Wnt-induced secreted protein-1 is a prohypertrophic and profibrotic growth factor.

Wnt1-induced secreted protein-1 (WISP-1) is a member of the cysteine-rich 61, connective tissue growth factor, and nephroblastoma overexpressed (CCN) family of growth factors and is expressed in the heart at low basal levels. The purpose of this study was to investigate whether WISP-1 is upregulated in postinfarct myocardium and whether WISP-1 exerts prohypertrophic and mitogenic effects stimulating myocyte hypertrophy, cardiac fibroblast (CF) proliferation, and collagen expression. Male C57Bl/6 (25 g) mice underwent permanent occlusion of the left anterior descending coronary artery. mRNA and protein levels were analyzed by Northern and Western blot analyses. Cardiomyocyte hypertrophy was quantified by protein and DNA synthesis. CF proliferation was quantified by CyQuant assay, and soluble collagen release by Sircol assay. A time-dependent increase in WISP-1 expression was detected in vivo in the noninfarct zone of the left ventricle, which peaked at 24 h (3.1-fold, P < 0.01). Similarly, biglycan expression was increased by 3.71-fold (P < 0.01). IL-1beta and TNF-alpha expression preceded WISP-1 expression in vivo and stimulated WISP-1 expression in neonatal rat ventricular myocytes in vitro. WISP-1-induced cardiomyocyte hypertrophy was evidenced by increased protein (2.78-fold), but not DNA synthesis, and enhanced Akt phosphorylation and activity. Treatment of primary CF with WISP-1 significantly stimulated proliferation at 48 h (6,966 +/- 264 vs. 5,476 +/- 307 cells/well, P < 0.01) and enhanced collagen release by 72 h (18.4 +/- 3.1 vs. 8.4 +/- 1.0 ng/cell, P < 0.01). Our results demonstrate for the first time that WISP-1 and biglycan are upregulated in the noninfarcted myocardium in vivo, suggesting a positive amplification of WISP-1 signaling. WISP-1 stimulates cardiomyocyte hypertrophy, fibroblast proliferation, and ECM expression in vitro. These results suggest that WISP-1 may play a critical role in post-myocardial infarction remodeling.

Over-expression of inducible HSP70 chaperone suppresses neuropathology and improves motor function in SCA1 mice.

Many neurodegenerative diseases are caused by gain-of-function mechanisms in which the disease-causing protein is altered, becomes toxic to the cell, and aggregates. Among these 'proteinopathies' are Alzheimer's and Parkinson's disease, prion disorders and polyglutamine diseases. Members of this latter group, also known as triplet repeat diseases, are caused by the expansion of unstable CAG repeats coding for glutamine within the respective proteins. Spinocerebellar ataxia type 1 (SCA1) is one such disease, characterized by loss of motor coordination due to the degeneration of cerebellar Purkinje cells and brain stem neurons. In SCA1 and several other polyglutamine diseases, the expanded protein aggregates into nuclear inclusions (NIs). Because these NIs accumulate molecular chaperones, ubiquitin and proteasomal subunits--all components of the cellular protein re-folding and degradation machinery--we hypothesized that protein misfolding and impaired protein clearance might underlie the pathogenesis of polyglutamine diseases. Over-expressing specific chaperones reduces protein aggregation in transfected cells and suppresses neurodegeneration in invertebrate animal models of polyglutamine disorders. To determine whether enhancing chaperone activity could mitigate the phenotype in a mammalian model, we crossbred SCA1 mice with mice over-expressing a molecular chaperone (inducible HSP70 or iHSP70). We found that high levels of HSP70 did indeed afford protection against neurodegeneration.

Combined and individual mitochondrial HSP60 and HSP10 expression in cardiac myocytes protects mitochondrial function and prevents apoptotic cell deaths induced by simulated ischemia-reoxygenation.

BACKGROUND: The mitochondrial heat-shock proteins HSP60 and HSP10 form a mitochondrial chaperonin complex, and previous studies have shown that their increased expression exerts a protective effect against ischemic injury when cardiac myocytes are submitted to simulated ischemia. The more detailed mechanisms by which such a protective effect occurs are currently unclear. We wanted to determine whether HSP60 and HSP10 could exert a protection against simulated ischemia and reoxygenation (SI/RO)-induced apoptotic cell death and whether such protection results from decreased mitochondrial cytochrome c release and caspase-3 activation and from the preservation of ATP levels by preservation of the electron transport chain complexes. In addition, we explored whether increased expression of HSP60 or HSP10 by itself exerts a protective effect. METHODS AND RESULTS: We overexpressed HSP60 and HSP10 together or separately in rat neonatal cardiac myocytes using an adenoviral vector and then subjected the myocytes to SI/RO. Cell death and apoptosis in myocytes were quantified by parameters such as enzyme release, DNA fragmentation, and caspase-3 activation. Overexpression of the combination of HSP60 and HSP10 and of HSP60 or HSP10 individually protected myocytes against apoptosis. This protection is accompanied by decreases in mitochondrial cytochrome c release and in caspase-3 activity and increases in ATP recovery and activities of complex III and IV in mitochondria after SI/RO. CONCLUSIONS: These results suggest that mitochondrial chaperonins HSP60 and HSP10 in combination or individually play an important role in maintaining mitochondrial integrity and capacity for ATP generation, which are the crucial factors in determining survival of cardiac myocytes undergoing ischemia/reperfusion injury.

Role of protein kinase C-epsilon in hypertrophy of cultured neonatal rat ventricular myocytes.

Using adenovirus (Adv)-mediated overexpression of constitutively active (ca) and dominant-negative (dn) mutants, we examined whether protein kinase C (PKC)-epsilon, the major novel PKC isoenzyme expressed in the adult heart, was necessary and/or sufficient to induce specific aspects of the hypertrophic phenotype in low-density, neonatal rat ventricular myocytes (NRVM) in serum-free culture. Adv-caPKC-epsilon did not increase cell surface area or the total protein-to-DNA ratio. However, cell shape was markedly affected, as evidenced by a 67% increase in the cell length-to-width ratio and a 17% increase in the perimeter-to-area ratio. Adv-caPKC-epsilon also increased atrial natriuretic factor (ANF) and beta-myosin heavy chain (MHC) mRNA levels 2.5 +/- 0.3- and 2.1 +/- 0.2-fold, respectively, compared with NRVM infected with an empty, parent vector (P < 0.05 for both). Conversely, Adv-dnPKC-epsilon did not block endothelin-induced increases in cell surface area, the total protein-to-DNA ratio, or upregulation of beta-MHC and ANF gene expression. However, the dominant-negative inhibitor markedly suppressed endothelin-induced extracellular signal-regulated kinase (ERK) 1/2 activation. Taken together, these results indicate that caPKC-epsilon overexpression alters cell geometry, producing cellular elongation and remodeling without a significant, overall increase in cell surface area or total protein accumulation. Furthermore, PKC-epsilon activation and downstream signaling via the ERK cascade may not be necessary for cell growth, protein accumulation, and gene expression changes induced by endothelin.

Heat shock factor 1-mediated thermotolerance prevents cell death and results in G2/M cell cycle arrest.

Mammalian cells respond to environmental stress by activating heat shock transcription factors (eg, Hsf1) that regulate increased synthesis of heat shock proteins (Hsps). Hsps prevent the disruption of normal cellular mitosis, meiosis, or differentiation by environmental stressors. To further characterize this stress response, transformed wild-type Hsf1+/+ and mutant Hsf1-/- mouse embryonic fibroblasts (MEFs) were exposed to (1) lethal heat (45 degrees C, 60 minutes), (2) conditioning heat (43 degrees C, 30 minutes), or (3) conditioning followed by lethal heat. Western blot analysis demonstrated that only Hsf1+/+ MEFs expressed inducible Hsp70s and Hsp25 following conditioning or conditioning and lethal heat. Exposure of either Hsf1+/+ or Hsf1-/- MEFs to lethal heat resulted in cell death. However, if conditioning heat was applied 6 hours before lethal heat, more than 85% of Hsf1+/+ MEFs survived, and cells in G2/M transiently increased 3-fold. In contrast, conditioned Hsf1-/- MEFs neither survived lethal heat nor exhibited this G2/M accumulation. Coinfection with adenoviral Hsp70 and Hsp25 constructs did not fully recreate thermotolerance in either Hsf1+/+ or Hsf1-/- MEFs, indicating other Hsf1-mediated gene expression is required for complete thermotolerance. These results demonstrate the necessity of Hsf1-mediated gene expression for thermotolerance and the involvement of cell cycle regulation, particularly the G2/M transition, in this thermotolerant response.

Functional properties of skeletal muscle from transgenic animals with upregulated heat shock protein 70.

The influence of inducible heat stress proteins on protecting contracting skeletal muscle against fatigue-induced injury was investigated. A line of transgenic mice overexpressing the inducible form of the 72-kDa heat shock protein (HSP72) in skeletal muscles was used. We examined the relationship between muscle contractility and levels of the constitutive (HSC73) and inducible (HSP72) forms of the 72-kDa heat shock protein in intact, mouse extensor digitorum longus (EDL), soleus (SOL), and the diaphragm (DPH). In all transgenic muscles, HSP72 was expressed at higher levels compared with transgene-negative controls, where HSP72 was below the level of detection. At the same time, HSC73 levels were downregulated in all transgenic muscle types. Shipment-related stress caused an elevation in the levels of HSP72 in all muscles for 1 wk after arrival of the animals. We also found that, although no statistical differences in response to intermittent fatiguing stimulation in the contractile properties of intact transgene-positive muscles compared with their transgene-negative counterparts were observed, the response of intact transgene-positive EDL muscles to caffeine was enhanced. These findings demonstrate that elevated HSP72 does not protect EDL, SOL, or DPH muscles from the effects of intermittent fatiguing stimulation. However, HSP72 may influence the excitation-contraction coupling (ECC) process, either directly or indirectly, in EDL muscle. If the effects on ECC were indirect, then these results would suggest that manipulation of a specific gene might cause functional effects that seem independent of the manipulated gene/protein.

Mice overexpressing rat heat shock protein 70 are protected against cerebral infarction.

Increased expression of heat shock protein 70 (HSP70) in the brain has been extensively documented in association with a variety of insults, including ischemia, and is suggested to play a role in cell survival and recovery after ischemic injury. To more directly assess the protective role of HSP70 during ischemic brain damage, we used transgenic mice overexpressing the rat HSP70 (HSP70tg mice). In contrast to wild-type (wt) littermates, high levels of HSP70 messenger RNA and protein were detected in brains of HSP70tg mice under normal conditions, and immunohistochemical analysis revealed primarily neuronal expression of HSP70. Heterozygous HSP70tg mice and their wt littermates were subjected to permanent focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Cerebral infarction after 6 hours of ischemia, as evaluated by Nissl staining, was significantly less in HSP70tg mice compared with wt mice. This reduction in infarction volume in HSP70tg mice was not attributable to an altered cardiovascular anatomy or to initial differences in body temperature or hemodynamic parameters. The HSP70tg mice were still protected against cerebral infarction 24 hours after permanent focal ischemia. The data suggest that HSP70 can markedly protect the brain against ischemic damage and that approaches aimed at inducing HSP70 may lead to new therapeutic interventions in cerebrovascular injuries.

Protection against endotoxemia by HSP70 in rodent cardiomyocytes.

Clinical and experimental studies have shown that myocardial dysfunction is an early event during endotoxemia or septic shock. Several reports have shown that rodents submitted to a mild heat shock become resistant to lipopolysaccharides (LPS) or sepsis. The most abundant of the heat shock proteins (HSP), the HSP70, has been postulated to be the principal mediator of the observed protection against endotoxemia. We have tested the hypothesis that a protective effect against endotoxemia is achievable by the increased presence of the HSP70 in rodent cardiomyocytes. We have found that a transgenic mouse line overexpressing the rat HSP70 gene in the heart exhibits an increased tolerance to LPS treatment (control estimated survival function [S(t)] = 0.538, transgenic S(t) = 0.787, P < 0.05). Interestingly, the increased presence of the HSP70 in the hearts of these mice results in a decrease in the activation of the inducible nitric oxide synthase (iNOS) after LPS treatment. We conclude that HSP70 protection against LPS is most probably mediated through the modulation of iNOS activation and the subsequent decreased synthesis of nitric oxide in cardiomyocytes.

Modulation of tolerance by mutant heat shock transcription factors.

It ought to be possible to recruit normal cellular defenses to mitigate ischemia/reperfusion damage and to reduce toxicity of chemotherapeutic drugs. Stress-preconditioned cells acquire a tolerant state characterized by increased resistance to such insults. This state is widely believed to be mediated, partially, by heat shock proteins (Hsps). Indirect evidence suggests that stress-induced Hsp expression is controlled by heat shock transcription factor 1 (Hsf1), which factor may therefore represent a preferred target for therapeutic modulation of tolerance. In support, positively acting (Hsf1(+)) and negatively acting (Hsf1(-)) mutants of Hsf1 were identified. Inhibition of endogenous Hsf1 activity by Hsf1(-) prevents stress-induced Hsp synthesis and development of tolerance. Hsf1(+) drastically enhances expression of major Hsps in the absence of stress and induces tolerance against heat, simulated ischemia and toxicity by cyclophosphamide. Where compared, tolerance induced was slightly better than that produced by heat preconditioning. Thus, development of the tolerant state is dependent on increased levels of the cohort of Hsps induced by stress preconditioning, and Hsf1 can induce accumulation of a typical set of Hsps, which proteins are alone capable of providing tolerance at a similar level as heat preconditioning. These findings make Hsf1 a preferred target for pharmacological intervention to deliberately induce tolerance.

Influence of phosphorylation and oligomerization on the protective role of the small heat shock protein 27 in rat adult cardiomyocytes.

Recent reports have demonstrated that the heat shock proteins (hsp) and in particular the hsp70 confer protection against cardiac ischemic damage. More recently, we have shown that increased expression of another heat shock protein, the hsp27, through an adenovirus vector system protects adult cardiomyocytes against ischemic injury. This small heat shock protein undergoes phosphorylation when the cell is under stress. This has led many to speculate that phosphorylation of hsp27 is required for the protective role this protein plays in the cell. In order to investigate this possibility, we have mutated the serines that are the sites of phosphorylation on the hsp27, to glycines or alanines. These nonphosphorylatable mutants of hsp27 were cloned into adenoviral vectors and used to infect adult rat cardiomyocytes to assess their ability in protecting against ischemic injury. In addition, we used a specific inhibitor of p38 MAP kinase that is a key member of the kinase pathway responsible for phosphorylating the hsp27. Our present results show that the nonphosphorylated hsp27 forms larger oligomeric complexes than the phosphorylated hsp27. Interestingly, phosphorylation of hsp27 seems not to play a role in its ability to protect adult rat cardiomyocytes against ischemic damage.

Specific heat shock proteins protect microtubules during simulated ischemia in cardiac myocytes.

The protective effects of heat shock proteins (HSPs) during myocardial ischemia are now well documented, but little is known about the mechanisms of protection and the specificity of different HSPs. Because cytoskeletal injury plays a crucial role in the pathogenesis of irreversible ischemic damage, we tested whether overexpression of specific HSPs protects the integrity of microtubules during simulated ischemia in rat neonatal cardiac myocytes. Overexpression of specific HSPs was achieved by adenovirus-mediated transgene expression. Damage was assessed by comparing control cells to cells that were subjected to a simulated ischemia protocol. Microtubular integrity was measured by indirect immunofluorescence, confocal microscopy, and image analysis. Within 14 h of simulated ischemia, microtubular integrity decreased significantly in uninfected myocytes (from 24.6 +/- 1.2 to 13.2 +/- 0.4) and in myocytes infected with a control virus that expressed no transgene (from 25.9 +/- 1.8 to 13.1 +/- 1.4). Microtubular integrity after ischemia was significantly better preserved in cells overexpressing constitutive Hsp70 (21.7 +/- 1.6) or alphaB-crystallin (18.0 +/- 2.7) but not in cells overexpressing inducible Hsp70 (11.5 +/- 0.8) or Hsp27 (14.0 +/- 2.2). We conclude that specific HSPs protect the microtubules during simulated cardiac ischemia.

Protection against myocardial dysfunction after a brief ischemic period in transgenic mice expressing inducible heat shock protein 70.

Brief ischemic periods lead to myocardial dysfunction without myocardial infarction. It has been shown that expression of inducible HSP70 in hearts of transgenic mice leads to decreased infarct size, but it remains unclear if HSP70 can also protect against myocardial dysfunction after brief ischemia. To investigate this question, we developed a mouse model in which regional myocardial function can be measured before and after a temporary ischemic event in vivo. In addition, myocardial function was determined after brief episodes of global ischemia in an isolated Langendorff heart. HSP70-positive mice and transgene negative littermates underwent 8 min of regional myocardial ischemia created by occlusion of the left descending coronary artery, followed by 60 min of reperfusion. This procedure did not result in a myocardial infarction. Regional epicardial strain was used as a sensitive indicator for changes in myocardial function after cardiac ischemia. Maximum principal strain was significantly greater in HSP70-positive mice with 88+/-6% of preischemic values vs. 58+/-6% in transgene-negative mice (P < 0.05). Similarly, in isolated Langendorff perfused hearts of HSP70-positive and transgene-negative littermates exposed to 10 min of global ischemia and 90 min of reperfusion, HSP70 transgenic hearts showed a better-preserved ventricular peak systolic pressure. Thus, we conclude that expression of HSP70 protects against postischemic myocardial dysfunction as shown by better preserved myocardial function.

Simultaneous overexpression of two stress proteins in rat cardiomyocytes and myogenic cells confers protection against ischemia-induced injury.

BACKGROUND: Mitochondria are known to be a major target during ischemic cardiac injury. Previous studies have shown that in rodent myogenic cells and in the hearts of transgenic mice in which the heat shock or stress protein 70 is increased, there is a marked tolerance to ischemia/reperfusion injury. Two other heat shock proteins (HSP60 and HSP10) are known to form, within the mitochondria, a chaperonin complex that is important for mitochondrial protein folding and function. We were then interested in investigating whether increased expression of these two stress proteins is able to protect myogenic cells against ischemia/reperfusion injury. METHODS AND RESULTS: We generated recombinant adenoviral vectors containing HSP60, HSP10, or a combination of the two genes. These adenoviral constructs overexpress significant amounts of these stress proteins in both rat neonatal cardiomyocytes and the myogenic H9 c2 cell line. Cells infected with an adenoviral construct overexpressing both HSP60 and HSP10 were found to be protected against simulated ischemia, whereas cells infected with adenoviral constructs overexpressing only HSP60 or HSP10 alone were not rendered tolerant to simulated ischemic injury. CONCLUSIONS: These results suggest that the simultaneous expression of these two proteins that form a chaperonin complex in the mitochondria plays an important role in the survival of myogenic cells after ischemia/reperfusion injury.

Induction of heat shock proteins by tyrosine kinase inhibitors in rat cardiomyocytes and myogenic cells confers protection against simulated ischemia.

Previous studies have shown that in rodent myogenic cells and in the hearts of transgenic mice in which heat shock protein expression is increased there is a marked tolerance to ischemic/reperfusion injury. Furthermore, a recent study has shown that the benzoquinoid ansamycin antibiotic and tyrosine kinase inhibitor, herbimycin A, is capable of inducing the expression of heat shock proteins in fibroblasts. Our intention, in the present study, was to investigate if exposure of rat cardiomyocytes and the myogenic cell line H9c2 to herbimycin A would induce these proteins and, thus, confer protection against ischemic stress. For this purpose, we exposed both rat neonatal cardiomyocytes and H9c2 cells to herbimycin A and another related benzoquinoid ansamycin antibiotic, geldanamycin. We found that cells exposed to these compounds overexpressed heat shock proteins and are also rendered more tolerant to simulated ischemia as measured by the release of cytoplasmic enzymes. In addition, we found that the mechanism of induction of heat shock proteins by these compounds is similar, if not identical, to that of a heat shock (42 degrees C, 60 min). These results suggest that these benzoquinoid ansamycin antibiotics, or closely related analogues, may offer a pharmacological means of increasing the level of heat shock proteins in cardiac tissue and thus protect the heart against ischemic/reperfusion injury.

Small heat shock proteins and protection against ischemic injury in cardiac myocytes.

BACKGROUND: Overexpression of the inducible hsp70 protects against ischemic cardiac damage. However, it is unclear whether the small heat shock proteins hsp27 and alphaB-crystallin protect against ischemic injury. METHODS AND RESULTS: Our aim was to examine whether the overexpression of hsp27 and alphaB-crystallin in neonatal and adult rat cardiomyocytes would protect against ischemic injury. Recombinant adenovirus expressing hsp27 or alphaB-crystallin under the control of the cytomegalovirus promoter was used to infect cardiac myocytes at high efficiency as assessed by immunostaining. Overexpression was confirmed by Western blot analysis. Cardiomyocytes were subjected to simulated ischemic stress, and survival was estimated through assessment of lactate dehydrogenase and creatine phosphokinase release. The hsp27 overexpression decreased lactate dehydrogenase release by 45+/-7.5% in adult cardiomyocytes but had no effect in the neonatal cells. In contrast, alphaB-crystallin overexpression was associated with a decrease in cytosolic enzyme release in both adult (29+/-6.6%) and neonatal (32+/-5.4%) cardiomyocytes. Decreased endogenous hsp25 with an antisense adenovirus produced a 29+/-9.9% increase in damage with simulated ischemia. Overexpression of the inducible hsp70 in adult cardiomyocytes was associated with a 34+/-4.6% decrease in lactate dehydrogenase release and is in line with our previous results in neonatal cardiomyocytes. CONCLUSIONS: The increased expression of hsp27 and alphaB-crystallin through an adenovirus vector system protects against ischemic injury in adult cardiomyocytes. Likewise, the overexpression of alphaB-crystallin protects against ischemic damage in neonatal cardiomyocytes. Decreasing the high levels of endogenous hsp25 present in neonatal cardiomyocytes renders them more susceptible to damage caused by simulated ischemia.

Adenovirus-mediated gene transfer of a heat shock protein 70 (hsp 70i) protects against simulated ischemia.

We have recently shown that the overexpression of a heat shock protein 70 (hsp 70) in a rat myogenic cell line confers protection against simulated ischemia. We also developed and demonstrated that overexpression of this protein, in the hearts of transgenic mice, protects against ischemia/reperfusion injury. We have now inserted the hsp70 gene in an adenoviral vector and show that we are able to transfer and achieve overexpression of this protein in neonatal cardiomyocytes and in the rat myogenic cell line H9c2. We find that cells infected with the adenoviral-hsp70i construct are rendered tolerant to simulated ischemia as compared to cells infected with a control recombinant adenoviral construct. In conclusion, our results demonstrate the feasibility of using adenoviral vectors to overexpress the hsp70 in myogenic cells, specially in cardiomyocytes, and the efficiency of this approach for providing protection against myocardial ischemia.

Overexpression of heat shock protein 72 in transgenic mice decreases infarct size in vivo.

BACKGROUND: Previous studies have demonstrated that induction of heat shock protein (HSP) 72 by whole-body hyperthermia reduces infarct size in an in vivo model of ischemia and reperfusion. Furthermore, hearts obtained from transgenic mice that overexpress HSP72 demonstrate improved functional recovery and decreased infarct size in vitro after global ischemia and reperfusion. METHODS AND RESULTS: To test the hypothesis that overexpression of HSP72 in transgenic mice reduces infarct size in vivo, transgenic mice that were heterozygous for a rat HSP70i gene ([+]HSP72) and transgene-negative littermate controls ([-]HSP72) were subjected to 30 minutes of left coronary artery occlusion followed by 120 minutes of reperfusion. Core body temperature was monitored with a rectal thermometer and maintained between 36.5 degrees C and 37.0 degrees C with a heating pad. Infarct size, determined by dual staining with triphenyltetrazolium chloride and phthalocyanine blue dye, was smaller in [+]HSP72 mice compared with [-]HSP72 mice (12.7 +/- 2.8% [n = 7] versus 33.4 +/- 4.5% [n = 6], infarct size/risk area, respectively; P < .05; mean +/- SEM). CONCLUSIONS: Overexpression of HSP72 reduces infarct size in this in vivo transgenic mouse model of myocardial ischemia and reperfusion.

Stable expression of a human HSP70 gene in a rat myogenic cell line confers protection against endotoxin.

Recent reports show that a pre-heat shock has a protective effect against endotoxin "in vivo" in rodents. It has remains unclear what actually confers the protection against endotoxin. One candidate for this protective effect is the heat shock protein of 70 kDa (HSP70). We found that a mild heat shock pretreatment is the rat myogenic cell line, H9c2(2-1), confers resistance to a subsequent exposure to endotoxin. A myogenic rat cell line stably transfected with the human inducible HSP70 exhibits an increased survival rate compared with cells stably transfected solely with the selectable neomycin marker gene or the parental cell line H9c2(2-1) when exposed to endotoxin. The mechanism of endotoxin-induced cell injury is postulated to be through the generation of nitric oxide in these myogenic cells during exposure to endotoxin. We conclude that HSP70, regardless of the particular mechanism of cytotoxicity, plays a role in protecting the cell against the deleterious effects of endotoxin.

Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury.

Myocardial protection and changes in gene expression follow whole body heat stress. Circumstantial evidence suggests that an inducible 70-kD heat shock protein (hsp70i), increased markedly by whole body heat stress, contributes to the protection. Transgenic mouse lines were constructed with a cytomegalovirus enhancer and beta-actin promoter driving rat hsp70i expression in heterozygote animals. Unstressed, transgene positive mice expressed higher levels of myocardial hsp70i than transgene negative mice after whole body heat stress. This high level of expression occurred without apparent detrimental effect. The hearts harvested from transgene positive mice and transgene negative littermates were Langendorff perfused and subjected to 20 min of warm (37 degrees C) zero-flow ischemia and up to 120 min of reflow while contractile recovery and creatine kinase efflux were measured. Myocardial infarction was demarcated by triphenyltetrazolium. In transgene positive compared with transgene negative hearts, the zone of infarction was reduced by 40%, contractile function at 30 min of reflow was doubled, and efflux of creatine kinase was reduced by approximately 50%. Our findings suggest for the first time that increased myocardial hsp70i expression results in protection of the heart against ischemic injury and that the antiischemic properties of hsp70i have possible therapeutic relevance.