Journal: Journal of Sport and Health Science

Article Title: Influence of diet-induced obesity and voluntary exercise training on cardiac lipids and mitochondrial function in mice

doi: 10.1016/j.jshs.2025.101095

Figure Lengend Snippet: Voluntary exercise alters left ventricle gene and protein expression related to mitochondrial biogenesis and dynamics in high fat- and chow-fed mice. (A) Western blot analysis to assess purity of crude mitochondria isolation where left ventricle mitochondrial pellet expresses mitochondrial marker TOM70 and COXIV and supernatant expresses calnexin. Protein expression of (B) LCLAT1 and (C) MFN2 in left ventricle-isolated crude mitochondria and (D) LCLAT1, (E) PGC-1α, and (F) MFN2 in left ventricle tissue from male VET or sedentary mice fed an HFD or chow diet. Left ventricle mRNA expression of (G) OPA1 and (H) DRP1 in male VET or sedentary mice fed an HFD or chow diet. Values were calculated relative to 18S housekeeper. Calnexin and β-actin were used as internal controls of protein loading in left ventricle samples and COXIV as internal control of protein loading in crude mitochondria samples. Analysis was performed using two-way analysis of variance with Tukey’s post hoc test for multiple comparisons. Data are expressed as mean ± standard error of the mean. (B–C) n : 5–6 per group. (D–F) n = 7 per group. (G and H) n = 9 per group. CE = chow exercise; CS = chow sedentary; COXIV = cytochrome c oxidase subunit 4; DRP1 = dynamin-related protein 1; HE = high fat diet exercise; HFD = high fat diet; HS = high fat diet sedentary; LCLAT1 = lysocardiolipin acyltransferase 1; MFN2 = mitofusin-2; OPA1 = optic atrophy 1; PGC-1α = peroxisome proliferator-activated receptor gamma coactivator-1α; TOM70 = translocase of outer mitochondria membrane 70; VET = voluntary exercise training.

Article Snippet: The following TaqMan assay gene transcripts were used: fatty acid-binding protein 3 (FABP3, Mm02342495_m1), platelet glycoprotein 4 (CD36, Mm00432403_m1), beta myosin heavy chain (β-MHC, Mm00600555_m1), atrial natriuretic peptide (ANP, Mm01255747_g1), OPA1 (Mm01349707_g1), DRP1 (Mm01342903_m1), robosomal18S (18S, Mm03928990_g1).

Techniques: Expressing, Western Blot, Isolation, Marker, Control, Membrane

Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Cardiac DAMPs inhibit fibroblast-to-cardiomyocyte reprogramming (A) Raw264.7 macrophages were exposed to the indicated ligands for 24 h. After 24 h, the cells were washed and incubated for a further 24 h in standard growth media. The media was collected, and debris was removed. Once cleaned, macrophage media was added to cardiac fibroblasts immediately after their transfection with a control non-targeting miRNA or miR combo. Complexes were removed one day later. Fourteen days after transfection, expression of the indicated cardiomyocyte-specific genes were determined by qPCR. Expression values were normalized to the house-keeping gene Gapdh. The heatmap shows the average of 3 independent experiments expressed as a Z score. Significances were determined by t test. Significant genes (control macrophage conditioned media versus heart extract stimulate macrophage conditioned media) are shown in bold. Raw data for two representative genes are shown in the left image. (B) Macrophages were exposed to the indicated ligands for 6 h. Expression of the indicated cytokines was determined by qPCR, normalized to Gapdh, and shown on the heatmap as a Z score. N = 3. (C) Cardiac fibroblasts were transfected with either miR combo or a non-targeting control miR. The day after transfection, LPS, heart extract, or vehicle were added to the cells for 24 h. Fourteen days after miR transfection, qPCR was used to measure the expression of the indicated cardiomyocyte genes. Expression values were normalized to Gapdh. The heatmap shows the average of 7 independent experiments as a Z score. One-sample t tests were used to compare groups to the control group. Significant genes (miR combo versus miR combo and heart extract) are shown in bold.

Article Snippet: The qPCR primers for Gapdh (Mm99999915_m1, catalog no. 4331182), Actn2 (Mm00473657_m1, catalog no. 4331182), Myh6 (Mm00440359_m1, catalog no. 4331182), Tnni3 (Mm00437164_m1, catalog no. 4331182), Tnnc1 (Mm00437111_m1, catalog no. 4331182), Acta2 (Mm00725412_s1, catalog no. 4331182), Postn (Mm01284919_m1, catalog no. 4331182), Col1a1 (Mm00801645_g1, catalog no. 4351372), Scna5(Mm01342518_m1, catalog no. 4331182), Cacna1c (Mm01188822_m1, catalog no. 4331182), and Ryr2 (Mm00465877_m1, catalog no. 4331182) were obtained from Thermo Fisher Scientific.

Techniques: Incubation, Transfection, Control, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming (A) Macrophages were incubated with either vehicle or heart extract for 24 h. After 24 h, the cells were washed and incubated for a further 24 h in standard growth media. The media was collected and debris removed. The media from three individual experiments were combined and analyzed by mass spectrometry. The relative protein abundance is shown on the right-hand side y axis. Larger proteins produce more peptides, and hence more signal, even if molar abundance is identical. Therefore, to remove size bias, the relative abundance was divided by protein molecular weight (left hand y axis). (B) ELISA measurements of Hb concentration in control media and in five different heart extracts (He). N = 5. (C) Cardiac fibroblasts were transfected with either miR combo or a non-targeting control miR. The day after transfection, the indicated doses of Hb were added to the cells for 24 h. Fourteen days after miR transfection, cells were analyzed for expression of the cardiomyocyte marker Actn2 by qPCR. N = 3. One-sample t tests were used to compare groups to the normalized control group (∗ p < 0.05; ns, not significant). (D–G) Cardiac fibroblasts were transfected with either miR combo or a non-targeting control miR. The day after transfection, hemoglobin (Hb, 5 mg/mL) was added to the cells for 24 h. Fourteen days after miR transfection, cells were analyzed for cardiomyocyte gene expression (D and E), cardiomyocyte numbers (F), and functional maturation (G). With respect to gene expression, qPCR was used to evaluate the expression of both cardiomyocyte sarcomere genes (D) and (cardiomyocyte ion channels (E). Expression values were normalized to the housekeeping gene Gapdh. N = 15. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗ p < 0.05; ns, not significant). A t test was used to determine the significance between the two miR combo (miRC) groups (### p < 0.001, ## p < 0.01; ns, not significant). (F) Cardiomyocyte numbers were determined by immunostaining for the cardiomyocyte marker Actn2 (red). Nuclei were visualized by DAPI (blue). Representative images are shown (scale bar, 50 microns). N = 4. (G) Functional maturation was determined by counting the number of spontaneous beating cells. N = 4. For (F) and (G), significances were determined by one-way ANOVA with Tukey post-hoc testing. Comparisons were made to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01; ns, not significant) as well as to the miR combo + Hb group (### p < 0.001, ## p < 0.01).

Article Snippet: The qPCR primers for Gapdh (Mm99999915_m1, catalog no. 4331182), Actn2 (Mm00473657_m1, catalog no. 4331182), Myh6 (Mm00440359_m1, catalog no. 4331182), Tnni3 (Mm00437164_m1, catalog no. 4331182), Tnnc1 (Mm00437111_m1, catalog no. 4331182), Acta2 (Mm00725412_s1, catalog no. 4331182), Postn (Mm01284919_m1, catalog no. 4331182), Col1a1 (Mm00801645_g1, catalog no. 4351372), Scna5(Mm01342518_m1, catalog no. 4331182), Cacna1c (Mm01188822_m1, catalog no. 4331182), and Ryr2 (Mm00465877_m1, catalog no. 4331182) were obtained from Thermo Fisher Scientific.

Techniques: Incubation, Mass Spectrometry, Quantitative Proteomics, Molecular Weight, Enzyme-linked Immunosorbent Assay, Concentration Assay, Control, Transfection, Expressing, Marker, Gene Expression, Functional Assay, Immunostaining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Blood mimics the inhibitory effects of Hb (A) Cardiac fibroblasts were incubated with Hb (5 mg/mL) for 24 h. Expression of the indicated fibroblast-specific genes was determined by qPCR and expression values normalized to the housekeeping gene Gapdh. A t test was used to determine the significance between the two groups (∗∗∗ p < 0.001; ns, not significant). (B) Cardiac fibroblasts were transfected with either miR combo or a non-targeting control miR. The day after transfection, blood (10 μL per ml of media) was added to the cells for 24 h. Fourteen days after miR transfection, cells were analyzed for expression of the indicated cardiomyocyte-specific genes by qPCR. N = 4. Expression values were normalized to the housekeeping gene Gapdh. For the heatmap, expression values are shown as Z scores for the four individual experiments. A t test was used to compare the two miR combo groups ∗∗∗ p < 0.001).

Article Snippet: The qPCR primers for Gapdh (Mm99999915_m1, catalog no. 4331182), Actn2 (Mm00473657_m1, catalog no. 4331182), Myh6 (Mm00440359_m1, catalog no. 4331182), Tnni3 (Mm00437164_m1, catalog no. 4331182), Tnnc1 (Mm00437111_m1, catalog no. 4331182), Acta2 (Mm00725412_s1, catalog no. 4331182), Postn (Mm01284919_m1, catalog no. 4331182), Col1a1 (Mm00801645_g1, catalog no. 4351372), Scna5(Mm01342518_m1, catalog no. 4331182), Cacna1c (Mm01188822_m1, catalog no. 4331182), and Ryr2 (Mm00465877_m1, catalog no. 4331182) were obtained from Thermo Fisher Scientific.

Techniques: Incubation, Expressing, Transfection, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Hemoglobin activates TLR signaling in cardiac fibroblasts (A) Cardiac fibroblasts were incubated with Hb (5 mg/mL) for the indicated times. Protein extracts were analyzed by immunoblotting for p-NFκB and p-MAPK. Gapdh was used as a loading control. Representative immunoblots are shown on the left-hand side. Quantification was performed by normalizing p-NFκB and p-MAPK band densities with those of the loading control. N = 4. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant). (B) Cardiac fibroblasts were incubated either vehicle or with varying concentrations of a TLR4 pharmacological inhibitor for 3 h. Hb (5 mg/mL) was then added. After 1 h, protein extracts were isolated and subsequently analyzed by immunoblotting for p-NFκB. Gapdh was used as a loading control. Representative immunoblots are shown on the left-hand side. Quantification was performed by normalizing p-NFκB band densities with those of the loading control. N = 4. One-sample t tests were used to compare groups to the control group (∗ p < 0.05; ns, not significant). (C) Cardiac fibroblasts were incubated with either LPS or Hb for 24 h, after which expression of the indicated pro-inflammatory cytokines was determined by qPCR. Expression values are shown relative to the housekeeping gene Gapdh. N = 6. ANOVA with Tukey post-hoc tests were used to determine significance (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant).

Article Snippet: The qPCR primers for Gapdh (Mm99999915_m1, catalog no. 4331182), Actn2 (Mm00473657_m1, catalog no. 4331182), Myh6 (Mm00440359_m1, catalog no. 4331182), Tnni3 (Mm00437164_m1, catalog no. 4331182), Tnnc1 (Mm00437111_m1, catalog no. 4331182), Acta2 (Mm00725412_s1, catalog no. 4331182), Postn (Mm01284919_m1, catalog no. 4331182), Col1a1 (Mm00801645_g1, catalog no. 4351372), Scna5(Mm01342518_m1, catalog no. 4331182), Cacna1c (Mm01188822_m1, catalog no. 4331182), and Ryr2 (Mm00465877_m1, catalog no. 4331182) were obtained from Thermo Fisher Scientific.

Techniques: Incubation, Western Blot, Control, Isolation, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Hemoglobin mediates gene repression through TLR2 and TLR4 (A and B) A study was conducted to determine the effect of hemoglobin (Hb) on (A) fibroblast-to-cardiomyocyte reprogramming and (B) fibroblast gene expression. With respect to fibroblast-to-cardiomyocyte reprogramming, cardiac fibroblasts were transfected with either miR combo or a non-targeting control miR. 24 h later, the cells were incubated with vehicle, a TLR2 pharmacological inhibitor, a TLR4 pharmacological inhibitor, or a combination of both pharmacological inhibitors for 3 h. After incubation with the indicated pharmacological inhibitors, Hb was added (5 mg/mL) to the media. All media was replaced the next day. Fourteen days after miR transfection, cells were analyzed for expression of the indicated cardiomyocyte specific genes by qPCR. Expression values were normalized to the housekeeping gene Gapdh. N = 6–10. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant). t test was used to determine the significance between the miR combo groups (## p < 0.01, # p < 0.05; ns, not significant). With respect to fibroblast gene expression, cardiac fibroblasts were incubated with vehicle, a TLR2 pharmacological inhibitor, a TLR4 pharmacological inhibitor, or a combination of both pharmacological inhibitors for 3 h. After incubation with the indicated pharmacological inhibitors, Hb was added (5 mg/mL) to the media. All media was replaced the next day. Expression of the indicated fibroblast-specific genes was determined by qPCR and normalized to the housekeeping gene Gapdh. N = 10. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant).

Article Snippet: The qPCR primers for Gapdh (Mm99999915_m1, catalog no. 4331182), Actn2 (Mm00473657_m1, catalog no. 4331182), Myh6 (Mm00440359_m1, catalog no. 4331182), Tnni3 (Mm00437164_m1, catalog no. 4331182), Tnnc1 (Mm00437111_m1, catalog no. 4331182), Acta2 (Mm00725412_s1, catalog no. 4331182), Postn (Mm01284919_m1, catalog no. 4331182), Col1a1 (Mm00801645_g1, catalog no. 4351372), Scna5(Mm01342518_m1, catalog no. 4331182), Cacna1c (Mm01188822_m1, catalog no. 4331182), and Ryr2 (Mm00465877_m1, catalog no. 4331182) were obtained from Thermo Fisher Scientific.

Techniques: Gene Expression, Transfection, Control, Incubation, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Hemoglobin reshapes transcription factor networks during reprogramming Motif enrichment analysis was performed on each of the four DAR groups to identify transcription factor-binding motifs ( q < 0.01). (A) Motifs are plotted by statistical significance (–log10 q value) and expression level of the corresponding transcription factor. The dotted line represents the expression of the housekeeping gene Gapdh. (B) Motifs are plotted by statistical significance and the effect of miR combo on the expression of their associated transcription factor. N = 3. A DARs, regions that became accessible during reprogramming; B DARs, regions that were closed during reprogramming; C DARs, reprogramming regions that lost accessibility with Hb; D DARs, regions that became accessible with Hb.

Article Snippet: The qPCR primers for Gapdh (Mm99999915_m1, catalog no. 4331182), Actn2 (Mm00473657_m1, catalog no. 4331182), Myh6 (Mm00440359_m1, catalog no. 4331182), Tnni3 (Mm00437164_m1, catalog no. 4331182), Tnnc1 (Mm00437111_m1, catalog no. 4331182), Acta2 (Mm00725412_s1, catalog no. 4331182), Postn (Mm01284919_m1, catalog no. 4331182), Col1a1 (Mm00801645_g1, catalog no. 4351372), Scna5(Mm01342518_m1, catalog no. 4331182), Cacna1c (Mm01188822_m1, catalog no. 4331182), and Ryr2 (Mm00465877_m1, catalog no. 4331182) were obtained from Thermo Fisher Scientific.

Techniques: Binding Assay, Expressing