Modellistica Computazionale dell'Elettrofisiologia Cardiaca: dalla Cellula al Paziente

Heart diseases are the leading cause of death worldwide, both for men and women. However, the ionic mechanisms underlying many cardiac arrhythmias and genetic disorders are not completely understood, thus leading to a limited efficacy of the current available therapies and leaving many open questions for cardiac electrophysiologists. On the other hand, experimental data availability is still a great issue in this field: most of the experiments are performed in vitro and/or using animal models (e.g. rabbit, dog and mouse), even when the final aim is to better understand the electrical behaviour of in vivo human heart either in physiological or pathological conditions. Computational modelling constitutes a primary tool in cardiac electrophysiology: in silico simulations, based on the available experimental data, may help to understand the electrical properties of the heart and the ionic mechanisms underlying a specific phenomenon. Once validated, mathematical models can be used for making predictions and testing hypotheses, thus suggesting potential therapeutic targets. This PhD thesis aims to apply computational cardiac modelling of human single cell action potential (AP) to three clinical scenarios, in order to gain new insights into the ionic mechanisms involved in the electrophysiological changes observed in vitro and/or in vivo. The first context is blood electrolyte variations, which may occur in patients due to different pathologies and/or therapies. In particular, we focused on extracellular Ca2+ and its effect on the AP duration (APD). The second context is haemodialysis (HD) therapy: in addition to blood electrolyte variations, patients undergo a lot of other different changes during HD, e.g. heart rate, cell volume, pH, and sympatho-vagal balance. The third context is human hypertrophic cardiomyopathy (HCM), a genetic disorder characterised by an increased arrhythmic risk, and still lacking a specific pharmacological treatment.Le malattie cardiache e cardiovascolari sono ad oggi la causa principale di morte nel mondo. Tuttavia, i meccanismi ionici responsabili di aritmie o di altre malattie cardiache non sono ancora del tutto conosciuti: questo spesso porta a una minore o mancata efficacia delle terapie attualmente disponibili, e lascia numerose domande aperte per gli elettrofisiologi. Inoltre, la difficoltà di acquisizione dei dati sperimentali rimane ancora uno dei problemi più grandi in questo campo. Infatti la maggior parte dei dati vengono raccolti in vitro e/o utilizzando modelli animali come coniglio, ratto o cane, sebbene l’obiettivo ultimo sia quello di una più completa comprensione del comportamento elettrico del cuore in vivo e nell’uomo, in condizioni sia fisiologiche sia patologiche. In questo contesto, la modellistica computazionale costituisceuno strumento indispensabile: infatti, le simulazioni in silico permettono di superare, almeno in parte, i limiti sperimentali, e di investigare i meccanismi ionici alla base di specifici fenomeni a diversi livelli (singola cellula, tessuto, intero cuore). Una volta validati sui dati sperimentali, i modelli matematici possono essere dunque utilizzati per fare predizioni, testare ipotesi e valutare l’efficacia di eventuali interventi farmacologici. Lo scopo di questa tesi di dottorato è stato quello di applicare tecniche di modellistica matematica a problemi di elettrofisiologia cardiaca, in particolare utilizzando modelli di potenziale d’azione (PA) umano in tre diversi contesti: variazioni elettrolitiche nel sangue, effetti della terapia dialitica sul cuore e cardiomiopatia ipertrofica

Diagnostic and prognostic implications of myocardial Gremlin-1 expression in patients with structural myocardial disease

Gremlin-1, an antagonist of bone morphogenetic proteins, is involved in fibrotic tissue formation in kidney, lung and liver. The impact of myocardial Gremlin-1 expression is unknown. Therefore, we investigated the diagnostic and prognostic value of Gremlin-1 in structural myocardial diseases

Reprogramming and transdifferentiation for cardiovascular development and regenerative medicine: where do we stand?

Heart disease remains a leading cause of mortality and a major worldwide healthcare burden. Recent advances in stem cell biology have made it feasible to derive large quantities of cardiomyocytes for disease modeling, drug development, and regenerative medicine. The discoveries of reprogramming and transdifferentiation as novel biological processes have significantly contributed to this paradigm. This review surveys the means by which reprogramming and transdifferentiation can be employed to generate induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and induced cardiomyocytes (iCMs). The application of these patient-specific cardiomyocytes for both in vitro disease modeling and in vivo therapies for various cardiovascular diseases will also be discussed. We propose that, with additional refinement, human disease-specific cardiomyocytes will allow us to significantly advance the understanding of cardiovascular disease mechanisms and accelerate the development of novel therapeutic options

Distinct properties of the atrial voltage-gated sodium channel

Sodium handling properties of left atria (LA) were compared to those of left ventricle (LV), with the aim to understand the atria’s susceptibility to arrhythmia and improve therapy. Mouse LA sodium channels displayed distinctive activation, inactivation and recovery kinetics compared to LV sodium channels. Distinctive voltage dependence of LA sodium channel inactivation was instrumental in reducing INa in LA compared to LV, when initiated from physiological holding potentials. Flecainide sodium channel inhibition was greater in LA than LV, likely also due to differences in kinetic properties of the sodium channels between chambers. Additionally, the greater inhibitory effect of flecainide at more positive membrane potentials could result in even greater LA sodium channel inhibition in vivo. Activation and inactivation distinctions observed between LA and LV sodium channels were conserved between chambers in the Plako+/- mouse. However, there was no difference in physiological INa density, sodium channel recovery or flecainide inhibition between Plako+/- LA and LV chambers. The novel Langendorff-free isolation method produced high yields of viable mouse cardiomyocytes comparable in morphology, signalling, calcium handling and sodium channel electrophysiology to cardiomyocytes isolated using the traditional Langendorff method. This maintained that injection isolation is a valuable method for obtaining cardiomyocytes for cardiac research

Endocrine system dysfunction and chronic heart failure: a clinical perspective

Chronic heart failure (CHF) leads to an excess of urgent ambulatory visits, recurrent hospital admissions, morbidity, and mortality regardless of medical and non-medical management of the disease. This excess of risk may be attributable, at least in part, to comorbid conditions influencing the development and progression of CHF. In this perspective, the authors examined and described the most common endocrine disorders observed in patients with CHF, particularly in individuals with reduced ejection fraction, aiming to qualify the risks, quantify the epidemiological burden and discuss about the potential role of endocrine treatment. Thyroid dysfunction is commonly observed in patients with CHF, and sometimes it could be the consequence of certain medications (e.g., amiodarone). Male and female hypogonadism may also coexist in this clinical context, contributing to deteriorating the prognosis of these patients. Furthermore, growth hormone deficiency may affect the development of adult myocardium and predispose to CHF. Limited recommendation suggests to screen endocrine disorders in CHF patients, but it could be interesting to evaluate possible endocrine dysfunction in this setting, especially when a high suspicion coexists. Data referring to long-term safety and effectiveness of endocrine treatments in patients with CHF are limited, and their impact on several “hard” endpoints (such as hospital admission, all-cause, and cardiovascular mortality) are still poorly understood

Cardioprotective Actions of Relaxin

Relaxin, a hormone of pregnancy, has shown broad cardioprotective effects including anti-fibrotic (reversed excess TGFβ signaling), anti-arrhythmic (Nav1.5 and INa upregulation and Cx43 phosphorylation and trafficking to intercalated disks) and anti-inflammatory properties (reduced IL-1β and IL-6). While relaxin’s anti-fibrotic effects are thought to occur through the SMAD2/3/TGFβ pathway, there is a general lack of understanding of relaxin’s mode of action to increase sodium current and alter connexin43 localization to suppress arrhythmias. Based on a rat model of aging, we tested the hypothesis that relaxin acts through activation of Wnt/β-catenin signaling to mediate its effects on Nav1.5 and Cx43 and to regulate collagen expression. We show for the first time that relaxin activates canonical Wnt signaling (increased nuclear β-catenin) to increase Nav1.5 in isolated cardiomyocytes. Block of canonical Wnt signaling (via Dikkopf-1) abrogated relaxin’s effect on both Nav1.5 in cardiomyocytes and suppression of excess collagen from fibroblasts. Further, we show that relaxin significantly suppressed expression of Dikkopf-1 in isolated cardiomyocytes and whole LV tissue. In addition, show that relaxin suppressed the age-associated genetic upregulation in inflammatory markers in a sex-dependent manner. Finally, we tested the hypothesis that relaxin would be an effective therapy in a rat model of pulmonary arterial hypertension and show that it reduced occlusion of small pulmonary arteries, myocardial and lung fibrosis and reversed the cardiac phenotype of ventricular arrhythmia or arrest. These results suggest that relaxin signals through multiple pathways to achieve its myriad effects and that relaxin has great potential as a therapy for multiple cardiopulmonary diseases

A Pharmacological Approach towards Myocardial Protection: New Perspectives in Acute and Chronic Cardiac Disease

Several cardiac diseases include myocardial ischaemia (acute or chronic), heart failure (systolic or diastolic) and left ventricular hypertrophy (either as a “primary” cause or developed secondary to other diseases) share the commonality of myocardial energetic deficiency or suboptimal myocardial metabolism. Therefore, approaches to modify myocardial metabolism in order to improve energetics present as an attractive therapeutic option. This is particularly useful when other options are limited: for example, lack of optimal symptom control with “maximal” treatment, or contraindications to other pharmacological treatment (by virtue of impairment of left ventricular systolic function and/or hypotension). The objective of this thesis is to examine the biochemical effects of various pharmacological agents towards modulation of myocardial metabolism, both in the acute (e.g. acute coronary syndrome) and chronic cardiac disease settings (e.g. diabetic heart). In particular, the effects of perhexiline, an interesting drug known to possess not only metabolic effects (by virtue of inhibiting carnitine palmitoyl transferase-1 [CPT-1], thereby shifting myocardial fatty acid oxidation towards glycolysis) but also anti-inflammatory effects, will be further explored. First, the pharmacokinetics and myocardial uptake profile of the individual perhexiline enantiomers were examined. This study showed that the myocardial uptake of both perhexiline enantiomers in patients were slow; and that in multivariate backward stepwise analysis, (-)-perhexiline was inversely correlated with on-treatment heart rate. This finding suggested that the weak calcium antagonist effect of perhexiline may potentially lie predominantly within the (-)- enantiomer. Additionally, other aspects of myocardial metabolism, including the nexus between inflammatory activation and metabolic effect, were investigated. In a study involving 12 patients presenting with acute coronary syndrome and hyperglycaemia, rapid reversal of hyperglycaemia with insulin infusion in 12 hours improved the anti-aggregatory effect of platelets, independent of the platelet content of the pro-inflammatory marker thioredoxin-interacting protein (TXNIP). Furthermore, this thesis also investigated the potential insulin sensitization effect of perhexiline in diabetic patients. This is a corollary of increased glucose utilization, which appears to be relevant even against the background of concomitant therapy with other insulin-sensitizing agents such as AMPK activators or ACE-inhibitors. Furthermore, platelet content of TXNIP tended to fall slightly (but not significantly) after perhexiline treatment, implying its lack of significant critical role in the improvement of both nitric oxide responsiveness and insulin sensitization. However, its overall contribution still cannot be completely ruled out. Lastly, in an in vitro experiment, the potency of inhibition of CPT-1 by both perhexiline enantiomers was investigated. It was found that the 50% inhibitory concentrations of both enantiomers were not significantly different. This provided evidence that the (differential) toxicity seen with the individual enantiomers (in previous studies) might be independent of CPT-1 inhibition. The CPT-1 inhibitory potency of several other cardiac drugs, including fluorinated perhexiline (developed by collaborators in Aberdeen, UK) and dronedarone (a benzofluranyl compound, structurally similar to amiodarone) was also determined in this thesis, and it was shown in particular that dronedarone was a potent CPT-1 inhibitor. The overall thrust of this work reinforces the concept that CPT-1 inhibition is seen with a large number of cardiovascular drugs, and is retained by enantiomers and structural analogues of perhexiline. The myocardial uptake of perhexiline and its enantiomers indicates a relatively slow process of equilibration with its primary sites of action.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201

Paracrine crosstalk between endothelial cells and cardiomyocytes during inflammation

Endothelial cells (EC) release paracrine factors which can modulate the survival, morphology, and function of neighbouring cardiomyocytes (CM), e.g. contraction and relaxation. In multiple cardiomyopathies, as well as in heart failure, EC dysfunction correlates strongly with severity and prognosis, and moreover, endothelial inflammation is thought to precede this dysfunction. While it is known that EC functions are modified by inflammation, the paracrine effects of this response on neighbouring CM are poorly understood and too often confused with those of endothelial dysfunction. I hypothesised that calcium handling and contractile properties of CM were differently regulated by inflamed EC. To investigate this, a co-culture system was first validated as a model of the paracrine EC-CM crosstalk. A pro-inflammatory cocktail of TNFα, IL-1β and hIL-6 (“Cytomix”) was then validated as a pre-conditioning treatment for cardiac microvascular EC. Adult ventricular CM were co-cultured with untreated or Cytomix-treated EC. Calcium transients were then analysed in CM using Fluo-4 and Fura-2. Cell contractility and relaxation were also studied using an automated CytoCypher™ system. Finally, living rat myocardial slices were used to investigate the effects of Cytomix in a more complex heterocellular model. Co-culture with Cytomix-treated EC induced a significant shortening of calcium transients in CM compared to untreated EC (thus validating the hypothesis). This was due to an increase in SERCA activity. However, the cell shortening amplitude and rate of relaxation were unaffected by co-culture or Cytomix treatment, while data also suggests that the myofilament sensitivity to calcium was unchanged. In cultured myocardial slices, Cytomix treatment increased force but not the passive tension, although this was limited to high levels of stretch and kinetics were unchanged compared to untreated slices. Further work is required to better define and understand these mechanisms. This could help determine the therapeutic potential of controlling EC function in inflammatory heart diseases.Open Acces

Atrial Fibrillation and Sudden Cardiac Death in Obesity: An Investigation of the Arrhythmogenicity of Epicardial Fat

Atrial fibrillation and sudden cardiac death are two burgeoning cardiac disorders caused by arrhythmic events in the heart. Patients with atrial fibrillation are at an increased risk of severe cardiovascular complications, hospitalisation, thromboembolic events, clinical morbidity, and mortality. Premature death resulting from sudden cardiac death is a significant cause of cardiovascular mortality, often occurring in patients without apparent high-risk conditions and with normal heart functions. The emergence of obesity epidemic in the community is implicated in the rising burdens of atrial fibrillation and sudden cardiac death, but this has not been well characterised. More recently, the ectopic cardiac fat depot “epicardial adipose tissue” is postulated to mediate the pro-arrhythmic sequalae of obesity. In this thesis, investigations were undertaken to characterise these relations in a meta-analysis; and to evaluate the cardiac electrophysiological and structural substrates due to epicardial fat in ovine sheep models of chronic weight gain and weight fluctuations. In chapter 2, a comprehensive systematic review of the literature and a meta-analysis were conducted to define the association of the fibrotic biomarker galectin-3 and atrial fibrillation. The findings demonstrated significant associations of high serum galectin-3 and risk and severity of atrial fibrillation. In chapter 3, a comprehensive systematic review of the literature and a meta-analysis were conducted to define the clinical associations of epicardial fat and atrial fibrillation, arrhythmia progression, recurrent atrial fibrillation following curative catheter ablation, and post-operative atrial fibrillation after cardiac surgery. The findings demonstrated significant associations of increased expansions of total cardiac and peri-atrial epicardial adipose tissue with greater risk of atrial fibrillation; severity of atrial fibrillation; atrial fibrillation recurrence post-ablation; and de novo incidence after cardiac surgery. Next, the underlying mechanisms were explored in chronic ovine models and presented in chapters 4 & 5. The results demonstrated that obesity induces expansion of epicardial fat and fibro-fatty replacement of atrial myocytes and deterioration of myocyte contractile apparatus, which may drive impairments of atrial electrical properties. Despite having comparable epicardial fat quantity with reference controls, weight fluctuation, induced similar abnormalities, albeit less severe, with stable obesity, thus highlighting an explanation for the increased atrial arrhythmias risks often seen with periodic fluxes in weight. Chapter 6 reports findings from a systematic review and meta-analysis undertaken to define the association between obesity and sudden cardiac death. The pooled analyses involving over 1.4 million patients demonstrated that, after correcting for traditional high-risk risk factors: underweight body mass index (<18.5 kg.m-2) associates with an increased risk of sudden cardiac death; overweight shows no significant association with sudden cardiac death; obesity (BMI: ≥30 kg.m-2) predicts an exaggerated risk for sudden cardiac death. Similarly, unit increment in body mass index was shown to demonstrate a greater risk for sudden cardiac death, further implicating the role of increased adiposity in the risk of sudden cardiac death. In chapter 7, the molecular and structural substrates for ventricular arrhythmias that lead to sudden cardiac death in a model of chronic obesity are presented. Obesity demonstrated two-and-half-fold expanded ventricular epicardial fat depot with a consequent extensive and severe fat cell infiltrations; significant reduction in ventricular desmosomal cadherin desmoglein-2, which demonstrated significant negative correlation with the degree of fatty infiltration; and induction of diffuse ventricular interstitial fibrosis. The findings further demonstrated that obesity results in significant abnormal modulation of fibrotic pathways, including an alternative component of the central transforming growth factor-beta 1 pathway, angiotensin II, endothelin and aldosterone signalling pathways. The observations of epicardial fat expansion and subsequent fibro-fatty infiltrations are particularly noteworthy. Epicardial fat adds an important extra layer to the stratification of patients at risk of atrial fibrillation and sudden cardiac death. Fibro-fatty infiltrates alone are sufficient to induce re-entrant tachyarrhythmias, leading to atrial fibrillation and sudden cardiac death. Clinical assessment of fibro-fatty infiltrates could help improve sudden cardiac death risk profiling of patients in the low-risk communities, who paradoxically have high absolute mortality rates. More importantly, the fibro-fatty deposits could form a key element in substrate mapping as a guide for ablation of lethal arrhythmias.Thesis (Ph.D.) -- University of Adelaide,Adelaide Medical School, 201