HSF1 Is Essential for the Resistance of Zebrafish Eye and Brain Tissues to Hypoxia/Reperfusion Injury

Abstract

Ischemia and subsequent reperfusion (IR) produces injury to brain, eye and other tissues, contributing to the progression of important clinical pathologies. The response of cells to IR involves activation of several signaling pathways including those activating hypoxia and heat shock responsive transcription factors. However, specific roles of these responses in limiting cell damage and preventing cell death after IR have not been fully elucidated. Here, we have examined the role of heat shock factor 1 (HSF1) in the response of zebrafish embryos to hypoxia and subsequent return to normoxic conditions (HR) as a model for IR. Heat shock preconditioning elevated heat shock protein expression and protected zebrafish embryo eye and brain tissues against HR-induced apoptosis. These effects were inhibited by translational suppression of HSF1 expression. Reduced expression of HSF1 also increased cell death in brain and eye tissues of embryos subjected to hypoxia and reperfusion without prior heat shock. Surprisingly, reduced expression of HSF1 had only a modest effect on hypoxia-induced expression of Hsp70 and no effect on hypoxia-induced expression of Hsp27. These results establish the zebrafish embryo as a model for the study of ischemic injury in the brain and eye and reveal a critical role for HSF1 in the response of these tissues to HR. Our results also uncouple the role of HSF1 expression from that of Hsp27, a well characterized heat shock protein considered essential for cell survival after hypoxia. Alternative roles for HSF1 are considered.

Maternal immune activation by LPS selectively alters specific gene expression profiles of interneuron migration and oxidative stress in the fetus without triggering a fetal immune response

“…Bnip3 is a prominent and early responding hypoxia-induced cell death gene (Chinnadurai et al, 2008), and silencing of the gene using an siRNA suppresses hypoxia-induced autophagy (Bellot et al, 2009). Members of the heat shock family of proteins, which respond to cellular stress signals including oxidative stress and hypoxia (Chi and Karliner, 2004; Tucker et al, 2011; Welch, 1992) were also significantly up-regulated in fetal brain further suggestive of intermittent fetal hypoxia. Caveolin1 ( Cav1 ), while not a BH3-containing molecule, also regulates apoptosis (Gargalovic and Dory, 2003).…”

Eye-specific gene expression following embryonic ethanol exposure in zebrafish: Roles for heat shock factor 1

“…To verify reactivity of the anti-human Hsf-1 antibody with zebrafish Hsf-1, a full-length cDNA encoding zebrafish Hsf-1 was cloned into the plasmid pEGFP-C1 (Clontech, Mountain View, CA) as in [49]. HeLa cells were transfected with pEGFP-C1-Hsf-1, or with pEGFP-C1, as previously [49].…”

“…HeLa cells were transfected with pEGFP-C1-Hsf-1, or with pEGFP-C1, as previously [49]. Cells were incubated overnight, washed in PBS, and collected in extraction buffer containing 1% nonidet P-40, 0.5% SDS, 50 mM Tris-HCl, and EDTA-free protease inhibitor cocktail (one tablet/7 mL; Roche Diagnostics, Indianapolis, IN) in PBS.…”

“…Antisense morpholino (MO) oligonucleotide injection was performed as described previously [50-53]. In brief, MOs were purchased from GeneTools LLC (Corvallis, OR), dissolved in a buffer (0.4 mM MgSO 4 , 0.6 mM CaCl 2 , 0.7 mM KCl, 58 mM NaCl, 25 mM HEPES pH 7.1), and heated to 65°C for 5 minutes prior to use.…”

“…In brief, MOs were purchased from GeneTools LLC (Corvallis, OR), dissolved in a buffer (0.4 mM MgSO 4 , 0.6 mM CaCl 2 , 0.7 mM KCl, 58 mM NaCl, 25 mM HEPES pH 7.1), and heated to 65°C for 5 minutes prior to use. The MO targeting the start codon for hsf-1 was described previously [40, 53]. The control MO was a non-specific control MO (with no complementary targets in zebrafish) described previously [54].…”