Cardiac-specific overexpression of serum response factor regulates age-associated decline in mitochondrial function

Fig. 1. Overexpression of SRF reduces lifespan and induces cardiomyopathy. A DNA construct for the generation of SRF transgenic mice. SRF, the full-length cDNA of human SRF gene; α-MHC, α-myosin heavy chain; Hgh, the polyA sequence of human growth hormone gene. B Kaplan–Meier graph of the maximum lifespan in SRF-Tg mice (gray, n = 20) compared with non-Tg littermate mice (black, n = 20). The differences between groups were evaluated using the log-rank test. C Hearts of SRF-Tg and non-Tg mice at 6 months of age. The ruler shows inches and centimeters markings. D Ratio of heart weight to body weight (mg/g) in non-Tg (NTg) and SRF-Tg mice at 3 months and 6 months of age. All data are presented as means ± SD, n = 20, where n represents biological replicates. **P < .005, ***P < .001, using two-way ANOVA with Bonferroni multiple comparisons test

Fig. 2. Histological and western blot analysis of cardiac hypertrophy and fibrosis. A Left ventricular cardiac myocytes of SRF-Tg and non-Tg mice at 6 months of age stained with hematoxylin and eosin stain. B Masson trichrome staining of the left ventricle section of SRF-Tg and non-Tg mice at 6 months of age. Collagen stains blue with trichrome. C Quantitative analysis of cell surface cross-sectional area of cardiomyocytes. Values are mean ± SD (n = 6). D Quantitative analysis of fibrosis area. The fibrotic area was measured as blue stained-positive area normalized to the high-power field of total myocardial area. Values are mean ± SD (n = 6). E Representative western blot images of BNP, a marker for left ventricle hypertrophy and total SRF expression at 3 months and 6 months of age. GAPDH was used as a loading control. F, G Quantification of relative protein levels normalized against GAPDH. Data are mean ± SD (n = 3). N represents independent biological repeats. A 20 × objective is used; scale bars indicate 20 μm. *P < .05, **P < .01, using two-way ANOVA with Bonferroni multiple comparisons test

Fig. 3. Transmission electron microscopy and western blot analysis of proteins involved in mitochondrial function and dynamics. A Detailed transmission electron microscopy of left ventricular tissue taken from SRF-Tg and non-Tg mice at 6 months of age. The red box in non-Tg section was magnified below; red arrows show regular arrangement of myofibrils and well-packed, regular cristae in the mitochondria. The magnified section of the yellow box in SRF-Tg below shows yellow arrows pointing toward shortened and disarrayed myofibrils and arrows indicating damaged mitochondria with loss of cristae. Scale bars indicate 1 μm (B) The average percentage of cristae in total mitochondrial area of different EM sections at 6 months of age (n = 6). C Percentage of damaged mitochondria of different EM sections at 6 months of age (n = 6). D Fold amount of mtDNA copy number at 3 months and 6 months of age (n = 5). E Representative immunoblots of PGC-1α, NT-PGC-1α, PGC-1β, Opa1, and Drp1 in SRF-Tg and non-Tg mice hearts at 3 months and 6 months of age. (F, G, H, I, J) Quantification of the relative changes in protein levels normalized against GAPDH. Data are mean ± SD (n = 3). N represents independent…

Fig. 4. Functional analysis of mitochondrial respiratory chain. A Representative trace of high-resolution respirometry with a multiple substrate-inhibitor titration protocol in mice hearts of SRF-Tg comparing to non-Tg littermates at 6 months of age. Oxygen consumption is represented as a function of time. Blue lines indicate times of titrations of substrates and inhibitors. The protocol includes the following steps: malate/glutamate (Complex I-linked substrates), ADP (OXPHOS capacity), rotenone (inhibition of Complex I), succinate (Complex II), antimycin A (inhibition of Complex III), ascorbate/tetramethyl-p-phenylenediamine (Complex IV), and sodium azide (inhibition of Complex IV). B Quantification of complexes activity (n = 3). C Representative western blot of OXPHOS mitochondrial complexes (I–V). Antibody cocktail against complexes was used to examine the expression of mitochondrial proteins in SRF-Tg mouse hearts at 3 and 6 months of age. D, E, F, G, H Relative quantification of protein levels normalized against GAPDH. Data are mean ± SD (n = 3). N represents independent biological repeats. *P < .05, **P < .01, ***P < .001, using two-way ANOVA with Bonferroni multiple comparisons test

Fig. 5. Western blot analysis of proteins involved in oxidative stress and calcium handling. A Representative western blot images of 4-HNE, a marker for oxidative stress, glutathione reductase, and MnSOD, an antioxidant enzyme, in SRF-Tg and non-Tg mouse hearts at 3 and 6 months of age. B, C, D Relative quantification of protein levels of 4-HNE, glutathione reductase, and MnSOD normalized against GAPDH in 3 months and 6 months of age. E Representative western blot images of SERCA2, which pumps calcium back into the sarcoplasmic reticulum, and RYR2, a calcium release channel, in SRF-Tg and non-Tg mouse hearts at 3 months and 6 months of age. F, G Relative quantification of protein levels of SERCA2 and RYR2 normalized against GAPDH. Data are mean ± SD (n = 5), where n represents independent biological repeats. *P < .05; **P < .01; ***P < .001; ns, P > .05, using two-way ANOVA with Bonferroni multiple comparisons test

Fig. 6. Overexpression of SRF activates the MAPK signaling pathway. A Representative immunoblots of total and phosphorylation of MEK, ERK1/2, JNK1/2, p38 MAPK in SRF-Tg and non-Tg mouse hearts at 3 months and 6 months of age. B, C, D, E Quantification of the relative changes in phosphorylation of MEK, ERK1/2, JNK1/2, and p38 from total protein. F Representative immunoblots of total and phosphorylated MEK, ERK1/2, JNK1/2, p38 in young (6 month) versus old (24 months) Wild-type mice hearts. G, H, I, J Quantification of the relative changes in phosphorylation of MEK, ERK1/2, JNK1/2, and p38 from total protein expression. Molecular weight (kDa) are indicated. GAPDH was used as a loading control. Data are expressed as mean ± SD (n = 5), where n represents independent biological repeats. *P < .05; **P < .01; ***P < .001; ns, P > .05, using two-way ANOVA with Bonferroni multiple comparisons test

Fig. 7. Overexpression of SRF alters mitochondrial bioenergetics. AC16 cells were transfected with either an SRF plasmid or an empty vector. Various mitochondrial assays were performed. A Seahorse XF Cell Mito Stress Test results of cells transfected with either empty vector or SRF vector shown as mean ± SD normalized to equal number of cells. Oligomycin was injected to inhibit ATP-linked mitochondrial respiration as a measure of mitochondrial ATP production. To determine maximal respiration, the mitochondrial uncoupler FCCP was injected. To determine the non-mitochondrial respiration to the oxygen consumption rate, rotenone was injected, inhibiting mitochondrial respiration. B Quantification of basal respiration, maximal respiration, and spare respiratory capacity calculated from Seahorse XF Cell Mito Stress Test (n = 5). C Glycolytic rate assay shown as mean ± SD normalized to equal number of cells. Rotenone/Antimycin A mix was injected to block mitochondrial activity and 2-DG (to inhibit glycolysis) as an internal control. D Graphs show the calculated glycolytic parameters of basal and compensatory glycolysis (n = 5). E Real-time ATP rate assay in AC16 cells (n = 5). F Representative…