Fig 2: High level of Trx protects against I/R mediated LV dysfunction and infarction of aged myocardium. (A). Representative M-mode images taken from NT, Trx-Tg and dnTrx-Tg hearts of sham (top) and I/R (bottom). Ejection fraction; EF (B) and fractional shortening; FS (C), an index of cardiac contractile function, was determined by echocardiographic analysis. *p <0.05 versus NT sham; **p <0.05 versus NT or dnTrx-Tg I/R hearts, n=3-5. (D) NT, Trx-Tg and dnTrx-Tg mice were subjected to 60 min ischemia and 30 min reperfusion and then TTC staining was performed as described in the methods section. TTC stains viable tissue brick red and necrotic tissue as white. (E) Infarct area in relation to area-at-risk (AAR). *p <0.05 versus NT or dnTrx-Tg I/R; **p <0.05 versus NT or Trx-Tg I/R, n=4. Statistical significance was determined with the Student’s t-test.

Fig 3: High level of Trx prevents I/R-induced mitochondrial cristae and DNA damage. (A) Ultrastructural analysis of sham or I/R hearts from NT, Trx-Tg and dnTrx-Tg. Representative transmission electron microscopic images showing cristae structure and density. Calculated cristae density (B), percent damaged mitochondria (loss of =50% cristae density) (C), and width of mitochondria (D) and plotted as bar graph. Values are means ± SD (n = 25). *, p < 0.01 versus Sham; **, p <0.01 versus NT or Trx-Tg IR.

Fig 4: Trx protects against I/R-induced mitochondrial dysfunction. Effect of I/R on coupling and electron flow in mitochondria isolated from mice heart: Mitochondria were isolated from AAR region of sham or I/R subjected NT, Trx-Tg and dnTrx-Tg mice hearts as described in Materials and methods. (A) representative figure of XF24 coupling assay of sham or I/R mice mitochondria from (A) NT; (E) Trx-Tg; (I) dn-Trx-Tg mice, and OCR response to ADP, oligomycin, FCCP and antimycin A using point-to-point measurements. Graph of basal (state 2), state 3, and state 40 respirations in sham and I/R mice heart mitochondria from (B) NT; (F) Trx-Tg; (J) dn-TrxTg. A representative OCR measurement figure of mitochondria from (C) NT; (G) Trx-Tg; (K) dn-TrxTg mice with pyruvate and malate as substrates, and the effect of rotenone, succinate, antimycin A and Asc/TMPD. Sham and I/R mouse mitochondria from (D) NT; (H) Trx-Tg; (L) dn-TrxTg mice with basal, rotenone, succinate, antimycin A and Asc/TMPD mediated OCR (sham = 3, I/R n = 3). Statistical significance was determined with one-way ANOVA followed by Tukey’s post-hoc multiple comparisons test. *P<0.05.

Fig 5: Trx prevents I/R-induced loss of mitochondrial proteins, by upregulating transcription of PGC1a. (A). Mitochondria was isolated from sham or I/R subjected NT, Trx-Tg and dnTrx-Tg mice. The mitochondrial extracts were analyzed for oxidative phosphorylation complex subunits by western blot using Abcam OXPHOS cocktail antibody. (B) Western blot analysis of ACO2, MFN1, MFN2, TFAM, Hexokinase 1 (HK-1), Sod2 and COX IV in sham or I/R mitochondrial extracts from NT, Trx-Tg and dnTrx-Tg mice. (C, D). Protein levels were quantified and expressed as fold change. *p <0.05 versus NT sham; **p <0.05 versus NT or dnTrx-Tg I/R. (E) Aconitase 2 activity and (F). MnSOD activity was determined in sham and I/R myocardium obtained from NT, Trx-Tg, or dnTrx-Tg mice as described in materials and methods. *p <0.05 versus respective sham; **p <0.05 versus NT or dnTrx-Tg I/R. n=3. (G) AAR region of sham or I/R myocardium from NT, Trx-Tg and dnTrx-Tg were lysed using M-PER lysis buffer and analyzed for PGC1a and GAPDH by western blotting. (H). Level of PGC1a was quantified and expressed as fold change. *p <0.05 versus NT sham; **p <0.05 versus NT or dnTrx-Tg I/R. (I). RT-PCR analysis of PGC1a, ACO2 and TFAM in sham and I/R myocardium (J). mRNA levels of PGC1a, ACO2 and TFAM were quantified and expressed as fold change. *p <0.05 versus NT sham; **p <0.05 versus NT or dnTrx-Tg I/R. Statistical significance was determined with the Student’s t test (C, D, H, and J) and one-way ANOVA followed by Tukey’s post-hoc multiple comparisons test (E, and F).