Our findings provide the first-ever global description of the UPR transcriptome of the heart and liver following T/HS. We demonstrated that T/HS leads to significant cardiomyocyte and hepatocyte apoptosis, which is prevented through the Stat3-dependent actions of IL-6. We examined the UPR transcriptome to identify candidate gene transcripts responsible for T/HS-induced apoptosis. By utilizing an expanded repertoire of UPR members, both canonical and non-canonical, and the reproducible and measurable outcome of IL-6-preventable apoptosis in our model of T/HS, we were able to identify potential UPR modulators that significantly impact T/HS-induced hepatocyte and cardiomyocyte apoptosis.
In the liver, members of the heat shock family of protein folding chaperones, Hsp70 and Hsp40, emerged as significant potential non-canonical UPR modulators of hepatocyte apoptosis in our model of T/HS. This compares with findings in other models of organ injury, such as work done by Wang et al., which demonstrated that Hsp70 and its induction with geranylgeranylacetone (GGA) can protect against primary proximal tubule apoptosis and acute kidney damage in an ischemic injury model27
, and work done by Kuboki et al., which demonstrated in a partial liver I/R model that induction of Hsp70 with sodium arsenite reduced liver injury, as determined by transaminase levels and histology28
. Besides their role in protein folding in the cytoplasmic space, heat shock proteins have been linked to the canonical UPR pathways of the endoplasmic reticulum. One example is Hsp72, a Hsp 70 family member, which has been shown to interact with the cytosolic domain of IRE1α, enhancing XBP1 splicing, and attenuating apoptosis in vitro29
. Heat shock protein chaperones have also been shown to prevent CHOP-induced apoptosis through the Hsp70-DnaJ chaperone pair inhibiting translocation of Bax to mitochondria in vitro30
Our findings provide in vivo
data linking the heat shock protein family to hepatocyte apoptosis possibly via a Stat3-dependent mechanism in T/HS. These findings are supported by previous work linking IL-6/Stat3 transcriptional regulation of heat shock protein family members31
. Hsp70 and Hsp40 appear to contribute to an adaptive and protective process in the liver, demonstrating upregulation in T/HS and further upregulation in livers of IL-6-resuscitated animals, correlating with prevention of apoptosis. However, when animals were pretreated with a Stat3 inhibitor that blocked IL-6's prevention of apoptosis, these chaperone transcripts were downregulated. Thus, these findings suggest that IL-6, via a Stat3-dependent pathway, acts to superinduce Hsp70 and 40 transcripts in T/HS. These findings are supported by the work of Masumichi et al32
, which demonstrate that IL-6 is necessary for upregulation of heat shock protein members, including Hsp70/40, in a model of acetaminophen-induced hepatic injury.
Interestingly, the canonical members of the UPR, while altered, were not the most dysregulated transcripts in the liver. CCAAT/-enhancer-binding protein homologous protein (CHOP), PERK, alpha subunit of eukaryotic initiation factor 2 (Eif2α), activating transcription factor 4 (ATF4), and calreticulin were significantly dysregulated (6 to −1.2 fold change) in T/HS (Supplemental Table 1). When considering those entities altered > 2-fold and taking into account known UPR and apoptotic functions of the canonical UPR members, only the transcriptional profile of ATF4 suggested a maladaptive contribution to hepatocyte apoptosis. However, this maladaptive role does not appear to be mediated through Stat3.
The heart demonstrated a distinctly different UPR transcriptional profile in comparison to the liver. When one considers the nature and functions of these organs, this is not unexpected. The liver is the largest glandular mass of tissue in the body and is highly secretory with both exocrine and endocrine function, whereas the heart, with myocyte predominance, is largely non-secretory with maintenance of biophysical function more paramount. The impact of T/HS on the canonical UPR transcriptome was even less in magnitude in the heart than in the liver. Significantly dysregulated canonical UPR transcripts included CHOP, PERK, X-box binding protein 1 (XBP1), Eif2α, and calreticulin (2.5 to −1.2 fold change) with only ATF4 dysregulated by more than 2 fold in response to T/HS (Supplemental Table 2). When taking into account known UPR and apoptotic function, CHOP, XBP1, and GADD34 exhibit transcriptional profiles suggestive of an adaptive role in T/HS-induced cardiomyocyte apoptosis. Given the fold-change was nominal (1.4 to 2-fold) however, further investigation is required to determine their true contribution to T/HS-induced apoptosis.
The protein folding chaperones, Hsp70 and Hsp40, which proved important modulators of apoptosis in the liver, were upregulated in the heart following T/HS, but were downregulated in animals in which IL-6 prevented cardiomyocyte apoptosis, suggesting these chaperones may play a maladaptive role in T/HS-induced cardiomyocyte apoptosis. The dichotomous nature of these chaperones' roles in the liver and heart in T/HS is supported by work in other models of organ injury. Indeed, previous studies have suggested that Hsp70 family proteins may serve to augment cardiac inflammation and contractile dysfunction33,34
, and its downregulation in IL-6 treated animals would support this hypothesis, as we have previously demonstrated that IL-6 acts to preserve contractile function following T/HS3
. However, Yao et al., have recently shown that Hsp70 upregulation may contribute to the cardioprotection against ischemia/reperfusion injury observed with lipopolysaccharide (LPS) pretreatment35
. Thus, the role of Hsp70 in myocardial ischemia/reperfusion injury may be specific to the insult and requires further study to clarify the adaptive versus maladaptive role it may play in ischemia/reperfusion events such as resuscitated hemorrhagic shock.
In addition to providing a global description of the UPR transcriptome of the heart and liver following T/HS, our findings demonstrate that IL-6, when utilized as a resuscitation adjuvant, may augment a physiologic protective role of Hsp70 and Hsp40 via a Stat3-dependent mechanism, thereby protecting against hepatocyte apoptosis. These findings support the concept that modulators of Hsp70 or 40 may offer a therapeutic strategy for prevention of apoptosis and ultimately hepatic dysfunction following T/HS.