Abstract 106: A Novel Small Molecule Protects Against Myocardial Ischemia and Doxorubicin-Induced Cardiomyopathy Through Heat Shock Response and Proteostasis Regulation In Vivo

Background: A potentially new non-cell-based therapy is the systemic delivery of small molecules to revive or protect injured myocardium. By screening a drug library, we identified a novel small molecule, sk421, and tested its effectiveness in two myocardial injury models in vivo: a myocardial infarction model and doxorubicin (DOX)-induced cardiomyopathy model. Results: Sk421 significantly increased cellular viability of DOX-injured cardiomyocytes by preventing apoptosis in vitro, demonstrated by assays for MTT (p=0.042), annexin-V, and active caspase-3 (p=0.004). Sk421 increased the concentrations of the GATA4 and the heat shock proteins (HSPs) in cardiomyocytes (by 1.5-fold), and fibroblasts (by 1.2-fold), but was unable to rescue DOX-injured fibroblast cells depleted of HSPs. Sk421 maintained the total proteosome by a 3.5-fold inhibition of ubiquitinized GATA4 degradation, as shown by immunoprecipitation assays. Oral administration of sk421(4mg/kg) rescued DOX-injured mice by significantly preserving cardiac function, weight gain, cardiomyocyte lesion size and abundance, and by reducing overall mortality (20% in treated groups) compared to control DOX-injured mice (80% in untreated groups, p<0.03). The levels of GATA4 and HSPs were increased in the sk421-rescued mice. In the MI model, (n=5/group), sk421 reduced myocardial infarct size, and protected heart function evaluated by ejection fraction 45.8 ± 2.4 vs. 34.8 ± 3.3, p=0.005 (day7), and 44.3 ± 2.8 vs. 34.4 ± 4.1, p=0.011 (day28). Sk421 treatment of MI rats resulted in a higher fraction of viable tissue (within the LV (70.20 ± 0.5024, n=3) as compared to only MI group (56.38 ± 3.879%, n=3) and the infiltration of CD68+ cells was found to be more in the infarct region of LV of MI group rats as compared to MI+CEL. Quantitative analysis showed a significantly higher proportion of TUNEL positive cells and mRNA levels of IL-1β, IL-6, TNF-α and MMP2 in the myocardium of MI rats compared to MI+SK421 rats. Conclusion: This study demonstrates the broad therapeutic benefit of sk421 in animal model of myocardial injury by mechanism of inducing the heat shock response and preserving GATA levels. The ease of oral delivery makes sk421 a potentially clinically useful adjunct for myocardial preservation.

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Induction of Heat Shock Protein 70 in Mouse RPE as an In Vivo Model of Transpupillary Thermal Stimulation

International Journal of Molecular Sciences ◽

10.3390/ijms21062063 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2063

Author(s):

Mooud Amirkavei ◽

Marja Pitkänen ◽

Ossi Kaikkonen ◽

Kai Kaarniranta ◽

Helder André ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Response ◽

Pigment Epithelium ◽

Retinal Pigment ◽

In Vivo Model ◽

Tunel Staining ◽

Long Duration ◽

Shock Response

The induction of heat shock response in the macula has been proposed as a useful therapeutic strategy for retinal neurodegenerative diseases by promoting proteostasis and enhancing protective chaperone mechanisms. We applied transpupillary 1064 nm long-duration laser heating to the mouse (C57Bl/6J) fundus to examine the heat shock response in vivo. The intensity and spatial distribution of heat shock protein (HSP) 70 expression along with the concomitant probability for damage were measured 24 h after laser irradiation in the mouse retinal pigment epithelium (RPE) as a function of laser power. Our results show that the range of heating powers for producing heat shock response while avoiding damage in the mouse RPE is narrow. At powers of 64 and 70 mW, HSP70 immunostaining indicates 90 and 100% probability for clearly elevated HSP expression while the corresponding probability for damage is 20 and 33%, respectively. Tunel staining identified the apoptotic regions, and the estimated 50% damaging threshold probability for the heating (ED50) was ~72 mW. The staining with Bestrophin1 (BEST1) demonstrated RPE cell atrophy with the most intense powers. Consequently, fundus heating with a long-duration laser provides an approachable method to develop heat shock-based therapies for the RPE of retinal disease model mice.

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In vivo heat-shock response in the brain: signalling pathway and transcription factor activation

Molecular Brain Research ◽

10.1016/j.molbrainres.2003.08.018 ◽

2003 ◽

Vol 119 (1) ◽

pp. 90-99 ◽

Cited By ~ 32

Author(s):

Paola Maroni ◽

Paola Bendinelli ◽

Laura Tiberio ◽

Francesca Rovetta ◽

Roberta Piccoletti ◽

...

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Heat Shock Response ◽

Signalling Pathway ◽

Transcription Factor Activation ◽

Shock Response ◽

The Brain

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HSF1-dependent and -independent regulation of the mammalian in vivo heat shock response and its impairment in Huntington's disease mouse models

Scientific Reports ◽

10.1038/s41598-017-12897-0 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 8

Author(s):

Andreas Neueder ◽

Theresa A. Gipson ◽

Sophie Batterton ◽

Hayley J. Lazell ◽

Pamela P. Farshim ◽

...

Keyword(s):

Heat Shock ◽

Huntington's Disease ◽

Huntington’S Disease ◽

Mouse Models ◽

Heat Shock Response ◽

Shock Response

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Heat-shock protein expression is absent in the antarctic fish Trematomus bernacchii (family Nototheniidae)

Journal of Experimental Biology ◽

10.1242/jeb.203.15.2331 ◽

2000 ◽

Vol 203 (15) ◽

pp. 2331-2339 ◽

Cited By ~ 19

Author(s):

G.E. Hofmann ◽

B.A. Buckley ◽

S. Airaksinen ◽

J.E. Keen ◽

G.N. Somero

Keyword(s):

Protein Synthesis ◽

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Response ◽

Solid Phase ◽

Antarctic Fish ◽

Shock Response ◽

Enhanced Expression ◽

Trematomus Bernacchii

The heat-shock response, the enhanced expression of one or more classes of molecular chaperones termed heat-shock proteins (hsps) in response to stress induced by high temperatures, is commonly viewed as a ‘universal’ characteristic of organisms. We examined the occurrence of the heat-shock response in a highly cold-adapted, stenothermal Antarctic teleost fish, Trematomus bernacchii, to determine whether this response has persisted in a lineage that has encountered very low and stable temperatures for at least the past 14–25 million years. The patterns of protein synthesis observed in in vivo metabolic labelling experiments that involved injection of (35)S-labelled methionine and cysteine into whole fish previously subjected to a heat stress of 10 degrees C yielded no evidence for synthesis of any size class of heat-shock protein. Parallel in vivo labelling experiments with isolated hepatocytes similarly showed significant amounts of protein synthesis, but no indication of enhanced expression of any class of hsp. The heavy metal cadmium, which is known to induce synthesis of hsps, also failed to alter the pattern of proteins synthesized in hepatocytes. Although stress-induced chaperones could not be detected under any of the experimental condition used, solid-phase antibody (western) analysis revealed that a constitutively expressed 70 kDa chaperone was present in this species, as predicted on the basis of requirements for chaperoning during protein synthesis. Amounts of the constitutively expressed 70 kDa chaperone increased in brain, but not in gill, during 22 days of acclimation to 5 degrees C. The apparent absence of a heat-shock response in this highly stenothermal species is interpreted as an indication that a physiological capacity observed in almost all other organisms has been lost as a result of the absence of positive selection during evolution at stable sub-zero temperatures. Whether the loss of the heat-shock response is due to dysfunctional genes for inducible hsps (loss of open reading frames or functional regulatory regions), unstable messenger RNAs, the absence of a functional heat-shock factor or some other lesion remains to be determined.

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Genetic differences in the duration of the lymphocyte heat shock response in mice.

Genetics ◽

10.1093/genetics/124.4.949 ◽

1990 ◽

Vol 124 (4) ◽

pp. 949-955

Author(s):

V K Mohl ◽

G D Bennett ◽

R H Finnell

Keyword(s):

Protein Synthesis ◽

Heat Shock ◽

Heat Shock Response ◽

Mouse Strains ◽

Adult Mice ◽

Shock Response ◽

Increased Sensitivity ◽

Shock Proteins ◽

The Relationship

Abstract Lymphocytes from adult mice bearing a known difference in genetic susceptibility to teratogen-induced exencephaly (SWV/SD, and DBA/2J) were evaluated for changes in protein synthesis following an in vivo heat treatment. Particular attention was paid to changes indicative of the heat shock response, a highly conserved response to environmental insult consisting of induction of a few, highly conserved proteins with simultaneous decreases in normal protein synthesis. The duration of heat shock protein induction in lymphocytes was found to be increased by 1 hr in the teratogen-sensitive SWV/SD strain as compared to the resistant DBA/2J strain. Densitometric analysis revealed a significant decrease in the relative synthesis of at least two non-heat shock proteins (36 kD and 45 kD) in the SWV/SD lymphocytes as compared to DBA/2J cells. The increased sensitivity of protein synthesis to hyperthermia in the SWV/SD lymphocytes were lost in the F1 progeny of reciprocal crosses between SWV/SD and DBA/2J mouse strains. Sensitivity to hyperthermia-induced exencephaly is recessive to resistance in these crosses. The relationship between altered protein synthesis and teratogen susceptibility is discussed.

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Modulation of inflammation in vivo through induction of the heat shock response, effects on NF-κB activation

Inflammation Research ◽

10.1007/bf02684012 ◽

2002 ◽

Vol 51 (2) ◽

pp. 108-109 ◽

Cited By ~ 6

Author(s):

T. Lawrence

Keyword(s):

Heat Shock ◽

Heat Shock Response ◽

Shock Response

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Surgical stress and the heat shock response: In vivo models of stress conditioning

Pathophysiology ◽

10.1016/s0928-4680(98)80409-4 ◽

1998 ◽

Vol 5 ◽

pp. 34

Author(s):

G.A. Perdrizet ◽

J.C. Garcia ◽

C.J. Lena ◽

D.S. Shapiro ◽

M.J. Rewinski

Keyword(s):

Heat Shock ◽

Heat Shock Response ◽

Surgical Stress ◽

In Vivo Models ◽

Shock Response ◽

Stress Conditioning

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Analysis of RNA for transcripts for catalase and SP71 in rat hearts after in vivo hyperthermia

Biochemistry and Cell Biology ◽

10.1139/o91-057 ◽

1991 ◽

Vol 69 (5-6) ◽

pp. 375-382 ◽

Cited By ~ 58

Author(s):

R. William Currie ◽

Robert M. Tanguay

Keyword(s):

Heat Shock ◽

Heat Shock Response ◽

Catalase Activity ◽

Mrna Level ◽

Regulation Of Transcription ◽

Rat Tissues ◽

Mrna Levels ◽

Control Heart ◽

Shock Response ◽

Human Hsp70

Hyperthermic stress induces synthesis of the major inducible (heat) stress protein (SP71) in all rat tissues. In addition, there is an increase in catalase activity in hearts at 24 and 48 h after the induction of the heat shock response. To more precisely define some of the molecular aspects of the induction of the heat shock response in hearts, we examined mRNA levels for the catalase, SP71 and HSP27. RNA was isolated from control hearts and at various time periods (0–24 h) of recovery after brief hyperthermic treatment and was analyzed by Northern blot analysis using as probes cDNA sequences for rat liver catalase, human HSP70 (inducible), and human HSP27. There was no detectable change in mRNA for catalase after heat shock or during recovery. Hyperthermic stress has no apparent effect on the regulation of transcription of mRNA coding for catalase, indicating that the increase in catalase activity is either translationally or post-translationally regulated. The human HSP70 cDNA did not hybridize to control heart RNA, but did hybridize to SP71 transcripts at 0, 1.5, and 3 h post heat shock. The mRNA level for SP71 peaked at 1.5 h, was reduced at 3 h, and became almost undetectable at 6 h post heat shock. Similarly, the human HSP27 cDNA did not hybridize to control heart RNA, but did hybridize to transcripts for HSP27 at 0, 1.5, 3, and up to 15 h post heat shock. Maximal signal for HSP27 was at 3 h post heat shock and was sharply reduced at 6 h post heat shock. The accumulation of transcripts for SP71 and HSP27 after hyperthermic stress is rapid, and degradation of the transcripts is almost complete by 6 h post heat shock.Key words: heat shock proteins, hyperthermia, catalase, heart, RNA.

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The heat shock response, determined by QuantiGene multiplex, is impaired in HD mouse models and not caused by HSF1 reduction

Scientific Reports ◽

10.1038/s41598-021-88715-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Casandra Gomez-Paredes ◽

Michael A. Mason ◽

Bridget A. Taxy ◽

Aikaterini S. Papadopoulou ◽

Paolo Paganetti ◽

...

Keyword(s):

Heat Shock ◽

Mouse Models ◽

Heat Shock Response ◽

Inclusion Bodies ◽

Neurodegenerative Disorder ◽

Comprehensive Analysis ◽

Mouse Tissue ◽

Brain Hemispheres ◽

Shock Response

AbstractHuntington’s disease (HD) is a devastating neurodegenerative disorder, caused by a CAG/polyglutamine repeat expansion, that results in the aggregation of the huntingtin protein, culminating in the deposition of inclusion bodies in HD patient brains. We have previously shown that the heat shock response becomes impaired with disease progression in mouse models of HD. The disruption of this inducible arm of the proteostasis network is likely to exacerbate the pathogenesis of this protein-folding disease. To allow a rapid and more comprehensive analysis of the heat shock response, we have developed, and validated, a 16-plex QuantiGene assay that allows the expression of Hsf1 and nine heat shock genes, to be measured directly, and simultaneously, from mouse tissue. We used this QuantiGene assay to show that, following pharmacological activation in vivo, the heat shock response impairment in tibialis anterior, brain hemispheres and striatum was comparable between zQ175 and R6/2 mice. In contrast, although a heat shock impairment could be detected in R6/2 cortex, this was not apparent in the cortex from zQ175 mice. Whilst the mechanism underlying this impairment remains unknown, our data indicated that it is not caused by a reduction in HSF1 levels, as had been reported.

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