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Cardiovascular Research 2003 57(1):147-157; doi:10.1016/S0008-6363(02)00695-8
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Copyright © 2003, European Society of Cardiology

Mitochondrial DNA mutations activate the mitochondrial apoptotic pathway and cause dilated cardiomyopathy

Dekui Zhanga, Justin L. Motta, Patricia Farrarb, Jan S. Ryersec, Shin-Wen Changa, Melissa Stevensa, Grace Dennigera and Hans Peter Zassenhausa,*

aDepartment of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, 1402 South Grand Boulevard, St. Louis, MO 63104, USA
bDepartment of Comparative Medicine, Saint Louis University Health Sciences Center, 1402 South Grand Boulevard, St. Louis, MO 63104, USA
cDepartment of Pathology, Saint Louis University Health Sciences Center, 1402 South Grand Boulevard, St. Louis, MO 63104, USA

* Corresponding author. Tel.: +1-314-577-8444; fax: +1-314-773-3403. zassenp{at}slu.edu

Received 25 March 2002; accepted 5 August 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: To determine whether low frequency mitochondrial DNA (mtDNA) mutations are pathogenic. Methods: We studied mice that express a proofreading-deficient mitochondrial DNA polymerase in the heart and develop cardiac mtDNA mutations. Results: At 4 weeks of age, when point mutation levels had risen to on average two per mitochondrial genome, these mice developed severe dilated cardiomyopathy. Interstitial fibrosis first became apparent at 4 weeks of age and progressed with age. Sporadic myocytic death occurred in all regions of the heart, apparently due to apoptosis as assessed by histological analysis and TUNEL staining. The frequency of TUNEL-positive cells peaked at 4–5 weeks of age and then gradually declined. While mitochondrial respiratory function, ultrastructure, and number remained normal, cytochrome c was released from mitochondria, a known apoptotic signal. Conclusion: mtDNA mutations therefore are pathogenic, and seem to trigger apoptosis through the mitochondrial pathway.

KEYWORDS Aging; Apoptosis; Cardiomyopathy; Mitochondria; Sequence (DNA/RNA/prot)


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Advancing age is associated with increased damage to mitochondrial DNA (mtDNA) and higher frequencies of mutations in the mitochondrial genome [1]. This molecular pathology is particularly pronounced in the heart, skeletal muscle, and brain, all of which are highly dependent on mitochondrial respiration for physiological function [2]. The rising frequency of mtDNA mutations has been attributed to a combination of at least two factors. First, continuing oxidative damage to mtDNA from reactive oxygen species generated as a byproduct of mitochondrial respiration gives rise to mutations if not repaired [3]. Second, post-mitotic cells have no known mechanism for eliminating mutations once they are produced. Indeed, it has been suggested that mtDNAs with deletion mutations may have a replicative advantage over wild-type genomes and thereby increase in prevalence with age [4].

mtDNA mutations have the potential to significantly affect mitochondrial function and in turn cellular health. There is no doubt that at high frequencies (>20%) mtDNA mutations cause human disease, for example, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like symptoms) and Kearns-Sayre Syndrome. These diseases are thought to arise from a deficiency in oxidative phosphorylation [4] caused by the reduction in mitochondrial-encoded electron transport chain complexes. In old age or age-related disease, the frequency of mutations is usually much lower, in the range of 0.01–1% [5,6]. It is not known whether such low mutation frequencies cause cellular dysfunction, disease, or senescence.

To test the pathogenesis of low levels of mtDNA mutations, we studied transgenic mice that rapidly accumulated mutations specifically in heart mtDNA. Mutations arose due to heart-specific expression of a proofreading-deficient mitochondrial DNA polymerase [7]. Characterization of those mice revealed no abnormalities in mitochondrial DNA levels, gene expression, or mitochondrial protein content suggesting that the mutant transgene did not cause a general perturbation of mitochondrial activity [7]. Here we report that these mice developed severe dilated cardiomyopathy and had increased cell death as mtDNA mutations accumulated. Interestingly, they evidenced no deficiency in mitochondrial respiration or in the content of ATP in the heart.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
The transgenic mice (line 4, FVB; line 13, C57BL6/FVB) were maintained as two independent lines [7]; all results were consistent in both lines. Transgenic mice were used as hemizygotes, and controls were always non-transgenic littermates. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).

2.2 Histopathology and electron microscopy
For histology, hearts were fixed in 10% neutral buffered formalin (NBF) overnight, embedded in paraffin, and 5–7-µm thick sections were stained with either hematoxylin/eosin or Gomori's modified trichrome [8]. For ultrastructural cytology, heart wedges were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.25, 2% sucrose, and 2 mM calcium chloride at 4 °C. Following post-fixation with osmium tetroxide and dehydration through graded ethanol, the tissue was embedded in PolyBed resin (PolySciences). Semi-thin (0.5 µm) sections were stained with toluidine blue for light microscopy and stained with uranyl acetate and lead citrate for transmission electron microscopy.

For morphometric measurements, hearts were perfused via the aorta with 1 ml of 100 mM cadmium chloride to arrest the heart in diastole. Then the heart was washed with PBS followed by 10% NBF. The heart was fixed overnight in NBF and transferred to PBS for at least 12 h, and sectioned on a vibratome. Measurements of left ventricular area were made on every third cross-section (200 µm each). Eight separate measurements were made of the free wall and septal thickness on the largest section of the heart. Area measurements were converted to volume by multiplying the sum of the areas times 600 µm (three times 200 µm).

2.3 Northern and Western blots
Whole cell RNA was prepared using Trizol (Gibco BRL) and poly(A) RNA analyzed for transgene expression [9].

For Western blotting, isolated mitochondria [10] were boiled in Laemmli sample buffer [11] and equal amounts of protein (BCA, Pierce) were fractionated by SDS–PAGE. Primary antibodies were used according to the manufacturer's instructions (anti-cytochrome c, PharMingen; anti-coxIV, Molecular Probes); secondary antibodies were from Jackson ImmunoResearch. Blots were visualized by enhanced chemiluminescence (ECL, Amersham).

2.4 Mutation analysis
Long-extension PCR was used to analyze whole cell DNA for mitochondrial deletion mutations as described previously [7]. Briefly, reactions contained 0.1 µg genomic DNA, 0.5 µM forward and reverse primers representing genomic positions 1953–1924 and 2473–2505, respectively, and Buffer 3 of the Expand Long Template PCR kit from Roche. Amplification conditions were as specified by the supplier using either 5- or 12-min extension times as indicated in the legend to Fig. 2. Nested PCR was performed with primers representing genomic positions 1794–1772 and 2501–2525 and 1 µl of a 1:100 dilution of the primary reaction mixture as template.


Figure 2
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  		Fig. 2 Analysis of MPW transgenic mice expressing a wildtype DNA pol {gamma}. (a) mtDNA deletion mutations (del) were not detected in either 1-month-old control (lanes 2 and 5) or 3-month-old MPW mice (lanes 4 and 7) (cf. with MHC mice, lanes 3 and 6), by long extension PCR using a 12-min extension time to amplify both full-length (16 kb) mtDNA and deletion molecules (lanes 2–4). Nested PCR (lanes 5–7), using a 5-min extension time to selectively amplify deletion molecules, verified the mitochondrial origin of the deletion molecules. (b) Ventricular cross-sections (H&E) demonstrate the normal left ventricular (LV) chamber size of mice expressing the wildtype mitochondrial pol {gamma} (MPW), compared to the dilation evident in mice with a mutagenic mitochondrial pol {gamma} (MHC). Representative results of at least five animals per group examined.

 
2.5 Immunohistochemistry
TUNEL staining was performed on paraffin-embedded tissue using Apoptag (Oncor). Nuclei were counter-stained with DAPI (1 µg/ml in PBS). The number of TUNEL-positive cells was determined by counting all such cells in three widely spaced cross-sections taken from each of three to six animals. The number of DAPI-stained nuclei was estimated by averaging the number per 400x field from counts of three such fields and then determining the number of fields in the cross-section. The frequency of TUNEL-positive cells was expressed as the percentage of DAPI-stained nuclei.

When TUNEL staining was combined with antibody staining, tissue sections were processed through the terminal deoxynucleotide transferase step, washed briefly in PBS, immersed in antigen unmasking solution (Vector), and heated in a pressure cooker for 6 min. After cooling to room temperature, sections were washed with PBS (3x, 5 min), incubated with blocking buffer (PBS containing 2% BSA, 0.2% non-fat dried milk, and 0.4% Triton X-100) for 1 h at room temperature, and then reacted with anti-troponin C (1:20 dilution; Novocastra Laboratories) in blocking buffer overnight at 4 °C. Slides were washed in PBS and incubated with rhodamine-conjugated anti-digoxigenin supplied with the Apoptag kit and fluorescein-conjugated goat anti-mouse IgG (1:100 dilution; Jackson ImmunoResearch) for 1 h at room temperature in the blocking buffer supplied with the Apoptag kit. Following three washes in PBS, nuclei were counterstained with DAPI.

2.6 Cytochrome c release
The high-speed supernatant after pelleting the mitochondria was subjected to further centrifugation at 100,000 g for 1 h. The resultant supernatant (S100 cytosol) was applied to a Sephacryl-S300 column equilibrated and run in 10 mM Tris–HCl, pH 8.0, 50 mM KCl, 0.1% CHAPS. Fractions were analyzed by Western blotting for cytochrome c.

2.7 Mitochondrial respiratory function
Isolated mitochondria were analyzed by polarography [10] using a YSI Model 5300 Biological Oxygen Monitor (Yellow Springs Instrument) equipped with a micro oxygen chamber. Difference spectroscopy [12] was performed to quantitatively measure mitochondrial hemoproteins. Cytochrome oxidase and citrate synthetase activities were measured on isolated mitochondria according to Trounce et al. [10].

2.8 Tissue ATP content
ATP levels in the heart were measured by HPLC from perchloric acid extracts of quick frozen tissue [13]. In all cases the ratio of ATP-to-ADP was greater than 20:1 indicating that little hydrolysis had occurred during sample preparation. Hearts from four to six animals per group were individually analyzed to calculate mean±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
The construction of transgenic mice with accelerated accumulation of mtDNA mutations specifically in the heart has been described [7]. Briefly, mutations accumulated rapidly with age starting shortly after birth. By 1 month of age the frequency of point mutations had climbed to 1.4 per 10,000 bp of mtDNA, representing at least a 20-fold elevation. Over the next 2 months the frequency of mutations climbed another 50%.

3.1 Congestive heart failure and dilated cardiomyopathy in mice with mtDNA mutations
Starting at 4 weeks of age, some 10–20% of the transgenic mice in the F1 generation suffered severe dyspnea, systemic edema (Fig. 1a), or cardiac death. Upon autopsy, they revealed pulmonary, hepatic, and splenic congestion, as well as accumulation of fluid in the pleural and abdominal cavities, indicative of congestive heart failure. In severely dyspnic mice, symptoms were improved dramatically by digoxin (1 µg/20 g body weight, bid orally), a cardiac glycoside that increases contractility in the failing heart. By the F3 generation, the incidence of congestive heart failure and death at early ages declined to ~5%. While clinical signs of heart failure were only observed in a minority of animals, nearly all transgenic mice showed cardiac disease by 4–5 weeks of age, characterized by bilateral ventricular and atrial dilation. Similar cardiac pathologies occurred in two independent transgenic lines, termed MHC line 4 and 13, indicating that non-specific integration effects of the transgene were not responsible for the cardiomyopathy.


Figure 1
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  		Fig. 1 Congestive heart failure and dilation in MHC transgenic mice. (a) Clinically apparent congestive heart failure in a 6-week-old line 13 transgenic mouse (right animal). Note that the animal appears to be twice the size of its non-transgenic littermate because of severe subcutaneous edema. (b) Hearts from 5-week-old line 13 mice (left, control; middle and right, transgenic). Note the enlarged ventricle (V) and atria (A) in the middle heart, whereas the right heart had primarily enlarged atria. (c) Cross-sections (12.5x) of perfusion-fixed hearts (left, control; right, transgenic) arrested in diastole. Note the dilated ventricle (LV) and the thinner free (F) and septal (S) muscle walls. (d) Morphometric measurements from sections as shown in panel c. Presented are the averages (±S.E.M.) from measurements of the indicated number (n) of mice. (e) Heart-to-body weight ratio (mass index; mean±S.E.M.) of line 4 mice with age (at least five animals per group). *P<0.05 for a comparison of transgenic versus age-matched control. (f) Multiple organizing atrial thrombi (Th), characterized by hemorrhage, necrosis, metastatic mineralization and calcification, mixed inflammatory cell infiltrates, and fibrosis. Note also the greatly enlarged atria (A) in comparison to the ventricle (V); hematoxylin/eosin, 12.5x. Line 13 mouse, 8 weeks of age, in end-stage heart failure.

 
Fig. 1b shows typically enlarged hearts from line 13 mice at 5 weeks of age in comparison to a control littermate. On cross-section (Fig. 1c), both ventricles were dilated while the muscle walls were thinner, indicating that little hypertrophy had occurred. In comparison to age matched littermates, the volume of the left ventricular chamber in transgenic hearts was 38% greater at 4 weeks of age, whereas the thickness of the left ventricular free wall and septum were each 20% thinner, consistent with a dilated cardiomyopathy (Fig. 1d). We did not quantify right ventricular volumes. The onset of pathology was determined to be between 3 and 4 weeks of age, based on dilation (Fig. 1d), and heart-to-body-weight ratio (Fig. 1e), as well as subjective inspection.

Cardiac pathology was observed in atria as well as ventricles. This is a logical consequence of the observation that the transgene was expressed and mutations were detected in both the ventricles and the atria [7]. Accordingly, atria were dilated and in some cases atrial dilation was the most prominent feature observed on gross necropsy (Fig. 1b). Multifocal thrombi in the enlarged atria were a frequent finding (>95% of animals examined, n100), and most animals in congestive heart failure exhibited multiple massive, organized atrial thrombi (Fig. 1f). The early onset and severity of dilation in these transgenic mice suggest that abnormalities of atrial muscle function played a primary role in the atrial dilation.

3.2 Cardiac abnormalities were not due to either over-expression of DNA pol {gamma} or acute toxicity of the mutant DNA pol {gamma}
Transgenic mice were also constructed expressing a wild-type pol {gamma} driven by the {alpha}-myosin heavy chain promoter; the same promoter used for the mutant transgene. Mice expressing this wildtype polymerase (termed MPW) expressed transgene mRNA at levels comparable to MHC mice, measured by Northern blot (not shown). However, MPW animals neither had elevated levels of mtDNA mutations (Fig. 2a) nor pathologic (Fig. 2b) or clinical signs of cardiac disease. These results suggested that over expression of DNA pol {gamma} by itself was not toxic to cardiomyocytes.

It remained possible that the mutant DNA pol {gamma} was uniquely toxic, thus causing the cardiac pathology by mechanisms unrelated to the accumulation of mitochondria DNA mutations. The delayed onset of cardiac dilation in MHC mice, however, indicated that expression of the proofreading-deficient DNA pol {gamma} was not by itself acutely toxic. While transgene expression was maximal by 1–2 days after birth [7], transgenic hearts were not dilated until 4 weeks of age. Normal development was also observed in measurements of the heart mass index (Fig. 1e). At 3 weeks of age, histological analysis revealed no abnormalities of myofibril architecture or myocytic structure (data not shown). It appeared, therefore, that over-expression of DNA pol {gamma} per se, whether as a wild-type or mutant enzyme, was not sufficient to cause cardiomyopathy, and disease likely follows the accumulation of mtDNA mutations. Because of the central role that mitochondria play in apoptosis we asked whether disease included cell death.

3.3 Rising frequencies of mtDNA mutations are associated with positive TUNEL staining
TUNEL staining revealed a greater frequency of DNA damage in myocytes in the transgenic compared to the normal heart. This increased frequency was first detectable when mice were ~3 weeks of age, 1 week before the onset of ventricular or atrial dilation. Fig. 3a shows TUNEL staining combined with DAPI staining to visualize nuclear morphology and anti-troponin C (a muscle-specific antigen) staining to identify the cell type. The majority of TUNEL(+) cells identified by this method were myocytes. Their frequency began to increase in transgenic compared to control hearts at 3 weeks of age (Fig. 3b). The frequency of TUNEL(+) cells was relatively high immediately after birth and declined to very low levels by 3 weeks of age in control hearts, a pattern that has been described [14]. By 4 weeks of age the frequency of TUNEL(+) cells in transgenic hearts peaked at ~1 TUNEL(+) cardiomyocyte per 250 DAPI-stained nuclei and gradually declined thereafter to levels still significantly higher than control mice at 10 weeks of age (Fig. 3b). Similar to the pan-cardiac pattern of transgene expression, TUNEL(+) cells were found in both ventricular and atrial tissue (data not shown). Likewise, no difference was found in the frequency of TUNEL(+) cells in the left versus right ventricular free wall, suggesting that TUNEL staining was not related to either mitochondrial respiratory activity or muscle work load.


Figure 3
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  		Fig. 3 TUNEL analysis. (a) A paraffin-embedded section from a 3-week-old line 13 heart was triple stained with TUNEL (to reveal apparent apoptotic cells), DAPI (to visualize nuclei), and anti-troponin C (to identify cardiomyocytes). The arrow points to the same cell in each panel; 400x. (b) The apoptotic index (% TUNEL-positive relative to DAPI-stained nuclei) with age in transgenic (solid circles) versus control (open circles) mice. Each time point represents the average (±S.E.M.) of at least three separate animals. From 3 weeks onward, P≤0.05 for the comparison of transgenic versus age-matched control.

 
3.4 TUNEL staining reflects apoptotic cell death
Since the TUNEL reaction may stain not only dying cells but also those with non-fatal nuclear damage [15], we examined cardiac tissue for morphological evidence of cell death. Histological examination of over 100 animals taken from either transgenic line and of various ages revealed a consistent finding of sporadic degenerating myocytes in an otherwise normal myocardium. At medium power (Fig. 4a,b) there was no gross disarray of tissue architecture in the transgenic heart. Higher magnification (Fig. 4c,d) of the transgenic tissue revealed signs of myocytic cell death and myocyte dropouts, which were not seen in the control heart. Fig. 4d shows vacuolated, degenerating myocytes in the left ventricular free wall. Degenerating myocytes were also seen in the atria (not shown). Like the tissue distribution of TUNEL(+) cells, the localization of these cellular abnormalities was scattered and not confined to a specific layer or region of the myocardium.


Figure 4
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  		Fig. 4 Myofibril architecture and myocytic degeneration. (a–d) Cross-sectional photomicrographs of 2-month-old line 13 transgenic heart and an age matched control littermate. Medium power (100x) view of the left ventricular free wall of a control (a) and transgenic (b) heart showing normal myofibril architecture; hematoxylin/eosin. Scattered myocyte degeneration (arrows) was found in the transgenic heart, left ventricular free wall shown (d), compared to the healthy control heart (c); hematoxylin/eosin, 400x. (e, f) Trichrome-staining of cardiac tissue from 2-month-old line 4 transgenic heart and an age matched control littermate. Note the absence of fibrosis in the control (e) compared to the intercellular fibrosis in a transgenic heart (f). The blue staining identifies the extracellular matrix characteristic of fibrosis; Gomori's modified trichrome, 400x. The density of nuclei in transgenic hearts is increased (panels b, d, f) and is consistent with an increase in cardiac fibrocytes, but was not due to increased T-cell or neutrophil/monocyte infiltration.

 
In the heart, dead myocytes are replaced by fibrotic tissue, which stains blue with Mason's trichrome [16]. In the transgenic heart, signs of fibrosis were first detected at 4 weeks of age, that is, ~1 week after increased frequencies of TUNEL(+) cells were first detected. For instance, Fig. 4f shows extensive, diffuse interstitial fibrosis in an 8-week-old heart; control animals of similar ages rarely showed any fibrosis (Fig. 4e).

A variety of signs suggested that the TUNEL staining reflected apoptosis in cardiomyocytes. First, degenerating cells did not provoke a cellular inflammatory response. For instance, a single degenerating cell (Fig. 5a) was surrounded by normal appearing myocytes in the left ventricle from a 6-week-old animal. Note the absence of lymphocytic or neutrophilic infiltration. Immunofluorescent analysis of frozen sections stained with anti-CD3 antibody (a T-cell specific probe) also indicated the absence of a pronounced generalized or focal myocitis (data not shown). Moreover, the pyknotic and occasionally fragmented appearance of TUNEL-positive nuclei (Fig. 3a) was consistent with apoptosis.


Figure 5
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  		Fig. 5 Apoptosis and cytochrome c release. (a) Semi-thin section of a 6-week-old line 4 mouse heart. Note the single degenerating myocyte (arrow) surrounded by normal appearing cells; toluidine blue, 600x. (b) Release of cytochrome c in the transgenic heart. Cytosol (prepared from eight to ten animals per group; Tg, line 4 transgenics, Ct, control littermates) was subjected to Sephacryl-S300 chromatography and fractions were probed for cytochrome c by Western blot. Molecular weights (Mr) of standards and the included volume (Vc) are indicated at the top; column fraction numbers are on the bottom. The individual chromatograms (Tg versus Ct) are juxtaposed for comparison.

 
The co-existence of other biochemical markers for apoptosis was not observed. Western blotting of heart lysates failed to show cleavage of either PARP or caspase 3 (data not shown), which occurs during apoptosis in the heart [18]. Neither could activated caspase-3 enzymatic activity be detected in heart lysates using a fluorogenic substrate, nor could DNA laddering be seen by gel electrophoresis (data not shown). Since the frequency of TUNEL positive cells was low, it may be that these methods lacked sufficient sensitivity.

3.5 Mitochondrial apoptotic signaling
When apoptotic signaling involves mitochondria, the biochemical hallmark is the release of cytochrome c from mitochondria into the cytoplasm and its subsequent association with Apaf-1 to form complexes with a size greater than 500 kDa [19]. Western blotting of fractions generated by gel permeation chromatography of cytosolic preparations revealed 5–10-fold increased levels of high molecular weight cytochrome c in transgenic hearts at 5 weeks of age compared to control littermates (Fig. 5b). Cytosolic citrate synthase activity (a mitochondrial matrix enzyme) was identical in control and transgenic hearts, indicating that the release of cytochrome c in transgenic hearts was not due to more fragile mitochondria in transgenic hearts. Furthermore, control experiments using extracts subjected to freeze–thaw cycles in order to deliberately lyse mitochondria showed that cytochrome c released from broken mitochondria fractionated as low molecular weight material. The absence of such low molecular weight signal in the S-300 chromatograms (Fig. 5) further indicated that the increased cytosolic cytochrome c in transgenic hearts did not arise from mitochondria broken during preparation. The total level of cytochrome c in transgenic compared to control hearts was not significantly elevated and neither was the mitochondrial content of cytochrome c significantly lower in transgenic hearts (see below); consequently, released cytochrome c represents a small fraction of total cytochrome c. Previously, it was also shown that the amount of mitochondria was not different in transgenic compared to normal hearts [7]. Thus, most mitochondria in transgenic hearts had not appeared to have lost so much cytochrome c as would be expected to affect mitochondrial respiration (see also further data presented below).

3.6 Mitochondrial respiratory function and structure are normal
Polarographic studies using isolated mitochondria revealed no abnormalities in State III respiration using succinate as a substrate, the TCA cycle substrates pyruvate or glutamate, or the fatty acid substrates palmitoyl- or acetyl carnitine (Table 1). The latter pair was included in the analyses because in the adult heart mitochondrial respiration primarily uses substrates derived from β-oxidation of long-chain fatty acids [20]. Respiratory control ratios in transgenic mitochondria were as high as controls indicating normal coupling between the electron transport chain and the ATP synthetase. Likewise, the amount of ADP consumed per mole of oxygen (P/O ratio) was as expected, indicating that no specific defect was present in the respiratory enzyme complexes from transgenic mitochondria. Presumably, those cells undergoing apoptosis had altered mitochondrial respiratory function but the contribution of their mitochondria to the total pool was undetectable due to the low frequency of apoptotic cells at any one time.


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  		Table 1 Mitochondrial respiratory capacity in transgenic heartsa

 
To confirm the absence of mitochondrial respiratory dysfunction, the amounts and activity of selected respiratory chain components were measured. By difference spectroscopy, no changes were detected in the relative amounts of cytochromes a+a3, cytochrome b, and cytochromes c+c1 from either of the two transgenic lines in comparison to control mice (Fig. 6a). Direct measurement of cytochrome oxidase activity also failed to reveal a deficiency (data not shown). By Western blotting of isolated mitochondria, the amounts of cytochrome c and cytochrome oxidase subunit IV were similar in transgenics versus controls (Fig. 6b). The tissue ATP content of transgenic hearts also revealed no deficits (Fig. 6c), implying that no deficiency existed in the capacity of mitochondria to carry out oxidative phosphorylation and thereby to supply the energy needs of the heart. Finally, by electron microscopy no structural alterations were evident in mitochondria from the transgenic heart in comparison to controls (Fig. 6d). Note also that cellular architecture of the contractile apparatus appeared normal in the transgenic heart.


Figure 6
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  		Fig. 6 Mitochondrial respiratory apparatus. (a) Difference spectra of isolated heart mitochondria from 8 week-old control and transgenic line 4 and 13 animals (pools of at least eight animals per group) for quantification of cytochromes. The TG13 spectrum, which was identical to the control, is plotted with a downward offset for clarity. Spectrums representative of three separate experiments). (b) Western blot analysis for COX IV and Cyt c in mitochondria isolated from 6 week-old mice (pools of four to six animals per group). (c) ATP content (mean±S.E.M.) in 3 month-old CT or TG13 littermate hearts. (d) Mitochondrial and cellular ultrastructure of the heart of a 2-month-old line 4 mouse.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Mutation frequencies in human cardiac mtDNA increase hundreds- to thousands-of-fold with aging [1]. They are also higher, compared to age-matched, healthy individuals, in ischemic [6] and dilated cardiac disease [21]. The absolute level of mtDNA mutations, however, is rarely higher than 1% when expressed as an average over the entire tissue and more commonly in the range of 0.01–0.1% [22,23]. It has been shown that there is cell-to-cell variation in the level of mtDNA mutations causing some cells to have much higher frequencies [24]. Still, it is not clear if rare mutations contribute to disease. By engineering mice that rapidly accumulate mitochondrial DNA mutations in the heart, we have a convenient model to study the pathogenesis of mtDNA mutations. The mutation frequency in the hearts of these mice was comparable to that reported in diseases and in aging [23], and these mice developed heart disease.

The cardiac pathologies in these transgenic mice were striking. Gross cardiac dilation occurred in all mice by 4–5 weeks of age. This included both ventricular and atrial dilation. In the atria, multifocal thrombi were common. One particularly dramatic presentation was the progression of some mice to acute congestive heart failure. Within 1–2 days, a seemingly healthy transgenic mouse would become severely dyspnic from pulmonary congestion, and often had peripheral organ (liver, spleen) congestion as well.

Microscopically, interstitial fibrosis of the heart developed and increased as disease progressed. Within the fibrosis, non-cardiomyocyte nuclei were common and likely represented fibrocytes. Sporadic myocytic cell death was observed in all chambers of the heart. Based on nuclear morphology and the lack of inflammation, this cell death was apoptotic. The release of cytochrome c supported this hypothesis, and implicated mitochondria in the cell death pathway.

The mechanisms whereby mtDNA mutations cause disease are unknown. Hypothesized mechanisms include impaired respiratory function, increased production of oxygen-free radicals, and increased apoptosis [25]. For instance, mice with a high frequency of a mtDNA deletion mutation showed mitochondrial respiratory dysfunction and disease [26]. In our mice, mitochondrial respiration remained normal, which was expected given the low frequency of mutations. In addition, mutations were generated in the absence of oxidative stress, which remained absent throughout disease [17]. Our results are consistent with activation of the mitochondrial apoptotic pathway by mtDNA mutations. The release of cytochrome c from the mitochondria supports this conclusion. Moreover, within a week after TUNEL staining reported increased cell death, the level of the anti-apoptotic protein, Bcl-2, increased some 10-fold in transgenic hearts [17] suggesting that the stimulus inciting cell death was pro-apoptotic. That Bcl-2 remained high after 4 weeks of age is coincident with a persistently elevated frequency of TUNEL(+) cells, possibly indicating that up-regulation of Bcl-2 represents a response to counteract pro-apoptotic signaling coming from mitochondria.

Evidence indicates that the pathologies observed in these mice were the consequence of increased mtDNA mutations rather than a non-specific toxicity caused by the transgene-encoded proofreading-deficient mtDNA polymerase. Mice expressing a similar level of the wild-type enzyme encoded in a transgene neither accumulated mtDNA mutations nor developed disease. The time course of disease also suggests that transgene expression per se was not toxic. The {alpha}-MHC promoter that drives the transgene is activated upon birth [27], so that by day 2–3 greater than 90% of all pol {gamma} derives from the transgene [7]. However, cardiac pathology is not observed until 4–5 weeks of age and the earliest cellular abnormalities, i.e. apoptosis, are not seen until ~3 weeks of age. During the first month of life, the heart undergoes significant hypertrophy with a weight increase of at least 10-fold. In the transgenic mice, the growth of the heart over the first 3 weeks of age, as measured by the mass index, was similar to that in controls. Histologically, transgenic hearts appear normal up until at least 3 weeks of age. Thus, expression of the mutant DNA polymerase did not appear to be acutely toxic. Rather, the delayed onset of cardiomyopathy relative to the time when transgene expression begins supports the hypothesis that disease resulted from the activity of the mutant transgene, namely, its generation of mtDNA mutations.

Over-expressed pol {gamma} is unlikely to function outside the mitochondria. No reports have demonstrated a function for DNA pol {gamma} unrelated to mtDNA synthesis in any eukaryote, including yeast where null mutants have been constructed and characterized [28]. Reports of its localization in the nucleus (and by inference of a nuclear function) have been difficult to confirm [29,30]. Experimentally, HEK293T cells transfected with either a myc-tagged pol {gamma} or a fusion of pol {gamma} and green fluorescent protein demonstrated that the enzyme was appropriately sorted to the mitochondria and could not be found in the nucleus [31]. This was also observed for cells stably over expressing a proofreading-deficient mutant constructed by the same site-specific mutation in the human enzyme as we used for the mouse enzyme. In our mice, we have shown by cell fractionation that mutant DNA pol {gamma} activity likewise is found in mitochondria [7] and the accumulation of mtDNA mutations confirms that the mutant enzyme is functional in mitochondria.

Furthermore, it appears unlikely that the transgenic pol {gamma} caused non-specific toxicity to the mitochondria, since the mitochondria are normal both functionally (respiration (see Results) and membrane potential [7]) and morphologically (EM). Elsewhere we have shown that the levels of the TCA cycle enzymes isocitrate synthetase, fumarase, and aconitase were normal [7] implying that import of nuclear encoded mitochondrial enzymes was not impaired. There was no disruption of mitochondrial DNA replication, gene expression or mitochondrial protein levels [7]. Finally, increased oxidative stress, which happens when mitochondrial function is disrupted [32], was also absent in these mice [17]. Taken together, these observations strongly suggest that the mutant DNA pol {gamma} did not have non-specific toxic effects on mitochondrial function.

The spectrum and frequency of the mtDNA mutations in our mice were comparable to that seen in human aging, in which both point and deletion mutations accumulate. The confounding effect of mtDNA mutations, decreased respiration, and increased oxidative damage in the aging heart make it difficult to sort out the cause and effect among these factors. Our results demonstrate the pathogenic potential of low frequency mtDNA mutations and support the notion that mtDNA mutations play a role in the aging process through increased apoptosis.

Time for primary review 30 days.


   Acknowledgements
 
We thank the personnel in the histopathology laboratory in the Department of Pathology for their help, and the expert technical assistance of Zehua Feng, Meribeth Broadway, and Jacqueline Spencer. This research was supported by grants from the Alzheimer Association, the American Heart Association, the American Diabetes Association, and the Aging and Heart, Lung, and Blood Institutes of the NIH. Fig. 1c was reproduced with permission [33].


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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K. G. Bensch, J. L. Mott, S.-W. Chang, P. A. Hansen, M. A. Moxley, K. T. Chambers, W. de Graaf, H. P. Zassenhaus, and J. A. Corbett
Selective mtDNA mutation accumulation results in {beta}-cell apoptosis and diabetes development
Am J Physiol Endocrinol Metab, April 1, 2009; 296(4): E672 - E680.
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