This study was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (No. KY2021-363). First, hearts of newborn Kunming mice were isolated and subjected to trypsin (T1300, Solarbio, Beijing, China) digestion. The digest was centrifuged (4°C, 1500 rpm, 5 min) after filtration, and the precipitate was collected. The precipitate was added to the complete culture solution, including Dulbecco’s modified Eagle’s medium (DMEM; D6429; Sigma, USA), 10% fetal bovine serum (Gibco, Grand Island, USA), and 1 × penicillin and streptomycin (Life Technologies, Grand Island, USA), spread evenly, transferred to cell bottles, and incubated at 37°C, and 5% CO₂, in 95% humid air for 90 min. The upper layer of unadhered cells was collected, and the cell density was adjusted by adding the culture solution. This was then spread to a plate covered with gelatin wells and incubated in an incubator for 24 h before treatment.

A 630 nm light emitting diode with a spot area of about 10 cm² was placed vertically above the cell well plate. Next, the power density of the light source was measured using a light source power density meter, and the vertical distance between the light source and the cell well plate was adjusted so that the final power density was maintained at 2.5 mW/cm² and irradiated for 10 min. The Non-LED group was illuminated under the same environmental conditions as the LED-Red group but without turning on the switch of the light source.

To analyze cellular ATP content, the ATP assay kit (S0027, Beyotime, Beijing, China) was used. The culture solution was aspirated from the cell well plate, and 200 µL of lysate were added to each well. After lysis, the mix was centrifuged at 4°C and 12000 g for 5 min, and the supernatant was removed for subsequent assays. An appropriate amount of ATP assay reagent was adjusted according to the manufacturer’s instructions. Then, 100 µL of ATP assay working solution was added to the assay wells and left at room temperature for 3–5 min. To the test wells, 20 µL of the sample was added, mixed rapidly, and the RLU value was measured with a luminometer after an interval of at least 2 s.

The working assay solution was prepared according to the SDH assay reagent instructions (BC0955, Solarbio, Beijing, China). After cell digestion and centrifugation, the supernatant was aspirated and used to detect SDH activity. The absorbance values were measured at 600 nm on a visible spectrophotometer, and the SDH activity was calculated and analyzed for each group.

CMs were cultured in 12-well plates, and the mitochondrial membrane potential was analyzed using the JC-1 assay kit (C2003S, Beyotime, Beijing, China). After light stimulation, the culture solution in the well plates was discarded, and the cells were washed three times with PBS. Then, the JC-1 solution was added, incubated for 20 min at 37°C, washed again with PBS, and the changes in mitochondrial membrane potential were observed under fluorescence microscopy. The red fluorescence represents an increase, and the green fluorescence indicates a decrease in mitochondrial membrane potential. Then, the mitochondrial membrane potential was quantified by fluorescence analysis using the software image J. The red and green fluorescence values were measured separately for each group, and then the red fluorescence value/sum of red and green fluorescence was calculated as the relative increase of mitochondrial membrane potential for each group.

CMs were inoculated in 96-well plates and ROS were detected using the Reactive Oxygen Species Assay Kit (S0033S, Beyotime, Beijing, China). The assay was performed according to the manufacturer’s instructions. Then, the cells were washed three times with PBS, and DMEM and DCFH-DA probes were prepared in a ratio of 1:1000. Appropriate volumes of probes were added to the well plates and incubated for 20 min at 37°C. Excess unloaded probes were then removed, and the fluorescence of each group was detected using a fluorescent enzyme marker.

The TUNEL Apoptosis Assay Kit (C1089, Beyotime, Beijing, China) was used to analyze apoptosis. CMs were first washed once with PBS after LED-Red stimulation. The cells were fixed in 4% paraformaldehyde for 30 min and then washed once with PBS. Next, the cells were permeabilized with PBS containing 0.3% Triton X-100 for 90 min. 50 µL TUNEL assay solution was added to each well according to the manufacturer’s instructions and incubated for 60 min at 37°C in a dark environment. Finally, the cell nuclei were labeled with DAPI, and apoptosis was observed by fluorescence microscopy.

Cells cultured in glass dishes received LED-Red stimulation. The culture medium was removed, and the cells were washed three times with Hanks buffer. Cells were incubated in 500× MitoTracker (22675, AAT Bioquest, California, USA) stain at 37°C with 5% CO₂ for 40 min; then, the stain was discarded. Hanks buffer and Hoest3342 stain were added to re-stain and label the nuclei. Mitochondrial division and fusion were observed by a confocal laser scanning microscope (FV10i; Olympus, Japan). Labeled mitochondria appeared green, and the labeled nuclei appeared blue.

Total RNA of CMs was extracted using TRIzol reagent (Invitrogen, California, USA). The RNA samples were then reverse transcribed to cDNA. SYBR Green PCR Master Mix (Applied Biosystems, USA) was used for qRT-PCR. The sequences of the main primers are indicated in Table S1.

Cells were scraped by adding an appropriate volume (20–60 µL) of cell lysate depending on cell density and state. Protein concentration was determined using the BCA Protein Concentration Assay Kit (P0012, Beyotime, Beijing, China). An appropriate concentration of SDS-polyacrylamide gel was used for electrophoresis according to the corresponding molecular weight of the incubated antibody. Membranes were then blocked in 5% non-fat milk for 90 min at room temperature and incubated with antibody against Platelet glycoprotein 4 (CD36; 1:1000; YT5585, Immunoway, USA), Recombinant Carnitine Palmitoyltransferase 1A (CPT1A; 1:1000; DF12004, Affinity, USA), Phosphorylated Dynamin-related protein 1 (p-DRP1; 1:500; YP1318, Immunoway, USA), Peroxisome proliferator-activated receptor gamma coactivator 1-alp (PGC-1α; 1:1000; YM0519, Immunoway, USA), Solute carrier family 2 facilitated glucose transporter member 4 (Glut4; 1:1000; YT5523, Immunoway, USA), Transcription factor A mitochondrial (TFAM; 1:1000; YT2916, Immunoway, USA), and pyruvate dehydrogenase kinase isozyme 4 (PDK4; 1:1000; DF7169; Affinity, USA) 4°C overnight. The membrane was finally incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature and scanned by Odyssey (LI-COR Biosciences, Lincoln, NE, USA).

Data are expressed by means ± SEM. Comparisons between two groups were made using Student’s t-test, and comparisons between multiple groups were made using one-way ANOVA and Bonferroni-corrected t-test (version 8.0, GraphPad Software, Inc., USA). P values < 0.05 were considered statistically significant.

Mitochondria are subcellular organelles that serve as the main source of energy supply for CMs. The efficiency and amount of ATP synthesis are critical for the contractile activity of CMs. Therefore, we irradiated mammalian CMs using 630 nm light-emitting diodes. The results showed that low-energy LED-Red irradiation for 10 min led to an increase in intracellular ATP content (Fig. 1A). Among the key enzymes that regulate the process of the mitochondrial tricarboxylic acid cycle, SDH is the only multi-subunit enzyme integrated into the mitochondrial membrane, which is essential for regulating the ATP synthesis of the mitochondrial tricarboxylic acid cycle (Bezawork-Geleta et al., 2017). Consistent with the upregulation of ATP content by LED-Red, SDH activity in CMs also exhibited upregulation after stimulation (Fig. 1B). In addition, JC-1 staining showed a significant increase in mitochondrial membrane potential (MMP) in CMs stimulated by LED-Red, compared with the group without irradiation treatment (Fig. 1C). Thus, the above results showed that LED-Red elevated SDH enzyme activity in mitochondria of CMs increased MMP, and promoted ATP synthesis.

Mammalian CMs synthesize ATP in the mitochondria, and aerobic respiration is predicated on the oxidative phosphorylation of fatty acids and glycolysis. The above-mentioned experiments revealed that LED-Red promotes ATP synthesis in CMs. Therefore, we next explored whether ATP production correlates with the uptake and consumption of energy substrates by CMs. In the subsequent experiments, we examined the expression of enzymes related to glycolipid metabolism in CMs. qRT-PCR results showed that lipid metabolism genes CD36 and PDK4 expression were significantly upregulated after LED-Red irradiation, compared with the Non-LED group. Among the glucose metabolism genes, CPT1A expression was up-regulated. In addition, the expression of PGC1-α, which regulates mitochondrial biosynthesis, and energy metabolism, also increased due to LED-Red stimulation (Fig. 2A). We examined the expression of proteins expressed by the above-mentioned genes. Interestingly, LED-Red promoted only CD36 and PDK4 protein expression and PGC1-α expression, although the increase was not statistically significant (Fig. 2B). These results suggest that LED-Red stimulation increased the expression of some glycolipid metabolizing RNAs and proteins in CMs and accelerated the transport of metabolic substrates in CMs, which is important for ATP production.

PBM alters the process of mitochondrial oxidative stress (Li et al., 2020; Feng et al., 2020). However, no study has verified the regulation of this process using LED-Red in CMs. A DCFH-DA probe showed that LED-Red increased ROS levels in CMs (Fig. 3A). An abnormal increase in ROS is often considered a marker of myocardial injury, although ROS can act as a signaling molecule for intercellular communication. Therefore, we examined the apoptosis of CMs after LED-Red stimulation. The results showed no significant difference in the number of TUNEL-positive CMs in the LED-Red group compared with the Non-LED group (Fig. 3B), suggesting that the slight rise in ROS induced by LED-Red may only function as a signaling molecule, acting as an intercellular communication transmitter, without causing damage to CMs.

Mitochondria are dynamic organelles that control the number and size of mitochondria through constant division and fusion (Chan, 2020). This has an important role in maintaining cellular activities. LED-Red also shows some involvement in this dynamic process of mitochondria. MitoTracker analysis of living cells showed that LED-Red stimulation-induced mitochondrial division. In addition, LED-Red also affected the distribution of mitochondria, shifting from the normal perinuclear distribution to diffusion into the cytoplasm (Fig. 4A). The dynamic balance of mitochondrial fusion is regulated by DRP1, which is considered the major pre-mitochondrial fission protein. Ser-616 phosphorylation of DRP1 is necessary to activate mitochondrial fission. Western blotting results showed that LED-Red stimulation promoted the phosphorylation of DRP1 compared to those in the unirradiated group (Fig. 4B). In conclusion, LED-Red was involved in mitochondrial kinetic processes, promoting DRP1 phosphorylation and accelerating mitochondrial division. It also changed the distribution of mitochondria in CMs, which facilitated the energy production and transport of CMs.

Mitochondria have a separate genome, and the integrity of the mitochondrial genome is critical for multicellular organisms. TFAM was originally cloned as a transcription factor and is essential for the maintenance of mitochondrial DNA (Zhang et al., 2018). In recent years, TFAM has been found to play a key role in several aspects of mitochondrial DNA transcription, replication, linkage formation, damage perception, and DNA repair (Kang and Hamasaki, 2005). Overexpression of TFAM in mice increased the amount of mitochondrial DNA and significantly improved cardiac dysfunction caused by myocardial infarction (Kang et al., 2007). Maintenance of mitochondrial DNA integrity is important for maintaining normal cellular function under both physiological and pathological conditions. Analysis of frontal TFAM in mitochondria of CMs after LED-Red treatment revealed that LED-Red stimulation caused a significant increase in TFAM expression in mitochondria (Fig. 5A). However, the protein level of TFAM was not altered (Fig. 5B).