Influence of heart failure on nucleolar organization and protein expression in human hearts

We investigate for the first time the influence of heart failure (HF) on nucleolar organization and proteins in patients with ischemic (ICM) or dilated cardiomyopathy (DCM). A total of 71 human hearts from ICM (n=38) and DCM (n=27) patients, undergoing heart transplantation and control donors (n=6), were analysed by western-blotting, RT-PCR and cell biology methods. When we compared protein levels according to HF etiology, nucleolin was increased in both ICM (117%, p<0.05) and DCM (141%, p<0.01). Moreover, mRNA expression were also upregulated in ICM (1.46-fold, p<0.05) and DCM (1.70-fold, p<0.05. Immunofluorescence studies showed that the highest intensity of nucleolin was into nucleolus (p<0.0001), and it was increased in pathological hearts (p<0.0001). Ultrastructure analysis by electron microscopy showed an increase in the nucleus and nucleolus size in ICM (17%, p<0.05 and 131%, p<0.001) and DCM (56%, p<0.01 and 69%, p<0.01). Nucleolar organization was influenced by HF irrespective of etiology, increasing fibrillar centers (p<0.001), perinucleolar chromatin (p<0.01) and dense fibrillar components (p<0.01). Finally, left ventricular function parameters were related with nucleolin levels in ischemic hearts (p<0.0001). The present study demonstrates that HF influences on morphology and organization of nucleolar components, revealing changes in the expression and in the levels of nucleolin protein.     

Virus-induced dilated cardiomyopathy is characterized by increased levels of fibrotic extracellular matrix proteins and reduced amounts of energy-producing enzymes

The most relevant clinical phenotype resulting from chronic enteroviral myocarditis is dilated cardiomyopathy (DCM). Mice of the susceptible mouse strain A.BY/SnJ mimick well human DCM since they develop as a consequence of persistent infection and chronic inflammation a dilation of the heart ventricle several weeks after coxsackievirus B3 (CVB3) infection. Therefore, this model is well suited for the analysis of changes in the heart proteome associated with DCM. Here, we present a proteomic survey of the dilated hearts based on differential fluorescence gel electrophoresis and liquid chromatography-mass spectrometric centered methods in comparison to age-matched non-infected hearts. In total, 101 distinct proteins, which belong to categories immunity and defense, cell structure and associated proteins, energy metabolism and protein metabolism/modification differed in their levels in both groups. Levels of proteins involved in fatty acid metabolism and electron transport chain were found to be significantly reduced in infected mice suggesting a decrease in energy production in CVB3-induced DCM. Furthermore, proteins associated with muscle contraction (MLRV, MLRc2, MYH6, MyBPC3), were present in significantly altered amounts in infected mice. A significant increase in the level of extracellular matrix proteins in the dilated hearts indicates cardiac remodeling due to fibrosis.     

Induction of oxidative stress and disintegrin metalloproteinase in human heart end-stage failure

Collagen degradation is required for the creation of new integrin binding sites necessary for cell survival. However, a complete separation between the matrix and the cell leads to apoptosis, dilatation, and failure. Previous studies have demonstrated increased metalloproteinase activity in the failing myocardium. To test the hypothesis that disintegrin metalloproteinase (DMP) is induced in human heart end-stage failure, left ventricle tissue from ischemic cardiomyopathic (ICM, n = 10) and dilated cardiomyopathic (DCM, n = 10) human hearts were obtained at the time of orthotopic cardiac transplant. Normal (n = 5) tissue specimens were obtained from unused hearts. The levels of reduced oxygen species (ROS) were 12 +/- 2, 25 +/- 3, and 16 +/- 2 nmol (means +/- SE, P < 0.005) in normal, ICM, and DCM, respectively, by spectrofluorometry. The percent levels of endothelial cells were 100 +/- 15, 35 +/- 19, and 55 +/- 11 in normal, ICM, and DCM, respectively, by CD31 labeling. The levels of nitrotyrosine by Western analysis were significantly increased, and endothelial nitric oxide (NO) by the Griess method was decreased in ICM and DCM compared with normal tissue. The synthesis and degradation of beta(1)-integrin and connexin 43 were significantly increased in ICM and DCM compared with normal hearts by Western analysis. Levels of DMP were increased, and levels of cardiac inhibitor of metalloproteinase (CIMP) were decreased. Aggrecanase activity of DMP was significantly increased in ICM and DCM hearts compared with normal. These results suggest that the occurrence of cardiomyopathy is significantly confounded by the increase in ROS, nitrotyrosine, and DMP activity. This increase is associated with decreased NO, endothelial cell density, and CIMP. In vitro, treatment of CIMP abrogated the DMP activity. The treatment with CIMP may prevent degradation of integrin and connexin and ameliorate heart failure.     

Proteomic analysis reveals significant elevation of heat shock protein 70 in patients with chronic heart failure due to arrhythmogenic right ventricular cardiomyopathy

As proteins are the ultimate biological determinants of phenotype of disease, we screened altered proteins associated with heart failure due to arrhythmogenic right ventricular cardiomyopathy (ARVC) to identify biomarkers potential for rapid diagnosis of heart failure. By 2-dimensional gel electrophoresis and mass spectrometry, we identified five commonly altered proteins with more than 1.5 fold changes in eight ARVC failing hearts using eight non-failing hearts as reference. Noticeably, one of the altered proteins, heat shock protein 70 (HSP70), was increased by 1.64 fold in ARVC failing hearts compared with non-failing hearts. The increase of cardiac HSP70 was further validated by Western blot, immunochemistry, and enzyme-linked immunosorbent assay (ELISA) in failing hearts due to not only ARVC, but also dilated (DCM, n = 18) and ischemic cardiomyopathy (ICM, n = 8). Serum HSP70 was also observed to be significantly increased in heart failure patients derived from the three forms of cardiomyopathies. In addition, we observed hypoxia/serum depletion stimulation induced significantly elevation of intracellular and extracellular HSP70 in cultured neonatal rat cardiomyocytes. For the first time to our knowledge, we revealed and clearly demonstrated significant up-regulation of cardiac and serum HSP70 in ARVC heart failure patients. Our results indicate that elevated HSP70 is the common feature of heart failure due to ARVC, DCM, and ICM, which suggests that HSP70 may be used as a biomarker for the presence of heart failure due to cardiomyopathies of different etiologies and may hold diagnostic/prognostic potential in clinical practice.     

Content of myosin-activating protein kinases in myocardium of patients with dilated cardiomyopathy and in the animal heart

The skeletal myosin light chain kinase (skMLCK) was identified in human and chicken embryo myocardium but not in embryo and adult rat heart using western blotting. The content of skMLCK and myosin-activating protein kinases: RhaA-activated protein kinase (ROCK), integrin-linked protein kinase (ILK), and zipper-interacting protein kinase (ZIPK) was compared in normal human myocardium and the hearts of patients with dilated cardiomyopathy (DCM). It was demonstrated that the content of skMLCK, ROCK and ILK increases in DCM whereas the content of ZIPK decreases. The results obtained may reflect compensatory processes in cardiomyocytes in DMC, which are aimed at increasing their viability and contractility.     

Cardiac protein changes in ischaemic and dilated cardiomyopathy: a proteomic study of human left ventricular tissue

The development of heart failure (HF) is characterized by progressive alteration of left ventricle structure and function. Previous works on proteomic analysis in cardiac tissue from patients with HF remain scant. The purpose of our study was to use a proteomic approach to investigate variations in protein expression of left ventricle tissue from patients with ischaemic (ICM) and dilated cardiomyopathy (DCM). Twenty-four explanted human hearts, 12 from patients with ICM and 12 with DCM undergoing cardiac transplantation and six non-diseased donor hearts (CNT) were analysed by 2DE. Proteins of interest were identified by mass spectrometry and validated by Western blotting and immunofluorescence. We encountered 35 differentially regulated spots in the comparison CNT versus ICM, 33 in CNT versus DCM, and 34 in ICM versus DCM. We identified glyceraldehyde 3-phophate dehydrogenase up-regulation in both ICM and DCM, and alpha-crystallin B down-regulation in both ICM and DCM. Heat shock 70 protein 1 was up-regulated only in ICM. Ten of the eleven differentially regulated proteins common to both aetiologies are interconnected as a part of a same network. In summary, we have shown by proteomics analysis that HF is associated with changes in proteins involved in the cellular stress response, respiratory chain and cardiac metabolism. Although we found altered expression of eleven proteins common to both ischaemic and dilated aetiology, we also observed different proteins altered in both groups. Furthermore, we obtained that seven of these eleven proteins are involved in cell death and apoptosis processes, and therefore in HF progression.     

Expression and localization of Hsps in the heart and blood vessel of heat-stressed broilers

The objective of this study was to investigate the kinetics of Hsp60, Hsp70, Hsp90 protein, and messenger RNA (mRNA) expression levels and to correlate these heat shock protein (Hsp) levels with tissue damage resulting from exposure to high temperatures for varying amounts of time. One hundred broilers were heat-stressed for 0, 2, 3, 5, and 10 h, respectively, by rapidly increasing the ambient temperature from 22 +/- 1 degrees C to 37 +/- 1 degrees C. Obvious elevations of plasma creatine kinase indicate damage to myocardial cells after heat stress. Hsp70 and Hsp90, and their corresponding mRNAs in the heart tissue of heat-stressed broilers, elevated significantly after 2 h of heat exposure and decreased quickly with continued heat stress. However, the levels of hsp60 mRNA in the heart of heat-stressed broilers increased sharply (P < 0.01) at 2 h of heat stress but then decreased quickly after 3 h, while the level of Hsp60 protein in the heart increased (P < 0.01) at 2 h of heat stress and maintained a high level throughout heat exposure. The results indicate that the elevation of the three Hsps, especially Hsp60 in heart, may be important markers at the beginning of heat stress and act as protective proteins in adverse environments. The reduction of Hsp signals in the cytoplasm of myocardial cells implies that myocardial cell lesions may have an adverse impact on the function of Hsps during heat stress. Meanwhile, the localization of Hsp70 in blood vessels of broiler hearts suggests another possible mechanism for protection of the heart after heat exposure.     

Diabetic animal fed with high‐fat diet prevents the protective effect of myocardial ischemic preconditioning effect in isolated rat heart perfusion model

Diabetic heart (diabetes mellitus [DM]) has been shown to attenuate the beneficial effect of ischemic preconditioning (IPC) in rat heart. But the effect of IPC on diabetic rat heart that develops myopathy remains unclear. This study was designed to test the impact of IPC on diabetic cardiomyopathy (DCM) rat heart. Male Wistar rats were grouped as (a) normal, (b) DM (streptozotocin: 65 mg/kg; fed with normal diet), and (c) DCM (streptozotocin: 65 mg/kg; fed with high‐fat diet). Isolated rat hearts from each group were randomly subjected to (a) normal perfusion, (b) ischemia‐reperfusion (I/R), and (c) IPC procedure. At the end of the perfusion experiments, hearts were analyzed for injury, contractile function, mitochondrial activity, and oxidative stress. The results obtained from hemodynamics, cardiac injury markers, and caspase‐3 activity showed that DCM rat displayed prominent I/R‐associated cardiac abnormalities than DM rat heart. But the deteriorated physiological performance and cardiac injury were not recovered in both DM and DCM heart by IPC procedure. Unlike normal rat heart, IPC did not reverse mitochondrial dysfunction (determined by electron transport chain enzymes activity, ATP level, and membrane integrity, expression levels of genes like PGC‐1ɑ, GSK3β, complex I, II, and V) in DCM and DM rat heart. The present study demonstrated that IPC failed to protect I/R‐challenged DCM rat heart, and the underlying pathology was associated with deteriorated mitochondrial function.     

Cellular redistribution of inducible Hsp70 protein in the human and rabbit heart in response to the stress of chronic hypoxia: role of protein kinases,

Many infants who undergo cardiac surgery have a congenital cyanotic defect where the heart is chronically perfused with hypoxemic blood. Infant hearts adapt to chronic hypoxemia by activation of intracellular protein kinase signal transduction pathways. However, the involvement of heat shock protein 70 in adaptation to chronic hypoxemia and its role in protein kinase signaling pathways is unknown. We determined expression of message and subcellular protein distribution for inducible (Hsp70i) and constitutive heat shock protein 70 (Hsc70) in chronically hypoxic and normoxic infant human and rabbit hearts and their relationship to protein kinases. In chronically hypoxic human and rabbit hearts message levels for Hsp70i were elevated 4- to 5-fold compared with normoxic hearts, Hsp70i protein was redistributed from the particulate to the cytosolic fraction. In normoxic infants Hsp70i protein was distributed almost equally between the cytosolic and particulate fractions. Hsc70 message and subcellular distribution of Hsc70 protein were unaffected by chronic hypoxia. We then determined if protein kinases influence Hsp70i protein subcellular distribution. In rabbit hearts SB203580 and chelerythrine reduced Hsp70i message levels, whereas SB203580, chelerythrine, and curcumin reversed the subcellular redistribution of Hsp70i protein caused by chronic hypoxia, with no effect in normoxic hearts, indicating regulation of Hsp70i message and subcellular distribution of Hsp70i protein in chronically hypoxic rabbit hearts is influenced by protein kinase C and mitogen-activated protein kinases, specifically p38 MAPK and JNK. We conclude the Hsp70 signal transduction pathway plays an important role in adaptation of infant human and rabbit hearts to chronic hypoxemia.     

Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice

We recently reported that mice deficient in the programmed cell death-1 (PD-1) immunoinhibitory coreceptor develop autoimmune dilated cardiomyopathy (DCM), with production of high-titer autoantibodies against a heart-specific, 30-kDa protein. In this study, we purified the 30-kDa protein from heart extract and identified it as cardiac troponin I (cTnI), encoded by a gene in which mutations can cause familial hypertrophic cardiomyopathy (HCM). Administration of monoclonal antibodies to cTnI induced dilatation and dysfunction of hearts in wild-type mice. Monoclonal antibodies to cTnI stained the surface of cardiomyocytes and augmented the voltage-dependent L-type Ca2+ current of normal cardiomyocytes. These findings suggest that antibodies to cTnI induce heart dysfunction and dilatation by chronic stimulation of Ca2+ influx in cardiomyocytes.     

Heat shock proteins in cell signaling and cancer

Heat Shock Proteins (HSPs) and their co-chaperones have well-established roles in regulating proteostasis within the cell, the nature of which continues to emerge with further study. To date, HSPs have been shown to be integral to protein folding and re-folding, protein transport, avoidance of protein aggregation, and modulation of protein degradation. Many cell signaling events are mediated by the chemical modification of proteins post-translationally that can alter protein conformation and activity, although it is not yet known whether the changes in protein conformation induced by post-translational modifications (PTMs) are also dependent upon HSPs and their co-chaperones for subsequent protein re-folding. We discuss what is known regarding roles for HSPs and other molecular chaperones in cell signaling events with a focus on oncogenic signaling. We also propose a hypothesis by which Hsp70 and Hsp90 may co-operate to facilitate cell signaling events that may link PTMs with the cellular protein folding machinery.     

Altered interleukin-1 receptor antagonist and interleukin-18 mRNA expression in myocardial tissues of patients with dilatated cardiomyopathy

Interleukin-1 (IL-1) is a potent regulator of cell proliferation, inflammation, and contraction of cardiovascular cells. It has been proposed that the IL-1/IL-1ra (IL-1 receptor antagonist) ratio influences these functions. Other members of the IL-1 family and the related caspase-1 also contribute to regulation of IL-1-mediated functions. We determined the mRNA expression of caspase-1, caspase-3, IL-1alpha , IL-1beta , IL-18, IL-1 receptor type I (IL-1-RI), and IL-1ra in left ventricle tissue of hearts from patients with ischemic or dilated cardiomyopathy (ICM or DCM) and in control tissues from unused donor transplant hearts in RT-PCR experiments. We show that the expression of caspase-1, caspase-3, IL-1beta , and IL-1-RI mRNA was not different between patients and control tissues. Furthermore, we did not find detectable amounts of IL-1alpha mRNA in any of these adult myocardial tissues. On the other hand, expression of IL-18 RNA was lower in myocardium of both patient groups compared with control hearts. Furthermore, IL-1ra mRNA expression was significantly lower in tissues of DCM patients compared with ICM patients and controls. This was in line with a trend towards lower IL-1ra protein levels in myocardial tissues of DCM patients. In contrast with the adult tissues discussed above, which did not express IL-1alpha mRNA, commercially available human fetal tissue expressed IL-1alpha mRNA. On the other hand IL-1beta mRNA was present in fetal and in adult human heart tissue. Our data provide evidence for an altered ratio of IL-1/IL-1ra in DCM patients. This dysregulation may contribute to pathogenesis and/or progression of heart disease by modulating the otherwise balanced IL-1-mediated functions in cardiovascular cells.     

Cell type-specific functions of the lysosomal protease cathepsin L in the heart

Deficiency of the lysosomal cysteine protease cathepsin L (Ctsl) in mice results in a phenotype affecting multiple tissues, including thymus, epidermis, and hair follicles, and in the heart develops as a progressive dilated cardiomyopathy (DCM). To understand the role of Ctsl in the maintenance of regular heart morphology and function, it is critical to determine whether the DCM in Ctsl-/- mice is primarily because of the lack of Ctsl expression and activity in the cardiomyocytes or is caused by the additional extracardiac pathologies. Cardiomyocyte-specific expression of Ctsl in Ctsl-/- mice, using an alpha-myosin heavy chain promoter-Ctsl transgene, results in improved cardiac contraction, normal mRNA expression of atrionatriuretic peptide, normal heart weight, and regular ultrastructure of cardiomyocytes. Epithelial expression of cathepsin L2 (CTSL2) by a K14 promoter-CTSL2-transgene resulted in rescue of the Ctsl-/- hair loss phenotype. In these mice, cardiac atrionatriuretic peptide expression and end systolic heart dimensions were also significantly attenuated. However, cardiac contraction was not improved, and increased heart weight as well as the typical changes in lysosomal ultrastructure of Ctsl-/- hearts persisted. Myocardial fibrosis was detected in all Ctsl-/- mice irrespective of transgene-mediated cardiac Ctsl expression or extracardiac CTSL2 expression. Expression of collagen 1 was not enhanced in Ctsl-/- hearts, but a reduced collagenolytic activity suggests a role for Ctsl in collagen turnover by cardiac fibroblasts. We conclude that the DCM of Ctsl-/- mice is primarily caused by absence of the protease in cardiomyocytes, whereas the complex gross phenotype of Ctsl-deficient mice, i.e. the fur defect, results in additional stress to the heart.     

Proteomic analysis of rat heart in ischemia and ischemia-reperfusion using fluorescence two-dimensional difference gel electrophoresis

Ischemia-reperfusion injury is a major complication occurring in acute myocardial infarction, cardiopulmonary bypass surgery, and heart transplantation. The aim of this study was to identify proteins that were involved in ischemia-reperfusion injury using fluorescence two-dimensional difference gel electrophoresis. We compared the 100,000 x g precipitate fractions of normal, ischemic and ischemia-reperfused rat hearts and detected six spots which changed more than two-fold in expression level and two additional spots related to these spots. Using peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, we identified five of these spots as protein disulfide isomerase A3 (PDA3), one as 60 kDa heat shock protein (HSP60) and two as elongation factor Tu (EF-Tu). HSP60 was increased during ischemia and decreased to normal expression level after reperfusion. EF-Tu was increased in ischemia but not decreased by reperfusion. We also found that several protein spots of PDA3 shifted towards a higher isoelectric point in ischemia and ischemia-reperfusion. Our data strongly suggested that PDA3 underwent dephosphorylation during ischemia and reperfusion and serine 343 of PDA3 was one of the phosphorylation sites.     

几种热激蛋白在细胞凋亡信号通路中的调控作用

热激蛋白(heat shock proteins, HSPs)作为进化保守的蛋白家族 之一,普遍存在于各种生物体中,并在生物体内发挥着重要的生理功能.大 量的实验证据表明,热激蛋白与细胞凋亡密切相关,参与细胞凋亡信号通 路的多个环节. 近年来有关该领域的研究已获得了重要的突破与进展.一方 面,热激蛋白主要起着抑制细胞凋亡、促进细胞存活的作用;另一方面, 某些热激蛋白又能够作为凋亡蛋白的分子伴侣,促进细胞凋亡,比如HSP70 能够激活DNase来促使细胞凋亡,线粒体内HSP60能够促进caspase依赖的细 胞凋亡途径.本文在阐明细胞凋亡信号通路的基础上,综述了近年来几种不 同热激蛋白家族（HSP90、 HSP70 、HSP60和小分子HSPs）在细胞凋亡调控 中作用的研究进展,重点阐述了几种主要热激蛋白与细胞凋亡信号通路上 相关因子的相互作用,并绘制了热激蛋白在细胞凋亡信号通路中的调控图 ,为进一步完善细胞凋亡调控网络研究提供一定的参考.