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Heart failure due to chronic volume overload (VO) in rats and humans is characterized by disorganization of the cardiomyocyte desmin/mitochondrial network. Here, we tested the hypothesis that desmin breakdown is an early and continuous process throughout VO. Male Sprague-Dawley rats had aortocaval fistula (ACF) or sham surgery and were examined 24 h and 4 and 12 wk later. Desmin/mitochondrial ultrastructure was examined by transmission electron microscopy (TEM) and immunohistochemistry (IHC). Protein and kinome analysis were performed in isolated cardiomyocytes, and desmin cleavage was assessed by mass spectrometry in left ventricular (LV) tissue. Echocardiography demonstrated a 40% decrease in the LV mass-to-volume ratio with spherical remodeling at 4 wk with ACF and LV systolic dysfunction at 12 wk. Starting at 24 h and continuing to 4 and 12 wk, with ACF there is TEM evidence of extensive mitochondrial clustering, IHC evidence of disorganization associated with desmin breakdown, and desmin protein cleavage verified by Western blot analysis and mass spectrometry. IHC results revealed that ACF cardiomyocytes at 4 and 12 wk had perinuclear translocation of αB-crystallin from the Z disk with increased α, β-unsaturated aldehyde 4-hydroxynonelal. Use of protein markers with verification by TUNEL staining and kinome analysis revealed an absence of cardiomyocyte apoptosis at 4 and 12 wk of ACF. Significant increases in protein indicators of mitophagy were countered by a sixfold increase in p62/sequestosome-1, which is indicative of an inability to complete autophagy. An early and continuous disruption of the desmin/mitochondrial architecture, accompanied by oxidative stress and inhibition of apoptosis and mitophagy, suggests its causal role in LV dilatation and systolic dysfunction in VO. NEW & NOTEWORTHY This study provides new evidence of early onset (24 h) and continuous (4−12 wk) desmin misarrangement and disruption of the normal sarcomeric and mitochondrial architecture throughout the progression of volume overload heart failure, suggesting a causal link between desmin cleavage and mitochondrial disorganization and damage.

Protein phosphorylation is reversibly regulated by the interplay between kinases and phosphatases. Recent developments within the field of proteomics have revealed the extent of this modification in nature. To date there is still a lack of information about phosphatase specificity for different proteomes and their conditions to achieve maximum enzyme activity. This information is important per se, and in addition often requested in functional and biochemical in vitro studies, where a dephosphorylated sample is needed as a negative control to define baseline conditions. In this study, we have addressed the effectiveness of two phosphatases endogenously present in the heart (protein phosphatases 1 and 2A) and two generic phosphatases (alkaline phosphatase and lambda protein phosphatase) on three cardiac subproteomes known to be regulated by phosphorylation. We optimized the dephoshorylating conditions on a cardiac tissue fraction comprising cytosolic and myofilament proteins using 2DE and MS. The two most efficient conditions were further investigated on a mitochondrial-enriched fraction. Dephosphorylation of specific proteins depends on the phosphatase, its concentration, as well as sample preparation including buffer composition. Finally, we analyzed the efficiency of alkaline phosphatase, the phosphatase with the broadest substrate specificity, using TiO(2) peptide enrichment and 2DLC-MS/MS. Under these conditions, 95% of the detected cardiac cytoplasmic-enriched phospho-proteome was dephosphorylated. In summary, targeting dephosphorylation of the cardiac muscle subproteomes or a specific protein will drive the selection of the specific phosphatase, and each requires different conditions for optimal performance.

Cardiovascular disease is the leading cause of mortality in the US and in westernized countries with ischemic heart disease accounting for the majority of these deaths. Paradoxically, the improvements in the medical and surgical treatments of acute coronary syndrome are leading to an increasing number of "survivors" who are then developing heart failure. Despite considerable advances in its management, the gold standard for the treatment of end-stage heart failure patients remains heart transplantation. Nevertheless, this procedure can be offered only to a small percentage of patients who could benefit from a new heart due to the limited availability of donor organs. The aim of this review is to evaluate the safety and efficacy of innovative approaches in the diagnosis and treatment of patients refractory to standard medical therapy and excluded from cardiac transplantation lists. © 2015 Giulio Agnetti et al.

Chronic 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 modifications 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 and signaling 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.

Recent developments in the field of protein separation allows for the analysis of qualitative and quantitative global protein changes in a particular state of a biological system. Due to the enormous number of proteins potentially present in a cell, sub-fractionation and the enrichment of specific organelles are emerging as a necessary step to allow a more comprehensive representation of the protein content. The proteomic studies demonstrate that a key to understand the mechanisms underlying physiological or pathological phenotypes lies, at least in part, in post-translational modifications (PTMs), including phosphorylation of proteins. Rapid improvements in proteomic characterization of amino acid modifications are further expanding our comprehension of the importance of these mechanisms. The present review will provide an overview of technologies available for the study of a proteome, including tools to assess changes in protein quantity (abundance) as well as in quality (PTM forms). Examples of the recent application of these technologies and strategies in the field of kinase signalling will be provided with particular attention on the role of PKC in the heart. Studies of PKC-mediated phosphorylation of cytoskeletal, myofilament and mitochondrial proteins in the heart have provided great insight into the phenotypes of heart failure, hypertrophy and cardioprotection. Proteomics studies of the mitochondria have provided novel evidences for kinase signalling cascades localized to the mitochondria, some of which are known to involve various isoforms of PKC. Proteomics technologies allow for the identification of the different PTM forms of specific proteins and this information is likely to provide insight into the determinants of morphological as well as metabolic mal-adaptations, both in the heart and other tissues.

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6- 3 H]-glucose uptake was increased (57% above control levels, p

Hypoxia/reoxygenation (H/R) is one of the causes of the increased expression of inducible nitric oxide synthase (iNOS) in cardiomyocytes. Since an aberrant NOS induction has detrimental consequences, we evaluated the effect of a green tea extract (GTE) on the NOS induction and activity in H/R-cardiomyocytes to define a nutritional strategy. Cultured rat cardiomyocytes were exposed to H/R in the presence of two concentrations of a green tea extract (GTE), which is reported to inhibit NOS expression and activity in different cells. In cultured cardiomyocytes two NOS isoforms were constitutively expressed, but only iNOS was induced by H/R. GTE supplementation at the lowest concentration, comparable to that in human plasma after dietary consumption, was ineffective, while the highest, comparable to that achievable by dietary supplements, counteracted the effect of H/R on iNOS induction and activity. It is necessary to verify in humans the relationship between the modulation of NO production and green tea dietary consumption.

Study Aim Numerous technical advances have characterized the proteomic field since it was first named in 1994.1 Some of the techniques that have driven the field, such as mass spectrometry, have undergone considerable technical development over the years. For instance, time-of-flight (TOF) mass analyzers date back to the first half of the 20th century.2 In the study described by Newell and colleagues3 a newly developed hybrid instrument, which combines a flow-cytometer with a TOF analyzer (cyTOF), was employed to characterize CD8+ T lymphocytes in humans. Notably, contrary to traditional flow-cytometry, where multiplexing (monitoring multiple analytes at the same time) is limited by the partial overlap of excitation/emission spectra of the different fluorophores, in this case the reporters that can be measured are many and more selective (little to no cross-talk). Finally, although, to my knowledge, this cutting-edge technique has not been applied yet in the cardiovascular research field, perhaps 1 application can be anticipated, in the field of cardiac regeneration. ### How Was the Aim Achieved? Newell and colleagues went about describing the heterogeneity of CD8+ T lymphocytes by enriching them from the peripheral blood of platelet aphaeresis donors. Two different approaches were employed to characterize the CD8+ population. One used mass cytometry, also referred to as cyTOF, due to the mass analyzer employed. In proteomics, TOF analyzers are associated most commonly to matrix assisted laser …