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. 2009 Jun 2;119(21):2789-97.
doi: 10.1161/CIRCULATIONAHA.108.822403. Epub 2009 May 18.

Overexpression of catalase targeted to mitochondria attenuates murine cardiac aging

Dao-Fu Dai  1 , Luis F Santana, Marc Vermulst, Daniela M Tomazela, Mary J Emond, Michael J MacCoss, Katherine Gollahon, George M Martin, Lawrence A Loeb, Warren C Ladiges, Peter S Rabinovitch
Affiliations

Overexpression of catalase targeted to mitochondria attenuates murine cardiac aging

Dao-Fu Dai et al. Circulation. .

Abstract

Background: Age is a major risk for cardiovascular diseases. Although mitochondrial reactive oxygen species have been proposed as one of the causes of aging, their role in cardiac aging remains unclear. We have previously shown that overexpression of catalase targeted to mitochondria (mCAT) prolongs murine median lifespan by 17% to 21%.

Methods and results: We used echocardiography to study cardiac function in aging cohorts of wild-type and mCAT mice. Changes found in wild-type mice recapitulate human aging: age-dependent increases in left ventricular mass index and left atrial dimension, worsening of the myocardial performance index, and a decline in diastolic function. Cardiac aging in mice is accompanied by accumulation of mitochondrial protein oxidation, increased mitochondrial DNA mutations and deletions and mitochondrial biogenesis, increased ventricular fibrosis, enlarged myocardial fiber size, decreased cardiac SERCA2 protein, and activation of the calcineurin-nuclear factor of activated T-cell pathway. All of these age-related changes were significantly attenuated in mCAT mice. Analysis of survival of 130 mice demonstrated that echocardiographic cardiac aging risk scores were significant predictors of mortality. The estimated attributable risk to mortality for these 2 parameters was 55%.

Conclusions: This study shows that cardiac aging in the mouse closely recapitulates human aging and demonstrates the critical role of mitochondrial reactive oxygen species in cardiac aging and the impact of cardiac aging on survival. These findings also support the potential application of mitochondrial antioxidants in reactive oxygen species-related cardiovascular diseases.

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Figures

Figure 1
Figure 1. Echocardiography of mice from different age groups in the longevity cohort
Age related changes in: A) LV mass index (mg/g body weight); B) LA dimension (mm); C) Fractional shortening (%); D) Ea/Aa measured by tissue Doppler imaging of the mitral annulus; E) Proportion of mice with diastolic dysfunction, defined as Ea/Aa < 1; F) Myocardial Performance index (MPI). There were significant linear increase across ages for all outcomes (p <0.001 for all except for FS where p=0.005, fit linearly to a continuous age function). MCAT mice had a significantly lower rate of progression with age in all of the above measurements when compared to WT (p<0.001 for all except for FS, p=0.03, for an interaction between age-slope and genotype). 90 mCAT and 80 littermate WT were examined, approximately 20 mice in each age and genotype group.
Figure 2
Figure 2. Cross-sectional study of cardiac pathology
A) Old WT (OWT) mice had larger myocardial fiber width than old mCAT (OmCAT) mice (trichrome stain, 400x, scale bar:10μm). B) Quantitative analysis showed a significant increase in myocardial fiber-width in aged heart, which was significantly attenuated in OmCAT mice. C) OWT mice had more fibrosis (blue; trichrome , 20x) than OmCAT mice, as shown by quantitative image analysis of the percent fibrotic area in aged hearts, which was significantly attenuated in OmCAT mice (D).
Figure 3
Figure 3. Mitochondrial DNA mutations, oxidative damage and biogenesis in aged heart and the protective effect of mCAT
A) Mitochondrial DNA point mutation and B) deletion frequencies (both per million) increased significantly with age and were better preserved in OmCAT mouse hearts. C) Mitochondrial protein carbonyls (nmol/mg) increased significantly in old age, while OmCAT mice were better protected from oxidative damage to mitochondrial proteins. D) Mitochondrial DNA copy number (normalized to young WT) increased significantly with age, and old mCAT mice had significantly lower mitochondrial DNA copy number. E). Genes involved in mitochondrial biogenesis, PGC-1α, TFAM, NRF-1 and NRF-2, were upregulated in aged heart, and changes in PGC-1α and TFAM were attenuated in old mCAT mice (*p<0.025 between young vs. old WT, #p<0.025 between old WT vs. mCAT; n=9-12 each group; old: 26-28 months, young:4-5 months old).
Figure 4
Figure 4. Activation of calcineurin-NFAT and GATA4 in age-dependent left ventricular hypertrophy and the effect of mCAT
A) Calcineurin phosphatase activity (nmol phosphate / 5 ug cardiac protein) increased significantly in aged heart and was partially protected in old mCAT hearts. B) Mobility shift assay showed that NFAT DNA-binding activity increased in old WT and the increase was less obvious in old mCAT hearts. Competitive DNA binding with excess unlabelled NFAT probe (arrow) using the leftmost old WT sample confirmed the specificity of the bands (arrowhead). C) The relative mRNA levels of ANP, BNP and MCIP, target genes of NFAT, increased significantly in old WT but not old mCAT hearts. D) Age-dependent increased phosphorylation of GATA-4 at Ser105 was reduced in old mCAT heart. *p<0.025 between YWT and OWT, # p<0.025 between OWT and OmCAT.
Figure 5
Figure 5. Calcium handling proteins and cardiac angiotensin-II
Western blots of calcium handling proteins were quantified relative to GAPDH. A) SERCA2 protein was significantly reduced in aged WT heart, and better preserved in old mCAT heart; B) NCX protein was significantly increased in aged heart, and lower in old mCAT compared with old WT; C) Linear regression demonstrates an inverse correlation between SERCA2 and NCX protein levels (all ages: β= −0.61, R2=0.49, p<0.01; old mice only: β= −0.82, R2=0.52, p=0.001). D) Cardiac angiotensin II concentration (fmol/mg total protein) measured by mass spectrometry was significantly increased with age in both WT and mCAT mice. E) Phosphorylated and total protein levels of phospholamban did not change significantly with age or mCAT expression.
Figure 6
Figure 6. Plausible mechanisms of diastolic dysfunction in aged heart
A) Myocardial fiber width was significantly greater in old mice with diastolic dysfunction (DD), compared with those with normal diastolic function (N). B) Ventricular fibrosis (%) was higher in old mice with diastolic dysfunction. C) SERCA2 protein was significantly decreased in mice with diastolic dysfunction. D) Representative calcium transients of isolated cardiomyocytes from young and old mouse hearts loaded with Fluo-4 and paced at 1 Hz. The inset illustrates OWT vs. OmCAT decay rates. E) Quantitative analysis showed significant decreases in Ca-transient amplitude (F/F0) and decay rate constant λ (s−1) in old WT, and protection in old mCAT cardiomyocytes. Sarcoplasmic reticulum Ca2+ load was also significantly higher in OmCAT cardiomyocytes, as shown by mobilization of Ca2+ by caffeine. The protective effect of mCAT was completely abolished by treating the old cardiomyocytes with cyclopyazonic acid, a SERCA-2 inhibitor.*p<0.025
Figure 7
Figure 7. Kaplan Meier analysis of follow-up mortality stratified by cardiac aging risk score tertiles
Cardiac aging risk score was calculated as a linear combination of MPI and LVMI obtained from the multivariate Cox model 1 (ß2 MPI + ß3 LVMI). Mice with the higher tertile of cardiac aging score (T3) had significantly shorter survival than those with the lower tertile (T1), when scores were analyzed for WT and mCAT mice together (A) or separately for WT (B) and mCAT (C) mice.
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