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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.

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.

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

BACKGROUND: Depressed contractility is a key feature of the failing heart and has been linked to reduced Ca2+ availability due to decreased activity of the sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Yet, surprisingly, a conditional, cardiac specific SERCA 2 knock out (KO) mouse, has sustained cardiac contractility for several weeks despite dramatically altered cytosolic Ca2+ handling and SR function. The molecular mechanism behind this intriguing contractile compensation is not clear. AIM: To examine the protein alterations in the cytosolic sub-proteome and identify the biological processes altered in the KO myocardium that can compensate for the loss of SERCA2 in the heart. METHODS AND RESULTS: SERCA2 KO and age matched control mice analyzed 9 days after induced gene excision showed no differences in cardiac function (left atrial diameter, echocardiography), even though the quantity of SERCA2 protein was reduced to 30±5% in the left ventricles in KO vs. control mice (western blot). The cytosolic-enriched protein extracts from KO and control hearts (n=6) was analyzed by two dimensional gel electrophoresis (pH 4–7 and 6–11) and 33 cytosolic proteins were identified as being altered (Redfin, Ludesi). The majority of the proteins were identified (tandem mass spectrometry, MS2) and analyzed further using seeded Bayesian networks. The main changes occurred to the cytoskeleton composition with alterations in vinculin, actin, gelsolin and coffilin-2. These were accompanied by regulation of a subset of heat shock proteins (HSPs), HSPB1, alpha-B-crystallin and HSPA5. Further analysis of the HSPs (Immobilized metal affinity chromatography and MS2) showed an increased level of the phosphorylated (activated) forms. Finally, these results were compared to effects in acute thapsigargintreated neonatal cardiomyocytes to examine whether the observed proteomic changes in SERCA2 KO hearts occur in vivo as a direct consequence of altered Ca2+ homeostasis.

Desmin constitutes the intermediate filaments (IFs) cytoskeleton in cardiac myocytes. Novel roles of the IFs, including the formation of toxic aggregates similar to those found in neurodegenerative diseases, have been recently linked to heart failure (HF). Post-translational modifications (PTMs) control desmin assembly and immunoreactivity, in vitro. Therefore PTMs likely play a role in desmin aggregation and could explain the controversy on desmin immuno-based quantitation in human HF. In the present study we explore the occurrence of desmin PTMs in canine and human HF and control specimens. We found a significant increase of three desmin forms, compatible with the mono- and bi-phosphorylated forms and a protein fragment (all 2-fold, p0.05), in a canine pacing model of dyssynchronous HF (DHF) compared to non-paced shams, using difference gel electrophoresis (DIGE). Importantly, these alterations were reverted by treatment (bi-ventricular or bi-V pacing for resynchronization). Two novel phosphorylation sites (Ser-27 and Ser-31) were identified by tandem mass spectrometry (MS2). Phosphorylated desmin species were also increased in the heart of class III NYHA patients when compared with healthy donors and phosphorylation of Ser-27 was observed, for the first time, in humans by MS2 and confirmed by multiple reaction monitoring. Finally, desmin-positive oligomers ( three bands, ~50, ~200 and ~600 kDa) were increased in DHF hearts compared to controls by blue-native gel electrophoresis (30%, p0.02). This is the first characterization of desmin phosphorylation in the heart, in vivo with identification of novel sites in both animal models and humans. We show that the levels of PTM-forms of desmin are modified in canine and human HF. Finally, these alterations correlate with the formation of oligomers and are reversed by bi-V pacing in dogs, suggesting that PTMs may regulate desmin-induced toxicity in HF.

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Cardiac resynchronization therapy (CRT) has become one of the major clinical therapies that improve long term survival in heart failure (HF) patients. The beneficial effects of CRT have been suggested, including upregulation of sarcoplamic reticulum ATPase, β1-adrenergic receptor expression, and improved chamber mechanoenergetics. Here, we reveal that CRT also acts as a calcium sensitizer and restores maximum force when applied in a dog model of dyssynchronous HF (DHF, 6 wks rapid atrial pacing with LBBB). Trabeculae (in mm: 0.35±0.02 wide, 0.22±0.01 thick, 1.74±0.13 long, n=27) from the ventricles of control, DHF, always synchronous HF (HF-synch, atrial rapid pacing for 6 wks), and CRT (BiV rapid pacing for latter 3 wks) were dissected, mounted between a force transducer and a motor arm, and chemically skinned in 1% Triton. Force-pCa relations were obtained with varied [Ca2+]. The Table compares maximal Ca2+- activated force (Fmax), the [Ca2+] for 50% of Fmax (Ca50), and the Hill coefficient in these groups. In parallel, myofibrillar ATPase activity as a function of Ca2+ activation were studied in these four groups (n=4, and 3 runs in each dog sample). DHF displayed a substantial downward shift with depression of minimum and maximal Ca2+-activated myofibrillar ATPase compared with control or CRT, and HF-synch myofibrils also yielded a response quite similar to that of DHF. Finally, 2D-DiGE and mass spectrometry of the myofilament proteins showed increased phosphorylation of troponin I, myosin light chain 2, and - tropomyosin (at a novel site, T237), while troponin T had less phosphorylation. These results demonstrate that (1) myofilament Ca2+ responsiveness is markedly depressed in HF and this myofilament abnormality occurs in HF per se (i.e. regardless of synchrony), (2) CRT significantly improves myofilament Ca2+ responsiveness, and (3) post-translational modification of myofil-ament proteins may underlie the beneficial myofilament 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.

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

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.

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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.

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.

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.