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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.

Lo studio COMPANION ha dimostrato che i pazienti con insufficienza cardiaca cronica, in fase avanzata, e con un intervallo QRS prolungato, possono trarre beneficio dalla terapia di risincronizzazione cardiaca (Cardiac Resynchronization Therapy o CRT). Il gruppo del Professor Kass, Department of Cardiology, Johns Hopkins Medicine (Baltimore, MD, USA) ha recentemente sviluppato un modello animale per monitorare gli effetti della CRT a partire da un ben caratterizzato modello canino di insufficienza cardiaca (Dissynchrony-induced Heart Failure, DHF). Il presupposto è stato lo studio del modello di Kass mediante metodologie di proteomica rivolgendosi in particolare a la frazione mitocondriale del tessuto cardiaco canino. Nel presente lavoro è stato possibile applicare un protocollo innovativo per la separazione delle proteine in frazioni mitocondriali ottenute dal tessuto cardiaco di cani con DHF alternativamente sottoposti a CRT. La separazione delle frazioni mitocondriali è stata ottenuta su due diversi intervalli di pH: da 4 a 7 (proteine acide) e da 6 a 11 (proteine basiche). Nel database di proteine mitocondriali MitoProteome sono raccolte circa 400 proteine, il 66% delle quali mostra un pI maggiore di 7. Almeno un terzo (32%) di queste hanno un pI maggiore di 9. Questo rende ragione del fatto che per poter considerare il proteoma di mitocondrio in maniera consistente bisogna utilizzare intervalli di pH basici in prima dimensione. Infine la metodica, utilizzata per la prima volta in questo laboratorio ha consentito una risoluzione mai conosciuta per la frazione basica rendendo possibile l’analisi dell’espressione differenziale di 1200 proteine con pI da 4 a 11, delle quali circa 800 nell’intervallo da 4 a 7 e circa 400 nell’intervallo da 6 a 11. I risultati di questa analisi hanno indicato la diversa espressione di 49 e 15 specie proteiche rispettivamente negli intervalli di pH 4-7 e 6-11 nei campioni CRT rispetto ai DHF.

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.