Heat Stress Recovery Research Articles

Glucose-Regulated HLP1 Acts as a Key Molecule in Governing Thermomemory.

Induction of heat shock proteins (HSPs) in response to heat stress (HS) is indispensable for conferring thermotolerance. Glc, a fundamental signaling and metabolic molecule, provides energy to stressed seedlings to combat stress. The recovery of stressed plants from detrimental HS in response to Glc is largely mediated by HSPs, but the mechanistic basis of this thermotolerance is not well defined. In this study, we show that Glc has a prominent role in providing thermotolerance. Glc-mediated thermotolerance involves HSP induction via the TARGET OF RAPAMYCIN (TOR)-E2Fa signaling module. Apart from HSPs, TOR-E2Fa also regulates the Arabidopsis (Arabidopsis thaliana) ortholog of human Hikeshi, named HIKESHI-LIKE PROTEIN1 (HLP1). Expression of proHLP1::GUS in the shoot apical meristem (SAM) after HS coincides with TOR-E2Fa expression, substantiating a role for TOR-E2Fa-HLP1 in providing thermotolerance. We also demonstrate that Glc along with heat could induce proliferation activity in the SAM after HS recovery, which was arrested by the TOR inhibitor AZD-8055. Molecular and physiological studies suggest that HS-activated heat stress transcription factor A1s also positively regulate HLP1 transcription, suggesting convergence of the Glc and HS signaling pathways. Loss of functional HLP1 causes HS hypersensitivity, whereas HLP1 overexpressors display increased thermotolerance. HLP1 binds to the promoters of Glc-regulated HS-responsive genes and promotes chromatin acetylation. In addition, Glc modifies the chromatin landscape at thermomemory-related loci by promoting H3K4 trimethylation (H3K4me3). Glc-primed accumulation of H3K4me3 at thermomemory-associated loci is mediated through HLP1. These findings reveal the novel function of Glc-regulated HLP1 in mediating thermotolerance/thermomemory response.

Effects of zinc amino acid complex on biomarkers of gut integrity and metabolism during and following heat stress or feed restriction in pigs.

Study objectives were to determine the effects of zinc (Zn) amino acid complex (Availa Zn, Zinpro Corporation, Eden Prairie, MN) on metabolism, biomarkers of leaky gut, and inflammation during and following heat stress (HS) and nutrient restriction. Crossbred gilts (n = 50; 50 ± 2 kg BW) were blocked by initial BW and randomly assigned to one of five treatments: 1) thermoneutral (TN) and ad libitum fed a control diet (TNCtl), 2) TN and pair-fed a control diet (PFCtl), 3) TN and pair-fed a Zn-supplemented diet (PFZn), 4) HS and ad libitum fed a control diet (HSCtl), and 5) HS and ad libitum fed a Zn-supplemented diet (HSZn). The study consisted of 3 experimental periods (P): during P1 (7 d), all pigs were fed their respective diets ad libitum and housed in TN conditions (20.84 ± 0.03 °C, 47.11 ± 0.42% relative humidity). During P2 (7 d), HSCtl and HSZn pigs were exposed to progressive cyclical HS conditions (27 to 30 °C, 41.9 ± 0.5% relative humidity), while TNCtl, PFCtl, and PFZn pigs remained in TN conditions and were fed ad libitum or pair-fed to their respective HSCtl and HSZn counterparts. During P3 (5 d; "recovery phase"), all pigs were housed in TN conditions and fed ad libitum. Pigs exposed to HS had overall increased rectal temperature, skin temperature, and respiration rate (0.33 °C, 3.76 °C, and 27 bpm, respectively; P < 0.01). Relative to TN controls, HS decreased ADFI and ADG (28 and 35%, respectively; P < 0.05), but these variables were unaffected by dietary treatment. Additionally, circulating insulin did not differ between HS and TN pigs (P = 0.41), but was decreased in PF relative to TN pigs (P < 0.01). During recovery, no differences were observed in rectal temperature or respiration rate across treatments, but HSZn pigs had decreased skin temperature relative to TN, PF, and HSCtl pigs (P < 0.01). During P3, no Zn effects were observed in production parameters; however, PF pigs had increased ADFI and ADG relative to TN and HS treatments (P < 0.01). During P3, circulating insulin was increased in pigs that were HS relative to TN and PF pigs (75%, P < 0.05). Interestingly, tumor necrosis factor alpha (TNFα) levels were decreased during P3 (P = 0.04) in Zn relative to Ctl-fed pigs. Circulating lipopolysaccharide-binding protein was not different among periods (P > 0.10). In summary, Zn reduced TNFα (regardless of HS), and the stimulatory effect of HS on insulin secretion is amplified during HS recovery.

The effect of recovery from heat stress on circulating bioenergetics and inflammatory biomarkers.

Heat stress (HS) jeopardizes animal productivity and health. The intestinal barrier is sensitive to HS and heat-induced hyperpermeability plays a key role in its pathophysiology. However, the biology of recovery following HS is less understood. Thus, study objectives were to determine the temporal pattern of metabolic, inflammatory, and intestinal histological parameters during HS recovery. Female pigs (n = 32; 19.5 ± 0.5 kg BW) were sacrificed following exposure to 1 of 4 environmental treatments: 1) constant thermoneutral (TN) conditions (TNC; 24.2 ± 0.5°C), 2) no TN recovery post HS (0D), 3) 3 d of TN recovery post HS (3D), and 4) 7 d of TN recovery post HS (7D). The HS protocol was cyclical (33.6 ± 1.8 to 37.4 ± 2.1°C) and lasted for 3 d for all HS treatments. During the 3 d of HS, rectal temperature, skin temperature, and respiration rates were increased (1.3°C, 4.8°C, and 77 breaths/min, respectively; P < 0.01) and ADFI was decreased (27%; P < 0.01) compared to TNC pigs. Skin temperature tended to be decreased 0.6°C in 3D pigs during days 1-3 of recovery (P = 0.06) and was decreased 1.6 and 0.7°C during days 1-3 and 4-7 of recovery, respectively, in 7D pigs (P ≤ 0.03) compared to TNC. Relative to TNC pigs, ADFI remained 14% decreased during days 1-3 of recovery in both 3D and 7D pigs, and 17% decreased during days 4-7 in 7D pigs (P ≤ 0.01). Plasma glucose was decreased (10%; P = 0.03) for 0D and 3D relative to TNC pigs. Circulating lipopolysaccharide-binding protein was increased in 3D and 7D vs. TNC pigs (110 and 147%, respectively; P = 0.01) and tended to increase linearly with increasing recovery time (P = 0.08). Circulating tumor necrosis factor alpha was decreased (15%) in 0D pigs and increased linearly with advancing recovery time (P < 0.01). Jejunum and ileum villus height were reduced 17 and 11% in 0D vs. TNC pigs and increased linearly with progressive recovery time (P < 0.01). Jejunum and ileum mucosal surface areas were reduced 17 and 9% in 0D pigs and remained decreased in the jejunum while the ileum recovered to TNC levels by day 3 of recovery. Relative to TNC pigs, goblet cell area was similar in jejunum and colon of 0D pigs but was reduced in the ileum of 0D pigs and in jejunum, ileum, and colon of 3D and 7D relative to TNC pigs (P < 0.01). In summary, HS has deleterious effects on intestinal morphology that seem to improve with recovery time. In contrast, feed consumption remained suppressed and inflammatory biomarkers indicative of leaky gut increased following the heat load.

RT-LAMP assay: an alternative approach for profiling of bovine heat shock protein 70 gene in PBMC cultured model.

The purpose of this study is to develop a novel Reverse Transcriptase Loop-mediated isothermal amplification (RT-LAMP) based assay for in vitro profiling of heat shock protein 70 (Hsp70) in bovine peripheral blood mononuclear cell (PBMC) culture model utilizing the absorbance level of magnesium pyrophosphate-a by-product of LAMP reaction. A set of bovine Hsp70 specific RT-LAMP primers were designed to detect the differential absorbance level of magnesium pyrophosphate by-product which signifies the degree of Hsp70 amplification from cDNA of thermally induced cultured cells at different recovery periods. The study revealed significant (P < 0.05) correlation between absorbance level and the fold change of Hsp70 transcripts at different kinetic intervals of heat stress recovery in bovine PBMC cell culture models. RT-LAMP based absorbance assay can be used as an indicator to measure the degree of bovine Hsp70 transcripts produced during thermal stress and can be used as an alternative to the traditional Real time PCR assay. Developed RT-LAMP assay can be used as a cost-effective method for profiling of bovine HSP70 gene.

The Effects of Recovery Time from Heat Stress on Circulating Bioenergetic Variables and Biomarkers of Leaky Gut

The metabolic consequences of heat stress (HS) are atypical (i.e. hyperinsulinemia) considering the animal's nutritional status and a compromised intestinal barrier may initiate the pathophysiological consequences of HS. However, the temporal pattern of metabolic and leaky gut variables during HS recovery is not well‐understood. Female pigs (n=32; 19.5±1.3 kg BW) were sacrificed following exposure to one of four environmental protocols: 1) constant thermoneutral conditions (TN; 24.2°C and 57.2% RH), 2) no TN recovery following 3 d of HS (0d), 3) 3 d of TN recovery post 3 d of HS (3d), 4) 7 d of TN recovery post 3 d of HS (7d). The HS protocol was cyclical (33.6 to 37.4°C, 29.5% RH). All pigs were fed ad libitum. During the heat load, HS increased rectal temperature (P&lt;0.01; 1.0ºC), and respiration rate (P&lt;0.01; 63 bpm), but decreased feed intake (P&lt;0.01; 24%) and growth (P&lt;0.01; 33%) compared to TN controls. Blood glucose was decreased (P&lt;0.05; 10%) for the 0 d pigs, but returned to TN levels by 3 and 7d. Recovery length did not affect circulating NEFA (P&gt;0.73), insulin concentrations or the insulin:glucose (P=0.80). However, the insulin:feed intake was increased in the 0d compared to the TN controls (P=0.06; 49%). Lipopolysaccharide binding‐protein was markedly increased (P&lt;0.05) with advancing recovery time: 67, 110, and 150% in 0, 3, and 7d pigs compared to TN controls, respectively. In summary, HS did not have a lasting effect on energetic variables, but advancing recovery length was associated with progressively increased biomarkers of leaky gut.

Comparing the effects of rapid and gradual cooling following acute hyperthermia on body temperature and inflammatory response

Hyperthermia negatively impacts human and animal health and may cause morbidity and mortality if recovery is not properly managed. The study objective was to evaluate the effects of two cooling methods on body temperature and the inflammatory response after acute heat stress (HS) exposure in a pig model for human hyperthermia. Male pigs (88.7 ± 1.6 kg BW) were exposed to thermoneutral conditions (TN; n = 12; 19.5 ± 0.1°C) for 6 hrs, or HS (36.4 ± 0.1°C) for 3 hrs, followed by a 3 hr recovery period of either rapid cooling (HSRC; n = 12; immediate TN exposure and ice water dousing) or gradual cooling (HSGC; n = 12; gradual decrease to TN temperature). Rectal (TR) and gastrointestinal temperatures (TGI) were obtained every 15 min. Blood samples were collected from 6 pigs per treatment at 60, 180, 210 and 240 min to determine TNFα and IL‐1ß concentrations. TR was increased (p &lt; 0.01) in HSRC and HSGC pigs compared to TN controls during HS (1.2°C) and recovery (0.6°C), and was reduced (p &lt; 0.01; 0.4°C) in HSRC versus HSGC pigs during the recovery period. TGI was increased (p &lt; 0.01) in HSRC and HSGC compared to TN pigs during HS (1.6°C and 1.5°C, respectively) and recovery (1.2°C and 1.3°C, respectively), but was similar (40.8°C) in HSRC and HSGC pigs during both periods. During HS, HSRC and HSGC pigs had increased TNFα concentrations (p &lt; 0.04; 27.5%), but IL‐1ß concentrations were similar (p &gt; 0.60; 10.2 pg/mL) to TN controls. In the recovery period, HSRC pigs tended to have increased TNFα concentrations (p &lt; 0.07; 41.2%) compared to HSGC pigs. In summary, although rapid cooling was more effective than gradual cooling in reducing TR during HS recovery it had no effect on TGI and this differential may be linked to the increased inflammatory response observed in HSRC pigs.

Cardiovascular Dysregulation during Heat Stroke Recovery in Rats

We showed previously that cardiovascular adjustments during uncompensable heat stress (HS) provide insight into the severity of pathophysiological responses in rats. The present analysis evaluated the association between hemodynamic and thermoregulatory patterns and morbidity and pathophysiology during 24h of HS recovery. Conscious male F344 rats (N=22), radiotelemetered for continuous HR, BP and core temperature (Tc) measurements, were monitored during exposure to ambient temperature (Ta) of 37°C to TcMax of 41.9 ± 0.1°C and during 24h of recovery at Ta of 20°C. Control (CON) rats were maintained at Ta of 20°C during experimentation. Rats with MILD and MODERATE HS responses demonstrated sustained tachycardia (p<0.01 vs CON), normal BP (p=0.995 vs CON), and hyperthermia ~1°C above CON values (p< 0.01) during recovery. Rats with SEVERE responses developed hypothermia ~1°C below CON values (p<0.01) and hypotension ~20mmHg below CON (p<0.01) during recovery. The SEVERE group demonstrated a ~51-fold increase in cardiac IL-6 gene expression at 24h (p<0.01), suggesting an inflammatory response that was substantially greater than that observed in MODERATE animals (7-fold; p<0.05); MILD were not different from CON (p<0.35). At 24h, cardiac gene expression of inducible nitric oxide synthase (iNOS) was elevated 14-fold in the SEVERE group. The increase in iNOS could contribute to the hypotension and hypothermia via promotion of peripheral vasodilation. These findings extend our understanding of HS-induced cardiovascular dysregulation and provide mechanistic insight into pathophysiology contributing to multi-organ failure in HS patients. Funded by USAMRMC; author views not official US Army or DOD policy

Comparative Study of Susceptible and Tolerant Genotype Reveals Efficient Recovery and Root System Contributes to Heat Stress Tolerance in Rice

In changing climate scenario, heat stress caused by increased atmospheric CO2 is a major concern for rice productivity. There is a need to decipher the mechanisms of heat stress susceptibility and tolerance response of rice cultivars considering that high temperature is detrimental to growth and development of rice crop. The present study was designed to understand the heat stress response in heat-susceptible (Vandana) and heat-tolerant (N22) cultivars of rice. Rice seedlings were subjected to short-duration (24 h, SDS) and long-duration (5 days, LDS) heat stress (42 °C/36 °C, day/night). Besides the heat stress, recovery response (REC) of both the cultivars was also studied. Physiological parameters (chlorophyll content and membrane thermostability) and root/shoot length analysis revealed that N22 has better efficiency in recovering from heat stress. In particular, root tissue of N22 showed increased thermotolerance during SDS and LDS when compared with Vandana. In addition to physiological studies, gene expression pattern of 13 genes including heat shock transcription factors and heat shock proteins and 9 microRNAs (miRNAs) was analyzed in root and shoot of both the genotypes during various treatments. Gene and miRNA expression studies showed that root tissue of N22 was more responsive during SDS and LDS, suggesting important function of roots in heat stress tolerance. Further, during recovery, root tissue of both the genotypes showed more significant change in gene expression than shoot which signifies the vital role of plant root system in heat stress recovery response. Very high expression of an unknown iron-sulfur cluster-binding protein OsFd involved in electron transport activity was observed in root tissue of N22 during all the stress treatments. This study shows that better recovery and efficient root system play an important role in heat tolerance trait of N22.

Coordination of Translational Control and Protein Homeostasis during Severe Heat Stress

Exposure of cells to severe heat stress causes not only misfolding and aggregation of proteins but also inhibition of translation and storage of mRNA in cytosolic heat stress granules (heat-SGs), limiting newly synthesized protein influx into overloaded proteome repair systems. How these two heat stress responses connect is unclear. Here, we show that both S.cerevisiae and D.melanogaster heat-SGs contain mRNA, translation machinery components (excluding ribosomes), and molecular chaperones and that heat-SGs coassemble with aggregates of misfolded, heat-labile proteins. Components in these mixed assemblies exhibit distinct molecular motilities reflecting differential trapping. Wedemonstrate that heat-SG disassembly and restoration of translation activity during heat stress recovery is intimately linked to disaggregation of damaged proteins present in the mixed assemblies and requires Hsp104 and Hsp70 activity. Chaperone-driven protein disaggregation directly coordinates timing of translation reinitiation with protein folding capacity during cellular protein quality surveillance, enabling efficient protein homeostasis.

Hysteresis in the heart rate‐core temperature relationship during acute heat stress in rats: implications for systemic hemodynamics

Heart rate (HR) increases during hyperthermia, even in the absence of exercise and contributes to increased cardiac output necessary for dissipation of excess heat. It is unknown whether the onset and recovery from heat stress have different cardiovascular profiles. We hypothesized that hyperthermia‐induced tachycardia would exhibit hysteresis. Core temperature (Tc), HR and MAP were measured via radiotelemetry in conscious male Fischer 344 rats (n=8; 262±8 gms) during exposure to 37°C until a Tc of 41.8°C was reached and during recovery at 20°C. Tc, HR and MAP significantly increased during heat exposure (ascending phase; P&lt;0.01 vs. control) and HR was significantly lower for a given Tc compared to recovery (descending phase; p&lt;0.001). For example, at Tc = 39.5°C, HR was 365±27 bpm during the ascending phase and 527±50 bpm during the descending phase (P&lt;0.001). Hysteresis during heat stress recovery may be a compensatory, baroreflex‐mediated response to maintain MAP during thermoregulatory vasodilation and decreased peripheral vascular resistance. These findings may have implications for understanding cardiovascular stress during recovery from heat exposure. Research supported by MRMC. Author views not official US Army or DoD policy.

Expression analysis of a heat-inducible, Myo-inositol-1-phosphate synthase (MIPS) gene from wheat and the alternatively spliced variants of rice and Arabidopsis

Molecular dissection and a deeper analysis of the heat stress response mechanism in wheat have been poorly understood so far. This study delves into the molecular basis of action of TaMIPS, a heat stress-inducible enzyme that was identified through PCR-select subtraction technology, which is named here as TaMIPS2. MIPS (L-Myo-inositol-phosphate synthase) is important for the normal growth and development in plants. Expression profiling showed that TaMIPS2 is expressed during different developing seed stages upon heat stress. Also, the transcript levels increase in unfertilized ovaries and significant amounts are present during the recovery period providing evidence that MIPS is crucial for its role in heat stress recovery and flower development. Alternatively spliced forms from rice and Arabidopsis were also identified and their expression analysis revealed that apart from heat stress, some of the spliced variants were also inducible by drought, NaCl, Cold, ABA, BR, SA and mannitol. In silico promoter analysis revealed various cis-elements that could contribute for the differential regulation of MIPS in different plant systems. Phylogenetic analysis indicated that MIPS are highly conserved among monocots and dicots and TaMIPS2 grouped specifically with monocots. Comparative analyses was undertaken by different experimental approaches, i.e., semi-quantitative RT-PCR, quantitative RT-PCR, Genevestigator as a reference expression tool and motif analysis to predict the possible function of TaMIPS2 in regulating the different aspects of plant development under abiotic stress in wheat.

Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae

A variety of results has been obtained consistent with activation of neutral trehalase in Saccharomyces cerevisiae through direct phosphorylation by cAMP-dependent protein kinase (PKA). A series of neutral trehalase mutant alleles, in which all evolutionarily conserved putative phosphorylation sites were changed into alanine, was tested for activation in vitro (by PKA) and in vivo (by glucose addition). None of the mutations alone affected the activation ratio, whereas all mutations combined resulted in an inactive enzyme. All mutant alleles were expressed to similar levels, as shown by Western blotting. Several of the point mutations significantly lowered the specific activity. Using this series of mutants with different activity levels we show an inverse relationship between trehalase activity and heat-shock survival during glucose-induced trehalose mobilization. This is consistent with a stress-protective function of trehalose. On the other hand, reduction of trehalase activity below a certain threshold level impaired recovery from a sublethal heat shock. This suggests that trehalose breakdown is required for efficient recovery from heat shock, and that the presence of trehalase protein alone is not sufficient for efficient heat-stress recovery.