Fig 2: Mitochondrial fission was increased during CF activation. (A) MitoTracker Deep Red staining was used to observe the mitochondrial morphology of cardiac fibroblasts (CF). Mitochondrial volume and the mean (area/perimeter)/circularity index was calculated using ImageJ. (B) Transmission electron microscopy (TEM) was employed to directly observe the fragmented mitochondria. Mitochondrial area and length were also measured using ImageJ. (C) The expression of fission-related and fusion-related proteins following TGF-ß1 plus mdivi-1 cotreatment was measured in whole-cell lysates via immunoblotting. (D) Immunoblotting, with quantification, of the same proteins in mitochondrial and cytoplasmic fractions. COXIV was used as the loading control for mitochondrial proteins. (E) The translocation of Drp1 to the mitochondrial membrane was observed by confocal microscopy. Colocalization was analyzed using the Coloc 2 plugin in ImageJ. Data are shown as mean ± standard error of the mean (n = 3 independent cell isolations per group). Means were compared using one-way ANOVA, followed by the Student–Newman–Keuls (SNK) post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: Expression of mitochondrial dynamics regulatory proteins in Sigmar1-/- mouse hearts at 6 months of age. A, Representative Western blot and (B) densitometric quantification of the expression of mitochondrial dynamic regulatory proteins in the whole-cell fraction of Wt and Sigmar1-/- hearts: pDrp1 Ser637, pDrp1 Ser616, Drp1, and OPA1. C, Representative Western blot and (D) densitometric quantification of the expression of mitochondrial dynamic regulatory proteins in the mitochondrial fraction of Wt and Sigmar1-/- hearts: Drp1 and OPA1. Drp1-overexpressing HEK293 cells were used as a positive control for Drp1, and ß-actin was used on the same membrane. The cytosol fraction was used as a positive control to confirm GAPDH detection. COXIV was used on the same membrane, confirming the mitochondrial extracts. Ponceau S protein staining of the transfer membrane confirmed approximately equal loading and transfer across the gel (n=5-6 mice per group). P values were determined by Tukey post hoc test. Boxes represent interquartile ranges, lines represent medians, whiskers represent ranges, and P values were determined by a Kruskal-Wallis test. NS indicates not significant; Wt, wild type.

Fig 4: Acute kidney injury (AKI) induces damage to cardiomyocyte mitochondria. (A) ATP production was measured through Enhanced ATP Assay Kit. (B,C) Cardiomyocytes were isolated from mice after AKI. Then, mitochondrial membrane potential was determined through JC-1 probe in primary cardiomyocytes. The ratio of red-to-green fluorescence intensity was used to evaluate mitochondrial membrane potential. (D,E) In cardiomyocytes, DCFH-DA was used to observe the levels of intracellular reactive oxygen species. (F,G) Immunofluorescence of the mitochondria. The number of fragmented mitochondria in cardiomyocytes was recorded. (H,I) Western blots were used to analyze the expression of phosphorylated Drp1 (p-Drp1). The relative expression of p-Drp1 was evaluated in cardiomyocytes. *p < 0.05 vs. sham group.

Fig 5: The mechanism diagram for the cognitive impairment by RH. Recurrent hypoglycemia reduces TRPC6-mediated transient Ca2+ intake, which induces excessive mitochondrial fission and mitochondrial dysfunction through increasing AMPK-dependent DRP1 phosphorylation on Ser616 and reducing it on Ser637, leading to neuronal death and cognitive impairment in diabetic mice. Treatment with hyperforin represses cognitive impairment in diabetic mice undergoing recurrent hypoglycemia by activating TRPC6