Conclusion
The aging myocardium is characterized by a number of structural,
biochemical and metabolic changes that contribute to a decline in
cardiac function18. An emerging characteristic of the
aging phenotype is a decline in autophagy, described in a number of
tissues19,20. This is a significant alteration as
autophagy is often cytoprotective and has been linked to improved
outcomes in response to IR injuries3,11,21–23.
Autophagy is also emerging as a quintessential mechanism involved in the
protective benefits of ischemic preconditioning5,24,
therapeutic hypothermia 4 and a number of
pharmaceuticals, such as statins25. Previous studies
have highlighted a reliance on autophagy in the response to IR in
patients undergoing cardioplegia during surgery15,22.
The goal of this study was to contribute to our understanding of how age
influences the autophagy response to IR insults in human cardiac tissue
by using samples from right atrial appendages of patients undergoing
CABG surgery.
Our results revealed no overt changes with age in several markers
indicative of mitochondrial content, with the notable exception of a
significant decrease in UQCRC2, a subunit of complex III. This is in
line with previous reports of functional impairments in complex III
activity in rodent models of aging2,26, and is
suggestive of a change in mitochondrial composition with age.
The critical end-stage steps of the autophagy pathway reside within
lysosomes, which serve to degrade dysfunctional cargo. Previous accounts
have described evidence of lysosomal dysfunction with age, as these
organelles tend to accumulate lipofuscin, indicative of a catabolic
blockade27–29. Strikingly, we observed sharp declines
in both TFEB and TFE3, transcriptional regulators of lysosomal
biogenesis, with age. Despite this decline, only a trend for decrease in
the lysosomal protein Cathepsin D was observed, a known target of TFEB.
However, the induction of v-ATPase, a robust effect observed with IR in
younger subjects, was completely blunted with age (Table 1). This
suggests that TFEB and TFE3 levels may be sufficient to maintain basal
levels of lysosomal proteins, but that the response to stress is
severely restricted with age. Interestingly, a trend for an increase in
the LC3 II:I ratio with age was observed without a change in the adaptor
proteins p62 and Optineurin. Together, these data point to a decline in
lysosomal function with age, since these proteins are normally consumed
in the autophagy process, and an increase in LC3-II represents an
accumulation of autophagosomes that have not fused to lysosomes for
degradation. This would be in line with numerous studies that support a
decrease in autophagy with age in other tissues, pointing potentially to
reduced degradative capacity of the lysosome, rather than impairments at
the initiation/tagging stages, or in the elongation of the
phagophore11,22,30. Our data also suggest a decrease
in mitophagy with age, evident from the marked decrements in both Parkin
and Nix (Table 1). This could lead to the accumulation of mitochondria
with altered composition, and possible dysfunction within the heart,
evident from the decrease in UQCRC2. It is well known from other studies
in muscle cells that the inhibition of lysosomal function leads to an
accumulation of mitochondrial content, but that these organelles have
reduced rates of respiration, as well as elevated ROS
emission31. Future measures of mitochondrial function
are required to verify this hypothesis.
Prior reports32 using the same surgical intervention
have demonstrated that perioperative IR promotes mitochondrial
biogenesis, likely by enhancing the translation of pre-existing mRNAs.
This is in line with our observations of increased COX I and VDAC
protein content. This increase in select mitochondrial markers may
represent a protective response, aimed at maintaining cardiac metabolic
capacity following an ischemic insult. It is important to note that not
all mitochondrial markers increased following IR, and this may reflect a
compositional change rather than an overall increase in content.
Interestingly, the aged cohort exhibited a blunted increase in COX I
protein content in comparison to young counterparts, further indicating
an inability of the aged heart to mount an appropriate response to
stress, as discussed above (Table 1). Increases in VDAC could also
contribute to an enhanced activity of the mitochondrial permeability
transition pore, and although increased pore formation to initiate
mitochondrial-mediated apoptosis is a common finding with
IR33–35, our data provide no evidence for enhanced
apoptotic signaling following this IR intervention in this cohort.
The ubiquitination “tagging” of mitochondria could be impaired
following IR in younger subjects, based on the marked decline in Parkin
levels following this bypass surgery. The interpretation of impaired
autophagy and mitophagy is supported by the modest trend for an increase
in LC3-II protein content observed in the younger patients, along with
previous accounts that have demonstrated poor clearance by the lysosomes
in rodent models of IR. As the nature of the autophagy process is highly
dynamic, researchers have relied on measurements of autophagy flux to
better understand this recycling process, although many of these
techniques rely on fluorescent tagging, or the use of inhibitors which
would not be appropriate for human trials. Thus, it is critical to rely
on data from animal models to fully appreciate the complexity of the IR
response.
During enhanced autophagy, LC3-II and p62 should be degraded along with
the dysfunctional cargo. The lack of decrease in these proteins, taken
together with the decline in Beclin-1, which is involved in the
initiation of autophagy, suggests that autophagy decreases following IR.
TFEB and TFE3 also decline, and as transcriptional regulators of a
number of autophagy-related genes 36, these data also
support this autophagy decrease. Interestingly, the postoperative
response in these parameters appeared to be blunted in the aged cohort
and this may be evidence of an inability to mount a stress response with
age (Table 1).
In summary, our data support the idea that autophagy declines with age
in human atria. It remains to be addressed whether this decrement lies
in the initiation of the recycling process, the ability to detect and
sequester damaged material, or in the capacity to breakdown cellular
components at the lysosome. Understanding the autophagic response is
also critical as it is thought to have repercussions for the activity of
cell death or apoptotic pathways3,15,37. Our findings
also demonstrate the influence of age on the response of the myocardium
to surgeries involving ischemia-reperfusion, as older subjects exhibit a
reduced resilience and adaptability compared to younger patients. Future
work will be needed to resolve the relative roles of apoptosis and
autophagy in the aging myocardium, and will be aided by measurements of
mitochondrial function.