the paper by stensløkken et al. (16) is an important investigation that examines the levels of expression of heat shock proteins (HSPs) in crucian carp (Carassius carassius) brain based on the prediction that there would be increases in expression of heat shock proteins in anoxia and that these alterations in expression would be of smaller magnitude at lower temperatures (8°C compared with 13°C). The crucian carp is a key model of anoxia tolerance as it survives in total oxygen deprivation for durations of days at room temperature to months at lower temperatures. By employing a real-time PCR analysis, Stensløkken et al. (16) demonstrated that levels of mRNAs for several heat shock proteins HSP70a, HSP70b, HSC70, HSP90, and HSP30 changed in anoxia with a dramatic increase in HSP70a mRNA expression in day 7 anoxic brains and hearts at 13°C but not at 8°C. Under normoxic conditions, a 7- and 11-fold higher HSP70a expression was found at 8°C in brain and heart, respectively, by comparison to 13°C. In anoxia, the differences in HSP70a expression at 8°C by comparison to 13°C were minimal (only 1.1 for brain and 1.9-fold for heart). These observations indicate that cold temperatures may play a preconditioning role in the brain and heart of the crucian carp to elicit tolerance for upcoming anoxia.
Models of anoxic survival.
The survival characteristics found in brain of crucian carp and in certain other anoxia-tolerant organisms, such as the freshwater turtle, can be viewed in sharp contrast to the responses of other nontolerant vertebrates, in which brain anoxia results in loss of ion gradients, a massive release of excitotoxic neurotransmitters, and catastrophic cell death (7). Different anoxia-tolerant organisms have developed alternative strategies for ensuring that ATP demand is successfully matched by ATP availability. The crucian carpapos;s primary strategy for survival in anoxia is increasing glycolysis but avoiding toxicity through the use of ethanol as the end product of anaerobic glycolysis. The crucian carp decreases metabolic rate but only moderately, and electrical activity in the brain is not suppressed, which is in contrast to the findings of altered excitability and channel arrest during anoxia observed in the brain of the anoxia-tolerant turtle (Trachemys scripta) (7).
The question of constitutive preconditioning in anoxic survival.
Previously, it has been proposed that anoxia-tolerant organisms may employ constitutive preconditioning as a general characteristic that is reflected in the normoxic expression of several prosurvival and protective cellular pathways. In crucian carp brain, for example, GABA neurotransmission is characterized by a brain GABA receptor composition with subunits that do not desensitize during sustained binding to GABA (1).
Another example of constitutive preconditioning is suggested in the case of the brain of the anoxia-tolerant turtle, in which high constitutive levels of HSP70 in normoxia may indicate a state of preparedness for the ensuing anoxic stress (11). By 4 h anoxia, HSP72 levels were significantly increased above normoxic levels, stayed elevated until 8 h, and declined at 12 h to baseline.
In the anoxia-tolerant western painted turtle (Chrysemys. picta), under forced-dive conditions, there was no increase in brain HSP72/73 protein levels from normoxia to the first 12 h of anoxia, which could indicate that normoxic HSP levels may be somewhat elevated in C. picta (12). HSP72 levels were found to be increased at later time points, reaching threefold by 30 h of anoxia. From the evidence pointing to constitutive preconditioning in different anoxia-tolerant species, it has been suggested that a broad examination of biological processes may be needed for an understanding of anoxic survival mechanisms rather than an analysis of individual gene products (1).
Mechanisms of protection by heat shock proteins against hypoxic and anoxic damage.
HSPs protect through acting as chaperones and through maintenance of correct folding of proteins, and in heart and brain, HSPs are capable of conferring protection against ischemic injury. Through interacting with key cellular proteins, the heat shock protein HSP90 contributes to functions, such as mitochondrial targeting of the ATP-sensitive potassium channel subunit Kir6.2 (3) and stabilization of the hypoxia-responsive transcriptional regulator HIF-1α (9). HSP90 is also reported to interact with other proteins, including steroid hormone receptors, tyrosine kinases, and components of the cytoskeleton (6). In mammalian systems HSP70 is recognized as a key component of a preconditioning response. Effects of HSP70 on preventing apoptotic processes are likely to be highly significant in this respect. The mechanisms of protection by HSP70 in preconditioning are reported to include inhibition of apoptotic processes in part through preventing recruitment by Apaf-1 of procaspase 9 (10). HSP70 has been also been found to interfere with caspase cleavage of a GATA transcription factor, thus maintaining prosurvival Bcl-XL expression (14). Other antiapoptotic effects have been reported, involving HSP70 interfering with target proteins other than Apaf1, one of which is the caspase-independent death effector apoptosis-inducing factor (13). Further noncaspase influences of HSP70 on apoptotic processes include the inhibition by HSP70 of c-Jun N-terminal kinase, which is proapoptotic in several systems (6).
A key proposal of the paper by Stensløkken et al. (16) is that hypothermia may be an important factor in preconditioning the crucian carp to activate mechanisms for survival during upcoming anoxia. The dramatically activated levels of HSP70 expression reported in brain and heart in normoxic crucian carp at 8°C compared with 13°C point to decreased temperature as a preconditioning stimulus. In a previous study on crucian carp, the transcription factor HIF-1 was reported to play a temperature-dependent role in activating hypoxia responses (15). The hypoxia-responsive transcription factor HIF-1α was proposed to be important for hypoxia-induced protection and adaptation in multiple tissues of crucian carp, where it is likely to activate important survival genes. In normoxic crucian carp, cold acclimation was found to increase HIF-1α activity in heart, gills, and kidney by greater than twofold by comparison to fish acclimated to lower temperatures (26°C compared with 8°C). In hypoxia, HIF-1 protein was elevated in several tissues at low temperatures, and HIF-1α activity was increased in the heart of 8°C acclimated and in the gills of 18°C acclimated fish relative to 26°C acclimated fish. In the same study, increased HSP70/HSC70 and HSP90 protein levels were found in liver, heart, gills, and kidney upon decreasing normoxic temperature from 26°C to 8°C (15).
Hypoxic preconditioning in mammalian systems and nonanoxia-tolerant organisms is reported to represent a HIF-1-induced response. The HIF-1 response may be activated by the hypoxic block in VHL-mediated degradation of HIF-1 transcription factor or alternatively by influences from redox modulation and/or prosurvival kinases such as Akt (2).
In the study by Stensløkken et al. (16), HSP90 mRNA levels were found to decrease in crucian carp brain and heart at 8°C in anoxia relative to normoxia (16). At 13°C, there was a decrease in HSP90 expression in brain but an increase in heart in anoxia by comparison to normoxia. As discussed above, HSP90 is known to contribute to the correct functioning of key proteins, including HIF-1α. The decrease in HSP90 expression in anoxia would be anticipated to correspond to a destabilization of HIF-1α, resulting in compromised HIF-1α-induced responses. Stensløkken et al. (16) point out the interesting possibility that a predicted decrease in HIF-1-induced responses by anoxia in anoxia-tolerant organisms could prevent induction of a number of processes that are overly costly in terms of ATP utilization.
In crucian carp, HSP70a mRNA levels were increased in heart and brain by >10-fold at 7 days anoxia relative to hypoxia, and this was independent of temperature. As mentioned above, increases in brain HSP72 have been reported at 17°C during a 30-h forced dive in the western painted turtle, pointing to a protective role for this protein in long-term anoxia (12). In the brain of the anoxia-tolerant turtle T. scripta at room temperature, HSP72 was increased over the first 4–8 h of anoxia, declining to baseline at 12 h of anoxia. Such a pattern of expression may relate to a role for HSP72 in stabilization of proteins during the orchestrated metabolic downregulation that occurs in the brain of T. scripta early in anoxia (8, 11).
HSC70 is increased after 24 h of anoxia in crucian carp and may be important at early stages of anoxic adaptation. High HSC70 may be associated with hypermetabolic rate, and HSC70 was more elevated in anoxic heart relative to brain, which may correspond to the high metabolic rate of the crucian carp heart in anoxia. In the anoxic freshwater turtle brain at room temperature, over the first 12 h of anoxia at room temperature, HSC70 protein is progressively increased, suggesting the protein may contribute to maintenance of neuronal network integrity during this period of reduced neuronal excitability (8, 11). Interestingly, in mammals, HSC70 forms part of a multiprotein complex associated with GABA synthesis and vesicular release pointing to a protective role for HSC70 in regulating the availability of this key inhibitory neurotransmitter (4). However, in crucian carp, HSC70 mRNA was reduced at 7 days in brain but not in heart, which may argue against a neuroprotective role for HSC70 in this model of anoxic survival.
The small heat shock proteins have been shown to contribute to protecting the cell from protein aggregation, and members of this class include HSP27, which may play an antiapoptotic role by interacting with components of the apoptotic machinery (6). Stensløkken et al. (16) found that in the crucian carp, HSP30 mRNA decreased in anoxic brains and hearts at both 13°C and 8°C. In freshwater turtle, white skeletal muscle HSP25 is induced significantly over 20 h anoxia, but no alteration in HSP25 levels is seen in heart over this time period (5). In kidney, HSP25 was found to be unaltered in anoxia but induced upon reoxygenation, which may be consistent with its reported role in protecting against cytoskeletal breakdown from oxidative stress. In mammalian systems, HSP27 has been reported to provide significant protection of neurons and cardiac myocytes against ischemic damage (6).
The crucian carp is a unique model for examining the contributions of HSPs to tissue protection in an organism that substantially maintains metabolic activity under conditions of anoxia. The article by Stensløkken et al. (16) reports alterations in levels of heat shock protein mRNAs under anoxic conditions and points to a role for lowered temperatures in normoxia as a preparative cue for enabling high-level HSP70 expression when needed. This paper indicates how heat shock proteins may contribute in a central manner to signaling for prosurvival and antiapoptotic events in anoxia, as well as to the maintenance of function of key proteins in a manner that that is appropriate to the level of metabolic activity of target tissues.
No conflicts of interest, financial or otherwise, are declared by the author.
- Copyright © 2010 the American Physiological Society