tratar células H9c2 expuestas a glucosa alta/ácido palmítico.
Solución de trabajo de Oil Red OServiceBioG1015-100MLUtilizado para teñir lípidos en cortes de tejido cardíaco congelado.
Kit Omni-ECL de Quimioluminiscencia EficienteEpiZymeSQ203LUtilizado para la visualización quimioluminiscente de bandas proteicas tras el western blot.
Ácido palmíticoSigma-AldrichP0500Utilizado a 0,25 mM con glucosa alta para establecer el modelo de células H9c2 tratadas con HG/PA.
ParaformaldehídoSigma-AldrichP6148Preparado como una solución al 4 % y utilizado para fijar tejido cardíaco y células antes de la tinción histológica o de lípidos.
Penicilina-estreptomicinaGibco, Thermo Fisher Scientific15140122Utilizado al 1% en medio de cultivo celular H9c2.
Solución salina tamponada con fosfato (PBS)Gibco, Thermo Fisher Scientific10010023Utilizado para lavados durante los procedimientos de tinción y como vehículo correspondiente en experimentos de tratamiento celular.
PrismGraphPad SoftwareVersión 8.0Utilizado para el análisis estadístico de datos experimentales.
membrana de transferencia de PVDF, 0.45 μmThermo Fisher Scientific88518Utilizado para la transferencia electroforética de proteínas durante el análisis de western blot.
RNeasy Mini KitQIAGEN74104Utilizado para el aislamiento/purificación de ARN total a partir de tejido cardíaco de ratón.
Solución de cloruro de sodio, 0,9 % (suero fisiológico)Sigma-AldrichS8776Utilizado para disolver Lac-Phe y como control vehicular para los ratones Control y DCM.
SPSSIBMVersión 20.0Utilizado para el análisis estadístico de datos experimentales.
Estreptozotocina (STZ)Sigma-AldrichS0130Administrado junto con dieta rica en grasas (HFD) para establecer el modelo de ratón de cardiomiopatía diabética.
Tampón TBS Tween-20 (20×)Thermo Fisher Scientific28360Utilizado para lavar membranas tras la incubación con anticuerpos primarios y secundarios durante el análisis de western blot.
Tissue-Tek O.C.T. CompoundSakura Finetek4583Empleada para incluir tejido cardíaco antes de la preparación de cortes congelados para la tinción con Oil Red O.
Microscopio electrónico de transmisiónHITACHIHT7800Utilizado para examinar cambios ultraestructurales en el tejido cardíaco de ratón.
TRIzol ReagenteInvitrogen15596026Utilizado para el aislamiento de ARN total a partir de tejido cardíaco de ratón.
Kit de preparación de bibliotecas VAHTS Universal V10 para RNA-seqVazymeNR616-01Utilizado para preparar bibliotecas de RNA-seq para análisis transcriptómico.
Aglutinina de germen de trigo (WGA)ServiceBioW7024Utilizado para la evaluación histológica del tejido cardíaco.

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