Cellular metabolism and oxidative stress are interrelated processes in the mitochondria implicated in a wide range of pathological conditions in the heart, including ischemia-reperfusion injury. The ubiquitous antioxidant thioredoxin and its endogenous inhibitor, Txnip, are emerging as a key link between mitochondrial function regulation and cellular redox balance. A previous study has shown that targeted degradation of Txnip
mRNA by injection of a small nucleotide-based catalytic enzyme directly into ischemic myocardium results in enhanced cardiomyocyte survival and reduced LV remodeling (34
). However, it remains unclear how Txnip mediated this adaptive response to ischemic injury. In the present study, we found that Txnip deficiency led to a substantial metabolic switch at the transcriptional level and repressed mitochondrial respiration, directing cardiomyocytes toward enhanced anaerobic glycolysis. The concept that defects in mitochondrial metabolism produce enhanced anaerobic metabolism is supported by our finding that the corresponding expression levels of the key enzymes controlling the aerobic metabolic pattern were downregulated. The RNA levels of 36 dehydrogenases and 71 oxidoreductases controlling OXPHOS (22 enzymes) and electron transport (36 enzymes) were decreased in Txnip-KO compared with WT hearts (Supplemental Table 1). Comparative analysis of the protein levels of key enzymes regulating the glycolytic and aerobic (TCA cycle) pathways supported the gene array data. For instance, the protein level of isocitrate dehydrogenase 2 participating in the TCA cycle (aerobic respiration) was significantly decreased in Txnip-KO cells. Furthermore, our experimental data confirmed that deletion of Txnip led to metabolic behavior that made substantial use of anaerobic glycolytic metabolism in association with the key metabolic switch controller PDH. Thus, defects in mitochondrial metabolism by deletion of Txnip may play a role in determining intracellular bioenergetic flux (aerobic or anaerobic). This metabolic shift was further accelerated by ischemia and was associated with ischemic tolerance in both ex vivo controlled conditions and in vivo physiological conditions. Given the central role that mitochondria serve in ischemia-reperfusion injury, we initially hypothesized that Txnip-KO hearts would manifest more severe cellular injury, reduced intracellular ATP levels, and cardiac dysfunction after ischemia and reperfusion. Surprisingly, deletion of Txnip afforded a protective advantage to the ischemic heart.
Adaptation to hypoxia requires a dramatic reduction in cellular oxygen consumption (32
) while maintaining an energy source to regenerate its ATP stores. This response involves a shift in cellular fuel utilization from mitochondrial respiration, which consumes oxygen, to generation of ATP outside of mitochondria by metabolizing glucose without the use of oxygen (anaerobic glycolysis) (32
). Anaerobic glycolysis appears to be the preferred means of replenishing ATP stores in hypoxic organs, and it requires almost 20 times more glucose to generate 1 mole of ATP by anaerobic glycolysis than by OXPHOS. Because lipids are not used as major precursors for anaerobic metabolism, glucose is the primary substrate to support anaerobic metabolism. Thus, anaerobic metabolism with concomitant increased use of glucose, as fostered by Txnip deletion, plays a significant role in preserving myocardial function and structure and in promoting recoverability of the hypoxic heart (35
Among antioxidant defense systems against mitochondrial ROS, Trx2 is the major contributor for respiration-dependent H2
removal in mitochondria (36
). Trx2 prevents ischemia-induced ROS production in a femoral artery ligation model (37
). Overexpression of Trx2-dependent peroxidase (peroxiredoxin-3) improves survival after myocardial infarction with an attenuation of mitochondrial oxidative stress (38
). Recently, Saxena et al. discovered a critically important interaction between mitochondrial Trx2 and Txnip (8
). Using a different technique, we also confirmed the direct interaction between Txnip and Trx2 (Supplemental Figure 2G). Under physiological conditions, Saxena et al. observed that Txnip was localized primarily in the nucleus of cells, but oxidative stress led to Txnip shuttling into mitochondria, where Txnip bound to and inhibited mitochondria Trx2 (8
). In the present study, we found that cytosolic Trx1 activity was not significantly affected; however, mitochondrial Trx2 activity was decreased by ischemia-reperfusion in WT hearts. In a mechanism analogous to that reported by Saxena et al., we suspect that Trx2 is inhibited by Txnip that translocates into mitochondria by ischemia-reperfusion–induced ROS formation. However, Txnip-KO hearts exhibited unperturbed Trx2 activity, even after ischemia and reperfusion. Taken together, these results suggest that a redox-sensitive interaction between Txnip and Trx2 contributes to the redox balance in mitochondria (Figure , C and D).
Stimuli that uncouple mitochondrial respiration prior to an ischemic insult have been suggested to underlie cardioprotection mediated by ischemic preconditioning (39
). The continuation of mitochondrial aerobic respiration in the absence of oxygen results in excessive or unneutralized ROS production, mitochondrial calcium overload, and the onset of mitochondrial permeability transition, which collectively promote mitochondrial-driven cardiomyocyte death (3
). Pharmacological inhibition of complex I and II has been found to be cardioprotective by limiting ROS production, resulting in reduced infarct size after reperfusion in a rat model of ischemia-reperfusion injury (40
). However, the hypoxic cell requires an energy source to regenerate its ATP stores. A previous report demonstrated that inclusion of glucose in the perfusate of isolated perfused rat heart preparations results in marked improvement in electrical and mechanical performance of the heart subjected to anoxia and in enhanced recovery during the subsequent period of reoxygenation (35
). While lactate production was 5-fold greater in the glucose-supported anoxic heart than in the anoxic heart without glucose, morphologic changes of mitochondria during anoxia were averted by inclusion of glucose in the perfusion fluid (35
). Thus, anaerobic metabolism, with concomitant increased use of glucose, can preserve myocardial function and structure and promote ischemic heart recovery. These cardioprotective mechanisms can be feasibly achieved by deletion of Txnip, which (a) limits myocardial ROS levels effectively by mitochondrial Trx2, (b) reduces oxygen consumption in mitochondria, and (c) greatly favors cellular glucose uptake and glycogen storage in the heart while simultaneously enhancing glucose flux toward anaerobic glycolytic metabolism. As a consequence, a high-flux glycolytic state coupled with the residual respiration provided sufficient ATP for survival and cellular protection in Txnip-KO hearts. As previously observed, these data reflect the physiology of tumor-genesis and the “Warburg effect” — physiology by which cancerous cells preferentially rely upon glycolytic metabolism, despite the availability of oxygen to support mitochondrial respiration (41
). Txnip acts as a mediator to integrate redox balance and energetic needs with cellular glucose supply: this may imply that the Warburg effect can be protective against ischemic insults in cardiomyocytes when ROS balance is secured by a potent antioxidant, such as mitochondrial Trx2.
We here identified Txnip as a regulator of mitochondrial function and demonstrated the role that Txnip serves during ischemia-reperfusion injury in the heart. However, it remains unclear how Txnip participates in the transcriptional regulatory circuits that control mitochondrial respiration. Mitochondrial Trx2 interacts with components of the mitochondrial respiratory chain and plays a role in the regulation of the mitochondrial membrane potential (42
). Trx2 deficiency is embryonic lethal at gestational day 10.5 in mice, which coincides with the transition period from anaerobic to aerobic metabolism in the embryo (43
). Thus, Txnip may regulate mitochondrial metabolism through mitochondrial Trx2.
To further identify a potential mechanistic role of Txnip by protein-protein interaction, we performed a proteomic screening using glutathione S-transferase (GST) tag protein interaction pulldown assays (Supplemental Results). Proteins from HEK 293F cells overexpressing Txnip with GST or GST alone (as a negative control) were pulled down from cellular lysates using magnetic glutathione beads, subjected to SDS-PAGE, and then analyzed by mass spectrometry. This proteomic screening revealed a putative interaction with several proteins involved in metabolism as candidates for binding targets of Txnip (Supplemental Table 3). These potential interactions of Txnip with other proteins may also be involved in a mechanistic role of Txnip as a controller of energy metabolism. Further studies will be necessary to define these roles.
In conclusion, we identified Txnip as a regulator of mitochondrial function and demonstrated that Txnip deficiency led to a substantial metabolic switch, directing cardiomyocytes toward enhanced glycolytic metabolism. Several potential pathways might be simultaneously involved in Txnip-KO mice; therefore, no single cause of mitochondrial dysfunction could be defined. Nonetheless, the present study provides insights into the role of myocardial Txnip in anaerobic metabolism that contributes to acute cardioprotection during ischemia-reperfusion injury.