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.