Hsf1 is essential for proteotoxic stress response in smyd1b-deficient embryos and fish survival under heat shock

Abstract

Molecular chaperones play critical roles in post-translational maintenance in protein homeostasis. Previous studies have shown that loss of Smyd1b function results in defective myofibril organization and dramatic upregulation of heat shock protein gene (hsp) expression in muscle cells of zebrafish embryos. To investigate the molecular mechanisms and functional importance of this stress response, we characterized changes of gene expression in smyd1b knockdown and knockout embryos using RNA-seq. The results showed that the top upregulated genes encode mostly cytosolic heat shock proteins. Co-IP assay revealed that the upregulated cytosolic Hsp70s associate with myosin chaperone UNC45b which is critical for myosin protein folding and sarcomere assembly. Strikingly, several hsp70 genes also display muscle-specific upregulation in response to heat shock-induced stress in zebrafish embryos. To investigate the regulation of hsp gene upregulation and its functional significance in muscle cells, we generated heat shock factor 1 (hsf^-/-) knockout zebrafish mutants and analyzed hsp gene expression and muscle phenotype in the smyd1b^-/-single and hsf1^-/-;smyd1b^-/- double-mutant embryos. The results showed that knockout of hsf1 blocked the hsp gene upregulation and worsened the muscle defects in smyd1b^-/- mutant embryos. Moreover, we demonstrated that Hsf1 is essential for fish survival under heat shock (HS) conditions. Together, these studies uncover a correlation between Smyd1b deficiency and the Hsf1-activated heat shock response (HSR) in regulating muscle protein homeostasis and myofibril assembly and demonstrate that the Hsf1-mediated hsp gene upregulation is vital for the survival of zebrafish larvae under thermal stress conditions.

Keywords: Smyd1b; heat shock factor 1; heat shock protein; myofibril; stress response.

Fig. 1.. Identification of differentially expressed genes in smyd1b deficient embryos by RNA-seq.

A. RNA-seq analysis was performed on smyd1b knockdown (KD) and knockout (KO) mutant embryos and their respective controls at 24hpf. Compared with embryos injected with the control-MO, knockdown of smyd1b resulted in upregulation of 469 gene expression and down-regulation of 157 gene expression. Similarly, compared with WT/Heterozygous mutant sibling, 396 genes were upregulated and 75 were downregulated in the smyd1b KO mutant embryos. Among the up- or downregulated genes in smyd1b-KD and KO groups, 15 commonly upregulated and 10 commonly downregulated genes were identified by Venn diagram in smyd1b-KD and smyd1b-KO groups. B. Heat map profiles show the fold changes of gene expression in the commonly up-regulated (red) or down-regulated (blue) genes. The top upregulated genes in smyd1b-KD and KO embryos encode mostly chaperones and co-chaperones. CTL1: Control-KD. CTL2:WT/heterozygotes. C. The fold changes of expression among the upregulated hsp40, hsp70s, hsp90s chaperone and unc45b co-chaperone genes in smyd1b-KD and smyd1b-KO embryos compared with the respective control. adjp: significant. adjusted p values.

Fig. 2.. Upregulation of heat shock protein gene expression in smyd1b mutant zebrafish embryos.

Hsp70 chaperone and cochaperone gene expression was analyzed in smyd1b^sa15678 mutant embryos by in situ hybridization and qRT-PCR at 24 hpf. Compared to the control groups (A, C, E, G, I, K and M), a muscle-specific upregulation of expression was detected for hsp70.1/2/l (B), hsp70.3 (D), hspa1b (F), hsc70 (H), hspa8b (J), hsph1 (L), and dnaja (N) in the smyd1b^sa15678 mutant embryos. All larvae are side view, with anterior left and dorsal up. O-T. Data of qRT-PCR analysis show quantified changes of hsp70.1/2/l (O), hsp70.3 (P), hspa1b (Q), hsc70 (R), hspa8b (S), hsph1 (T) gene expression in smyd1b mutant embryos compared with WT control. Scale bar = 100 μm.

Fig 3.. The upregulation of heat shock protein gene expression is a shared stress response in fish embryos with muscle defects from hsp90α1 and unc45b knockdown.

Hsp70 and cochaperone gene expression was analyzed in hsp90α1 and unc45b knockdown (KD) embryos by in situ hybridization at 24 hpf. Compared with the controls (A, D, G, J, M, P and S), a muscle-specific upregulation of hsp70.1/2/l (B, C), hsp70.3 (E, F), hspa1b (H, I), hsc70 (K, L), hspa8b (N, O), hsph1 (Q, R), and dnaja (T, U) was detected in hspa90α1-KD (B, E, H, K, N, Q and T) and unc45-KD (C, F, I, L, O, R and U) zebrafish embryos at 24 hpf. All larvae are side view, with anterior left and dorsal up. Scale bar = 100 μm.

Fig 4.. Analysis of hsp70 gene expression in response to heat shock (HS) in zebrafish embryos.

Wild type zebrafish embryos are exposed to heat shock at 37C for 1 hr at 24 hpf. hsp gene expression was analyzed in the control and heat shocked embryos by in situ hybridization. Compared to the control groups (A, C, E, G, I), a strong upregulation of expression was detected for hsp70.1/2/l (B), hsp70.3 (D), hspa1b (F), hsc70 (H), and hspa8b (J) in the heat shock (HS) treated embryos. Strikingly, hspa1b gene showed a muscle-specific upregulation in response to the HS stress. Side view. Anterior - left, dorsal - up. K-O. Data of qRT-PCR analysis show quantified changes of hsp70.1/2/l (K), hsp70.3 (L), hspa1b (M), hsc70 (N), hspa8b (O) gene expression in heat shocked embryos compared with non-HS control. Scale bar = 100 μm.

Fig. 5.. Coimmunoprecipitation (Co-IP) assay of Hsp70 binding with myosin chaperone Unc45b.

A. HEK293 cells were transfected with DNA plasmid expressing Myc-tagged Hsp70.3 or Hspa8 alone or co-transfected with plasmid expressing FLAG-tagged myosin chaperone UNC45b. Protein extract of the transfected cells was used for immunoprecipitation with anti-FLAG antibody and western analysis using anti-Myc and anti-FLAG antibodies. Co-IP results showed that Hsp70.3 and Hspa8 were coprecipitated with Unc45b. B. To assess whether this coprecipitation correlated with the unregulated Hsp70s, the Co-IP assay was performed with two upregulated cytosolic Hsp70s (Hspa1b and Hsc70), and the mitochondria Hspa9 which was not upregulated in smyd1b mutant embryos. The Co-IP results showed that Hspa1b and Hsc70 were coprecipitated with Unc45b. In contrast, the mitochondrial Hspa9 that was not coprecipitated with Unc45b.

Fig. 6.. Hsf1^−/− mutant zebrafish larvae are more susceptible to environmental stress from HS.

Fish embryos of WT (~25%), hsf1^+/− heterozygous (~50%) and hsf1^−/− homozygous (~25%) mutants were generated from the hsf1^+/− heterozygous in-crossing. The above embryos of mixed genotypes were subjected to heat shock treatment at 37.5°C for 4 hrs on 5, 7 or 15 dpf. Dead embryos resulting from HS treatment were collected and individually genotyped. The percentage of dead embryos among the three genotypes are shown in figure A. At day 5 and 7, all the dead embryos were hsf1^−/− homozygous mutants. On day 15, 85% of the dead embryos were hsf1^−/− homozygous mutants, while the wildtype and heterozygous mutants only represented 5% and 10% of the dead embryos, respectively. The numbers of dead embryos of the three genotypes and their percentages among the dead embryos are listed in figure B.

Fig. 7.. Hsf1 is required for the upregulation of hsp40, hspa1b and hspa8b gene expression in response to heat shock of zebrafish embryos.

Wild type and hsf1^−/− mutant zebrafish embryos of 24 hpf were exposed to heat shock at 37.5°C for 1 hr. Compared to the control groups (A-F) without heat shock treatment, expression of hsp40, hspa1b and hspa8b mRNAs was significantly upregulated in the heat shock treated WT embryos (G-I). However, this heat shock response of upregulating these hsp gene expression was blocked in hsf1^−/− mutant embryos (J-L). Scale bar = 100 μm.

Fig. 8.. Hsf1 is required for the muscle-specific upregulation of hsp chaperone and cochaperone gene expression in smyd1b mutant embryos.

Expression of three hsp70 genes (hsp70.1/2/l, hspa8b, and hsc70), hsp40, hsp90α1 and unc45b mRNAs was analyzed in the WT control (A-F), hsf1^−/− mutants (G-L), smyd1b^−/− mutants (M-R) and smyd1b^−/−; hsf1^−/− double mutant (S-X) embryos. No significant difference is visible between the WT (A-F) and hsf1^−/− mutant (G-L) embryos. In contrast, loss of smyd1b dramatically upregulated the hsp chaperone and cochaperone gene expression in muscle cells of zebrafish embryos at 24 hpf (M-R). However, this muscle-specific upregulation was blocked in the smyd1b^−/−; hsf1^−/− double mutant embryos (S-X). Scale bar = 100 μm.

Fig. 9.. Hsf1 activated heat shock stress response has a protective role in myofiber organization in smyd1b^−/− mutant embryos.

A-H. Myofiber organization of slow muscles was analyzed using anti-myosin antibody (F59) staining in WT control (A, E), hsf1^−/− mutant (B, F), smyd1b^−/− mutant (C, G) and hsf1^−/−; smyd1b^−/− double mutant (D, H) embryos at 28 hpf (A-D) and 48 hpf (E-H), respectively. Compared with the WT control (A, E), loss of Hsf1 function has no visible effects on myofiber organization in hsf1^−/− mutant embryos (B, F). In contrast, loss of Smyd1b function significantly disrupted the myofiber organization in smyd1b^−/− mutant embryos at 28 hpf (C). By 48 hpf, myofiber organization appeared to be improved (G). However, this improvement was significantly diminished in the hsf1^−/−; smyd1b^−/− double mutant embryos at 48 hpf (H). I-L. Myofiber organization of fast muscles was analyzed using anti-myosin antibody (MF20) staining in WT control (I), hsf1^−/− mutant (J), smyd1b^−/− mutant (K) and hsf1^−/−; smyd1b^−/− double mutant (L) embryos at 72 hpf. Scale bars = 50 μm.

Fig. 10.. A proposed model of Hsf1 activated muscle-specific HSR in smyd1b deficient fish embryos.

Loss of Smyd1b function in zebrafish embryos results in increased muscle protein misfolding and aggregation within muscle cells. The misfolded proteins and aggregates could trigger Hsf1-activated hsp gene expression in the muscle cells of these embryos. The upregulated cytosolic HSPs work together to control protein refolding or degradation in muscle cells of smyd1b mutant embryos. However, loss of hsf1 in the hsf1; smyd1b double mutants blocks the HSR, preventing the refolding of misfolded proteins and consequently directing them toward protein degradation pathways.