MtDNA point mutations are associated with deletion mutations in aged rat

Experimental Gerontology 40 (2005) 209–218 www.elsevier.com/locate/expgero

MtDNA point mutations are associated with deletion mutations in aged rat Jeong W. Pak, Fue Vang, Chad Johnson, Debbie McKenzie, Judd M. Aiken* Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1656 Linden Drive, Madison, WI 53706, USA Received 17 September 2004; received in revised form 3 December 2004; accepted 10 December 2004 Available online 18 January 2005

Abstract The age-dependent accumulation of point mutations in the control region of human mtDNA has been suggested to contribute to aging processes. We investigated whether mtDNA point mutations accumulate to detectable levels in this region of mtDNA from aged Fischer 344 X Brown Norway F1 hybrid rats. The control region and a portion of the major arc region (nucleotides 4386–7707) of the mtDNA were PCRamplified and directly sequenced from microdissected single cardiomyocytes and single skeletal muscle fibers of 36-month old rats. Point mutations were not observed in these regions of the full-length mtDNA. Point mutations were, however, associated with deletion mutations, especially in cardiac cells. Approximately 40% of the deletion mutations identified in heart contained a point mutation, whereas only 1.9% of deletion mutations in skeletal muscle contained a point mutation. Point mutations were located adjacent to the deletion breakpoints and each point mutation was unique. In aged rats, point mutations are clonally expanded only when associated with deletion events suggesting that there are important differences between rats and humans in the mechanisms that cause mtDNA abnormalities. q 2005 Elsevier Inc. All rights reserved. Keywords: Mitochondrial DNA; Point mutation; Deletion mutation; Laser-capture microdissection

1. Introduction The accumulation of damage to mitochondrial macromolecules as an underlying mechanism of aging was proposed decades ago (Harman, 1983). Screening for mitochondrial DNA (mtDNA) abnormalities identified age-induced deletion mutations in numerous species, primarily in nervous tissue, muscle and heart (Cortopassi et al., 1992; Zhang et al., 1992; Wanagat et al., 2001). The impact of these genomic alterations in skeletal muscle includes concomitant phenotypic expression of mitochondrial electron transport system (ETS) abnormalities and subsequent fiber atrophy and muscle fiber breakage (Cao et al., 2001; Wanagat et al., 2001). Single cell analysis has identified high levels of age-related somatic point mutations in some tissues (Jazin et al., 1996; Michikawa et al., 1999; Calloway et al., 2000; Murdock et al., 2000; Taylor et al., 2001; Wang et al., 2001; Nekhaeva et al., 2002). The majority of these studies have focused primarily on * Corresponding author. Tel.: C1 608 262 7362; fax: C1 608 262 7420. E-mail addresses: [email protected] (J.M. Aiken), jma@ ahabs.wisc.edu (J.M. Aiken). 0531-5565/$ - see front matter q 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2004.12.005

the control region (D-loop region, w1 kb) of the human mitochondrial genome. The total load of mtDNA point mutations in human cardiac and buccal cells, based on those identified in the control region, was postulated to be as high as one expanded point mutation per cell on average (Nekhaeva et al., 2002). Point mutations in the control region were not linked to other mtDNA mutations and accumulated to greater than 50% in individual cardiomyocytes. Since the control region is crucial for replication and transcription of the mitochondrial genome, the abundance and accumulation of point mutations in the control region from aged human tissues suggested that point mutations play a role in aging. Although single nucleotide mutations were shown to accumulate with age in humans, they have not been linked to abnormal phenotypes nor is it known if the age-associated clonal accumulation of point mutations occurs in other species. Transgenic mice expressing a proof-reading deficient version of the catalytic subunit of mtDNA polymerase have provided strong evidence that mtDNA rearrangements can cause aging phenotypes (Trifunovic et al., 2004). These mice exhibited increased levels of single nucleotide mtDNA mutations as well as mtDNA deletion mutations with age.

210

J.W. Pak et al. / Experimental Gerontology 40 (2005) 209–218

The mtDNA-mutator created a random set of point mutations in genes for respiratory chain subunits and the mutations were evenly distributed between tissues, accounting for deficient mitochondrial function in various tissues observed by histological analyses. Numerous severe phenotypes developed, including weight loss, osteoporosis, anemia and heart enlargement. We have previously demonstrated linkage between mtDNA deletion mutations in age-related mitochondrial enzymatic abnormalities and loss of fibers in skeletal muscle from rats and rhesus monkeys (Lopez et al., 2000; Cao et al., 2001; Wanagat et al., 2001; Bua et al., 2002). Deletion mutations accumulate focally and are concomitant with the ETS abnormal regions of affected muscle fibers. These abnormal fibers displayed a significant decline in crosssectional area within the ETS abnormal region of the fiber compared to the normal region (where no deletion mutations were present) of the same fiber. Longitudinal analysis of atrophied fibers (1000–2000 mm) demonstrated that some fibers diminished to where they were no longer observable and then reappeared several sections later, suggesting that they were broken. These data demonstrate that ETS abnormalities have a localized physiological impact on the cell and can result in fiber atrophy and, subsequently, fiber loss. Global changes in cardiac function and structure, with age, have been documented in the Fischer 344 X Brown Norway F1 hybrid rats (Wanagat et al., 2002). Hemodynamic, histological and mtDNA deletion analysis were performed on hybrid rats aged 5-, 18- and 38-months. Significant decreases in heart rate, left ventricle peak systolic pressure, diastolic pressure occur in the rats between 5 and 38 months of age. There are also significant declines in the rates of left ventricle pressure development and relaxation suggesting a global deterioration of cardiac function. Histological analysis indicated that the area of fibrosis in the hybrid rats increased 4-fold from 5 to 38 months of age, reaching a level of 40%. Increases in the number of mtDNA deletion mutations also occurred, correlating, temporally, with the age-related changes in structure and function (Wanagat et al., 2002). Since point mutations accumulate with age in human cardiomyocytes (Nekhaeva et al., 2002), we investigated whether point mutations were prevalent in aged rats. In our study, mtDNA point mutations were characterized in heart and skeletal muscle from 36-month old rats. These postmitotic tissues are most susceptible to mtDNA damage due to their heavy dependence on oxidative metabolism. Using single cells and direct sequencing, we scanned for point mutations in both non-deletion containing (referred to as ‘full-length’ in this work) and deletion-containing mtDNA genomes. Our data suggest that there is a significant difference regarding incidence of mtDNA point mutations between rodents and humans, as well as between skeletal and cardiac muscles in rodents.

2. Materials and methods 2.1. Animals and tissue samples Six 36-month male Fischer 344 x Brown Norway F1 hybrid rats were purchased from the National Institute on Aging colony maintained by Harlan Sprague Dawley (Indianapolis, IN). Rats were anesthetized with methoxyflurane (Metafanee; Abbott Laboratories, Abbott Park, IL) and sacrificed by exsanguination. The whole heart was removed and the atria and aorta were dissected from the ventricles. The right ventricle was dissected from the left ventricle. Each ventricle was cut transversely at the mid-ventricular level, embedded in optimal cutting temperature (OCT) media (Miles Inc., Elkhart, IN) and flash frozen in liquid nitrogen. Vastus lateralis and rectus femoris, two muscles of the quadriceps, were separated free from surrounding tissues, transected at the mid-belly, embedded and frozen as above. Tissues were stored at K80 8C until analyzed. 2.2. Tissue-sectioning and laser-capture microdissection (LCM) Tissue samples, embedded in OCT, were brought to the sectioning temperature of K20 8C. Ten-mm-thick serial sections were cut and placed on Probe-on Plus slides (Fisher Scientific, Pittsburgh, PA). Sections were stored at K80 8C until analyzed. LCM was performed as described previously (Cao et al., 2001; Wanagat et al., 2001; Gokey et al., 2004). Frozen sections were dehydrated through a series of ethanol and xylene, and individual cardiomyocytes and skeletal muscle fiber sections were captured on LCM transfer film on a CapSure cap using a PixCell II laser-capture microscope (Arcturus, Mountain View, CA). Settings for LCM were a laser spot size of 7.5 mm in diameter and a pulse power of 50 mV. Any extraneous captured material was removed from the cap using the CapSure pad (Arcturus). 2.3. DNA isolation and mtDNA amplification Total DNA was isolated from laser-dissected single cardiomyocytes and skeletal muscle fibers as described previously (Cao et al., 2001; Gokey et al., 2004). One microliter of digestion solution containing 2.0 mg/ml proteinase K, 0.5% SDS, 10 mM EDTA and 50 mM Tris– HCl (pH 8.0), was directly placed on the dissected single cells on the CapSure lid. The samples were incubated at 37 8C for 30 min in a humidified chamber. The extracted DNA was recovered with 10 ml ddH2O. To isolate total DNA from cardiac and skeletal muscle tissues, a whole tissue section on a slide was covered with the digestion solution, incubated at 37 8C for 30 min and diluted 10-fold with ddH2O to recover the extracted DNA. One microliter of the DNA solution from single cells or slide-scrape homogenates was used for subsequent PCR.

J.W. Pak et al. / Experimental Gerontology 40 (2005) 209–218 Table 1 Primers used for amplification of full-length (FL) and deleted (D) mtDNAs

tRNALys

amplify the two regions from full-length genomes and exclude amplification of deletion-containing genomes. The first primer set, FL1, amplifies nucleotides 14165–773 that includes the control region and the second primer set, FL2, was used to amplify nucleotides 4386–7707 including the ND2–COX2 genes (Fig. 1). These two regions of the fulllength genome were amplified (94 8C for 30 s; 56–60 8C depending on primer sets for 30 s; 72 8C for 2.5 min; 35 cycles) using high fidelity LA Taq polymerase (TaKaRa, Japan).

ND2

2.4. Direct sequencing

Seta

Primerb

Sequence (5 0 –3 0 )

Gene position

FL1

14165F

CAA CCA CTC CTT TAT CGA CCT ACC CCG CTT CAT TGG CTA CAC CTT GAC CGC CTG AGG AGG ACT TAA CCA GAC TAA AAG GTT AAC GCT CTA AGC TTC GCA ATG ACT CTA TAT CCC TCA CCA CAT TCC CCG CTT CAT TGG CTA CAC CTT GAC ACT TAC TGG CTT CAA TCT ACT TCT CC TAT GTG CTT GAT GCC CTC TCC TAT CCT GAC CGT GCA AAG GTA G TAT GTG CTT GAT GCC CTC TCC GGC AGA GCC AAG TAA TTG CGT GGC TAT GTT GAG GAA GGC ATC CG

Cytb

773R FL2

4386F 7707R

D1

4784F 773R

d1

5121F 190R

D2

1997F 190R

d2

2662F 15929R

211

12S rRNA ND2

12S rRNA tRNAAsn 12S rRNA 16S rRNA 12S rRNA tRNALeu D-loop

a The upper case letters indicate primer sets for primary PCR and the lower case letters for subsequent nested PCR. For full-length genome amplification, no nested PCR was performed. b The numbering system for each primer is based on rat mitochondrial genome sequence (1–16300 bp, GenBank accession no. X14848). Primers were named by the nucleotide number of the 5 0 end. F represents forward primer, while R represents reverse primer.

PCR primers are summarized in Table 1. Primers for amplifying mtDNA deletion mutations were positioned in the minor arc region (Fig. 1), based on our previous mtDNA deletion analysis with rat skeletal muscle (Cao et al., 2001). Deletion mutations were initially targeted using primer sets D1 and d1. Additionally, D2 and d2 primer sets were used to detect large deletions, which may remove the light strand origin (Fig. 1). Extension times during PCR amplification were adjusted to preferentially amplify deletion products. Although deleted mtDNA genomes could be amplified from a primary PCR, nested PCR was employed to enhance the specificity and quantity of the products. After 25 cycles of primary PCR amplification (94 8C for 30 s, 57–60 8C for 30 s, 72 8C for 5–8 min) with D1 or D2 primer sets, nested PCR (94 8C for 30 s, 58–62 8C for 30 s, 72 8C for 5–8 min) was often performed for additional 30 cycles with d1 or d2, respectively. GeneAmp PCR system 2400 was used (AB Applied Biosystems, Foster City, CA). Two regions of full-length mtDNA were amplified using primer sets FL1 or FL2, in which one primer of each set, 14165F in FL1 and 7707R in FL2, falls within an area of the genome that was typically lost in rat mtDNA deletion mutations (Cao et al., 2001). This strategy was used to

PCR-amplified products were gel-purified (Qiagen, Valencia, CA) and directly sequenced using BigDye on an ABI 3700 capillary-based DNA analyzer (Applied Biosystems International, Foster City, CA) at the University of Wisconsin-Madison DNA Sequencing Facility. To determine strain-specific differences between the published mtDNA sequence of Rattus norvegicus (X14848) and the hybrid rats, mtDNA was isolated from liver, skeletal muscle and heart tissues as well as from single muscle fibers and cardiomyocytes. The entire mtDNA genome was amplified in a series of overlapping 2–3 kb fragments and sequenced (GenBank accession no. AY769440). Sequencing was performed forward and reverse directions. Sequence differences between the hybrid rat mtDNA and the sequences obtained from individual cardiomyocytes were designated as point mutations. For each detected point mutation, PCR amplification and DNA sequence analysis from the same single cells were repeated to confirm the observed point mutation. The degree of heteroplasmy in a PCR-amplified product was estimated by titration series, in which plasmid constructs of wild-type mtDNA and known point mutation mtDNA were mixed in specific ratios and directly sequenced. The height of the mutant nucleotide peak relative to the corresponding wild-type nucleotide peak reflects the level of heteroplasmy. Detection of the point mutation and its heteroplasmy was reproducible.

3. Results 3.1. Point mutations are not observed in full-length mtDNA from aged rat single cardiomyocytes To investigate point mutation load in full-length mtDNA genomes, randomly selected single cardiomyocytes were isolated from histologic sections of cardiac tissue from aged (36-month) rats using laser-capture microdissection. Two regions of the full-length mtDNA were amplified and directly sequenced (Fig. 1). One region was a 2.9 kb fragment (nts 14165–773) that includes the entire mtDNA control region as well as the majority of the 12S rRNA and cytochrome b genes. A high incidence of point mutations

212

J.W. Pak et al. / Experimental Gerontology 40 (2005) 209–218

Fig. 1. Schematic representation of the rat mtDNA genome. Arrows represent primers for PCR amplification of deletion mutations. Curved bars indicate the two regions of full-length mtDNA that are amplified by primer sets, FL1 and FL2, and directly sequenced.

has been reported in this region of mtDNA from various human tissues (Michikawa et al., 1999; Nekhaeva et al., 2002). The other region was a 3.3 kb fragment (nts 4386– 7707) encompassing a portion of the ND2 gene, the origin of light strand replication and the genes encoding for tRNATrp, tRNAAla, tRNAAsn, tRNACys, tRNATyr, COX1, tRNASer, tRNAAsp and COX2. This region is susceptible to deletion events in ETS abnormal fibers from rat skeletal muscles (Cao et al., 2001). Together these two regions cover 38% of the mitochondrial genome. Analysis of 30 single cardiomyocytes (total 6.2 kb mtDNA sequenced/cell) isolated from left and right ventricles of four 36-month old rats revealed no point mutations in either region. 3.2. MtDNA point mutations are coupled with deletion mutations in individual cardiomyocytes To assess whether point mutations co-expand with deletion mutations in individual cells, randomly selected single cardiomyocytes (nZ103) were microdissected from 36-month old rats and screened for mtDNA deletion mutations. PCR-amplified mtDNA deletion products were gel-purified and directly sequenced. An average of 2.3 kb of

mtDNA was sequenced from each amplification product. Eleven out of the 103 cardiomyocytes (10.7%) analyzed contained at least one mtDNA deletion mutation. One cell (#58; Table 2) contained two deletion mutations. The average size of the deletions was 10.9G0.6 kb. Seven of the 12 deletion mutations (58.3%) contained at least one point mutation (Table 2). All point mutations were unique. The point mutations were confirmed by repeating the experiment with a completely independent PCR amplification from the DNA of the same cell and subsequent sequence analysis. DNA sequence chromatograms of point mutations detected in deleted genomes from single cardiomyocytes indicate that the point mutations are heteroplasmic within the mtDNA deletion mutation population (Fig. 2). Although the point mutations appeared to be randomly distributed, there was a tendency for them to be located near the deletion breakpoint (Fig. 3). The single cardiomyocyte study revealed the presence of an identical 11 kb mtDNA deletion mutation in six cardiomyocytes (#55, 58, 59, 60, 61 and 72; Table 2). All of these deletion mutations occurred in a single animal. In three of these deleted genomes (#58, 60 and 61; Table 2), point mutations occurred at different positions. Two other

J.W. Pak et al. / Experimental Gerontology 40 (2005) 209–218

213

Table 2 Analysis of point mutations from laser-captured single cardiomyocytes of 36-month rats Cell #

Deletion breakpointa

Size of deletion (kb)

No. of nucleotides sequenced (bp)

Point mutationa

Position of point mutation (gene)

5 26 55 58c 58c 59 60 61 72 88

5170/14902 2777/14684 3957/14981b 2700/14613 3957/14981b 3957/14981b 3957/14981b 3957/14981b 3957/14981b 5065/15136

9.7 11.9 11.0 11.9 11.0 11.0 11.0 11.0 11.0 10.0

3600 1400 1500 1300 2300 2300 2300 2300 2300 3300

Cytb

89 92

4598/15380d 4598/15380d

10.8 10.8

2500 2500

G14958A Not found Not found T15099C A3619G Not found A3588G T3893C Not found A4185G A15460G Not found G3959A

a b c d

Cytb ND1 ND1 ND2 ND2 D-loop ND2

The numbering system is based upon the rat mtDNA sequence (GenBank accession no. X14848). This deletion product was detected in six individual cardiomyocytes from the same tissue section. This cardiomyocyte contained two deletion mutations, each with a different point mutation. This deletion product was detected in two individual cardiomyocytes from the same tissue section.

cardiomyocytes (#89 and 92; Table 2) had an identical mtDNA deletion mutation (as defined by breakpoint location and sequence) but only one (#92) contained a point mutation. Cardiomyocytes containing deleted mtDNA genomes with a point mutation were further characterized to determine whether the same point mutation was detectable in full-length mitochondrial genomes from the same cell.

Primers were designed to specifically amplify full-length genomes, positioning one primer outside a deletion breakpoint while the other primer was located in the deleted region. Full-length genomes were amplified from all cells containing deletion mutations indicating that the deleted genomes were present in heteroplasmic states. The point mutations present in the deleted genomes were not detected in the full-length genomes from the same cell.

Fig. 2. Detection of point mutations associated with deletion mutations from single cardiomyocytes. Single nucleotide changes were detected by direct sequencing of PCR-amplified deletion products from single cardiomyocytes and confirmed by replicate PCR amplification and sequence analysis from the same cell. The # indicates the cardiomyocyte numbers shown in Table 2. (A) The single nucleotide change, T/C, at nt 3893 from the deleted genome (cardiomyocyte #61). (B) A/G change at nt 15460 from the deleted genome (cardiomyocyte #88). Note that the sequence chromatogram shows the complementary sequence. (C) G/A change at nt 3959 from the deleted genome (cardiomyocyte #92).

214

J.W. Pak et al. / Experimental Gerontology 40 (2005) 209–218

Fig. 3. Linear representation of mtDNA deletions and point mutations from 36-month rat heart homogenates, single cardiomyocytes and skeletal muscle homogenates. The deleted portion of the genome is indicated in white and the sequenced portion is in black. Numbers in parentheses indicate the distance of the point mutation (arrowhead) from the breakpoint in base-pair (bp). A single asterisk indicates the cardiomyocyte number shown in Table 2, while the double asterisk identifies the mtDNA deletion containing a point mutation from skeletal muscle in Table 4.

Additional mtDNA deletion mutations were obtained by analyzing thousands of cells from a histologic slide section as a homogenate. PCR amplification identified numerous amplification products, shorter than full-length ranging 400–4500 bp (Table 3). Eighteen of these deletion mutations were from the left ventricle and 15 from the right ventricle. The average deletion size was 10.3G1.1 kb from the left ventricle and 10.3G1.6 kb from the right ventricle. Loss of the light strand origin was observed in over 60% of deletion mutations from both left and right ventricles. Eleven of the deleted genomes contained 1–2 point mutation(s) within the amplified fragment (6/18 from left ventricle and 5/15 from right ventricle). The frequencies of both deletion and point mutations were not different between the left ventricle and the right ventricle. Different deletion mutations contained different point mutations. As observed with the laser-captured single cardiomyocytes,

the point mutations were located in close proximity to the deletion breakpoint (Fig. 3). 3.3. Tissue specificity of mtDNA point mutations: clonal point mutations are rare in skeletal muscle The link between mtDNA deletion mutations and single nucleotide mutations in cardiac tissue prompted us to examine the presence of point mutations in skeletal muscle fibers from aged rats. MtDNA deletion mutations in skeletal muscle can accumulate to high levels resulting in ETS enzymatic abnormalities and concomitant fiber atrophy (Wanagat et al., 2001). Fibers exhibiting ETS normal and ETS abnormal (i.e. COXK/SDHCC) phenotypes were individually dissected from vastus lateralis and rectus femoris of 36-month old rats (20 fibers for each phenotype). The control region and the ND2–COX2 region from

J.W. Pak et al. / Experimental Gerontology 40 (2005) 209–218

215

Table 3 Analysis of mtDNA mutations from heart slide-scrape homogenates of 36-month rats Deletion mutation #

Tissue

Deletion breakpoints

Size of deletion (kb)

No. of nucleotides sequenced (bp)

Point mutation

Position of point mutation (gene)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

LV LV LV LV LV LV LV LV LV LV LV LV LV LV LV LV LV LV RV RV RV RV RV RV RV RV RV RV RV RV RV RV RV

2688–15423 2820–15423 3910–15091 3957–14981 4684–15315 5063–15006 5141–15293 5152–15266 5157–15373 5158–16144 5163–14986 5173–15018 5188–15109 5451–15109 5531–14991 6020–14911 6061–14979 6224–14911 2687–15392 2741–15075 3121–15911 3134–15122 3994–15399 4420–14484 4949–15222 4974–15109 5143–14476 5250–14220 5389–15265 5457–14613 5569–15080 6879–16090 8254–15129

12.7 12.6 11.2 11.0 10.6 9.9 10.1 10.1 10.2 11.0 9.8 9.8 9.9 9.6 9.5 8.9 8.9 8.7 12.7 12.3 12.8 12.0 11.4 10.0 10.0 10.1 9.3 9.0 9.9 9.0 9.5 9.2 6.9

500 700 2000 1500 400 1100 1400 1200 800 400 1500 1500 1500 1700 1900 2500 2500 2700 600 900 500 1300 1200 900 1400 2500 2000 1100 1500 2100 1500 2100 4500

C2687 del 2820T ins

tRNALeu ND1

C15210T G47A

Cytb tRNAPhe

A6004T

COX1

C6154G G2672A

COX1 tRNALeu

A2878G

ND1

T4948 del

tRNATrp

T5242C A5246C T8251C

tRNATyr tRNATyr ATP6

LV, left ventricle; RV, right ventricle.

full-length mtDNA genomes were amplified, as described for the cardiac analysis, from 20 normal fibers to screen for point mutations. Single nucleotide changes were not observed in the full-length mitochondrial genomes. As expected, all 20 COXK/SDHCC fibers contained clonal mtDNA deletion mutations (average deletion size 7.8G 1.0 kb). Within the 2000–6100 bp PCR amplicons sequenced for each deletion mutation, no point mutations were observed (Table 4). Similar to the cardiac study, we also analyzed a large number of mtDNA deletion mutations from slide-scrape homogenates of muscle tissue. Thirty-three deletion mutations were identified (14 from vastus lateralis and 19 from rectus femoris) with average 6.7G2.6 kb of DNA lost. Approximately 15% of the mtDNA deletions from skeletal muscle involved the removal of the light strand origin, compared to the 60% observed in the heart homogenates. Only one of the 33 mtDNA deletion mutations analyzed from skeletal muscle contained a point mutation. This point mutation (insertion of an A at

nucleotide position 13213) was located 1 bp from the deletion breakpoint (Table 4, Fig. 3).

4. Discussion Three distinct mtDNA genotypes were identified in cardiac cells from aged rats. The majority of the cardiomyocytes contained full-length genomes with no evidence of single nucleotide mutations. Approximately 10% of the cardiomyocytes contained, in addition to fulllength mtDNA, mitochondrial genomes with deletion mutations. These deleted genomes were clonal within an affected cardiomyocyte. The third group consisted of fulllength mtDNA and deleted genomes heteroplasmic for single nucleotide mutation(s). Although all three mtDNA genotypes were present in aged rat skeletal muscle, deletion-linked point mutations were more prevalent in cardiomyocytes than skeletal muscle fibers. Clonal accumulation of point mutations was not observed in the full-length

216

Table 4 Analysis of mtDNA mutations from individual skeletal muscle fibers and slide-scrape homogenates of 36-month rats Single skeletal muscle fibers

Skeletal muscle slide-scrape homogenates

Tissue

Deletion breakpoints

Size of deletion (kb)

No. of nucleotides sequenced (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

VL VL VL VL VL VL VL VL VL VL VL VL VL VL RF RF RF RF RF RF

5242–14617 6345–15299 6099–14977 5534–14404 6248–14991 6316–14908 6099–14613 7170–15136 7454–15214 5250–12893 7169–14672 7878–15292 7525–14613 6514–13190 5658–13814 7552–14933 7686–14638 7382–14215 6749–13133 8631–13943

9.4 9.0 8.9 8.9 8.7 8.6 8.5 8.0 7.8 7.6 7.5 7.4 7.1 6.7 8.2 7.4 7.0 6.8 6.4 5.3

2000 2400 2500 2500 2700 2800 2900 3400 3600 3800 3900 4000 4300 4700 3200 4000 4400 4600 5000 6100

Point mutation

Deletion mutation #

Tissue

Deletion breakpoints

Size of deletion (kb)

No. of nucleotides sequenced (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

VL VL VL VL VL VL VL VL VL VL VL VL VL VL RF RF RF RF RF RF RF RF RF RF RF RF RF RF RF RF RF RF RF

2687–15392 3907–15088 5170–15209 5423–15353 6562–13621 6099–13095 6091–12806 7837–13660 7399–13212 7689–12973 9345–13801 9394–13812 10274–14612 10307–14607 3247–14915 4653–15356 5165–14929 5589–15120 6117–13315 6099–13178 6099–13054 6255–13315 6749–13133 7493–13221 7527–13100 8098–14920 8931–14920 9662–13731 10265–14219 10427–14616 10567–14610 10896–13639 11033–13707

12.7 11.2 10.0 9.9 7.1 7 6.7 5.8 5.8 5.3 4.5 4.4 4.3 4.3 11.7 10.7 9.8 9.5 7.2 7.1 7.0 7.1 6.4 5.7 5.6 6.8 6.0 4.1 4.0 4.2 4.0 2.7 2.7

600 2100 1400 1500 4300 4400 4700 5600 5600 1300 2100 2200 2900 2900 1600 2600 1600 1900 4200 4300 4400 4300 5000 900 1000 400 1200 3100 3200 3000 3200 4500 4500

Point mutation

Position of point mutation (gene)

13213A ins

ND5

For skeletal muscle slide-scrape homogenates, additional nested primer sets were used to amplify small deletions: 7238F/13821R, 8004F/15209R. VL, vastus lateralis; RF, rectus femoris.

J.W. Pak et al. / Experimental Gerontology 40 (2005) 209–218

Deletion mutation #

J.W. Pak et al. / Experimental Gerontology 40 (2005) 209–218

mtDNAs of either rat skeletal muscle fibers or cardiomyocytes. In aged human cardiac and buccal cells, the heteroplasmy of point mutations ranged 25–100% (Nekhaeva et al., 2002). In rat cells, we found no evidence of clonally accumulated point mutations in full-length mtDNAs. Our detection limit for clonally accumulated point mutations is 20% heteroplasmy based on direct sequencing of a titration series of plasmids containing wild-type mtDNA and point mutations. The lack of point mutations at this sensitivity level indicates that point mutations do not accumulate in rat cells to the level of clonal expansion observed in aged human cells (Nekhaeva et al., 2002). One possible explanation for the difference in mtDNA point mutation accumulation between rats and humans is the longevity of humans. In the absence of replicative advantage, mathematical models have predicted that relaxed replication of mtDNA can result in the clonal accumulation of point mutations within single post-mitotic cells with age in humans (Elson et al., 2001). According to the model generated by Elson et al. (2001), the earliest age in which a point mutation reached 20% heteroplasmy was approximately 10 years. In the lifespan of rats, if relaxed replication is the mechanism of point mutation accumulation, we would not expect to see 20% heteroplasmy in the lifetime of a rat. Our observations from the single cell study indicate that point mutations in rat originated at or following the initial deletion event. First, all mtDNA point mutations identified were associated with deleted genomes, however, not all deleted genomes had point mutations. Second, all point mutations were heteroplasmic in a residing deletion population. If a point mutation preceded a deletion event, the point mutation would be expected to be homoplasmic. Third, point mutations associated with deleted genomes are not detectable in the full-length genomes from the same cells. Finally, unique point mutations were associated with the same deletion mutations identified from different individual cardiomyocytes (Table 2). There are two possible explanations for heteroplasmic point mutations within the deletion population. The point mutation and the deletion mutation could occur at the same time. If an error in mtDNA replication caused both a deletion and a nearby base misincorporation, the base mismatch would not be converted to a point mutation until the next round of replication and would result in one deleted molecule with no point mutation (formed from the template strand with the correct base) and one deleted molecule with a point mutation (formed from the template strand with the mismatched base). This starts the deletion-associated point mutation at 50% heteroplasmy that could then drift randomly. Alternatively, point mutations could occur subsequent to the deletion event. This study suggests that, at least in rats, single nucleotide mutations in the deleted genomes are, in effect, passengers and that the detection of point mutations is the result of the replication of the mtDNA deletion mutations.

217

The frequency of point mutations in deleted mitochondrial genomes appears to be tissue-specific. Point mutations associated with deletion mutations were initially found in single cardiomyocytes and their abundance was verified by a large number of deletion mutations obtained from cardiac tissue slide-scrape homogenates. In skeletal muscle, the analysis of individual fibers did not identify any point mutations associated with deleted genomes. One deletion-associated point mutation was, however, detected from slide-scrape homogenates, indicating that deletion association is a potential mechanism for point mutation accumulation in skeletal muscle. The overall findings from individual cells and tissue homogenates showed that only 1.9% of the deletion mutations analyzed from skeletal muscle contained point mutations whereas 40% of the deletion mutations from heart contained single nucleotide changes. The differences in deletion-associated point mutations between cardiac and skeletal muscle may reflect different levels of oxidative stress in the two tissues. When mtDNA mutations are genetically induced, through a proof-reading deficient version of the catalytic subunit of mtDNA polymerase in transgenic mice, increased levels of aggregated individual mutations can cause reduced lifespan and numerous severe aging phenotypes (Trifunovic et al., 2004). Although we found no evidence of the clonal accumulation of naturally occurring single nucleotide mutations in full-length mitochondrial genome from aged rats, our results do not rule out non-clonal accumulation of individual point mutations. The results of our study using old rats contrasts with studies of mtDNA from aged humans in which high levels of clonal accumulation of point mutations were identified in buccal and cardiac cells (Nekhaeva et al., 2002). We sequenced a larger proportion of the mtDNA including the control region (characterized in the human study) as well as a 3.3 kb region of the genome containing COX subunit genes and found no point mutations. Although there are significant differences in the methodologies employed in the two studies, the discrepancy in abundance and localization of single nucleotide mutations most likely reflects speciesspecific differences. While mtDNA deletion mutations have been demonstrated to accumulate to high levels in a variety of tissues and species, our study indicates that the clonal accumulation of point mutations in mtDNA is not a universal aging phenomenon.

Acknowledgements We are very grateful to Susan McKiernan and Jody Johnson for critical reading of this manuscript. This work was supported by grant AG17543-04 from the National Institutes of Health.

218

J.W. Pak et al. / Experimental Gerontology 40 (2005) 209–218

References Bua, E.A., McKiernan, S.H., Wanagat, J., McKenzie, D., Aiken, J.M., 2002. Mitochondrial abnormalities are more frequent in muscle undergoing sarcopenia. J. Appl. Physiol. 92, 2617–2624. Calloway, C.D., Reynolds, R.L., Herrin Jr.., G.L., Anderson, W.W., 2000. The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age. Am. J. Hum. Genet. 66, 1384–1397. Cao, Z., Wanagat, J., McKiernan, S.H., Aiken, J.M., 2001. Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection. Nucleic Acids Res. 29, 4502–4508. Cortopassi, G.A., Shibata, D., Soong, N.-W., Arnheim, N., 1992. A pattern of accumulation of a somatic deletion of mitochondrial DNA in aging human tissues. Proc. Natl Acad. Sci. USA 89, 7370–7374. Elson, J.L., Samuels, D.C., Turnbull, D.M., Chinnery, P.F., 2001. Random intracellular drift explains the clonal expansion of mitochondrial DNA mutations with age. Am. J. Hum. Genet. 68, 802–806. Gokey, N.G., Cao, Z., Pak, J.W., Lee, D., McKiernan, S.H., McKenzie, D., Weindruch, R., Aiken, J.M., 2004. Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibres from aged rhesus monkeys. Aging Cell 3, 319–326. Harman, D., 1983. Free radical theory of aging: consequences of mitochondrial aging. Age 6, 86–94. Jazin, E.E., Cavelier, L., Eriksson, I., Oreland, L., Gyllensten, U., 1996. Human brain contains high levels of heteroplasmy in the noncoding regions of mitochondrial DNA. Proc. Natl Acad. Sci. USA 93, 12382–12387. Lopez, M.E., Van Zeeland, N.L., Dahl, D.B., Weindruch, R., Aiken, J.M., 2000. Cellular phenotypes of age-associated skeletal muscle mitochondrial abnormalities in rhesus monkeys. Mutat. Res. 452, 123–138. Michikawa, Y., Mazzucchelli, F., Bresolin, N., Scarlato, G., Attardi, G., 1999. Aging-dependent large accumulation of point mutations in the human mtDNA control region for replication. Science 286, 774–779.

Murdock, D.G., Christacos, N.C., Wallace, D.C., 2000. The agerelated accumulation of a mitochondrial DNA control region mutation in muscle, but not brain, detected by a sensitive PNAdirected PCR clamping based method. Nucleic Acids Res. 28, 4350–4355. Nekhaeva, E., Bodyak, N.D., Kraytsberg, Y., McGrath, S.B., Van Orsouw, N.J., Pluzhnikov, A., Wei, J.Y., Vijg, J., Khrapko, K., 2002. Clonally expanded mtDNA point mutations are abundant in individual cells of human tissues. Proc. Natl Acad. Sci. USA 99, 5521–5526. Taylor, R.W., Taylor, G.A., Durham, S.E., Turnbull, D.M., 2001. The determination of complete human mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations. Nucleic Acids Res. 29, E74. Trifunovic, A., Wredenberg, A., Falkenberg, M., Spelbrink, J.N., Rovio, A.T., Bruder, C.E., Bohlooly-Y, M., Gidlof, S., Oldfors, A., Wibom, R., Tornell, J., Jacobs, H.T., Larsson, N.G., 2004. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429, 417–423. Wanagat, J., Cao, Z., Pathare, P., Aiken, J.M., 2001. Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia. FASEB J. 15, 322–332. Wanagat, J., Wolff, M.R., Aiken, J.M., 2002. Age-associated changes in function, structure and mitochondrial genetic and enzymatic abnormalities in the Fisher 344 x Brown Norway F1 hybrid rat heart. J. Mol. Cell. Cardiol. 34, 17–28. Wang, Y., Michikawa, Y., Mallidis, C., Bai, Y., Woodhouse, L., Yarasheski, K.E., Miller, C.A., Askanas, V., Engel, W.K., Bhasin, S., Attardi, G., 2001. Muscle-specific mutations accumulate with aging in critical human mtDNA control sites for replication. Proc. Natl Acad. Sci. USA 98, 4022–4027. Zhang, C., Baumer, A., Maxwell, R.J., Linnane, A.W., Nagley, P., 1992. Multiple mitochondrial DNA deletions in an elderly human individual. FEBS Lett. 297, 34–38.