Quantification of Plasma Metabolites

Plasma was collected into anticoagulant tubes, gently inverted to mix, and centrifuged at 2,500-3,000 rpm for 15 min. Supernatant plasma (200 µL) was aliquoted and stored at −80 °C. Plasma Lac‑Phe levels were quantified by ultra‑high‑performance liquid chromatography–mass spectrometry (UHPLC‑MS).

Animals

Eight-week-old male C57/BL6 mice were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd. All mice were housed under controlled temperature conditions with a normal diet and maintained on a standard 12-hour light/dark cycle. Efforts were made to minimize the animals’ suffering, and the number of animals used was kept to a minimum during the euthanasia procedures.

All experimental procedures were approved by the Institutional Ethics Committee of Nanjing Drum Tower Hospital (Approval No. 2020AE01077) and performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament.

Transverse Aortic Constriction (TAC)

As previously described [17, 18]. Male mice (8–10 weeks) were anesthetized with intraperitoneal ketamine (100 mg/kg) and xylazine (5 mg/kg), placed supine, and secured on a surgical platform. A thoracotomy was performed to expose the aortic arch, and transverse aortic constriction was induced by tying a 4‑0 silk ligature around the arch between the left common carotid and innominate arteries using a 27‑gauge needle as a spacer; Sham mice underwent identical surgery without ligation. Two weeks post‑operatively, peak aortic arch flow velocity was assessed by Doppler echocardiography. Consistent doses were used according to previous references in the study by Li et al., published in Nature (2022) [10]. TAC model mice were intraperitoneally injected with 50 mg/kg N-Lac-Phe daily for 4 weeks.

Echocardiography

After anesthesia, mice underwent TAC or sham surgery, followed by imaging with the Vevo 3100LT ultrasound system (VisualSonics, Toronto, Ontario, Canada). All mice were under standard and stable light anesthesia at the time of echocardiography to minimize severe fluctuations in heart rate due to the depth of anesthesia or stress. Left ventricular ejection fraction (EF%) and fractional shortening (FS%) were measured according to the manufacturer’s instructions.

Cell Culture and Treatments

H9c2 cells (RRID: CVCL_0286) were cultured with 10% fetal bovine serum (Solarbio, A8020), high-glucose-DMEM (Solarbio, 11995) and antibiotics (Biosharp, BL505A). N-Lac-Phe was procured from MCE(HY-150012). Angiotensin II was from Aladdin (A107852). Antibodies against the following proteins were used: ANP (Abcam, ab181242, 1:1000), α-SMA (Abcam, ab5694, 1:1000), and Tubulin (Abcam, ab7291, 1:1000). BCA protein assay kit was purchased from Beyotime (P0011), and JC-1 was obtained from Beyotime (C2003S). Tunel was obtained from Beyotime (KTA2011). MPTP was obtained from Beyotime (C2003S).

Histology

The mouse hearts were perfused with saline and subsequently fixed with 4% paraformaldehyde. As described in previous studies, the hematoxylin and eosin (H&E) [19] staining and Wheat Germ Agglutinin (WGA) staining was used to measure myocardial cell cross-sectional area, while Masson’s trichrome staining was performed to assess cardiac fibrosis [20].

Western Blotting (WB)

Cell and mouse cardiac tissues were homogenized in lysis buffer to obtain whole‑cell lysates, and protein concentrations were determined using a BCA assay kit. As previously described [21], equal protein amounts were subjected to SDS‑PAGE, transferred to PVDF membranes, blocked with 5% milk, and incubated with primary antibodies overnight at 4 °C, followed by appropriate secondary antibodies for 1 h at room temperature. After washing, immunoreactive proteins were detected using a chemiluminescent substrate (Abbkine, BMU102-CN). The following antibodies were used: GPX4 (Abcam, ab252833, 1:1000), ACSL4 (Cell signaling, F6T3Z, 1:1000), MFN2 (Abcam, ab124773, 1:1000), ANP (Abcam, ab181242, 1:1000), α-SMA (Abcam, ab5694, 1:1000), AMPK (Proteintech,10929-2-AP, 1:5000), pAMPK (Proteintech, 83924-1-RR, 1:2000), PGC1a (66369-1-lg, 1:5000) and Tubulin (Abcam, ab7291, 1:1000).

Ultrastructure Observation

Cardiac ultrastructure was evaluated by TEM. Mouse hearts were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide, stained with 2% aqueous uranyl acetate, dehydrated through graded ethanol, and embedded in resin. Ultrathin sections were flattened in xylene vapor, mounted on nickel grids, and imaged using a JEM-1400 Plus transmission electron microscope (JEOL, Japan).

Determination of MDA and Iron Level in Serum

Mouse blood was collected and serum was collected by centrifugation, and serum levels of malondialdehyde (Elabscience, E-BC-K025-M) and iron (Elabscience, E-BC-K772-M) were measured using commercially available kits following the instructions provided by the manufacturer.

TUNEL Assay

Apoptosis in myocardial tissue was detected using the TUNEL assay. Myocardial tissue was fixed with 4% paraformaldehyde at 4 °C for 12 h, followed by incubation with the TUNEL kit reagents (Beyotime, KTA2011) for 30 min, according to the manufacturer’s instructions. Apoptotic nuclei were stained with green fluorescein, while total myocardial nuclei were counterstained with DAPI. Finally, the cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan).

CCK8 Assay

H9c2 cell viability was assessed using a CCK‑8 kit (Beyotime, C0038, Shanghai, China) per the manufacturer’s protocol. Briefly, 1,000 cells/well were seeded in 96‑well plates in 100 µL medium with 10% FBS. After treatment, 10 µL CCK‑8 solution was added and incubated in the dark for 2 h, and absorbance at 450 nm was measured using a multimode microplate reader.

Oxygen Consumption Rate (OCR)

A black, clear-bottom 96-well plate is prepared with blank, control, and sample wells. A cell suspension (≈ 5 × 10^5 cells/mL) is seeded at 100 µL per well (≈ 5 × 10^4 cells/well) and incubated overnight at 37 °C in 5% CO₂. The medium is then gently aspirated to preserve the monolayer. Blank wells receive 100 µL fresh medium, whereas control and sample wells are treated with 100 µL working solution from the OCR Fluorescence Assay Kit (Elabscience, E-BC-F068).

The plate is incubated for 30 min at 37 °C in either a microplate reader or a temperature-controlled incubator, followed by fluorescence measurement according to the manufacturer’s protocol. Fluorescence intensity (F) is plotted versus time (min), the linear phase is identified, and its slope is calculated to derive and compare OCR values among cell samples.

Statistical Analyses

The data are presented as the mean ± SD. Statistical analysis was based on Graphpad Prism software and a value of P < 0.05 was considered statistically significant. Student t-test was used for 2 sample comparisons; One-way ANOVA test was performed to compare the data between multiple groups and Two-way ANOVA was used to compare multiple groups when there were 2 experimental factors.