Chronic Apoptotic Signaling is Induced by Low Levels of Mitochondrial Dna Mutations in the Mouse Heart

. MtDNA mutations rise with age and are associated with a variety of degenerative diseases. Their pathogenesis has been questioned, however, in part because it is not clear by what mechanism low levels of mtDNA mutations cause cellular dysfunction. In order to study this issue, we constructed transgenic (tg) mice that rapidly accumulate both point and deletion mutations in cardiac mtDNA starting at birth due to the expression of a proofreading deficient DNA pol γ (1). By 1 month of age, mutations are at levels commonly seen in the aged or ischemic human heart (2). Tg mice develop dilated cardiomyopathy but pathogenesis does not appear to involve either mitochondrial respiratory dysfunction or increased oxidative stress (3). Rather, the rising frequency of mtDNA mutations induces chronic pro-apoptotic signaling in all cardiomyocytes, some of which succumb by apoptosis. This mitochondrial signaling is counteracted by a persistent upregulation of a number of antiapoptotic proteins. Although myocytic apoptosis is thereby blunted, we propose that the "compensated state" contributes to myocytic dysfunction. METHODS. Protein levels were quantified by western blotting of heart lysates or sub-cellular fractions. Cytochrome c (cyt c) release from mitochondria was characterized by gel filtration of S100 fractions. The proportion of cardiomyocytes showing elevated pro- and anti-apoptotic signaling was analyzed by immunohistochemistry and TUNEL staining. RESULTS. Apoptosis in the transgenic heart begins to rise relative to controls at 3 weeks of age, peaks 1 week later, and then declines at a time when Bcl2 levels rise and remain high (Fig. 1). Apoptosis is associated with the release of cyt c and translocation of BAX onto mitochondria (Fig. 2). Counteracting these pro-apoptotic signals is the upregulation of a number of anti-apoptotic proteins which inhibit different steps in mitochondrial apoptotic signaling (Fig. 2): Bfl1 (BAX activity {4}), Bcl2 and Bcl-xL (BAX-induced release of cyt c {5}), Hsp27 (activation of caspase 9 {6}), XIAP (activation of caspase 3 {7}). Although total levels of both Bcl2 and Bcl-xL are elevated, only Bcl2 is also upregulated in mitochondria (Fig. 2). Immunohistochemical analysis for Bcl2 and cyt c shows that by 5 weeks of age all cardiomyocytes in the transgenic heart show upregulation of Bcl2 and release of cyt c.