Mitorubin, berberrubine-based compounds that improve mitochondrial function, exhibit cardioprotective effects against age-related cardiac dysfunction

Figures

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  Fig. 1. Identification of berberrubine, a berberine metabolite, as a compound that enhances MITOL expression. a Schematic representation of the compound evaluation strategy. Various herbal extracts and previously reported mitochondria-activating bioactive compounds were individually administered to human dermal fibroblasts. Candidate compounds identified based on MITOL mRNA upregulation by qRT-PCR were subsequently administered to mice to assess MITOL expression in multiple organs. b Relative MITOL mRNA levels in human dermal fibroblasts after 24-hour treatment with each compound, quantified by qRT-PCR. mRNA levels were normalized to GAPDH and expressed as a percentage relative to the control group (without test substances). Data represent the average of three independent experiments (n = 3). c, d Increased MITOL expression in mouse tissues following herbal extract administration. Mice were administered each herbal extract (Phellodendron amurense, Coptis japonica, or Paeonia suffruticosa) intraperitoneally, and 9 days later, MITOL expression levels in various organs were assessed by western blot. d shows the graph of MITOL expression levels in the heart, normalized to tubulin (n = 5–6…

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  Fig. 2. Synthesis of quinoid-type berberrubine, berberrubine acetic acid adduct, and berberrubine acetic acid salt. a Quinoid-type berberrubine, shown as a reddish-black solid, was obtained by thermal decomposition of berberine chloride under reduced pressure (Step i). The resulting berberrubine powder was suspended in acetic acid, and the precipitate formed was collected by suction filtration, washed with diethyl ether, and dried under reduced pressure (Step ii), yielding the yellow solid berberrubine acetic acid adduct. Further drying (Step iii) produced the reddish-brown solid berberrubine acetic acid salt. b ¹H NMR spectra of the berberine-derived compounds in deuterated DMSO. From top to bottom: reddish-black solid (quinoid-type berberrubine), reddish-brown solid (berberrubine acetic acid salt), and yellow solid (berberrubine acetic acid adduct). The peaks indicated by the red arrow correspond to the protons of the methyl groups in acetic acid molecules.

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  Fig. 3. Enhancement of mitochondrial biogenesis and protein expression by berberrubine in C2C12 myoblasts. a, b C2C12 cells were treated with Q-BRB or AcA-BRB (5 μM or 10 μM) for 48 h, and representative western blots of mitochondrial-localized proteins (outer membrane: MITOL, Tom20, Mfn2; matrix: HSP60; inner membrane: ATP5a) are shown in (a). Signal intensities were normalized to Vinculin and expressed as values relative to the 0 μM group (n = 4 per group) (b). The 0 μM group indicates cells cultured without any additives, including solvents. c mRNA levels of MITOL and Tom20 were measured by qRT-PCR and normalized to GAPDH and expressed as fold change relative to the 0 μM group (n = 3 per group). d Mitochondrial content was quantified by real-time PCR based on the ratio of mtDNA (16S rRNA or ND1) to nuclear DNA (Hexokinase 2; HK2) and expressed as fold change relative to the 0 μM group (n = 3 per group). Statistical significance was determined using one-way ANOVA followed by Tukey’s HSD test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

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  Fig. 4. Mitochondrial elongation and increased oxygen consumption by berberrubine in C2C12 myoblasts. a C2C12 cells were treated with Q-BRB or AcA-BRB (10 μM) for 48 h, followed by immunostaining with an anti-Tom20 antibody to visualize mitochondrial morphology (green: anti-Tom20; blue: Hoechst). Representative images are shown in the upper panels. Enlarged views of the boxed regions are displayed in the lower panels. Scale bars: 20 μm (upper), 10 μm (lower). The None group indicates cells cultured without any additives, including solvents. b Mitochondrial length was quantified in 30 randomly selected cells per group using MiNA (Mitochondrial Network Analysis). Mitochondria were classified into four categories based on length: Short ( ≤ 1.855 μm), Middle (1.855–3.599 μm), Long (3.599–8.103 μm), and Very long ( ≥ 8.103 μm), and presented as the proportion of mitochondria in each category within each group. Representative images for each category are shown on the right. Scale bar: 20 μm. c, e C2C12 cells were treated with Q-BRB or AcA-BRB (30 μM) for 24 h, and mitochondrial oxygen consumption rate (OCR) was measured (n = 3 per group). d, f Basal respiration, ATP synthesis-linked…

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  Fig. 5. Cardioprotective effects of berberrubine in aged mice with age-related cardiac dysfunction. a Experimental scheme of berberrubine treatment in aged mice. b Representative M-mode echocardiography recordings in the indicated mice. M-mode images were obtained from the short-axis view at the mid-ventricular level. c Echocardiographic measurements of left ventricular end-diastolic diameter (LVDd, mm), left ventricular end-systolic diameter (LVDs, mm), percent fractional shortening (%FS) and Left Ventricular Ejection Fraction (LVEF) at 8 weeks after intervention (n = 7, control; n = 10, AcA-BRB). d Representative echocardiographic recordings of transmitral flow velocity, including early transmitral flow velocity (E), atrial systolic velocity (A) (top row), and early diastolic mitral annular velocity (e’) (bottom row) in the indicated groups. e E/e’ ratios in the indicated mice (n = 7, control; n = 10, AcA-BRB). f Gross appearance of whole hearts (top row; scale bar, 5 mm) and whole lungs (bottom row; scale bar, 5 mm) from the indicated mice. g Body weight (BW, g), tibial length (TL, mm), heart weight/tibial length ratio (HW/TL, mg/mm), and lung weight/tibial length ratio (LW/TL, mg/mm)…

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  Fig. 6. Effects of berberrubine on mitochondrial function and lifespan in aged mice with age-related cardiac dysfunction. Relative expression of genes associated with mitochondrial (a) biogenesis, (b) dynamics, and (c) mitophagy in heart tissues of the indicated mice (n = 6, control; n = 9, AcA-BRB). Relative mRNA expression levels were normalized to Rps18 and presented as fold changes relative to the control group. d OCR analysis of mitochondria isolated from heart tissues of the indicated groups (n = 5 per group). e Quantification of basal respiration (Basal), ATP-linked respiration in the presence of ADP (State 3), resting respiration in the presence of oligomycin (State 4o), and maximal uncoupled respiration in the presence of FCCP (State 3 u) (n = 5 per group) in the indicated groups. f Survival curve of the indicated groups (n = 33 per group). g Survival curve of the indicated groups (n = 13, control; n = 14, AcA-BRB). The timing of berberrubine administration is indicated by red arrowheads in (f, g). Statistical significance was determined using a two-sided unpaired Student’s t test (a, b, c, d, e) or the log-rank (Mantel-Cox) test (f, g).