Decreased temperature as a signal for regulation of heat shock protein expression in anoxic brain and heart.

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1 Articles in PresS. Am J Physiol Regul Integr Comp Physiol (April 21, 2010). doi: /ajpregu Editorial focus: April 2010 Decreased temperature as a signal for regulation of heat shock protein expression in anoxic brain and heart. Howard M. Prentice College of Biomedical Science, Florida Atlantic University, Boca Raton, Florida Address for correspondence: H. M. Prentice, College of Biomedical Science, Florida Atlantic University, Boca Raton, FL ( hprentic@fau.edu) The paper by Stenslokken 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 degrees C compared to 13 degrees C). The crucian carp is a key model of anoxia tolerance that survives in total oxygen deprivation for durations of days at room temperature to months at lower temperatures. By employing a real time PCR analysis the study demonstrates that levels of mrnas for several heat shock proteins HSP70a-b, HSC70 HSP90 and HSP30 changed in anoxia with a dramatic increase in HSP70a mrna expression in day 7 anoxic brains and hearts at 13 degrees C but not at 8 degrees C. Under normoxic conditions a 7 and 11 fold higher HSP70a expression was found at 8 degrees C in brain and heart respectively by comparison to 13 degrees C. In anoxia the differences in HSP70a expression at 8 degrees C by comparison to 13 degrees 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 non-tolerant vertebrates where 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 carp 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. It has previously 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). Copyright 2010 by the American Physiological Society.

2 Another example of constitutive preconditioning is suggested in the case of the brain of the anoxia tolerant turtle where 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 hours and declined at 12 hours to baseline. In the anoxia tolerant western painted turtle (C. picta) under forced dive conditions there was no increase in brain HSP72/73 protein levels from normoxia to the first 12 hours 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 three fold by 30 hours 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 transciptional regulator HIF-1-alpha (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 pro-caspase 9 (10). Hsp70 has been also been found to interfere with caspase cleavage of a GATA transcription factor thus maintaining prosurvival Bcl-X L expression (14). Other anti-apoptotic 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 (AIF) (13). Further non caspase influences of HSP70 on apoptotic processes include the inhibition by HSP70 of c-jun N-terminal kinase which is pro-death in several systems (6). A key proposal of the paper by Stenslokken 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 degrees C by comparison to 13 degrees 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-1 alpha 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-1 alpha activity in heart, gills and kidney by greater then two fold by comparison to fish acclimated to lower temperatures (26 degrees C compared to 8 degrees C). In hypoxia, HIF-1 protein was elevated in several tissues at low temperatures and HIF-1 alpha activity was increased in the heart of 8 degree C acclimated and in the

3 gills of 18 degree C acclimated fish relative to 26 degree 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 degrees C to 8 degrees C (15). Hypoxic preconditioning in mammalian systems and non- anoxia 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 pro-survival kinases such as Akt (2). In the study by Stenslokken et al (16) HSP90 mrna levels were found to decrease in crucian carp brain and heart at 8 degrees C in anoxia relative to normoxia (16). At 13 degrees 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-1 alpha. The decrease in HSP90 expression in anoxia would be anticipated to correspond to a destabilization of HIF-1 alpha resulting in compromised HIF-1 alpha induced responses. Stenslokken 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 degrees 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 hours of anoxia, declining to baseline at 12 hours of anoxia. Such a pattern of expression may relate to a role for HSP72 in stabilization of proteins during the orchestrated metabolic down-regulation that occurs in the brain of T. scripta early in anoxia (11, 8). 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 hours 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 (11, 8). 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 neuro-protective 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 anti-apoptotic role by interacting with components of the apoptotic machinery (6). Stenslokken et al., (16) found that in the crucian carp HSP30 mrna decreased in anoxic brains and hearts at both 13 degrees C and 8 degrees C. In freshwater turtle white skeletal

4 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 re-oxygenation which may be consistent with it s 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). Concluding remarks. 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 Stenslokken 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 pro-survival and anti-apoptotic 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. References: 1) Ellefsen S, Stensløkken KO, Fagernes CE, Kristensen TA, Nilsson GE. Expression of genes involved in GABAergic neurotransmission in anoxic crucian carp brain (Carassius carassius). Physiol Genomics. 2009;36(2): ) Jiang BH, Jiang G, Zheng JZ, Lu Z, Hunter T, Vogt PK. Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. Cell Growth Differ. 2001;12(7): ) Jiao JD, Garg V, Yang B, Hu K. Novel functional role of heat shock protein 90 in ATP-sensitive K+ channel-mediated hypoxic preconditioning. Cardiovasc Res. 2008;77(1): ) Jin H, Wu H, Osterhaus G, Wei J, Davis K, Sha D, Floor E, Hsu CC, Kopke RD, Wu JY. Demonstration of functional coupling between gamma -aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles. Proc Natl Acad Sci U S A. 2003;100(7):

5 5) Krivoruchko A, Storey KB. Regulation of the heat shock response under anoxia in the turtle, Trachemys scripta elegans. J Comp Physiol B. 2010;180(3): ) Latchman DS. Heat shock proteins and cardiac protection. Cardiovasc Res. 2001;51(4): ) Lutz PL, Nilsson GE. Contrasting strategies for anoxic brain survival--glycolysis up or down. J Exp Biol. 1997;200: ) Milton SL, Prentice HM. Beyond anoxia: the physiology of metabolic downregulation and recovery in the anoxia-tolerant turtle. Comp Biochem Physiol A Mol Integr Physiol. 2007;147: ) Minet E, Mottet D, Michel G, Roland I, Raes M, Remacle J, Michiels C. Hypoxiainduced activation of HIF-1: role of HIF-1alpha-Hsp90 interaction. FEBS Lett. 1999;460: ) Pandey P, Saleh A, Nakazawa A, Kumar S, Srinivasula SM, Kumar V, Weichselbaum R, Nalin C, Alnemri ES, Kufe D, Kharbanda S. Negative regulation of cytochrome c-mediated oligomerization of Apaf-1 and activation of procaspase-9 by heat shock protein 90. EMBO J. 2000;19(16): ) Prentice HM, Milton SL, Scheurle D, Lutz PL. The upregulation of cognate and inducible heat shock proteins in the anoxic turtle brain. J Cereb Blood Flow Metab. 2004;24: ) Ramaglia V, Buck LT. Time-dependent expression of heat shock proteins 70 and 90 in tissues of the anoxic western painted turtle. J Exp Biol. 2004;207:

6 13) Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Daugas E, Zamzami N, Mak T, Jäättelä M, Penninger JM, Garrido C, Kroemer G. Heat-shock protein 70 antagonizes apoptosis-inducing factor. Nat Cell Biol. 2001;3: ) Ribeil JA, Zermati Y, Vandekerckhove J, Cathelin S, Kersual J, Dussiot M, Coulon S, Moura IC, Zeuner A, Kirkegaard-Sørensen T, Varet B, Solary E, Garrido C, Hermine O. Hsp70 regulates erythropoiesis by preventing caspase-3-mediated cleavage of GATA- 1. Nature. 2007;445(7123): ) Rissanen E, Tranberg HK, Sollid J, Nilsson GE, Nikinmaa M. Temperature regulates hypoxia-inducible factor-1 (HIF-1) in a poikilothermic vertebrate, crucian carp (Carassius carassius). J Exp Biol. 2006;209: ) Stenslokken KO, Ellefsen S, Kile Larsen H, Vaage JI, Nilsson GE. Expression of heat shock proteins in anoxic crucian carp (Carassius carassius); support for cold as a preparatory cue for anoxia. Am J Physiol Regul Integr Comp Physiol Mar 24.