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There are limited options to quantify and characterize amyloid species from biological samples in a simple fashion. Thioflavin T (ThT) has now been used for decades to stain amyloid fibrils but to our knowledge we were the first to use it in-gel. Thioflavin T in-gel stain is convenient as it is fast, inexpensive, available to most labs, compatible with other fluorescent stains and downstream analyses such as mass spectrometry (MS).

BackgroundThough acute mortality by myocardial infarction (MI) has declined in past decades, MI still represents one of the leading causes of heart failure (HF) development. We recently demonstrated the accumulation of toxic desmin aggregates in patients with HF of ischemic origin. Since desmin aggregates are toxic for the heart we aimed to test whether their formation can be induced by oxidative stress as a proxy for reperfusion injury, as well as addressing the effects of therapeutic strategies aimed at reducing desmin aggregation with cardiac oxidative stress.Methods and ResultsWe demonstrate here that oxidative stress is able to induce desmin aggregation, acutely, in a cell-specific and dose-dependent fashion. We also show that elevation ofO-linked β-N-acetylglucosamine (O-GlcNAc) prior to or after oxidative stress reduces the formation of toxic desmin aggregates and its pro-aggregating desmin post-translational modifications (PTM). In addition, we show for the first time a role for the transmembrane protease serine 13 (TMPRSS13) with desmin cleavage in response to oxidative stress while desmin’s single cysteine plays a protective role from I/R injury, which is independent of gain or loss of desmin function.ConclusionsThe proliferation of desmin PTM-forms (i.e., proteoforms) and its aggregation hallmark acute and chronic cardiac stress and result in both loss of and gain of desmin function. We report here two novel mechanisms that could be targeted for therapy to preserve desmin homeostasis and cardiac function in the acute settings of oxidative stress and reperfusion injury.

Background Pulseless electrical activity (PEA) is a common initial rhythm in cardiac arrest. A substantial number of PEA arrests are caused by coronary ischemia in the setting of acute coronary occlusion, but the underlying mechanism is not well understood. We hypothesized that the initial rhythm in patients with acute coronary occlusion is more likely to be PEA than ventricular fibrillation in those with prearrest severe left ventricular dysfunction. Methods and Results We studied the initial cardiac arrest rhythm induced by acute left anterior descending coronary occlusion in swine without and with preexisting severe left ventricular dysfunction induced by prior infarcts in non–left anterior descending coronary territories. Balloon occlusion resulted in ventricular fibrillation in 18 of 34 naïve animals, occurring 23.5±9.0 minutes following occlusion, and PEA in 1 animal. However, all 18 animals with severe prearrest left ventricular dysfunction (ejection fraction 15±5%) developed PEA 1.7±1.1 minutes after occlusion. Conclusions Acute coronary ischemia in the setting of severe left ventricular dysfunction produces PEA because of acute pump failure, which occurs almost immediately after coronary occlusion. After the onset of coronary ischemia, PEA occurred significantly earlier than ventricular fibrillation (

Background: One of the molecular hallmarks in the development of heart failure (HF) is loss of ultrastructure within the cardiac myocyte. In addition, HF is increasingly recognized as a proteinopathy characterized by the accumulation of misfolded proteins similar to Alzheimer and Parkinson disease. However, despite its increasing prevalence and poor prognosis, the advances in the pharmacological treatment of HF have been limited, highlighting an urgent need for the discovery of new therapeutic targets. We reported a consistent accumulation of mono-phosporylated desmin in experimental and clinical models of HF. We also demonstrated how mono-phosphorylated desmin is more prone to cleavage and aggregation in isolated cardiac myocytes. Therefore, if on the one hand desmin cleavage could easily explain the loss of a cardiac myocyte’s ultrastructure, its high abundance and propensity to aggregate make it an ideal candidate as the seed generating pre-amyloid-oligomers (PAOs) and amyloid fibrils in the heart. Methods: Using a combination of novel and established protein biochemistry techniques, we aimed at demonstrating desmin’s identity as the seed starting the nucleation process which leads to the formation of cardiac PAOs and amyloid fibrils. Results: Desmin displayed common features shared by other established PAOs and fibrils (e.g. tinctorial properties) in experimental and clinical models of HF. Conclusions: The inherent propensity of intermediate filaments to aggregate, combined with the use of cardiac tissue as a model for repeated mechanical stretch, suggest that intermediate filaments aggregation could be used as a way to dissipate/scavange mechanical as well as chemical stress. We will therefore use the highly organized structure of cardiac myocytes to infer IFs function in mammalian cells.

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.

Deposition of amyloidogenic proteins leading to the formation of amyloid fibrils in the myocardium cause cardiac amyloidosis. Although any form of systemic amyloidosis can affect the heart, light-chain (AL) or transthyretin amyloidosis (ATTR) account for the majority of diagnosed cardiac amyloid deposition. The extent of cardiac disease independently predicts mortality. The reversal or arrest of adverse cardiac remodeling is the target of current therapies, as cardiac-related mortality worsens prognosis in patients where the underlying systemic amyloidosis was successfully treated. Here, we provide a condensed overview on the pathophysiology of AL and ATTR cardiac amyloidoses and describe treatments that are currently used or investigated in clinical or preclinical trials. We also briefly touch on acquired amyloid deposition in cardiovascular disease other than AL or ATTR.

Pulmonary vascular damage is a rare but possible complication of treatment with chemotherapeutic agents or bone marrow transplantation. The main clinical manifestations involving the pulmonary vessels are the development of pulmonary arterial hypertension or pulmonary veno-occlusive disease. In this chapter we describe the main mechanisms underlying the development of this form of toxicity, the screening algorithm, and its clinical management.

Background Recently, tangles and plaque-like aggregates have been identified in certain cases of dilated cardiomyopathy (DCM), traditionally labeled idiopathic (iDCM), where there is no specific diagnostic test or targeted therapy. This suggests a potential underlying cause for some of the iDCM cases. Objectives This study sought to identify the make-up of myocardial aggregates to understand the molecular mechanisms of these cases of DCM; this strategy has been central to understanding Alzheimer’s disease. Methods Aggregates were extracted from human iDCM samples with high congophilic reactivity (an indication of plaque presence), and the findings were validated in a larger cohort of samples. We tested the expression, distribution, and activity of cofilin in human tissue and generated a cardiac-specific knockout mouse model to investigate the functional impact of the human findings. We also modeled cofilin inactivity in vitro by using pharmacological and genetic gain- and loss-of-function approaches. Results Aggregates in human myocardium were enriched for cofilin-2, an actin-depolymerizing protein known to participate in neurodegenerative diseases and nemaline myopathy. Cofilin-2 was predominantly phosphorylated, rendering it inactive. Cardiac-specific haploinsufficiency of cofilin-2 in mice recapitulated the human disease’s morphological, functional, and structural phenotype. Pharmacological stimulation of cofilin-2 phosphorylation and genetic overexpression of the phosphomimetic protein promoted the accumulation of “stress-like” fibers and severely impaired cardiomyocyte contractility. Conclusions Our study provides the first biochemical characterization of prefibrillar myocardial aggregates in humans and the first report to link cofilin-2 to cardiomyopathy. The findings suggest a common pathogenetic mechanism connecting certain iDCMs and other chronic degenerative diseases, laying the groundwork for new therapeutic strategies.

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.

Traditional chemotherapeutics are essential tools in the management of cancer patients. Nevertheless, these drugs are burdened by some degree of cardiovascular toxicity. Anthracycline-induced toxicity has been historically the most studied, but also the use of other drugs can be limited by a certain risk of cardiac and vascular toxicities. Here we acknowledge the main mechanistic insights, and we describe the different aspects of cardiotoxicity of these drugs, highlighting the different cellular compartments and cardiovascular components affected.

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In the last two decades, newer biological drugs have been designed in order to “target” specific proteins involved in cancer proliferation and overcome the increased risk of cardiovascular toxicity associated with “broad-spectrum” classic chemotherapeutics. Unfortunately, these proteins are also important for the maintenance of cardiovascular homeostasis. The humanized anti-ErbB2 antibody, trastuzumab, is the prototypical biological drug first introduced in antineoplastic protocols for the treatment of ErbB2+ breast cancer. Indeed, not only is this protein overexpressed in several breast cancers, but also it plays a major role in the cardiovascular system in cell growth, including myocyte growth, and inhibition of apoptosis and can modulate the oxidative damage induced by anthracyclines. Hence, patients treated with trastuzumab developed systolic dysfunction, especially when administered with or shortly after doxorubicin.

Rationale: Disrupted proteostasis is one major pathological trait that heart failure (HF) shares with other organ proteinopathies, such as Alzheimer and Parkinson diseases. Yet, differently from the latter, whether and how cardiac preamyloid oligomers (PAOs) develop in acquired forms of HF is unclear. Objective: We previously reported a rise in monophosphorylated, aggregate-prone desmin in canine and human HF. We now tested whether monophosphorylated desmin acts as the seed nucleating PAOs formation and determined whether positron emission tomography is able to detect myocardial PAOs in nongenetic HF. Methods and Results: Here, we first show that toxic cardiac PAOs accumulate in the myocardium of mice subjected to transverse aortic constriction and that PAOs comigrate with the cytoskeletal protein desmin in this well-established model of acquired HF. We confirm this evidence in cardiac extracts from human ischemic and nonischemic HF. We also demonstrate that Ser31 phosphorylated desmin aggregates extensively in cultured cardiomyocytes. Lastly, we were able to detect the in vivo accumulation of cardiac PAOs using positron emission tomography for the first time in acquired HF. Conclusions: Ser31 phosphorylated desmin is a likely candidate seed for the nucleation process leading to cardiac PAOs deposition. Desmin post-translational processing and misfolding constitute a new, attractive avenue for the diagnosis and treatment of the cardiac accumulation of toxic PAOs that can now be measured by positron emission tomography in acquired HF.

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.

In this chapter we provide an overview of bottom-up proteomic approaches. These allow the identification and characterization of proteins and their amino acid sequences, including post-translational modifications, by proteolytic digestion prior to mass spectrometry (MS) analysis. Intact proteins can be separated by gel electrophoresis followed by in-gel protein digestion to generate peptides which are then analyzed by MS. Alternatively, complex protein mixtures can be digested directly (an approach referred to as “shotgun”) and the resulting peptides can be separated by liquid chromatography prior to MS. Following MS analysis, the comparison of the peptides’ spectra with those predicted from genomics/proteomics sequence databases, or annotated peptide spectral libraries, allows the identification of peptides which are finally assigned to corresponding proteins. After a description of the separation methods and MS acquisition modes, a relevant part of the chapter will be dedicated to data processing pointing to algorithms, computational tools and strategies useful for researchers in the discovery process. In particular, liquid-chromatography (LC) based approaches, including Multidimensinal Protein Identification Technology (MudPIT), will be taken as reference and different aspects, ranging from database search engines to protein-protein interaction (PPI) network analysis, will be addressed. Potential issues will be discussed in the context of cardiovascular research, and specifically the last section will focus on the translational applications (clinical proteomics) of cardiovascular proteomics.

Mass Spectrometry has potentially been one of the most powerful tool available to scientists. The future for mass spectrometry lies not only in deep technological advances in its inherent performance but also in increasing its robustness and ease of use, and by reductions in size and cost, that will make it increasingly accessible to more users.

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.

Background: Heart Failure (HF) is one of the main causes of morbidity and mortality in westernized countries but the molecular mechanisms underlying its development are still unclear. A paradigm-shifting view focuses on the accumulation of preamyloid oligomers (PAOs), similar to those observed in Alzheimer disease, as a potential mechanism of cardiac toxicity. We reported that differential desmin phosphorylation at serines (S) 27 and 31 could drive the formation of PAOs in the heart, in the absence of genetic mutations. We sought to establish the identity of the molecular seed triggering the nucleation of cardiac PAOs in an experimental model of HF and in cultured cardiac cells. Methods: Mice were subjected to transverse aortic constriction (TAC) for 4 weeks (FS% = 29.3±2.6, P=0.0001). Alternatively, neonatal rat ventricular myocytes were transduced with lentiviral vectors carrying alanine (A) or phospho-mimetic aspartate (D) desmin double mutants at S27 and S31, fused with GFP. Protein homogenates were subjected to western blot analysis with fluorescent co-staining using the A11 anti-PAOs and anti-desmin antibodies. Transduced cells were also subjected to live imaging to assess phenotype. Results: Co-western blot analysis of both TAC mice and phospho-mimetic mutant cells revealed the colocalization of PAOs with desmin modified (potentially cleaved) forms. Preamyloid oligomers and a desmin fragment were both increased in TAC mice vs. controls (2.8-fold, P=0.023 and 1.8-fold, P=0.038, respectively). The DD mutant, mimicking the doubly phosphorylated desmin that we hypothesized is the physiological form, showed a healthier phenotype in terms of number of spontaneously contracting cells (P=0.041) and incorporation of GFP-desmin at the Z-discs (P=0.0027), whereas the mono-phosphomimetic mutant (AD) resulted in the increase of desmin positive aggregates (P=0.0014). Conclusions: This preliminary evidence suggests that desmin modified forms represent the seed triggering the formation of cardiac PAOs, in the absence of genetic mutations. The accumulation of desmin PAOs could therefore represent an overarching mechanism underlying the deterioration of cardiac function in HF.

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.

The aim of our current special issue was to present a series of original researches and reviews on recent advances in the diagnosis, medical therapy, and surgical approaches of heart failure. As reported in the introductive review of Agnetti et al., 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 syndromes 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 authors reported in this comprehensive review the evaluation of 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. Among the studies included in this special issue, two of them investigated specific pathogenic aspects of heart failure. M. Kunin et al. studied the role of proinflammatory cytokines in congestive heart failure. In particular the authors evaluated the effect of peritoneal dialysis used in the long-term management of these patients on the peripheral-circulating levels of these cytokines. Interestingly, they found that peritoneal dialysis treatment caused a reduction in circulating inflammatory cytokines levels along with improvement in plasma markers of inflammation in patients with refractory chronic heart failure, concluding that this effect may be partly responsible for the efficacy of peritoneal dialysis for refractory heart failure. It is described that heart failure is accompanied by the development of an imbalance between oxygen- and nitric oxide-derived free radical production leading to protein nitration. To cast further light on this issue, A. Cabassi et al. investigated the relationship between plasma myeloperoxidase-related chlorinating activity, ceruloplasmin, and ferroxidase I and nitrosative stress and inflammatory, neurohormonal, and nutritional biomarkers in heart failure patients. This elegant study supported the conclusions that plasma myeloperoxidase chlorinated activity is increased in elderly patients who suffer from chronic heart failure and positively associated with ceruloplasmin and inflammatory, neurohormonal, and nitrosative parameters, suggesting a key role in heart failure progression. A major focus of studies on acute heart failure is the need for methods that allow the early detection of hemodynamic variables that can be a key prognostic role after cardiac surgery. F. Corradi et al. in their study investigated the Renal Doppler Resistive Index as a marker of oxygen supply and demand mismatch in 61 postoperative cardiac surgery patients. Interestingly, by multivariate analysis, Renal Doppler Resistive Index was significantly correlated with mixed-venous oxygen saturation, suggesting that, in mechanically ventilated patients after cardiac surgery, it could be used as a marker of early vascular response to tissue hypoxia. We must not forget that there are many patients suffering from heart failure secondary to extracardiac diseases. Among the causes of heart failure, an important place is held by cardiotoxicity due to antineoplastic treatments that has emerged as a clinically relevant problem as a consequence of the relevant improvement of survival after cancer. In the last decade recent advances have emerged in clinical and pathophysiological aspects of left ventricular dysfunction induced by the most widely used anticancer drugs. M. Molinaro et al., in their comprehensive review entitled “Recent Advances on Pathophysiology, Diagnostic and Therapeutic Insights in Cardiac Dysfunction Induced by Antineoplastic Drugs,” have particularly examined the role of early, sensitive markers of cardiac dysfunction, in order to predict this form of cardiomyopathy before left ventricular ejection fraction is reduced. It seems actually that this is increasingly important issue, along with the evaluation of novel therapeutic and cardioprotective strategies, to protect cardiooncologic patients from the development of congestive heart failure. As reported by A. Adegunsoye et al., there are also a consistent number of patients who die due to right heart failure and pulmonary hypertension secondary to fibrotic lung diseases. Significant factors which appear to play a role in the mechanism of progression of right heart dysfunction include chronic hypoxia, defective calcium handling, hyperaldosteronism, pulmonary vascular alterations, cyclic strain of pressure and volume changes, elevation of circulating TGF-β, and elevated systemic NO levels. The authors have reported an exhaustive review of novel therapeutic strategies for reducing right heart failure associated mortality in fibrotic lung diseases, because only “an early, effective and individualized therapy may prevent overt right heart failure in fibrotic lung disease leading to improved outcomes and quality of life.” Another major focus of studies on acute heart failure is related to the evaluation of new surgical alternatives to transplantation or new systems or new materials available for future cardiac assist devices. In this special issue the review of J. Anand et al. explores the evolution of mechanical circulatory support and its potential for providing long-term therapy, which may address the limitations of cardiac transplantation. The innovation progresses have led to a solution of current challenges involving device complications. Moreover outcomes continue to improve and further data from both small and large registries help to advance evidence-based practices: thus patients in the most advanced stages of heart failure appear to have more hope than ever before. On the other hand the high costs, expanding indications, and rapidly increasing number of devices implanted will ultimately require important decisions to be made on the part of society, clinicians, and administrative agencies in order to establish the potential amount of economic resources to spend on this expensive, yet effective, therapy. Patient selection will remain paramount, although a very large population of patients will have the potential to benefit. In particular, K. Unthan et al. reported a study on the design and evaluation of a fully implantable control unit for blood pumps. It is well known that pneumatic devices sufficiently supply the patients with blood flow, although the patient's quality of life is limited by the percutaneous pressure lines and the size of the external control unit. General requirements for any implantable control unit are defined from a technical and medical point of view: need for a Transcutaneous Energy Transmission, autonomous operation, safety, geometry, and efficiency. The authors described the development of the control unit of the ReinHeart, a fully implantable Total Artificial Heart that, in validation tests, is demonstrated to be a stable operation with a promising good efficiency. Finally P. Morillas-Sendin et al. assessed the effect of sevoflurane and propofol on organ blood flow in a porcine model with a left ventricular assist device. The authors demonstrated that, compared with propofol, sevoflurane increases blood flow in the brain, liver, and heart after implantation of a left ventricular assist device under conditions of partial support, giving interesting indications for the intensive pharmacological care of these high-risk patients. The modern approach to the diagnosis and treatment of heart failure is multidisciplinary and should be based on a close collaboration among researchers, clinicians, and cardiac surgeons particularly given that mandatory multiorgan attention is required in these high-risk patients. Future therapies for heart failure could include ventricular assist devices implantation or ventricular restoration techniques with the aim to obtain a reverse, positive remodeling in the unloaded heart. With an expanding “toolbox” of comprehensive basic, medical, surgical, and technological approaches, it is expected that these novel findings will soon be translated to the clinical practice. In fact, new therapeutic strategies are needed by the millions of patients suffering from heart failure. We hope that this special issue will help readers become familiarized with recent progress regarding the diagnosis and treatment of heart failure.

Background Recently, tangles and plaque-like aggregates have been identified in certain cases of dilated cardiomyopathy (DCM). This suggests a potential underlying cause for the one-third of cases, traditionally labeled idiopathic (iDCM), where there is no specific diagnostic test or targeted therapy. Objectives This study sought to identify the make-up of myocardial aggregates to understand the molecular mechanisms of these cases of DCM; this strategy has been central to understanding Alzheimer's disease. Methods Aggregates were extracted from human iDCM samples with high congophilic reactivity (an indication of plaque presence), and the findings were validated in a larger cohort of samples. We tested the expression, distribution, and activity of cofilin in human tissue and generated a cardiac-specific knockout mouse model to investigate the functional impact of the human findings. We also modeled cofilin inactivity in vitro by using pharmacological and genetic gain- and loss-of-function approaches. Results Aggregates in human myocardium were enriched for cofilin-2, an actin-depolymerizing protein known to participate in neurodegenerative diseases and nemaline myopathy. Cofilin-2 was predominantly phosphorylated, rendering it inactive. Cardiac-specific haploinsufficiency of cofilin-2 in mice recapitulated the human disease's morphological, functional, and structural phenotype. Pharmacological stimulation of cofilin-2 phosphorylation and genetic overexpression of the phosphomimetic protein promoted the accumulation of "stress-like" fibers and severely impaired cardiomyocyte contractility. Conclusions Our study provides the first biochemical characterization of prefibrillar myocardial aggregates in humans and the first report to link cofilin-2 to cardiomyopathy. The findings suggest a common pathogenetic mechanism connecting certain iDCMs and other chronic degenerative diseases, laying the groundwork for new therapeutic strategies. © 2015 American College of Cardiology Foundation.

A new concept in the field of heart failure research points to a role of misfolded proteins, forming pre-amyloid oligomers (PAOs), in cardiac toxicity. This is largely based on few studies reporting the presence of PAOs, similar to those observed in neurodegenerative disease, in experimental models and human heart failure. As the majority of proteinopathies are sporadic in nature, protein post-translational modifications (PTMs) likely play a major role in this growing class of diseases. In fact, PTMs are known regulators of protein folding and of the formation of amyloid species in well-established proteinopathies. Proteomics has been instrumental in identifying both chemical and enzymatic PTMs, with a potential impact on protein mis-/folding. Here we provide the basics on how proteins fold along with a few examples of PTMs known to modulate protein misfolding and aggregation, with particular focus on the heart. Due to its innovative content and the growing awareness of the toxicity of misfolded proteins an “Alzheimer’s theory of heart failure” is timely. Moreover, the continuous innovations in proteomic technologies will help pinpoint PTMs that could contribute to the process. This nuptial between biology and technology could greatly assist in identifying biomarkers with increased specificity as well as more effective therapies.

Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function also increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance myofilament calcium responsiveness. To test this hypothesis, we examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony (HFdys) or CRT. Myofilament force-calcium relationships were measured in skinned trabeculae and/or myocytes. Compared with control, maximal calcium-activated force and calcium sensitivity declined globally in HFdys; however, CRT restored both. Phosphatase PP1 induced calcium desensitization in control and CRT-treated cells, while HFdys cells were unaffected, implying that CRT enhances myofilament phosphorylation. Proteomics revealed phosphorylation sites on Z-disk and M-band proteins, which were predicted to be targets of glycogen synthase kinase-3beta (GSK-3beta). We found that GSK-3beta was deactivated in HFdys and reactivated by CRT. Mass spectrometry of myofilament proteins from HFdys animals incubated with GSK-3beta confirmed GSK-3beta-dependent phosphorylation at many of the same sites observed with CRT. GSK-3beta restored calcium sensitivity in HFdys, but did not affect control or CRT cells. These data indicate that CRT improves calcium responsiveness of myofilaments following HFdys through GSK-3beta reactivation, identifying a therapeutic approach to enhancing contractile function.

AIMS: The ultimate cause of heart failure (HF) is not known to date. The cytoskeletal protein desmin is differentially modified and forms amyloid-like oligomers in HF. We postulated that desmin post-translational modifications (PTMs) could drive aberrant desmin aggregation in HF. Therefore, we identified these PTMs and investigated their impact on desmin amyloidogenicity in human and experimental HF. METHODS AND RESULTS: We detected increased levels of selectively phosphorylated and cleaved desmin in a canine pacing model of dyssynchronous HF (DHF) compared with either controls or animals treated with cardiac resynchronization therapy (CRT). This unique animal model combines clinically relevant features with the possibility of a partly rescued phenotype. We confirmed analogous changes in desmin modifications in human HF and identified two phosphorylation sites within a glycogen synthase kinase 3 (GSK3) consensus sequence. Desmin-positive oligomers were also increased in DHF hearts compared with controls. Their amyloid properties were decreased by treatment with CRT or an anti-amyloid small molecule. Finally, we confirmed GSK3's involvement with desmin phosphorylation using an in vitro model. CONCLUSIONS: Based on these findings, we postulate a new mechanism of cardiac toxicity based on the PTM-driven accumulation of desmin amyloid-like oligomers. Phosphorylation and cleavage as well as oligomers formation are reduced by treatment (CRT) indicating a relationship between the three. Finally, the decrease of desmin amyloid-like oligomers with CRT or small molecules points both to a general mechanism of HF based on desmin toxicity that is independent of protein mutations and to novel potential therapies.

Background— 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-β 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.

We 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.

Cardiac resynchronization therapy (CRT) is a procedure used in the clinics to ameliorate the symptoms associated with heart failure-induced conduction disturbances and ventricular dyssynchrony. The molecular modifications underlying the beneficial effects of CRT have not been completely clarified. Recent investigations suggest that CRT may reduce apoptosis of cardiac myocytes. Mitochondria are likely to be major players in this benign transition due to their role in both energy production and apoptosis regulation. Therefore the proteome of cardiac mitochondria was investigated in a canine model for heart failure alternatively submitted to CRT. Methods and results: Mitochondria-enriched fractions obtained from sections of the left ventricular free wall of dyssynchrony-induced heart failure (DHF) dogs alternatively submitted to CRT were analyzed through two-dimensional gel electrophoreisis (2DE). Roughly 1200 protein spots were visualized on the 2D gels after silver staining. Software-assisted differential display analysis indicated that the density of almost 50 protein spots was significantly changed upon CRT. These spots were selected and identified through mass spectrometry. Among other changes six forms of ATP synthase beta subunit were decreased 2- to 3-fold (0.0001≤p≤0.047), as well as four forms of cytochrome C (2-fold decrease, 0.002≤p≤0.047). The existence of multiple forms of a protein suggests for the presence of post-translational modifications (PTM). Therefore the phosphorylation (a common PTM) status of mitochondrial proteome was also monitored by combining the differential-in-gel electrophoresis (DIGE) technology with alkaline phosphatase teatment. ATP synthase beta subunit was differently phosphorylated in CRT samples comparing to DHF as indicated by the change in its isoelectric point upon treatment with alkaline phosphatase. Conclusions: The proteomic investigation of mitochondria revealed a previously unseen presence of PTM mechanisms at the mitochondria level in heart failure and CRT. Phosphorylation mechanisms in the mitochondria may play a prominent role in modulating cardiac function, as observed for the beneficial effects of CRT.

Separation 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.

The key components to the molecular understanding of the pathophysiology of various forms of heart failure involve global and/or large-scale identifications of proteins, their patterns of expression, posttranslational modifications, and functional characterization. Particularly, proteins involved in the induction of cardiac (mal)adaptive hypertrophic growth, interstitial fibrosis, and contractile dysfunction are of interest. In general, with the accumulation of vast amounts of DNA sequences in databases, researchers have become aware that merely having complete sequences of genomes and transcriptional changes for thousands of genes simultaneously will not be sufficient to elucidate, in molecular terms, the etiology and pathophysiology of cardiovascular disease. In the last decade, a new technology called proteomics has become available that allows biological and (patho)physiological questions to be approached exclusively from the protein perspective. Proteomics may enable us to map the entire complement of proteins expressed by the heart at any time and condition. This approach creates the unique possibility to identify, by differential analysis, protein alterations associated with the etiology of heart disease and its progression, outcome, and response to therapy. To illustrate the true power of proteomics, most of the currently available methodologies are first reviewed, including their limitations. This review also deals with the current status and the perspectives of proteomics applications in research on heart failure in general. Furthermore, examples of our recent data on global protein profiling of the pressure-overloaded rat right ventricle and of endothelin-1-stimulated cultures of neonatal rat cardiac myocytes are provided. The last section is devoted to the continuous advances in proteomic technologies, including protein separation methods, mass spectrometric instrumentation, computational analysis, and bioinformatic tools, together with integrative databases.

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 …

BACKGROUND: Despite its prevalence the molecular bases of heart failure (HF) remain obscure. Our group recently reported a role for amyloid-like oligomers (ALOs) generated by mono-phosphorylated desmin as a potential mechanism underlying HF development and progression. We reported that differential desmin phosphorylation at Ser 27 and 31 may drive the formation of 600 kDa desmin ALOs. We first optimized a way to isolate these ALOs and tested their potential toxicity on cardiac sub-mitochondrial particles. METHODS: We implemented an electrophoretic method to separate and identify the ALOs in-gel. The procedure was optimized using a well-established murine model of cardiac proteotoxicity (R120G CryAB mice). We alternatively generated desmin ALOs by transducing cultured cardiac myocytes with a lentivirus carrying desmin double mutants that mimic or prevent desmin phosphorylation at Ser27/31, by substitution to Asp or Ala, respectively. ALOs at both 600 kDa and 1200 kDa, serving as a negative control, were purified using gel electrophoresis, followed by electroelution of the ALOs. We incubated cardiac sub-mitochondrial particles (SMPs) with 600 and 1200 kDa ALOs from double mutants and measured O2 consumption by polarographic analysis. RESULTS & CONCLUSIONS: Only the 600kDa oligomers showed amyloid-like properties as specified by Thioflavin T binding using our new optimized separation. Furthermore, only the mono-phosphomimetic desmin ALOs decreased O2 consumption in SMPs in a dose- (and sequence-) dependent fashion when SMPs were energized by NADH. These data point to a novel mechanism of mitochondrial toxicity for ALOs generated by mono- hosphorylated desmin, which is increased in both human and experimental HF. This work is supported by the American Heart Association (12SDG9210000), the NIH Proteomic innovation contract P01HL081427 and PPG on cardiac resynchronization therapy PO1-HL077180, Fondazione del Monte, and RFO University of Bologna (Bologna, Italy) to GA