Project data

Funding entity: Italian Ministry of University and Research

Call: PRIN 2017

Coordinator: UNIVERSITA’ DEGLI STUDI DI FIRENZE

UNISI Principal Investigator: Pier Leopoldo Capecchi

Department: Medicine, surgery and neuroscience

Start date: 29 August 2019 – End date: 29 August 2023

 

Description

The occurrence of lethal arrhythmic events and sudden cardiac death remains an unsolved medical burden. The consequences of unexpected cardiac arrest or severe ventricular arrhythmias are particularly disappointing, due to the neurologic sequelae in resuscitated patient. Moreover, arrhythmias often bring about a deterioration of the quality of life, especially in young people with implanted devices. Despite the undoubtful success of cardiovascular pharmacology and therapy in many clinical settings, prevention and treatment of arrhythmias is still an open issue and a short chapter in textbooks, with the exception a few old drugs designed as “channel blockers”. A large mass of investigation in the last twenty years helped to shed light on some possible explanation: we know the electrocardiographic hallmarks but the mechanistic basis for channel derangement, apart from rare channelopathies, is multifaceted, may develop over a large timescale and makes it difficult to link causes (“culprits”) to effect. Just to make a few examples, 1) high-risk, eventually asymptomatic genetic syndromes (cardiomyopathies) cause extensive remodelling and arrhythmias; 2) acquired non-cardiovascular conditions such as viral myocarditis and autoimmune inflammatory diseases (rheumatoid arthritis and systemic lupus erythematosus, and 3) a wide spectrum of comorbidities, including hypertension and obesity, are associated with an increased cardiovascular mortality due to tissue remodelling and electrical disturbances. Rhythm disturbances arise from the interplay of substrate alterations and triggers, favouring the onset of an arrhythmogenic mechanism called “re-entry”. Related mechanisms can alter ion currents in primary and subsidiary pacemakers, leading to Sick Sinus node Syndrome (SSS) or Atrio-Ventricular (AV) block. While alteration in the substrate (fibrosis, adipose tissue replacement, disarray) is a long-term process, triggers may consist of an abrupt or sub-acute electrical perturbance. Based on the recognized expertise of our individual Units, highly interdisciplinary, supported by a Coordinator with a long-standing experience in the field of arrhythmogenesis and cardiac cellular electrophysiology, we all identified specific, clinically relevant settings: A) Immunoinflammatory conditions and the occurrence of brady- and tachy-arrhythmias B) Cardiac remodelling resulting in non-homogenous impulse generation and propagation. With an integrate approach, we will focus on poorly explored mediators, such as i) autoantibodies and cytokines, mainly IL-6, for which a proarrhythmic role has been clearly identified by the pioneer, clinical work of UNISI, ii) the interplay of channel-modifying mechanisms, at cardiomyocyte level and involving intra- and inter-cellular signals. Each of these mechanisms founds on the expertise and recognized reputation of leading groups, either for: • well-established mediators: redox state, intracellular cyclic nucleotides and protein kinases, alterations in calcium homeostasis, • unexplored mediators or “hidden culprits”: Hydrogen sulphide, carbonic anhydrase and monoamine-oxidase, glucocorticoids and GILZ, • emerging arrhythmogenic substrates caused by fatty-fibrotic tissue infiltration and consequent overflow of proactive molecules (e.g., IL-6) joint to interposition of in-excitable obstacles. Units count on state-of-the-art methodologies, from in vivo to ex-vivo studies in animal models including knockout and transgenic mice for GILZ and H2S-synthetizing enzyme CSE, excellent immunohistochemistry and imaging apparatus, skills and laboratories for cell isolation, culture, and molecular biology, cutting edge set ups and devices for patch-clamp and recording intracellular ions (Ca2+, H+, Na+). The presence of clinicians will guarantee a “bedside to bench” approach to relevant clinical issues and the long-standing collaboration with cardiologists and cardiac surgeons the availability of human cardiac tissue for translational studies. The combination of reliable methodological approach, sound pre-existing background and novel hypothesis emerging from Units’ preclinical and clinical expertise, will drive us to well-defined proofs of conception novel therapeutic strategies, aimed to test unconventional “antiarrhythmic” strategies, acting on channel-modifying pathways (e.g., corticosteroids,) redox/metabolic (oleoylethanolamide) or ionic imbalance (e.g., carbonic anhydrase inhibitors). The identification of unforeseen mechanisms as causal factors for arrhythmias might open novel therapeutic avenues for tailored interventions and allow a better stratification of patients at risk. In particular: 1) Targeting the immune-inflammatory system may effectively reduce arrhythmic risk in selected patients; accordingly, we pursue and envisage that specific inflammatory mediators, such as IL-6, may represent an innovative approach. Anti-IL-6 monoclonal antibodies are currently used for the treatment of rheumatic arthritis (tocilizumab), where an antiarrhythmic potential has been already demonstrated. 2) The evidence that autoantibodies induce channelopathies by directly cross-reacting with specific amino acid sequences on ion channels, suggests a pioneering therapeutic approach based on the use of short decoy peptides (peptide-based therapy) distracting the pathogenic antibodies from channel binding-sites. 3) At this regard, an original interaction in our project consists in the study of the role of GILZ in the direct/indirect effects of immunoinflammatory mediators. This idea emerges from the observation that the clinical electrophysiological effect of glucocorticoids precedes their anti-inflammatory action. The presence of a Unit with recognized experience in this field may allow not only searching for a mechanistic interpretation of clinical findings, but also to prospectively investigate alterations in GILZ expression/function (e.g., exploiting available animal models) as a basis for the propensity to arrhythmias. 4) The identification of electrophysiological effects – either direct or indirect – of the H2S-mediated pathways in cardiomyocytes (CMs) over a large span of models, from cell cultures to human cells, and its crosstalk with the redox/metabolic homeostasis and immunoinflammatory response opens new potential strategies for co-morbidities with significant clinical impact, such as atrial fibrillation associated with cardiomyopathies or obesity. As recently pointed out, to date the development and exploitation of H2S donors against cardiovascular diseases has been prevented by the paucity of basic and translational studies. Moreover, while the efficacy as antithrombotic agents of H2S donors has been documented, their antiarrhythmic activity – despite recognised effects on ion channels – awaits for investigation. 5) Strictly related to gas transmitters (NO, H2S), the redox balance and ROS production will be studied from the perspective of mitochondria, and particularly MAO, based on our published and unpublished data showing over-activation/expression in human cardiomyopathies. The identification of signalling pathways from ROS to target oxidation and phosphorylation (CaMKII and sodium channel) may allow envisaging the exploitation of MAO inhibitors or testing the effect of drugs such as ranolazine on this pathway. 6) The carbonic anhydrase (CA) pathway is a further original topic in our project, and a neglected “culprit” in arrhythmogenesis. This might be surprising due to the long-standing evidence of the relevance of pH homeostasis in controlling cell excitability and intracellular calcium handling. Especially in human CMs, nothing has been attempted to identify CA isoforms, their subcellular localization and effect on excitation contraction coupling mechanisms in health and disease. We will exploit the promising circumstance of availability of human CMs, pioneer experience in studying their electrophysiological properties, and the established collaboration with Prof. Supuran’s lab (Dr. Berrino) in UNIFI providing isoform selective inhibitors of CA to explore this field. The collaboration with medicinal chemistry is of utmost importance for drug development. 7) Still in the track of unconventional antiarrhythmic strategies is the approach with the endogenous activator of PPAR-alpha, oleoylethanolamide (OEA), whose benefit in metabolic and psychiatric disorders has been demonstrated; however, no clear evidence exists concerning cardiac diseases. In view of the increasing appraisal of the arrhythmogenic burden caused by adipose tissue infiltration in atrial and ventricular tissue, it is mandatory to test the hypothesis of OEA efficacy also in a model of obesity-induced cardiomyopathy. Of course, adipose tissue may act by secreting proactive molecules (e.g. IL-6) able to modify CM electrical properties; as said, this cytokine is a crucial part of our project. However, it would be interesting to construct a model based on immunostaining analysis from DIO rats to simulate the impact of infiltration (i.e., adipocytes as in-excitable obstacles) on cardiac conduction. To this aim, we can exploit our skills in modelling combined to long-standing collaboration with LENS lab in Sesto Fiorentino.

 

This project has received funding from Ministry of University and Research (MUR) – PRIN 2017