A comparison among the tissue-specific effects of aging and calorie restriction on TFAM amount and TFAM-binding activity to mtDNA in rat

Abstract

Background: Mitochondrial Transcription Factor A (TFAM) is regarded as a histone-like protein of mitochondrial DNA (mtDNA), performing multiple functions for this genome. Aging affects mitochondria in a tissue-specific manner and only calorie restriction (CR) is able to delay or prevent the onset of several age-related changes also in mitochondria.

Methods: Samples of the frontal cortex and soleus skeletal muscle from 6- and 26-month-old ad libitum-fed and 26-month-old calorie-restricted rats and of the livers from 18- and 28-month-old ad libitum-fed and 28-month-old calorie-restricted rats were used to detect TFAM amount, TFAM-binding to mtDNA and mtDNA content.

Results: We found an age-related increase in TFAM amount in the frontal cortex, not affected by CR, versus an age-related decrease in the soleus and liver, fully prevented by CR. The semi-quantitative analysis of in vivo binding of TFAM to specific mtDNA regions, by mtDNA immunoprecipitation assay and following PCR, showed a marked age-dependent decrease in TFAM-binding activity in the frontal cortex, partially prevented by CR. An age-related increase in TFAM-binding to mtDNA, fully prevented by CR, was found in the soleus and liver. MtDNA content presented a common age-related decrease, completely prevented by CR in the soleus and liver, but not in the frontal cortex.

Conclusions: The modulation of TFAM expression, TFAM-binding to mtDNA and mtDNA content with aging and CR showed a trend shared by the skeletal muscle and liver, but not by the frontal cortex counterpart.

General significance: Aging and CR appear to induce similar mitochondrial molecular mechanisms in the skeletal muscle and liver, different from those elicited in the frontal cortex.

Keywords: Aging rat; Calorie restriction; Mitochondrial Transcription Factor A; Mitochondrial Transcription Factor A–mitochondrial deoxyribonucleic acid binding; Tissue-specificity.

Fig. 1

Age- and CR-related changes of TFAM amount in three rat tissues. Representative Western blot carried out in (A) the frontal cortex, (C) the soleus skeletal muscle, and (E) the liver. The bands from top to bottom show, respectively, the signals from β-actin and TFAM. (B) The histogram shows the relative amount of TFAM in AL-26 and CR-26 rats, compared to AL-6 rats, all normalized with respect to β-actin in frontal cortex samples. Bars represent the mean (±SD) of the values obtained, respectively, from analysis in triplicate of the protein extract, from each young and aged AL and CR-treated rat. *p < 0.05 versus the value of the AL-6 rats (fixed as 1); n, number of analyzed animals. (D) The histogram shows the results in the soleus samples. The details of the legend are as in (B). (F) The histogram shows the relative amount of TFAM in AL-28 and CR-28 rats, compared to that in AL-18 rats, all normalized with respect to β-actin in liver samples. *p < 0.05 versus the value of the AL-18 rats (fixed as 1); n, number of analyzed animals.

Fig. 2

Genetic map of rat mtDNA. Rat mtDNA consists of a heavy-strand (outer circle) and a light-strand (inner circle) that code for the 12S and 16S rRNA and 22 tRNA genes (single-letter codes indicated under the corresponding tRNA genes) and subunits of the oxidative phosphorylation complexes: NADH dehydrogenase subunits (ND1–6 and ND4L), cyto-chrome b (Cyt b), cytochrome c oxidase subunits (CO I–III), and ATP synthase subunits (A6, A8). Also represented are: the origin of replication of the H strand (O_H), the origin of replication of the L strand (O_L), and the promoter of the L strand transcription (LSP). The thick arches extending out of the circles represent the five mIP amplified regions delimited, respectively, by the primer pairs: 16.0 For/16.2 Rev (D-loop), 5.0 For/5.3 Rev (Ori-L), 7.1 For/7.3 Rev (CO II), 8.0 For/8.2 Rev (DR1), and 15.0 For/15.2 Rev (Cyt b), listed in Table 1, and corresponding to the indicated nucleotides. Numbering is according to GenBank™ accession number AY172581. The outmost arch represents the 4.8-kb deletion, delimited by direct repeat 1 and 2 (DR1, DR2).

Fig. 3

Age- and CR-related changes of TFAM-binding to rat mtDNA regions (D-loop, Ori-L, DR1, CO II, and Cyt b) by mIP assay and following semi-quantitative PCR of mIP templates from the frontal cortex samples. Evaluation of TFAM-binding results in the three groups of animals after the mIP assay performed at five mtDNA regions. The groups of assayed animals included, respectively, eight AL-6, seven AL-26, and seven CR-26 rats, and their values were represented in the scatter-plot format. The intensity of each DNA band, produced by the specific PCR and visualized on agarose gels (not shown), was analyzed by densitometry and used for detection of TFAM-binding by subtracting the value of the intensity of the aliquot precipitated without primary antibody from that of the TFAM-immunoprecipitated aliquot, both normalized to the value of the respective input aliquot made equal to 1.

Fig. 4

Age- and CR-related changes of TFAM-binding to rat mtDNA regions (D-loop, Ori-L, DR1, CO II, and Cyt b) by mIP assay and following semi-quantitative PCR of mIP templates from the soleus skeletal muscle samples. Evaluation of TFAM-binding results in the three groups of animals after the mIP assay was performed at five mtDNA regions. The groups of assayed animals included, respectively, five AL-6, six AL-26, and five CR-26 rats, and their values were represented in the scatter-plot format. The intensity of each DNA band, produced by the specific PCR and visualized on agarose gels (not shown), was analyzed by densitometry and used for detection of TFAM-binding by subtracting the value of the intensity of the aliquot precipitated without primary antibody from that of the TFAM-immunoprecipitated aliquot, both normalized to the value of the respective input aliquot made equal to 1.

Fig. 5

Age- and CR-related changes of TFAM-binding to rat mtDNA regions (D-loop, Ori-L, and DR1) by mIP assay and following semi-quantitative PCR of mIP templates from the liver samples. Evaluation of TFAM-binding results in the three groups of animals after the mIP assay performed at three mtDNA regions. The groups of assayed animals included, respectively, six AL-18, six AL-28, and six CR-28 rats, and their values were represented in the scatter-plot format. The intensity of each DNA band, produced by the specific PCR and visualized on agarose gels (not shown), was analyzed by densitometry and used for detection of TFAM-binding by subtracting the value of the intensity of the aliquot precipitated without primary antibody from that of the TFAM-immunoprecipitated aliquot, both normalized to the value of the respective input aliquot made equal to 1.

Fig. 6

Age- and CR-related changes of the relative mtDNA content. The histogram shows the mean value of the ratio mtDNA/nuclear DNA in AL and CR rats. Bars represent the mean (±SE) obtained, respectively, from analysis in triplicate of total nucleic acids from each AL-6, AL-18, AL-26, AL-28, CR-26 and CR-28 rat. The groups of assayed animals included, respectively, five AL-6, five AL-18, six AL-26, six AL-28, five CR-26 and five CR-28 rats. *p < 0.05 versus the value of the younger rats (AL-6 or AL-18 fixed as 1); **p < 0.05 versus the value of the old AL rats (AL-26 or AL-28).