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).