43 research outputs found
Divide and conquer: the application of organelle proteomics to heart failure.
No full textChronic heart failure is a worldwide cause of mortality and morbidity and is the final outcome of a number of different etiologies. This reflects both the complexity of the disease and our incomplete understanding of its underlying molecular mechanisms. One experimental approach to address this is to study subcellular organelles and how their functions are activated and synchronized under physiological and pathological conditions. In this review, we discuss the application of proteomic technologies to organelles and how this has deepened our perception of the cellular proteome and its alterations with heart failure. The use of proteomics to monitor protein quantity and posttranslational modificationsb has revealed a highly intricate and sophisticated level of protein regulation.
Posttranslational modifications have the potential to regulate organelle function and interplay most likely by targeting both structural andnsignaling proteins throughout the cell, ultimately coordinating their responses. The potentials and limitations of existing proteomic technologies are also discussed emphasizing that the development of novel methods will enhance our ability to further investigate organelles and decode intracellular communication
Modificazioni post-traduzionali dei filamenti intermedi: chiave di volta nell’insufficienza cardiaca?
No full textOptimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome.
No full textSeparation of basic proteins with 2-DE presents technical challenges involving protein precipitation, load limitations, and streaking. Cardiac mitochondria are enriched in basic proteins and difficult to resolve by 2-DE. We investigated two methods, cup and paper bridge, for sample loading of this subproteome into the basic range (pH 6-11) gels. Paper bridge loading consistently produced improved resolution of both analytical and preparative protein loads. A unique benefit of this technique is that proteins retained in the paper bridge after loading basic gels can be reloaded onto lower pH gradients (pH 4-7), allowing valued samples to be analyzed on multiple pH ranges
Genetics, genomics and proteomics in sudden cardiac death
No full textThis chapter describes the Genetics, genomic and proteomics aspect related to the Sudden Cardiac Deat
Modulation of mitochondrial proteome and improved mitochondrial function by biventricular pacing of dyssynchronous failing hearts.
No full textBACKGROUND: Cardiac resynchronization therapy (CRT) improves chamber mechanoenergetics and morbidity and mortality of patients manifesting heart failure with ventricular dyssynchrony; however, little is known about the molecular changes underlying CRT benefits. We hypothesized that mitochondria may play an important role because of their involvement in energy production.
METHODS AND RESULTS: Mitochondria isolated from the left ventricle in a canine model of dyssynchronous or resynchronized (CRT) heart failure were analyzed by a classical, gel-based, proteomic approach. Two-dimensional gel electrophoresis revealed that 31 mitochondrial proteins where changed when controlling the false discovery rate at 30%. Key enzymes in anaplerotic pathways, such as pyruvate carboxylation and branched-chain amino acid oxidation, were increased. These concerted changes, along with others, suggested that CRT may increase the pool of Krebs cycle intermediates and fuel oxidative phosphorylation. Nearly 50% of observed changes pertained to subunits of the respiratory chain. ATP synthase-beta subunit of complex V was less degraded, and its phosphorylation modulated by CRT was associated with increased formation (2-fold, P=0.004) and specific activity (+20%, P=0.05) of the mature complex. The importance of these modifications was supported by coordinated changes in mitochondrial chaperones and proteases. CRT increased the mitochondrial respiratory control index with tightened coupling when isolated mitochondria were reexposed to substrates for
both complex I (glutamate and malate) and complex II (succinate), an effect likely related to ATP synthase subunit modifications and complex quantity and activity.
CONCLUSIONS: CRT potently affects both the mitochondrial proteome and the performance associated with improved cardiac function
Novel human and canine desmin phosphorylations as potential regulators of oligomer formation and disease in heart failure
No full textCardiac troponins in intensive care
No full textThe cardiac troponins are integral components of the myofibrillary apparatus and they regulate muscle contraction. The measurement of cardiac troponins has replaced other biomarkers for the specific detection of myocardial necrosis and for the diagnosis of myocardial infarction. The tissue specificity plus sensitivity of the measurement technology has meant that cardiac damage can be detected in circumstances other than conventional acute coronary syndromes. The ability to specifically detect cardiac damage as part of multiple organ failure in intensive care patients has been shown to provide prognostic information, but it is unclear whether this is a dependent or an independent marker of outcome
Modified desmin pre-amyloid oligomers are increased in experimental heart failure
No full textObjectives: Heart Failure (HF) is the major cause of hospitalization in the US but the molecular mechanisms underlying its development are unclear to date. The increased formation of preamyloid oligomers (PAOs), similar to those observed in Alzheimer's disease, have been reported in several different models of HF. We first reported that the induction of desmin phosphorylation at serines (S) 27 and 31 associate with increased cardiac PAOs deposition in experimental, non-genetic HF. We now show that modified desmin is a likely candidate for the seed initiating the nucleation of toxic PAOs in the heart and isolated cardiac cells.
Materials and methods: We subjected mice to transverse aortic constriction (TAC) for 4 weeks (FS% = 29.3 ± 2.6, P = 0.0001) and transduced neonatal rat ventricular myocytes (NRVMs) using lentiviral vectors carrying alanine (A) or phospho-mimetic aspartate (D) desmin double mutants at S27 and S31, fused with GFP. We analyzed the formation of PAOs in cardiac protein extracts by western blot analysis combined with infrared detection, which enabled the contemporary measurement of PAOs and desmin. We also monitored the effects of phospho-mimetic desmin expression in NRVMs by live imaging.
Results: Co-staining for both desmin and PAOs in TAC mice and NRVMs confirmed the co-migration (by molecular weight) of PAOs with modified desmin, along with their increase in experimental HF (≈3-fold, P = 0.023 and ≈ 2-fold, P = 0.038, respectively). Cells expressing the doubly phospho-mimetic mutant, which we believe is the physiological form, displayed a “healthier” phenotype as docu- mented by the number of contracting cells (P = 0.041) and localiza- tion of GFP desmin at the Z-bands (P = 0.0027). On the contrary the expression of mono-phosphomimetic mutant (S27A, S31D) induced increased desmin aggregation (P = 0.0014).
Conclusions: These data strongly suggest that modified desmin constitutes the seed initiating the formation of cardiac PAOs in non- genetic HF. The increased levels of toxic desmin PAOs in the heart could, therefore, represent a novel mechanism of organ dysfunction in HF, in the absence of genetic mutations
The role of circulating transforming growth factor-beta in vascular Ehlers-Danlos syndrome: implications for drug therapy
No full textProteomic profiling of endothelin-1 stimulated hypertrophic cardiomyocytes reveals the increase of four different desmin species and alpha-beta-crystallin
No full textWe performed a proteomic investigation on primary cultures of neonatal rat cardiomyocytes after treatment with 10 nM endothelin-1 (ET1) for 48 h, an in vitro model for cardiac hypertrophy. Two-dimensional gel electrophoresis profiles of cell lysates were compared after colloidal Coomassie Blue staining. 12 protein spots that significantly changed in density due to ET1 stimulation were selected for in-gel digestion and identified through mass spectrometry. Of these, 8 spots were increased and 4 were decreased. Four of the increased proteins were identified as desmin, the cardiac component of intermediate filaments and one as alpha-B-crystallin, a molecular chaperone that binds desmin. All the desmins increased 2- to 5-fold, and alpha-B-crystallin increased 2-fold after ET1 treatment. Desmin cytoskeleton has been implicated in the regulation of mitochondrial activity and distribution, as well as in the formation of amyloid bodies. Mitochondria-specific fluorescent probe MitoTracker indicated mitochondrial redistribution in hypertrophic cells. An increase of amyloid aggregates containing desmin upon treatment with ET1 was detected by filter assay. Of the four proteins that showed decreased abundance after ET1 treatment, the chaperones hsp60 and grp75 were decreased 13- and 9-fold, respectively. In conclusion, proteomic profiling of ET1-stimulated rat neonatal cardiomyocytes reveals specific changes in cardiac molecular phenotype mainly involving intermediate filament and molecular chaperone proteins. (C) 2008 Elsevier B.V. All rights reserved
