Results
Mitochondrial markers following ischemia-reperfusion with age. No significant changes were found with age in the outer membrane protein VDAC, the inner membrane subunits COX I, and COX IV, as well as the matrix enzyme citrate synthase (Fig. 1 A-E). However, a 32% reduction in UQCRC2, a subunit of complex 3 of the electron transport chain (Fig. 1F; 0.74 vs 0.53, main effect of age, p=0.03), was observed with age, suggesting a compositional change that may be indicative of a complex III defect in the aging heart. This was also observed in a negative correlation between the UQCRC2 protein content and age (Fig. 1G; r=-0.61, p=0.06).
IR resulted in 30% increases in VDAC (0.75 vs 1.05, p=0.03) and COX-I (0.63 vs 1.10, p=0.03), in a manner that was attenuated with age. This suggests an acute accumulation of mitochondrial proteins that are not being degraded.
Autophagy markers following ischemia-reperfusion with age. LC3 is a component of the autophagosomal membrane, which undergoes lipidation as part of its maturation from LC3-I to LC3-II. No significant changes were found in the immature or mature forms basally (Fig. 2B), or in the ratio of LC3II to LC3I (0.62 vs 1.27, p=0.70), despite a trend toward an increase with age (Fig. 2C; 0.62 vs 1.18, p=0.82). Furthermore, a negative correlation was seen between Optineurin, an autophagy adaptor protein, with age (Fig. 2G; r=-0.62, p=0.057). These data support an impairment in autophagy with age.
A decrease in Optineurin (main effect of IR; 1.14 vs 0.65, p=0.003) was also observed post-reperfusion in the young subjects, however this was not seen in the aged cohort (Fig. 2D). When accompanied by the marked reduction in the autophagy initiator Beclin-1 (Fig. 2F; main effect of IR, 0.93 vs 0.50, p=0.001), and the lack of change in p62 (2.03 vs 1.54, p=0.10), these data suggest that the autophagy pathway was reduced post-IR.
Mitophagy markers following ischemia-reperfusion with age.Parkin, the E3 ubiquitin ligase, was reduced in aged atrial samples (Fig. 3D; r=-0.78, p=0.02), along with NIX, a receptor on the outer mitochondrial membrane that has roles in both mitophagy and in apoptosis. Following IR, Parkin levels were dramatically reduced in atrial samples of younger patients (Fig. 3B; 0.95 vs n.d., p=0.02), an effect that was not observed in the aged cohort (Fig. 3B; 0.49 vs 0.47, interaction effect, p=0.04; main effect of IR, p=0.02). This suggests a decline in mitophagy following IR in younger patients. However, this could have been compensated for by the elevation in NIX, by 3.8-fold in the young cohort (0.60 vs 1.8, p=0.002), but only 2.3-fold in the aged subjects (Fig. 3C; 0.21 vs 0.76, interaction effect, p=0.04; main effect of IR, p=0.002; main effect of age, p=0.004). These data highlight a blunted response in the aged cohort to the IR injury, suggesting a loss in the ability to mount an appropriate response to stress with age.
Lysosomal markers following IR with age. Dramatic 68% and 85% reductions in TFEB and TFE3, the transcription factors that regulate lysosomal- and autophagy-related genes, were observed with age (Fig. 4B-C; 0.63 vs 0.20, p=0.02; 1.00 vs 0.10, p=0.0001 respectively), which corresponded to negative correlations with age (Fig. 4F-G; r=-0.56, p=0.15; r=-0.95, p=0.001 respectively). Furthermore, a trend for reduced Cathepsin-D, a lysosomal protease, was found in the aged cohort (Fig. 4D; 0.54 vs 0.34, p=0.14), but no change was observed for V-ATPase, a proton pump found on the lysosomal membrane (Fig. 4E; 0.51 vs 0.54). TFEB and TFE3 were markedly reduced in the young patients following IR (Fig. 4B-C; 0.63 vs n.d., main effect of IR, p=0.02 for TFEB). Surprisingly, there was an increase in V-ATPase protein content in the atria of younger patients following IR (Fig. 4E; 0.51 vs 1.12, main effect of IR, p=0.005). These changes were dependent on age. TFEB content remained unchanged (Fig. 4B; 0.20 vs 0.25, interaction effect, p=0.02), while TFE3 content increased 3-fold (Fig. 4C; 0.10 vs 0.45, interaction effect, p=0.01), while V-ATPase did not respond in the aged cohort (Fig. 4E; 0.54 vs 0.71, interaction effect, p=0.05), supporting the notion of a blunted stress response in the atria of aged subjects, compared to the young (1.12 vs 0.71, p=0.004). This would also support declines in the capacity for autophagy in the aged, which was evident by the reduced response in autophagy markers in the aged patients.
Stress response markers following ischemia-reperfusion with age.No significant changes were observed in either HSP70 or caspase-3 with age, or following the IR (Fig. 5A-D).