Abstract 924: Aging Induces Titin Oxidation And Increases Passive Cardiomyocyte Stiffness
One of the hallmarks of aging is an increase in ventricular passive stiffness leading to diastolic heart failure, however, the molecular basis for this is unclear. The giant elastic protein titin, working as an entropic spring, is the major determinant of passive cardiomyocyte stiffness. We hypothesized that accumulation of oxidized titin in the aging heart results in increased cardiomyocyte passive stiffness and thereby contributes to diastolic dysfunction. We further hypothesized that an age-assocated disturbance in the ubiquitin/proteasome pathway plays a role in the accumulation of oxidized titin.
Methods: Cardiomyocytes from adult (4 month) and aging (34 month) mouse hearts were isolated and chemically skinned and the passive force-sarcomere length relationship in single cells were obtained. Post-translational modification of titin was assessed in whole heart homogenates by determining the content of 1) titin carbonyls, a marker of irreversible protein oxidation, and 2) titin ubiquitinylation, a marker for proteasomal degradation.
Results: Cardiomyocyte passive tension was significantly higher in the aging group (at SL=2.45 μm, 1008.4 ± 45.5 vs. 871.6 ± 48.3 μg; p<0.05). While the total amount of titin was unchanged between both groups, the carbonyl content of titin was increased 3-fold with aging (p<0.05). High molecular weight ubiquitin-conjugated proteins were increased with aging, which is consistent with a decrease in proteasomal activity with age. Surprisingly, titin ubiquitinylation was decreased with aging suggesting an impairment upstream of the proteasome, in titin-specific ubiquitin ligase activity. Furthermore, the muscle specific ubiquitin ligase MURF-2, which has been shown to interact with titin and play a role in muscle protein turnover, showed a significant shift from a high to low molecular weight isoform with aging.
Conclusion: Our data suggests that an age-associated impairment in titin turnover contributes to accumulation of oxidatively modified titins with consequent impairment of cardiomyocyte stiffness. We speculate that the oxidation of titin will change the elastic properties of titin, directly accounting for the change in passive tension and contributing to diastolic dysfunction associated with aging.