Genetic inhibition or activation of JNK1/2 protects the myocardium from ischemia-reperfusion-induced cell death in vivo

The c-Jun NH2-terminal kinase (JNK) branch of the mitogen-activated protein kinase signaling cascade has been implicated in the regulation of apoptosis in a variety of mammalian cell types. In the heart, disagreement persists concerning the role that JNKs may play in regulating apoptosis, since both pro- and antiapoptotic regulatory functions have been reported in cultured cardiomyocytes. Here we report the first analysis of cardiomyocyte cell death due to JNK inhibition or activation in vivo using genetically modified mice. Three separate mouse models with selective JNK inhibition were assessed for ventricular damage and apoptosis levels following ischemia-reperfusion injury. jnk1-/-, jnk2-/-, and transgenic mice expressing dominant negative JNK1/2 within the heart were each shown to have less JNK activity in the heart and less injury and cellular apoptosis in vivo following ischemia-reperfusion injury. To potentially address the reciprocal gain-of-function phenotype associated with sustained JNK activation, transgenic mice were generated that express MKK7 in the heart. These transgenic mice displayed elevated cardiac c-Jun kinase activity but, ironically, were also significantly protected from ischemia-reperfusion. Mechanistically, JNK-inhibited mice showed increased phosphorylation of the proapoptotic factor Bad at position 112, whereas MKK7 transgenic mice showed decreased phosphorylation of this site. Collectively, these results underscore the complexity associated with JNK signaling in regulating apoptosis, such that sustained inhibition or activation both elicit cellular protection in vivo, although probably through different mechanisms.     

Characterization of the Cardiac Overexpression of HSPB2 Reveals Mitochondrial and Myogenic Roles Supported by a Cardiac HspB2 Interactome

Small Heat Shock Proteins (sHSPs) are molecular chaperones that transiently interact with other proteins, thereby assisting with quality control of proper protein folding and/or degradation. They are also recruited to protect cells from a variety of stresses in response to extreme heat, heavy metals, and oxidative-reductive stress. Although ten human sHSPs have been identified, their likely diverse biological functions remain an enigma in health and disease, and much less is known about non-redundant roles in selective cells and tissues. Herein, we set out to comprehensively characterize the cardiac-restricted Heat Shock Protein B-2 (HspB2), which exhibited ischemic cardioprotection in transgenic overexpressing mice including reduced infarct size and maintenance of ATP levels. Global yeast two-hybrid analysis using HspB2 (bait) and a human cardiac library (prey) coupled with co-immunoprecipitation studies for mitochondrial target validation revealed the first HspB2 “cardiac interactome” to contain many myofibril and mitochondrial-binding partners consistent with the overexpression phenotype. This interactome has been submitted to the Biological General Repository for Interaction Datasets (BioGRID). A related sHSP chaperone HspB5 had only partially overlapping binding partners, supporting specificity of the interactome as well as non-redundant roles reported for these sHSPs. Evidence that the cardiac yeast two-hybrid HspB2 interactome targets resident mitochondrial client proteins is consistent with the role of HspB2 in maintaining ATP levels and suggests new chaperone-dependent functions for metabolic homeostasis. One of the HspB2 targets, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), has reported roles in HspB2 associated phenotypes including cardiac ATP production, mitochondrial function, and apoptosis, and was validated as a potential client protein of HspB2 through chaperone assays. From the clientele and phenotypes identified herein, it is tempting to speculate that small molecule activators of HspB2 might be deployed to mitigate mitochondrial related diseases such as cardiomyopathy and neurodegenerative disease.     

Use of gammaphos for protecting the brain from delayed radiation damage

The influence of a radioprotector, gammaphos, on the development of delayed vascular changes and necrosis in rat brain following local brain irradiation with 25 Gy was investigated. The radioprotective effect was manifested by both the morphometric parameters of vessels and the survival rate and relative number of animals with gross vascular abnormalities and brain necrosis. There was a causative relationship between the development of gross vascular abnormalities and the occurrence of brain necrosis after exposure to moderate radiation doses.     

Roles for Dnmt3b in mammalian development: a mouse model for the ICF syndrome

ICF (Immunodeficiency, Centromeric instability and Facial anomalies) syndrome is a rare autosomal recessive disease caused by mutations in the DNA methyltransferase gene DNMT3B. To investigate the function of Dnmt3b in mouse development and to create animal models for ICF syndrome, we have generated three mutant alleles of Dnmt3b in mice: one carrying a deletion of the catalytic domain (null allele) and two carrying ICF-like missense mutations in the catalytic domain. The Dnmt3b null allele results in embryonic lethality from E14.5 to E16.5 with multiple tissue defects, including liver hypotrophy, ventricular septal defect and haemorrhage. By contrast, mice homozygous for the ICF mutations develop to term and some survive to adulthood. These mice show phenotypes that are reminiscent of ICF patients, including hypomethylation of repetitive sequences, low body weight, distinct cranial facial anomalies and T cell death by apoptosis. These results indicate that Dnmt3b plays an essential role at different stages of mouse development, and that ICF missense mutations cause partial loss of function. These mutant mice will be useful for further elucidation of the pathogenic and molecular mechanisms underlying ICF syndrome.     

HDAC6 inhibitors reverse axonal loss in a mouse model of mutant HSPB1-induced Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system. Mutations in the 27-kDa small heat-shock protein gene (HSPB1) cause axonal CMT or distal hereditary motor neuropathy (distal HMN). We developed and characterized transgenic mice expressing two different HSPB1 mutations (S135F and P182L) in neurons only. These mice showed all features of CMT or distal HMN dependent on the mutation. Expression of mutant HSPB1 decreased acetylated α-tubulin abundance and induced severe axonal transport deficits. An increase of α-tubulin acetylation induced by pharmacological inhibition of histone deacetylase 6 (HDAC6) corrected the axonal transport defects caused by HSPB1 mutations and rescued the CMT phenotype of symptomatic mutant HSPB1 mice. Our findings demonstrate the pathogenic role of α-tubulin deacetylation in mutant HSPB1-induced neuropathies and offer perspectives for using HDAC6 inhibitors as a therapeutic strategy for hereditary axonopathies.     

Inhibition of cyclooxygenase-2 enhances myocardial damage in a mouse model of viral myocarditis

To determine critical role of cyclooxygenase-2 (COX-2) for development of viral myocarditis, a mouse model of encephalomyocarditis virus-induced myocarditis was used. The virus was intraperitoneally given to COX-2 gene-deficient heterozygote mice (COX-2+/-) and wild-type mice (WT). We examined differences in heart weights, cardiac histological scores, numbers of infiltrating or apoptotic cells in myocardium, cardiac expression levels of COX-2, tumor necrosis factor-alpha (TNF-alpha), and adiponectin mRNA, immunoreactivity of COX-2, TNF-alpha, and adiponectin in myocytes, cardiac concentrations of TNF-alpha and adiponectin, prostaglandin E2 (PGE2) levels in hearts, and viral titers in tissues between COX-2+/- and WT. We observed significantly decreased expression of COX-2 mRNA and reactivity in hearts from COX-2+/- on day 8 after viral inoculation as compared with that from WT, together with elevated cardiac weights and severe inflammatory myocardial damage in COX-2+/-. Cardiac expression of TNF-alpha mRNA, reactivity, and protein on day 8 was significantly higher in COX-2+/- than in WT, together with reciprocal expression of adiponectin mRNA, reactivity, and protein in hearts. Significantly reduced cardiac PGE2 levels on day 8 were found in COX-2+/- compared with those in WT. There was no difference in local viral titers between both groups on day 4. Infected WT treated with a selective COX-2 inhibitor, NS-398, also showed the augmented myocardial damage on day 8. These results suggest that inhibition of COX-2 may enhance myocardial damage through reciprocal cardiac expression of TNF-alpha and adiponectin in a mouse model of viral myocarditis.     

LC-MS-based untargeted metabolomics reveals the mechanism underlying prostate damage in a type 2 diabetes mouse model

Type 2 diabetes mellitus (T2DM) can cause prostate damage and affect male reproductive function, but the underlying mechanisms are not completely understood. In this study, we used liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics to identify endogenous metabolites in the prostate of a T2DM mouse model. The selected endogenous metabolites were then subjected to bioinformatics analysis and metabolic pathway studies to understand their role in the development of T2DM-induced prostate damage. We used male homozygous BTBR ob/ob mice (n = 12) and BTBR WT mice (n = 11) in this study. We monitored changes in blood glucose, body weight, prostate weight, and prostate index, as well as performed hematoxylin and eosin (H&E) staining and observed that the prostate of the BTBR ob/ob was damaged. We then used ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) for metabolomics analysis. The stability of the model was validated using principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Using variable importance in projection (VIP) > 1, false discovery rate (FDR) < 0.05, and coefficient of variation (CV) < 30 as criteria, a total of 149 differential metabolites (62 upregulated and 87 downregulated) were identified between the prostates of the two groups of mice. Topological pathway analysis showed that these differential metabolites were mainly involved in sphingolipid (SP) and glycerophospholipid (GP) metabolism. In conclusion, our study not only emphasizes the damage caused by T2DM to the prostate but also provides new insights into the potential mechanisms of T2DM-induced male reproductive dysfunction.     

Essential actions of melatonin in protecting the ovary from oxidative damage

Free radicals and other reactive species are involved in normal ovarian physiology. However, they are also highly reactive with complex cellular molecules (proteins, lipids, and DNA) and alter their functions leading to oxidative stress. Oxidative damage may play a prominent role in the development of disorders that considerably influence female fertility. Melatonin, because of its amphiphilic nature that allows for crossing morphophysiological barriers, is an effective antioxidant for protecting macromolecules against oxidative stress caused by reactive species. The balance between reactive oxygen species and antioxidants within the follicle seems to be critical to the function of the oocyte and granulosa cells and evidence has accumulated showing that melatonin is involved in the protection of these cells. Melatonin appears to have varied functions at different stages of follicle development, oocyte maturation, and luteal stage. Melatonin concentration in the growing follicle may be an important factor in avoiding atresia, because melatonin in the follicular fluid reduces apoptosis of critical cells. Melatonin also has protective actions during oocyte maturation reducing intrafollicular oxidative damage. An association between melatonin concentrations in follicular fluid and oocyte quality has been reported; this would allow a preovulatory follicle to fully develop and provide a competent oocyte for fertilization. The functional role of reactive species and the cytoprotective properties of melatonin on the ovary from oxidative damage are summarized in this brief review.     

Neuronal NOS and cyclooxygenase-2 contribute to DNA damage in a mouse model of Parkinson disease

DNA damage is a proposed pathogenic factor in neurodegenerative disorders such as Parkinson disease. To probe the underpinning mechanism of such neuronal perturbation, we sought to produce an experimental model of DNA damage. We thus first assessed DNA damage by in situ nick translation and emulsion autoradiography in the mouse brain after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 4 × 20 mg/kg, ip, every 2 h), a neurotoxin known to produce a model of Parkinson disease. Here we show that DNA strand breaks occur in vivo in this mouse model of Parkinson disease with kinetics and a topography that parallel the degeneration of substantia nigra neurons, as assessed by FluoroJade labeling. Previously, nitric oxide synthase and cyclooxygenase-2 (Cox-2) were found to modulate MPTP-induced dopaminergic neuronal death. We thus assessed the contribution of these enzymes to DNA damage in mice lacking neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), or Cox-2. We found that the lack of Cox-2 and nNOS activities but not of iNOS activity attenuated MPTP-related DNA damage. We also found that not only nuclear, but also mitochondrial, DNA is a target for the MPTP insult. These results suggest that the loss of genomic integrity can be triggered by the concerted actions of nNOS and Cox-2 and provide further support to the view that DNA damage may contribute to the neurodegenerative process in Parkinson disease.     

The Small Heat-Shock Proteins HSPB2 and HSPB3 Form Well-defined Heterooligomers in a Unique 3 to 1 Subunit Ratio

Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from Escherichia coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintaining a strict 3:1 HSPB2/HSPB3 subunit ratio. These complexes are thermally stable up to 40 °C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or αB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could associate efficiently with HSP20. Taken altogether, this study provides evidence that, despite the high level of sequence homology within the sHSP family the biochemical properties of the HSPB2/B3 complex are distinctly different from those of other sHSPs, indicating that the HSPB2/B3 assembly is likely to possess cellular functions other than those of its family members.     

Formation of a TBA reaction product from crown ether in the presence of KO2.

Potassium superoxide (KO2), which can be dissolved in dimethyl sulfoxide containing crown ether, has been used as a source of O2-. for superoxide reaction systems. We have found that crown ether reacts with thiobarbituric acid (TBA) in the presence of KO2 to form a red pigment, which is a well-known reaction product of lipoperoxide.     

A role for SENSITIVE TO FREEZING2 in protecting chloroplasts against freeze-induced damage in Arabidopsis

The SENSITIVE TO FREEZING2 ( SFR2 ) gene has an important role in freezing tolerance in Arabidopsis thaliana . We show that homologous genes are present, and expressed, in a wide range of terrestrial plants, including species not able to tolerate freezing. Expression constructs derived from the cDNAs of a number of different plant species, including examples not tolerant to freezing, are able to complement the freezing sensitivity of the Arabidopsis sfr2 mutant. In Arabidopsis the SFR2 protein is localized to the chloroplast outer envelope membrane, as revealed by the analysis of transgenic plants expressing SFR2 fusions to GFP, by confocal microscopy, and by the immunological analysis of isolated chloroplasts treated with thermolysin protease. Moreover, the chloroplasts of the sfr2 mutant show clear evidence of rapid damage after a freezing episode, suggesting a role for SFR2 in the protection of the chloroplast.     

PEP-1-heat shock protein 27 protects from neuronal damage in cells and in a Parkinson's disease mouse model

Heat shock proteins (HSPs) are a highly conserved family of proteins that are induced in response to various environmental stressors including reactive oxygen species. HSP27 is a chaperone protein with the ability to increase cell survival in response to oxidative stress. Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. Although the mechanism of PD remains unclear, oxidative stress is known to be important in its pathogenesis. This study investigated the protective effects of PEP-1-HSP27 on neuronal damage induced by 1-methyl-4-phenyl pyridinium (MPP(+) ) in SH-SY5Y cells and in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. PEP-1-HSP27 rapidly entered the cells and protected them against MPP(+) -induced toxicity by inhibiting the reactive oxygen species levels and DNA fragmentation. Furthermore, transduced PEP-1-HSP27 prevented dopaminergic neuronal cell death in the substantia nigra of MPTP-induced PD mouse models. These results demonstrate that PEP-1-HSP27 provides a potential strategy for therapeutic delivery against various diseases and is a potential tool for the treatment of PD.     

Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption

The mammalian small heat shock protein (sHSPs) family is comprised of 10 members and includes HSPB1, which is proposed to play an essential role in cellular physiology, acting as a molecular chaperone to regulate diverse cellular processes. Whilst differential roles for sHSPs are suggested for specific tissues, the relative contribution of individual sHSP family members in cellular and organ physiology remains unclear. To address the function of HSPB1 in vivo and determine its tissue-specific expression during development and in the adult, we generated knock-in mice where the coding sequence of hspb1 is replaced by a lacZ reporter gene. Hspb1 expression marks myogenic differentiation with specific expression first confined to developing cardiac muscles and the vascular system, and later in skeletal muscles with specific expression at advanced stages of myoblast differentiation. In the adult, hspb1 expression was observed in other tissues, such as stratified squamous epithelium of skin, oronasal cavity, tongue, esophagus, and uterine cervix but its expression was most prominent in the musculature. Interestingly, in cardiac muscle hsbp1 expression was down-regulated during the neonatal period and maintained to a relatively low steady-level throughout adulthood. Despite this widespread expression, hspb1-/- mice were viable and fertile with no apparent morphological abnormalities in tissues under physiological conditions. However, at the cellular level and under stress conditions (heat challenge), HSPB1 act synergistically with the stress-induced HSPA1 (HSP70) in thermotolerance development, protecting cells from apoptosis. Our data thus indicate a nonessential role for HSPB1 in embryonic development and for maintenance of tissues under physiological conditions, but also shows that it plays an important role by acting synergistically with other HSPs during stress conditions to exert cytoprotection and anti-apoptotic effects.     

Effectiveness of protecting the myocardium against ischemia with a normothermic cardioplegic solution and creatine phosphate

The protective effects of cardioplegic solutions (CS) containing creatine phosphate (CP) were studied in a rat heart model of cardiopulmonary bypass and ischemic cardiac arrest. Isolated rat hearts were subjected to a 3-minute coronary infusion with CS containing CP in normothermic (37 degrees C) and hypothermic (4-6 degrees C) regimes. In the normothermia group, the postischemic functional recovery was 70-75% of the preischemic control value, while the cellular ATP and CP content was reduced but insignificantly. By contrast, in the hypothermia group, the postischemic functional recovery was markedly depressed, with the tissue high-energy phosphate content being appreciably lowered. The data obtained confirm high efficacy of CP-containing cardioplegic solutions administered under normothermia conditions.     

Increased oxidative damage to DNA in a transgenic mouse model of Huntington's disease

Mitochondrial dysfunction and oxidative damage may play a role in the pathogenesis of Huntington's disease (HD). We examined concentrations of 8-hydroxy-2-deoxyguanosine (OH(8)dG), a well-established marker of oxidative damage to DNA, in a transgenic mouse model of HD (R6/2). Increased concentrations of OH(8)dG were found in the urine, plasma and striatal microdialysates of the HD mice. Increased concentrations were also observed in isolated brain DNA at 12 and 14 weeks of age. Immunocytochemistry showed increased OH(8)dG staining in late stages of the illness. These results suggest that oxidative damage may play a role in the pathogenesis of neuronal degeneration in the R6/2 transgenic mouse model of HD.     

Demonstration of autoimmunity in the tight skin-2 mouse: a model for scleroderma

The tight skin-2 (Tsk2/+) mouse has been proposed as an animal model of systemic sclerosis (SSc) because this animal exhibits increased collagen synthesis and accumulation in the dermis. The Tsk2/+ mouse also has been reported to have a mononuclear cell infiltrate in the dermis; however, to date no evidence of autoimmunity has been described in this animal model. We report here that Tsk2/+ mice harbor numerous autoantibodies in their plasma including some, which are similar to those, present in SSc patients. Immunofluorescence with HEp-2 cells revealed the presence of anti-nuclear Abs (ANAs) in the plasma of 92% of the Tsk2/+ mice. In contrast, <5% of cage-mated CAST/ei mice had a positive ANA and none of the C3H/HeJ age-matched controls were positive. Homogenous, speckled, rim, nucleolar, centromere as well as combinations of these patterns were observed. The proportion of Tsk2/+ animals with a positive ANA increased slightly with age. ELISAs showed that 93% of the Tsk2/+ animals were positive for anti-Scl70, 82% for anti-centromere, 5% for anti-RNP/Sm, and none were positive for anti-RNA-polymerase II Abs. Indirect immunofluorescence with Crithidia luciliae and ELISA for anti-dsDNA Abs showed that 76% of Tsk2/+ mice were positive for this autoantibody. The high frequency of anti-Scl70 and anti-centromere autoantibodies indicates that Tsk2/+ mice display some humoral immune alterations which are similar to those found in patients with SSc. However, the Tsk2/+ mice also develop autoantibodies to dsDNA and a majority of the mice develop multiple autoantibody specificities (anti-Scl70, anti-CENP-B, and anti-dsDNA) indicating that the mouse may be a useful model to study autoimmunity in a wider spectrum of connective tissue diseases.     

Protective mechanisms of resveratrol against ischemia-reperfusion-induced damage in hearts obtained from Zucker obese rats: the role of GLUT-4 and endothelin

The resveratrol-induced cardiac protection was studied in Zucker obese rats. Rats were divided into five groups: group 1, lean control; group 2, obese control (OC); group 3, obese rats treated orally with 5 mg kg(-1) day(-1) of resveratrol (OR) for 2 wk; group 4, obese rats received 10% glucose solution ad libitum for 3 wk (OG); and group 5, obese rats received 10% glucose for 3 wk and resveratrol (OGR) during the 2nd and 3rd wk. Body weight, serum glucose, and insulin were measured, and then hearts were isolated and subjected to 30 min of ischemia followed by 120 min of reperfusion. Heart rate, coronary flow, aortic flow, developed pressure, the incidence of reperfusion-induced ventricular fibrillation, and infarct size were measured. Resveratrol reduced body weight and serum glucose in the OR compared with the OC values (414 +/- 10 g and 7.08 +/- 0.41 mmol/l, respectively, to 378 +/- 12 g and 6.11 +/- 0.44 mmol/l), but insulin levels were unchanged. The same results were obtained for the OG vs. OGR group. Resveratrol improved postischemic cardiac function in the presence or absence of glucose intake compared with the resveratrol-free group. The incidence of ventricular fibrillation and infarct size was reduced by 83 and 20% in the OR group, and 67 and 16% in the OGR group, compared with the OC and OG groups, respectively. Resveratrol increased GLUT-4 expression and reduced endothelin expression and cardiac apoptosis in ischemic-reperfused hearts in the presence or absence of glucose intake. Thus the protective effect of resveratrol could be related to its direct effects on the heart.     

Roles of Hoxa1 and Hoxa2 in patterning the early hindbrain of the mouse

Early in its development, the vertebrate hindbrain is transiently subdivided into a series of compartments called rhombomeres. Genes have been identified whose expression patterns distinguish these cellular compartments. Two of these genes, Hoxa1 and Hoxa2, have been shown to be required for proper patterning of the early mouse hindbrain and the associated neural crest. To determine the extent to which these two genes function together to pattern the hindbrain, we generated mice simultaneously mutant at both loci. The hindbrain patterning defects were analyzed in embryos individually mutant for Hoxa1 and Hoxa2 in greater detail and extended to embryos mutant for both genes. From these data a model is proposed to describe how Hoxa1, Hoxa2, Hoxb1, Krox20 (Egr2) and kreisler function together to pattern the early mouse hindbrain. Critical to the model is the demonstration that Hoxa1 activity is required to set the anterior limit of Hoxb1 expression at the presumptive r3/4 rhombomere boundary. Failure to express Hoxb1 to this boundary in Hoxa1 mutant embryos initiates a cascade of gene misexpressions that result in misspecification of the hindbrain compartments from r2 through r5. Subsequent to misspecification of the hindbrain compartments, ectopic induction of apoptosis appears to be used to regulate the aberrant size of the misspecified rhombomeres.