Increased Heat Shock Protein Expression Decreases Inflammation in Skeletal Muscle During and after Frostbite Injury.

BACKGROUND: Frostbite injury results in serious skeletal muscle damage. The inflammatory response due to frostbite causes local muscle degeneration. Previous studies have shown that heat shock proteins (hsps) can protect against inflammation. In addition, our previous studies showed that increased expression of hsp70 is able to protect skeletal muscle against cryolesion. METHODS: Therefore, our aim was to determine if the induction of the heat shock proteins are able to minimize inflammation and protect skeletal muscle against frostbite injury. RESULTS: In the present study, we used the hsp90 inhibitor, 17-dimethylaminoethylamino- 17-demethoxygeldanamycin (17-DMAG), which was administered within 30 minutes following frostbite injury. Rat hind-limb muscles injected with 17-DMAG following frostbite injury exhibited less inflammatory cell infiltration as compared to control rat hind-limb muscles. In agreement with this observation, it has been observed that increased hsp expression resulted in decreased inflammatory cytokine expression. Additionally, we found that the administration of 17-DMAG after frostbite injury can preserve muscle tissue structure as well as function. CONCLUSION: It has been concluded that compounds such as 17-DMAG that induce the heat shock proteins are able to preserve skeletal muscle function and structure if injected within 30 minutes after frostbite injury. Our studies provide the basis for the development of a potential therapeutic strategy to treat the injury caused by frostbite.

Protein kinase C-α interaction with iHSP70 in mitochondria promotes recovery of mitochondrial function after injury in renal proximal tubular cells.

This study determined the role of PKC-α and associated inducible heat shock protein 70 (iHSP70) in the repair of mitochondrial function in renal proximal tubular cells (RPTCs) after oxidant injury. Wild-type PKC-α (wtPKC-α) and an inactive PKC-α [dominant negative dn; PKC-α] mutant were overexpressed in primary cultures of RPTCs, and iHSP70 levels and RPTC regeneration were assessed after treatment with the oxidant tert-butylhydroperoxide (TBHP). TBHP exposure increased ROS production and induced RPTC death, which was prevented by ferrostatin and necrostatin-1 but not by cyclosporin A. Overexpression of wtPKC-α maintained mitochondrial levels of active PKC-α, reduced cell death, and accelerated proliferation without altering ROS production in TBHP-injured RPTCs. In contrast, dnPKC-α blocked proliferation and monolayer regeneration. Coimmunoprecipitation and proteomic analysis demonstrated an association between inactive, but not active, PKC-α and iHSP70 in mitochondria. Mitochondrial iHSP70 levels increased as levels of active PKC-α decreased after injury. Overexpression of dnPKC-α augmented, whereas overexpression of wtPKC-α abrogated, oxidant-induced increases in mitochondrial iHSP70 levels. iHSP70 overexpression (1) maintained mitochondrial levels of phosphorylated PKC-α, (2) improved the recovery of state 3 respiration and ATP content, (3) decreased RPTC death (an effect abrogated by cyclosporine A), and (4) accelerated proliferation after oxidant injury. In contrast, iHSP70 inhibition blocked the recovery of ATP content and exacerbated RPTC death. Inhibition of PKC-α in RPTC overexpressing iHSP70 blocked the protective effects of iHSP70. We conclude that active PKC-α maintains mitochondrial function and decreases cell death after oxidant injury. iHSP70 is recruited to mitochondria in response to PKC-α dephosphorylation and associates with and reactivates inactive PKC-α, which promotes the recovery of mitochondrial function, decreases RPTC death, and improves regeneration.

Hyperthermia: from diagnostic and treatments to new discoveries.

Hyperthermia is an important approach for the treatment of several diseases. Hyperthermia is also thought to induce hypertrophy of skeletal muscles in vitro and in vivo, and has been used as a therapeutic tool for millennia. In the first part of our work, we revise several relevant patents related to the utilization of hyperthermia for the treatment and diagnostic of human diseases. In the second part, we present exciting new data on the effects of forced and natural overexpression of HSP72, using murine in vitro (muscle cells) and ex vivo (primary skeletal muscles) models. These studies help to demonstrate that hyperthermia effects are orchestrated by tight coupling between gene expression, protein function, and intracellular Ca2+ signaling pathways with a key role for calcium-induced calcium release. We hope that the review of current patents along with previous unknown information on molecular signaling pathways that underlie the hypertrophy response to hyperthermia in skeletal muscles may trigger the curiosity of scientists worldwide to explore new inventions that fully utilize hyperthermia for the treatment of muscle diseases.

Overexpression of inducible 70-kDa heat shock protein in mouse improves structural and functional recovery of skeletal muscles from atrophy.

Heat shock proteins play a key regulatory role in cellular defense. To investigate the role of the inducible 70-kDa heat shock protein (HSP70) in skeletal muscle atrophy and subsequent recovery, soleus (SOL) and extensor digitorum longus (EDL) muscles from overexpressing HSP70 transgenic mice were immobilized for 7 days and subsequently released from immobilization and evaluated after 7 days. Histological analysis showed that there was a decrease in cross-sectional area of type II myofiber from EDL and types I and II myofiber from SOL muscles at 7-day immobilization in both wild-type and HSP70 mice. At 7-day recovery, EDL and SOL myofibers from HSP70 mice, but not from wild-type mice, recovered their size. Muscle tetanic contraction decreased only in SOL muscles from wild-type mice at both 7-day immobilization and 7-day recovery; however, it was unaltered in the respective groups from HSP70 mice. Although no effect in a fatigue protocol was observed among groups, we noticed a better contractile performance of EDL muscles from overexpressing HSP70 groups as compared to their matched wild-type groups. The number of NCAM positive-satellite cells reduced after immobilization and recovery in both EDL and SOL muscles from wild-type mice, but it was unchanged in the muscles from HSP70 mice. These results suggest that HSP70 improves structural and functional recovery of skeletal muscle after disuse atrophy, and this effect might be associated with preservation of satellite cell amount.

Overexpression of HSP10 in skeletal muscle of transgenic mice prevents the age-related fall in maximum tetanic force generation and muscle Cross-Sectional Area.

Skeletal muscle atrophy and weakness are major contributors to frailty and impact significantly on quality of life of older people. Muscle aging is characterized by a loss of maximum tetanic force (P(o)) generation, primarily due to muscle atrophy, to which mitochondrial dysfunction is hypothesized to contribute. We hypothesized that lifelong overexpression of the mitochondrial heat shock protein (HSP) HSP10 in muscle of mice would protect against development of these deficits. P(o) generation by extensor digitorum longus muscles of adult and old wild-type and HSP10-overexpressing mice was determined in situ. Muscles were subjected to damaging lengthening contractions, and force generation was remeasured at 3 h or 28 days to examine susceptibility to, and recovery from, damage, respectively. Muscles of old wild-type mice had a 23% deficit in P(o) generation and a 10% deficit in muscle cross-sectional area compared with muscles of adult wild-type mice. Overexpression of HSP10 prevented this age-related fall in P(o) generation and reduction in cross-sectional area observed in muscles of old wild-type mice. Additionally, overexpression of HSP10 protected against contraction-induced damage independent of age but did not improve recovery if damage occurred. Preservation of muscle force generation and CSA by HSP10 overexpression was associated with protection against the age-related accumulation of protein carbonyls. Data demonstrate that development of age-related muscle weakness may not be inevitable and show, for the first time, that lifelong overexpression of an HSP prevents the age-related loss of P(o) generation. These findings support the hypothesis that mitochondrial dysfunction is involved in the development of age-related muscle deficits.

HSP72 protects against obesity-induced insulin resistance.

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of kappaB kinase, and tumor necrosis factor-alpha, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

Troponin I levels in patients with preeclampsia.

INTRODUCTION: Preeclampsia involves a diffuse inflammatory state and elevated levels of troponins in patients with preeclampsia have been anecdotally reported. It is, however, unknown whether it is attributable to the preeclampsia. OBJECTIVE: We sought to determine the troponin I levels at the time of delivery in pregnant women with and without preeclampsia. METHODS: Plasma samples were obtained at the time of delivery and serum troponin I was measured by ELISA method. RESULTS: Thirty-nine women were included (20 with preeclampsia and 19 without). Mean troponin I level was 0.008 ng/mL in patients with preeclampsia and 0.01 ng/mL in controls (P =.59). The highest troponin I level was 0.04 ng/mL for both patients with and without preeclampsia. CONCLUSIONS: Preeclampsia was not associated with a rise in troponin I levels in our study. Patients with preeclampsia and elevated troponin levels should have further cardiac investigations.

CHIP and HSPs interact with beta-APP in a proteasome-dependent manner and influence Abeta metabolism.

The C-terminus Hsp70 interacting protein (CHIP) has dual function as both co-chaperone and ubiquitin ligase. CHIP is increasingly implicated in the biology of polyglutamine expansion disorders, Parkinson's disease and tau protein in Alzheimer's disease. We investigated the involvement of CHIP in the metabolism of the beta-amyloid precursor protein and its derivative beta-amyloid (Abeta). Using immunoprecipitation, fluorescence localization and crosslinking methods, endogenous CHIP and betaAPP interact in brain and cultured skeletal myotubes as well as when they are expressed in stable HEK cell lines. Their interaction is confined to Golgi and ER compartments. In the presence of the proteasome inhibitor with MG132, endogenous and expressed betaAPP levels are significantly increased and accordingly, the interaction with CHIP enhanced. Concurrently, levels of Hsp70 were most consistently induced by proteasome inhibition among the various heat shock proteins (HSPs) tested. Thus, complexes of CHIP, Hsp70 and holo-betaAPP (as well as C-terminal fragments) were stabilized by the action of MG132. Moreover, CHIP itself is shown to both increase cellular holo-betaAPP levels and protect it from oxidative stress and degradation. Interestingly, CHIP also promotes the association of ubiquitin with betaAPP, implying that a smaller pool of betaAPP is destined for proteasomal processing. In neuronal cultures, CHIP and Hsp70/90 expression reduce steady-state cellular Abeta levels and hasten its degradation in pulse-chase experiments. The functional significance of CHIP and HSP interactions, especially with Hsp70, was tested using siRNA and in neuronal cells where protection from Abeta-induced toxicity is shown. We conclude that CHIP, as a bimolecular switch, interacts with HSP to stabilize normal holo-betaAPP on the one hand while also assisting in the ubiquitination of a subpopulation of betaAPP molecules that are destined for proteasome degradation. CHIP also hastens the clearance of Abeta in a manner consistent with its known neuroprotective properties.

Importance of small heat shock protein 20 (hsp20) C-terminal extension in cardioprotection.

Recent studies show that overexpression of small heat shock protein 20 (Hsp20) in mouse hearts reduces infarct size and improves cardiac performance. However, it is not known whether Hsp20 exerts its protective action through improved calcium handling or chaperone activity. The C-terminal extensions of small heat shock proteins, such as alphaB-crystallin and Hsp25, are implicated in chaperoning activity. Through adenovirus mediated overexpression of Hsp20 with C-terminal extension substitution, we delineated the mechanism of protection. Neonatal and adult rat cardiomyocytes overexpressing either the full-length Hsp20 or Hsp20 with a C-terminal extension substitution were subjected to simulated ischemia for 14-16 h followed by reperfusion 6-8 h. Overexpressing Hsp20 with a C-terminus extension substitution did not protect against simulated ischemia/reperfusion in either adult (98+/-8.8% LDH release of control) or neonatal cardiomyocytes (103+/-1.8% CK release of control) as measured by creatine kinase (CK) and lactate dehydrogenase (LDH) cell viability assays (n=4, P<0.05). However, this Hsp20 C-terminal substitution mutant increased calcium transients 33+/-11% and cell contraction amplitude 60+/-15% as quantified through epifluorescence microscopy (n=16 to 34 cells per heart from 4 to 5 hearts, P<0.05). In contrast, overexpression of the full-length Hsp20 protected cultured adult (53+/-8.5% LDH release of control) and neonatal rat (57+/-8.3% CK release of control) cardiomyocytes from simulated ischemia/reperfusion injury. This overexpression also increased calcium transients 30+/-10% and cell contraction amplitude 50+/-10%. These novel data suggest that the C-terminal extension of Hsp20 is essential for cardioprotection. Hsp20 renders this protection through its C-terminal extension protein domain, while this part of the protein is not involved in the Hsp20 ability to increase both calcium transients and cell contraction.

Influence of PKC-alpha overexpression on HSP70 and cardioprotection.

Recent research has indicated that the protein kinase C (PKC) isoforms and the heat shock proteins (HSPs) are involved in cardioprotection. We have investigated the possible interaction between these two protein families. We have found that adenoviral-mediated expression of PKC-alpha in neonatal rat ventricular myocytes (NRVM) not only increases the expression of HSP70 but also protects against simulated ischemia-reperfusion. In addition, Western blots of PKC-alpha-infected NRVM indicated that other HSPs are not induced in the same manner as HSP70. In an effort to determine the mechanism of induction of HSP70 by PKC-alpha, we tested a chimeric construct that linked the luciferase reporter gene to the 5'-promoter region of HSP70 in myogenic H9c2 cells. When PKC-alpha was expressed, the 5'-promoter region of the HSP70 responded robustly, indicating that PKC-alpha induction of HSP70 expression is through transcription activation. Electrophoretic mobility shift assay determined that overexpression of PKC-alpha, PKC-delta, or PKC-epsilon did not induce activation of heat shock factor-1 (HSF-1). Therefore, induction of HSP70 by PKC-alpha is independent of heat shock factor-1 activation. We also measured cellular injury by assessing creatine kinase (CK) release from NRVM after simulated ischemia to determine cardioprotection. NRVM infected with the wild-type adenoviral construct AdwtPKC-alpha released 54% less CK than control NRVM. Experiments using small interfering RNA against HSP70 indicate that loss of PKC-alpha-induced HSP70 expression results in increased CK release or a loss of protection. Our results show that there is a close interaction between PKC-alpha and HSP70, independent of heat shock factor-1 activation, and that the protection conferred by PKC-alpha overexpression is mediated by the transcriptionally induced expression of HSP70.

Downregulation of the constitutively expressed Hsc70 in diabetic myocardium is mediated by insulin deficiency.

The 70 kDa heat shock protein family plays important cardiac protective roles against myocardial injuries. Reduced myocardial protection is a common feature of diabetic myocardium. This study was carried out to define the changes in the 70 kDa heat shock protein family in the myocardium in the of streptozotocin-diabetes rats, and to explore the mechanisms through which diabetes alters the abundance of Hsp70/Hsc70 in cardiac muscle. In the diabetic myocardium, the abundance of Hsc70 was significantly reduced. The abundance of Hsp70 was low in cardiac muscle and was not induced in the diabetic myocardium. Unlike Hsp60, Hsp70 and Hsc70 did not augment insulin-like growth factor-I receptor signaling in cardiac muscle cells. In cultured cardiomyocytes, insulin directly increased the abundance of Hsc70, whereas insulin could not modulate Hsp70. Treating diabetic rats with insulin restored myocardial Hsc70 level, but phlorizin treatment failed to restore myocardial Hsc70. These in vivo and in vitro studies showed that downregulation of Hsc70 in diabetic myocardium was secondary to insulin deficiency. Thus, insulin played a major role in maintaining adequate expression of Hsc70 in cardiac muscle.

Differential effects of mitochondrial heat shock protein 60 and related molecular chaperones to prevent intracellular beta-amyloid-induced inhibition of complex IV and limit apoptosis.

Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been reported in Alzheimer disease tissue and in cultured cells that overexpress amyloid precursor protein. Mitochondrial dysfunction contributes to neurodegeneration in Alzheimer disease partly through formation of reactive oxygen species and the release of sequestered molecules that initiate programmed cell death pathways. The heat shock proteins (HSP) are cytoprotective against a number of stressors, including accumulations of misfolded proteins and reactive oxygen species. We reported on the property of Hsp70 to protect cultured neurons from cell death caused by intraneuronal beta-amyloid. Here we demonstrate that Hsp60, Hsp70, and Hsp90 both alone and in combination provide differential protection against intracellular beta-amyloid stress through the maintenance of mitochondrial oxidative phosphorylation and functionality of tricarboxylic acid cycle enzymes. Notably, beta-amyloid was found to selectively inhibit complex IV activity, an effect selectively neutralized by Hsp60. The combined effect of HSPs was to reduce the free radical burden, preserve ATP generation, decrease cytochrome c release, and prevent caspase-9 activation, all important mediators of beta-amyloid-induced neuronal dysfunction and death.

Ca2+/Calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes.

Previous studies in transgenic mice and with isolated ryanodine receptors (RyR) have indicated that Ca2+-calmodulin-dependent protein kinase II (CaMKII) can phosphorylate RyR and activate local diastolic sarcoplasmic reticulum (SR) Ca2+ release events (Ca2+ sparks) and RyR channel opening. Here we use relatively controlled physiological conditions in saponin-permeabilized wild type (WT) and phospholamban knockout (PLB-KO) mouse ventricular myocytes to test whether exogenous preactivated CaMKII or endogenous CaMKII can enhance resting Ca2+ sparks. PLB-KO mice were used to preclude ancillary effects of CaMKII mediated by phospholamban phosphorylation. In both WT and PLB-KO myocytes, Ca2+ spark frequency was increased by both preactivated exogenous CaMKII and endogenous CaMKII. This effect was abolished by CaMKII inhibitor peptides. In contrast, protein kinase A catalytic subunit also enhanced Ca2+ spark frequency in WT, but had no effect in PLB-KO. Both endogenous and exogenous CaMKII increased SR Ca2+ content in WT (presumably via PLB phosphorylation), but not in PLB-KO. Exogenous calmodulin decreased Ca2+ spark frequency in both WT and PLB-KO (K0.5 approximately 100 nmol/L). Endogenous CaMKII (at 500 nmol/L [Ca2+]) phosphorylated RyR as completely in <4 minutes as the maximum achieved by preactivated exogenous CaMKII. After CaMKII activation Ca2+ sparks were longer in duration, and more frequent propagating SR Ca2+ release events were observed. We conclude that CaMKII-dependent phosphorylation of RyR by endogenous associated CaMKII (but not PKA-dependent phosphorylation) increases resting SR Ca2+ release or leak. Moreover, this may explain the enhanced SR diastolic Ca2+ leak and certain triggered arrhythmias seen in heart failure.

Dynamic changes in free Ca-calmodulin levels in adult cardiac myocytes.

Despite its multifunctional role in cardiac myocyte function, little is known about dynamic changes in activation state of calmodulin (CaM). Thus, the purpose of this study was to develop a tool to measure Ca bound CaM (Ca-CaM) levels in intact cardiac myocytes. For dynamic measurements of Ca-CaM, we generated an adenoviral vector which expresses a cyan and a yellow fluorescent protein linked by a modified version of the Ca-CaM binding domain of avian smooth muscle myosin light chain kinase. Adult rabbit cardiac myocytes were infected with the Ca-CaM sensing probe or simultaneously infected with viruses containing CaM and the Ca-CaM sensing probe for 24-48 h. Myocytes were then field stimulated (1 Hz) and excited at 440 nm with emitted fluorescence measured at 485 and 535 nm. Changes in [Ca-CaM] are expressed as the ratio of 485 nm/535 nm. Small beat-to-beat changes of [Ca-CaM] were detected, but only when CaM was co-expressed with the sensor. However, upon beta-adrenergic stimulation with isoproterenol, there was an increase in the amplitude of the signals during each beat (parallel to the shortening, which is an indirect measure of [Ca]i) and also a rise in the diastolic [Ca-CaM]. Total [CaM] measured by both competitive ELISA and semi-quantitative Western blots was 5-6 microM in isolated adult ventricular myocytes. Our results indicate that there are dynamic changes in free Ca-CaM levels (a phasic component tracking [Ca]i) as well as system memory that integrates the [Ca]i signals (a tonic component).

Effect of lifelong overexpression of HSP70 in skeletal muscle on age-related oxidative stress and adaptation after nondamaging contractile activity.

Skeletal muscle aging is characterized by atrophy, a deficit in specific force generation, increased susceptibility to injury, and incomplete recovery after severe injury. The ability of muscles of old mice to produce heat shock proteins (HSPs) in response to stress is severely diminished. Studies in our laboratory using HSP70 overexpressor mice demonstrated that lifelong overexpression of HSP70 in skeletal muscle provided protection against damage and facilitated successful recovery after damage in muscles of old mice. The mechanisms by which HSP70 provides this protection are unclear. Aging is associated with the accumulation of oxidation products, and it has been proposed that this may play a major role in age-related muscle dysfunction. Muscles of old wild-type (WT) mice demonstrated increased lipid peroxidation, decreased glutathione content, increased catalase and superoxide dismutase (SOD) activities, and an inability to activate nuclear factor (NF)-kappaB after contractions in comparison with adult WT mice. In contrast, levels of lipid peroxidation, glutathione content, and the activities of catalase and SOD in muscles of old HSP70 overexpressor mice were similar to adult mice and these muscles also maintained the ability to activate NF-kappaB after contractions. These data provide an explanation for the preservation of muscle function in old HSP70 overexpressor mice.

Aberrant protein aggregation is essential for a mutant desmin to impair the proteolytic function of the ubiquitin-proteasome system in cardiomyocytes.

Aberrant protein aggregates in cardiomyocytes are frequently observed in many forms of cardiomyopathies and are often associated with impairment of proteolytic function of the ubiquitin-proteasome system (UPS). However, a causal relationship between mutant desmin (MT-des) induced aberrant protein aggregation and UPS impairment has not been established. The present study has tested the causal relationship. In cultured neonatal rat ventricular myocytes, modest overexpression of a human (cardio)myopathy-linked MT-des protein led to formation of desmin-positive aggregates and inhibited UPS proteolytic function in cardiomyocytes in a dose-dependent manner. Prevention or reduction of aberrant protein aggregation by co-expression of a heat shock protein (Hsp), alphaB-crystallin or inducible Hsp70, or by treatment of Congo red prevented and/or significantly attenuated the induction of UPS malfunction by MT-des. These findings prove for the first time that aberrant protein aggregation is not only sufficient but also required for MT-des to impair UPS proteolytic function in cardiomyocytes.

Impairment of the ubiquitin-proteasome system in desminopathy mouse hearts.

Protein misfolding and aberrant aggregation are associated with many severe disorders, such as neural degenerative diseases, desmin-related myopathy (DRM), and congestive heart failure. Intrasarcoplasmic amyloidosis and increased ubiquitinated proteins are observed in human failing hearts. The pathogenic roles of these derangements in the heart remain unknown. The ubiquitin-proteasome system (UPS) plays a central role in intracellular proteolysis and regulates critical cellular processes. In cultured cells, aberrant aggregation by a mutant (MT) or misfolded protein impairs the UPS. However, this has not been demonstrated in intact animals, and it is unclear how the UPS is impaired. Cross-breeding UPS reporter mice with a transgenic mouse model of DRM featured by aberrant protein aggregation in cardiomyocytes, we found that overexpression of MT-desmin but not normal desmin protein impairs UPS proteolytic function in the heart. The primary defect does not appear to be in the ubiquitination or the proteolytic activity of the 20S proteasome, because ubiquitinated proteins and the peptidase activities of 20S proteasomes were significantly increased rather than decreased in the DRM heart. Therefore, the defect resides apparently in the entry of ubiquitinated proteins into the 20S proteasome. Consistent with this notion, key components (Rpt3 and Rpt5) of 19S proteasomes were markedly decreased, while major components of 20S proteasomes were increased. Additional experiments with HEK cells suggest that proteasomal malfunction observed in MT-desmin hearts is not secondary to cardiac malfunction or to disruption of desmin filaments. Thus, UPS impairment may represent an important pathogenic mechanism underlying cardiac disorders with abnormal protein aggregation.

Overexpression of inducible 70-kDa heat shock protein in mouse attenuates skeletal muscle damage induced by cryolesioning.

Heat shock protein expression is elevated upon exposure to a variety of stresses and limits the extent of stress-induced damage. To investigate the putative role of inducible 70-kDa heat shock protein (HSP70) in skeletal muscle damage and regeneration, soleus and tibialis anterior (TA) muscles from HSP70-overexpressing transgenic mice were subjected to cryolesioning and analyzed after 1, 10, and 21 days. Histological analysis showed that the muscles from both HSP70 and wild-type mice treated with radicicol (a HSP inducer) had decreased necrosis after cryolesioning compared with controls. The decrease in muscle fiber cross-sectional area in both soleus and TA muscles in 10 days postlesioning was attenuated in HSP70 mice compared with wild-type mice. Glutathione peroxidase activity was increased 1 day after cryolesioning in both HSP70 and control mice and remained elevated for up to 21 days. Immunodetection of neuronal cell adhesion molecule (a satellite cell marker) and developmental/neonatal MHC were significantly lower in cryolesioned HSP70-overexpressing mice than in cryolesioned controls. These results suggest that HSP70 protects skeletal muscle against injury and radicicol might be useful as a skeletal muscle protective agent.

Phosphorylation and binding of AUF1 to the 3'-untranslated region of cardiomyocyte SERCA2a mRNA.

Experimental animals and patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of sarco(endo)plasmic reticulum calcium ATPase 2a (SERCA2a), the cardiomyocyte sarcoplasmic reticulum Ca2+ pump. We previously showed that SERCA2a downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the hypertrophic agonist phorbol myristate acetate (PMA) or by overexpression of the novel protein kinase C (PKC) isoenzymes PKCdelta and PKCepsilon. PKC activation, in turn, decreased SERCA2a promoter activity and destabilized the SERCA2a mRNA. Here we demonstrate by using an RSV beta-galactosidase reporter system that a 609-nt fragment of the SERCA2a mRNA 3'-untranslated region (UTR), containing five adenylate-uridylate (AU)-rich regions, may be responsible for destabilizing the message following PMA treatment. UV cross-linking analysis demonstrated that several proteins found in the NRVM cell extracts bind to the 609-nt fragment. In addition, protein binding was transiently increased in response to PMA stimulation. 3'-UTR mRNA pull-down assays and Western blot analysis indicated that the AU binding protein AUF1 interacted with the SERCA2a 3'-UTR. AUF1 binding activity was predominantly found in the nuclear fraction, and PMA-induced AUF1 binding was associated with increased threonine phosphorylation of AUF1. These data suggest that the phosphorylation, binding, and location of AUF1 affect the posttranscriptional regulation of the SERCA2a message in NRVM.

4-Tertiary butyl phenol exposure sensitizes human melanocytes to dendritic cell-mediated killing: relevance to vitiligo.

The trigger initiating an autoimmune response against melanocytes in vitiligo remains unclear. Patients frequently experience stress to the skin prior to depigmentation. 4-tertiary butyl phenol (4-TBP) was used as a model compound to study the effects of stress on melanocytes. Heat shock protein (HSP)70 generated and secreted in response to 4-TBP was quantified. The protective potential of stress proteins generated following 4-TBP exposure was examined. It was studied whether HSP70 favors dendritic cell (DC) effector functions as well. Melanocytes were more sensitive to 4-TBP than fibroblasts, and HSP70 generated in response to 4-TBP exposure was partially released into the medium by immortalized vitiligo melanocyte cell line PIG3V. Stress protein HSP70 in turn induced membrane tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and activation of DC effector functions towards stressed melanocytes. Melanocytes exposed to 4-TBP demonstrated elevated TRAIL death receptor expression. DC effector functions were partially inhibited by blocking antibodies to TRAIL. TRAIL expression and infiltration by CD11c+ cells was abundant in perilesional vitiligo skin. Stressed melanocytes may mediate DC activation through release of HSP70, and DC effector functions appear to play a previously unappreciated role in progressive vitiligo.