Cyclosporin A inhibits cardiac hypertrophy and enhances cardiac dysfunction during postinfarction failure in rats

Calcineurin has recently been implicated as a mediator in the signaling pathways that transform intracellular calcium signals to the phenotype of myocardial hypertrophy. The present study was conducted to examine the effects of cyclosporin A (CsA), an inhibitor of calcineurin, on myocardial hypertrophy and remodeling during congestive heart failure (CHF) in rats. After ligation of the left coronary artery, rats were randomized to treatment with CsA or vehicle for 14 days. Compared with vehicle, CsA substantially attenuated myocardial hypertrophy in the CHF rats as assessed by alterations in ventricular weight-to-tibial length ratios (P < 0.05). Myocardial gene expression of skeletal alpha-actin was also reduced in the failing left ventricle (LV) after treatment with CsA (P < 0. 05), although the mRNA levels were still substantially elevated relative to those of sham rats. CsA-induced inhibition of compensatory myocardial hypertrophy in the CHF rats led to increased dilatation of the LV cavity and reduced fractional shortening and peak positive and negative first derivatives of LV pressure (P < 0. 05). Plasma renin and endothelin-1 levels were increased in the CHF-CsA rats, providing humoral cues of aggravated cardiac function. Thus this study supports a crucial role of calcineurin-dependent pathways in the mechanisms of compensatory myocardial hypertrophy during CHF. In addition, our data indicate that inhibition of compensatory myocardial hypertrophy exerts detrimental effects on cardiac remodeling and function after myocardial infarction.     

Alterations induced by cyclosporine A in myocardial fibers and extracellular matrix in rat

Cyclosporine A (CsA) is the first choice immunosuppressant universally used in allotransplantation. However, it has been demonstrated that this drug produces unwanted side effects in several organs and in particular in the kidney and in the heart. While the cardiac toxicity, due to alteration of myocardial prostanoid has been reported, no data are available about the effects of CsA on myocardial cytoarchitecture. We studied the CsA induced alterations of the myocardial structure and of the extracellular matrix components (ECM). To test the ECM enzymatic chances we studied a family of enzymes (matrix metalloproteinase-MMP), responsible for the degradation of extracellular matrix components. In particular we investigated MMPI, MMP2 and MMP9. The study was carried out on two groups of Wistar rats. The group I animals served as a control and were injected subcutaneously daily with castor oil for 21 days. Group II: animals were subcutaneously injected daily with CsA (dose: 15 mg/Kg in castor oil) for 21 days. The group I animals (control) had normal heart architecture and low levels of MMPI, MMP2 and MMP9. The group II animals showed degenerative changes with myocardial fibrosis, low levels of MMP1 and MMP9 but a clear increase in MMP2. We suggest that the myocardial fibrosis was a consequence of the cardiotoxic effect of CsA determining the alteration of the balance between synthesis and degradation of ECM. The increase in MMP2 suggests that this enzyme could play a protective role during myocardial damage and represent a compensatory mechanism for the excessive accumulation of collagen.     

Inhibition of histone deacetylases promotes ubiquitin-dependent proteasomal degradation of DNA methyltransferase 1 in human breast cancer cells

Histone deacetylases (HDAC) play a critical role in chromatin modification and gene expression. Recent evidence indicates that HDACs can also regulate functions of nonhistone proteins by catalyzing the removal of acetylated lysine residues. Here, we show that the HDAC inhibitor LBH589 down-regulates DNA methyltransferase 1 (DNMT1) protein expression in the nucleus of human breast cancer cells. Cotreatment with the proteasomal inhibitor MG-132 abolishes the ability of LBH589 to reduce DNMT1, suggesting that the proteasomal pathway mediates DNMT1 degradation on HDAC inhibition. Deletion of the NH(2)-terminal 120 amino acids of DNMT1 diminishes LBH589-induced ubiquitination, indicating that this domain is essential for its proteasomal degradation. DNMT1 recruits the molecular chaperone heat shock protein 90 (Hsp90) to form a chaperone complex. Treatment with LBH589 induces hyperacetylation of Hsp90, thereby inhibiting the association of DNMT1 with Hsp90 and promoting ubiquitination of DNMT1. In addition, inactivation of HDAC1 activity by small interfering RNA and MS-275 is associated with Hsp90 acetylation in conjunction with reduction of DNMT1 protein expression. We conclude that the stability of DNMT1 is maintained in part through its association with Hsp90. Disruption of Hsp90 function by HDAC inhibition is a unique mechanism that mediates the ubiquitin-proteasome pathway for DNMT1 degradation. Our studies suggest a new role for HDAC1 and identify a novel mechanism of action for the HDAC inhibitors as down-regulators of DNMT1.     

Inhibition of heat shock protein (Hsp) 27 potentiates the suppressive effect of Hsp90 inhibitors in targeting breast cancer stem-like cells

Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44 + cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. In the present study, we discovered that the expression of Hsp90α was increased in ALDH + human breast cancer cells. Geldanamycin (GA), a Hsp90 inhibitor, could suppress ALDH + breast cancer cells in a dose dependent manner. We are interesting in the insufficiently inhibitory effect of low dose GA treatment. It was correlated with the upregulation of Hsp27 and Hsp70. By co-treatment with HSP inhibitors, quercetin or KNK437 potentiated BCSCs, which determined with ALDH+ population or mammosphere cells, toward GA inhibition, as well as anti-proliferation and anti-migration effects of GA. With siRNA mediated gene silencing, we found that knockdown of Hsp27 could mimic the effect of HSP inhibitors to potentiate the BCSC targeting effect of GA. In conclusion, combination of HSP inhibitors with Hsp90 inhibitors could serve as a potential solution to prevent the drug resistance and avoid the toxicity of high dose of Hsp90 inhibitors in clinical application. Furthermore, Hsp27 may play a role in chemoresistant character of BCSCs.     

Methylmercury Alters the Activities of Hsp90 Client Proteins,Prostaglandin E Synthase/p23 (PGES/23) and nNOS

Methylmercury (MeHg) is a persistent pollutant with known neurotoxic effects. We have previously shown that astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS) by altering glutamate signaling, generating oxidative stress, depleting glutathione (GSH) and initiating lipid peroxidation. Interestingly, all of these pathways can be regulated by the constitutively expressed, 90-kDa heat shock protein, Hsp90. As Hsp90 function is regulated by oxidative stress, we hypothesized that MeHg disrupts Hsp90-client protein functions. Astrocytes were treated with MeHg and expression of Hsp90, as well as the abundance of complexes of Hsp90-neuronal nitric oxide synthase (nNOS) and Hsp90-prostaglandin E synthase/p23 (PGES/p23) were assessed. MeHg exposure decreased Hsp90 protein expression following 12 h of treatment while shorter exposures had no effect on Hsp90 protein expression. Interestingly, following 1 or 6 h of MeHg exposure, Hsp90 binding to PGES/p23 or nNOS was significantly increased, resulting in increased prostaglandin E₂ (PGE₂) synthesis from MeHg-treated astrocytes. These effects were attenuated by the Hsp90 antagonist, geldanmycin. NOS activity was increased following MeHg treatment while cGMP formation was decreased. This was accompanied by an increase in •O₂ ⁻ and H₂O₂ levels, suggesting that MeHg uncouples NO formation from NO-dependent signaling and increases oxidative stress. Altogether, our data demonstrates that Hsp90 interactions with client proteins are increased following MeHg exposure, but over time Hsp90 levels decline, contributing to oxidative stress and MeHg-dependent excitotoxicity.     

Chronic Treatment with Novel Small Molecule Hsp90 Inhibitors Rescues Striatal Dopamine Levels but Not α-Synuclein-Induced Neuronal Cell Loss

Hsp90 inhibitors such as geldanamycin potently induce Hsp70 and reduce cytotoxicity due to α-synuclein expression, although their use has been limited due to toxicity, brain permeability, and drug design. We recently described the effects of a novel class of potent, small molecule Hsp90 inhibitors in cells overexpressing α-synuclein. Screening yielded several candidate compounds that significantly reduced α-synuclein oligomer formation and cytotoxicity associated with Hsp70 induction. In this study we examined whether chronic treatment with candidate Hsp90 inhibitors could protect against α-synuclein toxicity in a rat model of parkinsonism. Rats were injected unilaterally in the substantia nigra with AAV8 expressing human α-synuclein and then treated with drug for approximately 8 weeks by oral gavage. Chronic treatment with SNX-0723 or the more potent, SNX-9114 failed to reduce dopaminergic toxicity in the substantia nigra compared to vehicle. However, SNX-9114 significantly increased striatal dopamine content suggesting a positive neuromodulatory effect on striatal terminals. Treatment was generally well tolerated, but higher dose SNX-0723 (6–10 mg/kg) resulted in systemic toxicity, weight loss, and early death. Although still limited by potential toxicity, Hsp90 inhibitors tested herein demonstrate oral efficacy and possible beneficial effects on dopamine production in a vertebrate model of parkinsonism that warrant further study.     

Hsp10 and Hsp60 suppress ubiquitination of insulin-like growth factor-1 receptor and augment insulin-like growth factor-1 receptor signaling in cardiac muscle: implications on decreased myocardial protection in diabetic cardiomyopathy

We have investigated the effects of two heat shock proteins, Hsp10 and Hsp60, on insulin-like growth factor-1 receptor (IGF-1R) signaling in cardiac muscle cells. Neonatal cardiomyocytes were transduced with Hsp10 or Hsp60 via adenoviral vector. Compared with the cells transduced with a control vector, overexpression of Hsp10 or Hsp60 increased the abundance of IGF-1R and IGF-1-stimulated receptor autophosphorylation. Thus, Hsp10 and Hsp60 overexpression increased the number of functioning receptors and amplified activation of IGF-1R signaling. IGF-1 stimulation of MEK, Erk, p90Rsk, and Akt were accordingly augmented. Transducing cardiomyocytes with antisense Hsp60 oligonucleotides reduced Hsp60 expression, decreased the abundance of IGF-1R, attenuated IGF-1R autophosphorylation, and suppressed the pro-survival action of IGF-1 in cardiomyocytes. Using cycloheximide to inhibit protein synthesis did not alter the effect of Hsp60 on IGF-1R signaling, and IGF-1R mRNA levels were not up-regulated by Hsp10 or Hsp60. Additional experiments showed that Hsp10 and Hsp60 suppressed polyubiquitination of IGF-1 receptor. These data indicate that Hsp10 and Hsp60 can modulate IGF-1R signaling through post-translational modification. In animal models of diabetes, diabetic myocardium is associated with decreased abundance of Hsp60, increased ubiquitination of IGF-1R, and lower level of IGF-1R protein. Declined myocardial protection is a major feature of diabetic cardiomyopathy. These data suggest that decreased Hsp60 expression and subsequent decline of IGF-1R signaling may be a fundamental mechanism underlying the development of diabetic cardiomyopathy.     

Water-restraint stress enhances methamphetamine-induced cardiotoxicity

Methamphetamine (MAP) and stress both cause a variety of cardiovascular problems. Stress also increases stimulant drug-seeking or drug-taking behavior by both humans and animals. In addition to the physiological effects on circulation, metabolism, and excretion, stress affects subject's responses to stimulant drugs such as MAP. However, the mechanisms underlying the drug–stress interactions remain unknown. In the present study, we assessed the effects of stress on myocardial responses to MAP in mice. Mice were injected with MAP (30 mg/kg) immediately before exposure to water-restraint stress (WRS), which has often been used as a stressor in animal experiments. The combination of MAP with WRS produced a significant increase (p < 0.01) in the leakage of proteins specific to myocardial damage and the levels of cytokines IL-6, TNF-α, and IL-10. The histological findings indicated the possibility that a combination of MAP with WRS induced cardiac myocytolysis. We also examined the expression of heat shock proteins (Hsps), which have cardioprotective effects. Administration of MAP alone significantly stimulated the RNA expressions of Hsp32, 60, 70, and 90 and the protein Hsp70 in cardiac muscles, whereas the expressions due to WRS or MAP plus WRS were not increased. These results reveal the fact that exposure to WRS depresses the induction of Hsps, in particular Hsp70, due to MAP injection, following to enhance MAP-induced myocardial damage. We believe that interactions between MAP and severe stress, including environmental temperature, affect the induction of Hsps, following to susceptibility of hosts to cardiotoxicity due to the stimulant drug.     

Expression and distribution of heat shock proteins in the heart of transported pigs

The expression and localization of four heat shock proteins (Hsp70, Hsp86, Hsp90, and Hsp27) were shown in the heart tissue of pigs transported for 6 h. Immunostaining detected the consistent presence of all Hsps in the pig myocardial cells under both transported and normal housing conditions. Immunohistochemical analysis revealed predominance of Hsp70 (significantly highest levels) and Hsp27 in the cytoplasm of myocardial cells. Hsp90 and Hsp86 were expressed both in the cytoplasm and in the nucleus, preferentially in the cytoplasm, of the myocardial cells. In view of their abundant and uniform distributions in the myocardial cells, the expression and distribution patterns of all detected Hsps within the myocardial cells, mostly limited to the cytoplasm, could be related to their chaperone function for cells with important special activities in this study. The identification of all four Hsps in the blood vessel endothelial cells possibly implies that endothelial cells react to ischemia and hypoxia by expressing Hsps. Immunoblot findings suggest that the level of all Hsps decreased in response to stress due to a 6 h journey. The decrease in Hsp levels in the myocardial cells may indicate that the transport stress may have overcharged the repair mechanisms of the cells. Whether this distinct depletion of Hsps contributes to an increased susceptibility to acute heart failure and the sudden death syndrome in transported pigs should be elucidated in future experiments.     

Overexpression of angiopoietin-1 increases CD133+/c-kit+ cells and reduces myocardial apoptosis in db/db mouse infarcted hearts

Hematopoietic progenitor CD133(+)/c-kit(+) cells have been shown to be involved in myocardial healing following myocardial infarction (MI). Previously we demonstrated that angiopoietin-1(Ang-1) is beneficial in the repair of diabetic infarcted hearts. We now investigate whether Ang-1 affects CD133(+)/c-kit(+) cell recruitment to the infarcted myocardium thereby mediating cardiac repair in type II (db/db) diabetic mice. db/db mice were administered either adenovirus Ang-1 (Ad-Ang-1) or Ad-β-gal systemically immediately after ligation of the left anterior descending coronary artery (LAD). Overexpression of Ang-1 resulted in a significant increase in CXCR-4/SDF-1α expression and promoted CD133(+)/c-kit(+), CD133(+)/CXCR-4(+) and CD133(+)/SDF-1α(+) cell recruitment into ischemic hearts. Overexpression of Ang-1 led to significant increases in number of CD31(+) and smooth muscle-like cells and VEGF expression in bone marrow (BM). This was accompanied by significant decreases in cardiac apoptosis and fibrosis and an increase in myocardial capillary density. Ang-1 also upregulated Jagged-1, Notch3 and apelin expression followed by increases in arteriole formation in the infarcted myocardium. Furthermore, overexpression of Ang-1 resulted in a significant improvement of cardiac functional recovery after 14 days of ischemia. Our data strongly suggest that Ang-1 attenuates cardiac apoptosis and promotes cardiac repair by a mechanism involving in promoting CD133(+)/c-kit(+) cells and angiogenesis in diabetic db/db mouse infarcted hearts.     

PKA phosphorylation of the small heat-shock protein Hsp20 enhances its cardioprotective effects

The small heat-shock protein Hsp20 (heat-shock protein 20), also known as HspB6, has been shown to protect against a number of pathophysiological cardiac processes, including hypertrophy and apoptosis. Following β-adrenergic stimulation and local increases in cAMP, Hsp20 is phosphorylated on Ser16 by PKA (protein kinase A). This covalent modification is required for many of its cardioprotective effects. Both Hsp20 expression levels and its phosphorylation on Ser16 are increased in ischaemic myocardium. Transgenic mouse models with cardiac-specific overexpression of Hsp20 that are subject to ischaemia/reperfusion show smaller myocardial infarcts, and improved recovery of contractile performance during the reperfusion phase, compared with wild-type mice. This has been attributed to Hsp20's ability to protect against cardiomyocyte necrosis and apoptosis. Phosphomimics of Hsp20 (S16D mutants) confer improved protection from β-agonist-induced apoptosis in the heart, whereas phospho-null mutants (S16A) provide no protection. Naturally occurring mutants of Hsp20 at position 20 (P20L substitution) are associated with markedly reduced Hsp20 phosphorylation at Ser16, and this lack of phosphorylation correlates with abrogation of Hsp20's cardioprotective effects. Therefore phosphorylation of Hsp20 at Ser16 by PKA is vital for the cardioprotective actions of this small heat-shock protein. Selective targeting of signalling elements that can enhance this modification represents an exciting new therapeutic avenue for the prevention and treatment of myocardial remodelling and ischaemic injury.     

Myocardial accumulation and localization of the inducible 70-kDa heat shock protein, Hsp70, following exercise

Exercise increases the 70-kDa heat shock protein (Hsp70) in the myocardium, and this exercise-induced increase is associated with significantly improved cardiac recovery following insult. However, while heat shock has been shown to elevate Hsp70 primarily in the cardiac vasculature of the myocardium, the localization following exercise is unknown. Male Sprague-Dawley rats performed continuous treadmill running at 30 m/min for 60 min (2% incline) on either 1 or 5 consecutive days. At 30 min and 24 h following exercise, hearts were extirpated, and the left ventricle was isolated, OCT-cork mounted, and sectioned for immunofluorescent analysis. Whereas immunofluorescent analysis revealed little to no Hsp70 in control hearts and 30 min postexercise, the accumulation of Hsp70 24 h after a single exercise bout or 5 days of training was predominantly located in large blood vessels and, in particular, colocalized with a marker of smooth muscle. Furthermore, higher core temperatures attained during exercise led to more abundant accumulation in smaller vessels and the endothelium. It is concluded that the accumulation of myocardial Hsp70 following acute exercise predominantly occurs in a cell type-specific manner, such that changes in the cardiac vasculature account for much of the increase. This accumulation appears first in the smooth muscle of larger vessels and then increases in smaller vessels and the endothelium, as core temperature attained during exercise increases. This finding supports the observations after heat shock and further suggests that the vasculature is a primary target in exercise-induced cardioprotection.     

Selection Transforms the Landscape of Genetic Variation Interacting with Hsp90

The protein-folding chaperone Hsp90 has been proposed to buffer the phenotypic effects of mutations. The potential for Hsp90 and other putative buffers to increase robustness to mutation has had major impact on disease models, quantitative genetics, and evolutionary theory. But Hsp90 sometimes contradicts expectations for a buffer by potentiating rapid phenotypic changes that would otherwise not occur. Here, we quantify Hsp90’s ability to buffer or potentiate (i.e., diminish or enhance) the effects of genetic variation on single-cell morphological features in budding yeast. We corroborate reports that Hsp90 tends to buffer the effects of standing genetic variation in natural populations. However, we demonstrate that Hsp90 tends to have the opposite effect on genetic variation that has experienced reduced selection pressure. Specifically, Hsp90 tends to enhance, rather than diminish, the effects of spontaneous mutations and recombinations. This result implies that Hsp90 does not make phenotypes more robust to the effects of genetic perturbation. Instead, natural selection preferentially allows buffered alleles to persist and thereby creates the false impression that Hsp90 confers greater robustness.     

Geldanamycin, an inhibitor of Hsp90 increases cytochrome P450 2E1 mediated toxicity in HepG2 cells through sustained activation of the p38MAPK pathway

Cytochrome P450 2E1 (CYP2E1) can mediate reactive oxygen species (ROS) induced cell death through its catalytic processes. Heat shock protein 90 (Hsp90) is an important molecular chaperone which is essential for cellular integrity. We previously showed that inhibition of Hsp90 with Geldanamycin (GA), an inhibitor of Hsp90 increased CYP2E1 mediated toxicity in CYP2E1 over-expressing HepG2 cells (E47 cells) but not in C34-HepG2 cells devoid of CYP2E1 expression. The aim of the present study was to test the hypothesis that the potentiation of CYP2E1 toxicity in E47 cells with GA may involve changes in mitogen activated protein kinase signal transduction pathways. GA was toxic to E47 cells and SB203580, an inhibitor of p38 MAPK prevented this decrease in viability. The protective effects of SB203580 were effective only when SB203580 was added before GA treatment. GA activated p38 MAPK in E47 cells and this activation was an early and a sustained event. GA elevated ROS levels and lipid peroxidation and lowered GSH levels in E47 cells and these changes were blunted or prevented by treatment with SB203580. Apoptosis was increased by GA and prevented by pre-treatment with SB203580. The loss in mitochondrial membrane potential in E47 cells after GA treatment was also decreased significantly with SB203580 treatment. The activity and expression of CYP2E1 and Hsp90 levels were not altered by SB203580. In conclusion, the inhibition of Hsp90 with GA increases the toxicity of CYP2E1 in HepG2 cells through an early and sustained activation of the p38 MAPK pathway.     

Role of Hsp90 in Biogenesis of the β-Cell ATP-sensitive Potassium Channel Complex

The pancreatic β-cell ATP-sensitive potassium (K_ATP) channel is a multimeric protein complex composed of four inwardly rectifying potassium channel (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits. K_ATP channels play a key role in glucose-stimulated insulin secretion by linking glucose metabolism to membrane excitability. Many SUR1 and Kir6.2 mutations reduce channel function by disrupting channel biogenesis and processing, resulting in insulin secretion disease. To better understand the mechanisms governing K_ATP channel biogenesis, a proteomics approach was used to identify chaperone proteins associated with K_ATP channels. We report that chaperone proteins heat-shock protein (Hsp)90, heat-shock cognate protein (Hsc)70, and Hsp40 are associated with β-cell K_ATP channels. Pharmacologic inhibition of Hsp90 function by geldanamycin reduces, whereas overexpression of Hsp90 increases surface expression of wild-type K_ATP channels. Coimmunoprecipitation data indicate that channel association with the Hsp90 complex is mediated through SUR1. Accordingly, manipulation of Hsp90 protein expression or function has significant effects on the biogenesis efficiency of SUR1, but not Kir6.2, expressed alone. Interestingly, overexpression of Hsp90 selectively improved surface expression of mutant channels harboring a subset of disease-causing SUR1 processing mutations. Our study demonstrates that Hsp90 regulates biogenesis efficiency of heteromeric K_ATP channels via SUR1, thereby affecting functional expression of the channel in β-cell membrane.