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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 18, 2003 as doi:10.1096/fj.02-1009fje. |
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,2
* Division of Biology, California Institute of Technology, Pasadena, California, USA;
Institute of Clinical Neurology, University of Milan, Ospedale Maggiore Policlinico, Milan, Italy; and
Department of Medical Biochemistry and Biology, University of Bari, Bari, Italy
3Correspondence: Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA. E-mail: attardi{at}caltech.edu
SPECIFIC AIMS
The main objective of the present work was to carry out a comprehensive and in-depth analysis of the donor’s age-related changes that occur in various biochemical and bioenergetic parameters underlying the oxidative phosphorylation (OX-PHOS) capacity in fibroblast cultures, mostly skin-derived, from a large group of subjects (55), 20-week fetal–103 years of age.
PRINCIPAL FINDINGS
In the present work, pronounced differences were observed in several parameters between the fibroblast cultures derived from two 20-week fetuses and those derived from very young (1- to 19-year-old) subjects. Thus, the two fetal samples exhibited a much higher rate of mitochondrial protein synthesis than almost all postnatal samples (50 samples of 51) and, in particular, higher than observed in the fibroblasts from the six very young individuals (Fig. 1
). Furthermore, they showed very low rates of native or dinitrophenol (DNP)-uncoupled endogenous respiration, which approached the lowest values found in the samples from old individuals. The latter results could account also for the apparently relaxed control of respiration by cytochrome c oxidase (COX) in fetal fibroblasts, as revealed by the very high values in these cells of the COXRmax, i.e., of the ratio of maximum COX capacity to the endogenous DNP-uncoupled O2 consumption rate (COXRmax); these values were indeed close to the highest values observed in the samples from old subjects. These interesting properties of the fetal fibroblasts examined—which appeared to reflect a biochemical differentiation state expected for cells relying mostly on glycolysis as a bioenergetic source, but very active in mitochondrial biogenesis—obviously demand a more extensive analysis. In the present work, the corresponding data, because of their distinctive character, have not been included in the calculation of the statistical significance of the fibroblast age-related changes.
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1. Very significant aging-related decrease in rate of mitochondrial protein synthesis in skin fibroblasts
A striking aging-related change observed in 51 postnatal skin fibroblast samples in a fundamental process underlying mitochondrial biogenesis and function was the very significant decrease (P<0.0001) in rate of mitochondrial protein synthesis (Fig. 1A
). In particular, despite the considerable interindividual variability, an age-group plot revealed that the average rate of mitochondrial protein labeling remained fairly constant in fibroblasts from individuals in the age range of 1–39 years but decreased markedly in fibroblasts from subjects in the age range of 42–103 years (Fig. 1B
).
2. Aging-related decline in endogenous native or DNP-uncoupled respiration rate and increase in COXRmax in skin fibroblasts
The oxygen consumption rates were measured polarographically in a buffer in which 143B.TK– osteosarcoma cells, which we often use as reference cells, can respire, using endogenous substrates, at the same rate as in Dulbecco’s modified Eagle’s medium lacking glucose. The analysis of the native or DNP-uncoupled endogenous respiration rate in the large group of fibroblast cultures from differently aged postnatal individuals investigated here (51) showed a rather complex pattern. Despite the large scattering of the individual values, an age-group plot revealed a fairly constant average rate of native or uncoupled O2 consumption in the fibroblast cultures from individuals in the age range between 1 and 39 years, and a clear decrease in the cultures from individuals in the age range between 42 and 89 years, with a tendency to uncoupling in the subjects above 60 years. The regression lines of the endogenous native or uncoupled respiration data from the subjects 1–89 years old did indeed show a very significant age-related decrease (P<0.0001). The decrease in respiration rate was not apparent in the fibroblast cultures from individuals 
90 years of age, as a result of the presence among these of a subgroup of six highly respiring samples. Consistent with the decrease in DNP-uncoupled respiration rate in the fibroblasts from individuals 42–89 years of age, the analysis of the in vivo control of respiration by COX revealed a significant increase in the same donor’s age range in COXRmax.
3. Unusually high endogenous respiration rates in skin fibroblasts from 90-year-old subjects
A surprising finding was the occurrence, mentioned above, of a subgroup of six individuals 90 years old exhibiting unusually high rates of native and DNP-uncoupled endogenous respiration. The evidence obtained in the present work has indicated that the high rates of O2 consumption in cells derived from this subgroup of very old individuals were not a result of the tendency to uncoupling of endogenous respiration observed in fibroblasts from aged individuals nor of a higher mitochondrial (mt)DNA content per cell.
4. Marked aging-dependent decline in coupling efficiency of respiration and phosphorylation in skin fibroblasts
The most striking aging-related mitochondrial phenotypic changes observed in skin fibroblast cultures were those affecting the OX-PHOS capacity. Thus, intact fibroblasts from 46 individuals of different ages exhibited a very significant (P=0.0007) aging-dependent decrease in the ratio of maximum DNP-uncoupled endogenous respiration to respiration inhibited by oligomycin, a drug that inhibits adenosine 5'-triphosphate (ATP) synthase in mitochondria, bringing the cells to a state similar to state 4. This result pointed to a decline with age in the control of respiration by the mitochondrial membrane potential. A direct estimate of the aging-induced decrease in efficiency of adenosine 5'-diphosphate (ADP) phosphorylation in fibroblasts was obtained by determining their phosphorylation-to-oxygen (P:O) ratio, i.e., the amount of ATP in nmoles produced from ADP + inorganic phosphate (Pi) per nanogram atom of oxygen consumed, by a novel in situ method, i.e., by polarography in digitonin-permeabilized fibroblasts. As shown in Figure 2
, the efficiency of ADP phosphorylation in 32 postnatal samples decreased very significantly with the donor’s age (P=0.0006). The age-related changes in the efficiency of control of respiration by phosphorylation were also monitored by calculating the respiratory control ratio (RCR), i.e., the ratio of the rate of respiration in the presence of ADP (state 3) to the rate of respiration in the absence of ADP (state 4). Consistent with the aging-dependent decrease in the control of respiration by the mitochondrial membrane potential and in the efficiency of ADP phosphorylation, the RCR values showed a very significant decrease with the donor’s age (P<0.0001).
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CONCLUSIONS AND SIGNIFICANCE
This paper has presented a comprehensive analysis of the age-related biochemical and bioenergetic changes occurring in fibroblasts from human subjects 20-week fetal–103 years of age. An interesting outcome of this work has been the recognition that the developmental stage of the fibroblast donors, in the prenatal and the postnatal period, plays an important role in determining the level of several parameters that control the OX-PHOS capacity of fibroblasts.
The fundamental conclusion of this work is that several fundamental mitochondrial processes underlying the cell OX-PHOS capacity, i.e., mitochondrial protein synthesis, respiration, and coupling of respiration to ATP synthesis, deteriorate with aging in human skin fibroblasts, starting in general at approximately 40 years. As illustrated in Figure 3
, it seems very likely that reactive oxygen species (ROS), which are known to cause aging-dependent damage to DNA, proteins, and lipids, are mainly responsible for the OX-PHOS defects described above. In particular, these ROS could act directly on the OX-PHOS apparatus or indirectly, i.e., by causing mtDNA damage or nuclear damage, with the resultant synthesis of altered or quantitatively insufficient mitochondrial translation products or nuclear-encoded proteins. The OX-PHOS defects could, in turn, further activate the ROS production, thus creating a progressive, vicious cycle.
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In the past 15 years, numerous biochemical, histochemical, immunohistochemical, and in situ hybridization analyses on bioptic tissue samples or isolated cell populations have revealed a decline with aging in the activities of the human OX-PHOS apparatus. In the present study, for the first time, the measurements of a multiplicity of parameters have allowed us to correlate the various biochemical or bioenergetic changes observed in fibroblast cultures so as to formulate a plausible, general sequence of events, as illustrated in Figure 3
. They have also helped explain some of the biochemical events. Thus, the low DNP-uncoupled, endogenous respiration rate could account in good part for the high COXRmax in fibroblasts from fetuses and in fibroblasts from old individuals, supporting the conclusion that, in the cells from both types of donors, the upstream respiratory chain activities are more limiting.
The discovery of a subgroup of skin fibroblast cultures from very old individuals exhibiting an unusually high endogenous respiration rate has pointed to a possible compensatory phenomenon. If this interpretation is correct, the above observation would extend to the aging processes the cell capacity to compensate for a deficient respiratory chain activity, which has been previously observed in cellular and mouse models of mitochondrial myopathy, caused, respectively, by the mitochondrial tRNALeu(UUR) mutation responsible for the MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes) encephalomyopathy and by the mitochondrial transcription factor A (Tfam) disruption. However, no conclusion could be drawn in the present work as to whether this proposed compensatory phenomenon in aging occurs during the in vitro growth of the fibroblasts or already in vivo. An alternative explanation for the high respiration rate of the skin fibroblast cultures from some very old individuals may be the selection of a different cell type as a consequence of a more extensive dermal atrophy.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1009fje; doi: 10.1096/fj.02-1009fje 
2 Present address: Department of Biological and Environmental Science and Technology, University of Lecce, I-73100 Lecce, Italy.
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) were excluded. (B) Age group plot of the data in A. Each bar is the average value of mitochondrial protein synthesis rate ± SE for the considered 20-year age range.
