Cardiac microdomains in cyclic nucleotide signalling in mouse atrial cardiomyocytes: role of the caveolar compartments and popeye proteins

Transverse axial tubules (TAT) and caveolae are essential structural microdomains in cardiomyocytes that recruit components of various signalling pathways. Of particular interest, β- adrenergic receptors (β-ARs) are localised to these structures and in response to catecholamines elicit compartmentalised cyclic adenosine 3′,5′-monophosphate (cAMP) signals. Both Popeye domain-containing (POPDC) and Caveolin-3 (CAV3) proteins are localised to these membrane compartments. POPDC1 is a CAV3-interacting protein, and the Popeye domain acts as a highaffinity cAMP binding site. The function of these proteins in atrial myocytes (AMs) remains elusive. This study has investigated whether AMs isolated from CAV3 and POPDC null mutants display an altered TAT structure and aberrant cAMP response. The TAT structure was investigated in AMs isolated from the left and right atria of CAV3-/- and Popdc1-/- mice. In both mutants, the TAT structure of AMs originating from the right atria were stronger affected than from the left. cAMP compartmentation was studied with the help of a transgenic FRET sensor. In response to β2-AR-stimulation, phosphodiesterase (PDE) 4 is critical compared to PDE3 for cAMP compartmentation. To understand how changes in TAT structure and cAMP signalling might alter atrial function, sinoatrial pacemaking and atrial conduction were studied in Popdc1-/- and Popdc2-/- isolated atrial tissues. Both mutants demonstrated abnormal pacemaker activity, associated with depressed sinoatrial pacemaking, enhanced ectopy and tachycardia-bradycardia arrhythmias increasing HR lability in response to β-AR stimulation. Additionally, Popdc1-/- displayed slower atrial conduction, increased fibrosis and downregulated connexin-43 expression. Along with the altered fast sodium current and the elevated late sodium current, these facilitated the development of atrial tachyarrhythmias in Popdc1-/- mutants. These data suggest that Cav3-/- and Popdc1-/- mutants are associated with structural changes resulting in aberrant cAMP compartmentation, which may result in an increased risk of developing atrial arrhythmogenesis.Open Acces

Dynamic regulation of subcellular calcium handling in the atria:modifying effects of stretch and adrenergic stimulation

Atrial fibrillation is the fast and irregular heart rate that occurs when the upper chambers of the heart experience chaotic electrical activation. Three main factors contribute to the development of this disease: triggers, substrate and modifying factors. An arrhythmia is thus like a fire that needs a spark (Trigger) to ignite a pile of wood (Substrate) and depends on the humidity or accelerants (modifying factors) to burn faster or slower. This body of work takes a closer look at such modifying factors. The major finding of this thesis is that stretching atrial heart muscle cells releases Calcium ions from storage spaces within each cell. If these Calcium release events get frequent enough they can act as triggers for the arrhythmia. The thickness of the atrial muscle is heterogeneous, thus filling the atrium with blood distends thinner parts stronger than ticker portions. The varying degree of stretch might stimulate Calcium release predominantly from myocytes in thinner regions of the atria. This heterogeneity in spontaneous Calcium release can modify also the substrate. A comparable effect of stretch was previously described in the heart’s main chambers. However, it appears that the in the atria it depends on another mechanism, which could serve as a treatment target that mainly acts on the atria without negatively affecting the ventricle

Structural and functional differences between cardiomyocytes from right and left ventricles in health and disease

Several disorders including pulmonary hypertension (PH) and heart failure (HF) could lead to right ventricle (RV) hypertrophy and failure. RV failure is one of the most important prognostic factors for morbidity and mortality in these disorders. However, there is still no therapy to prevent the RV hypertrophy in PH. Treatments developed for the left ventricle (LV) failure do not improve the survival in patients with RV failure probably due to the significant differences in the chambers physiology and hemodynamic function. A better understanding of the cellular and molecular mechanisms of RV hypertrophy is needed. Our focus lies into the alterations of cellular microarchitecture that promotes functional changes in Ca2+ handling. Recently our group showed that reorganisation of the transverse-axial tubular system (TATS) in HF are of particular importance for Ca2+ mishandling and contractile impairment of failing cells. Rationale: This study aims to establish the differences in membrane organisation of Ca2+ handling between healthy RV and LV myocytes, and to investigate the remodelling of RV during disease. Specifically, the objectives are: (1) To study the membrane organisation of RV and LV myocytes by revealing the surface topography using Scanning Ion Conductance Microscopy and by studying the TATS using confocal microscopy. (2) To assess the contraction and Ca2+ transients in RV and LV myocytes. (3) To determine the spatial distribution and properties of single L-type Ca2+ channels (LTCC) in RV myocytes using \u201csmart patch clamp\u201d technique. (4) To describe the changes occurring in the RV and LV in the two disease rat models: PH induced by monocrotaline injection and HF induced by chronic myocardial infarction (MI). This thesis showed that in healthy samples the TATS of RV myocytes has a different organization as compared to LV. Two main Ca2+ channels for the excitation-contraction coupling: LTCC and ryanodine receptors (RyR) were studied by immunofluorescence staining. The density of LTCC was lower in RV than in LV myocytes. However, the density of RyR was similar between the chambers. Contraction duration was longer in RV than in LV myocytes. The distribution of functional LTCCs in RV myocytes was uniform along the cell surface, in contrast to LV myocytes, where LTCCs were primarily located in the T-tubules. Secondly, PH rats showed a reduction of the conduction velocity anisotropy throughout the RV as well as prolongation of the refractoriness of the tissue. The hypertrophy of RV myocytes in PH was accompanied by the reduction of the TATS organisation. The amplitude of contraction of RV PH myocytes was higher, the activation of Ca2+ transients was more desynchronised than in controls, and the rate of spontaneous Ca2+ activity was significantly elevated. Functionally, in PH the open probability (Po) of LTCC located in the T-tubules was significantly higher. On the other hand, PH LV myocytes had preserved TATS but still showed prolonged Ca2+ transients that could influence increased refractoriness of LV tissue. Thirdly, by studying RV myocytes from the MI model, a significant hypertrophy was found, accompanied by a reduction of TATS organisation. The study reports a prolongation of Ca2+ transients with more frequent local Ca2+ waves in MI versus control RV myocytes. Higher Po of LTCCs was shown in MI RV myocytes could be associated with the PKA-mediated phosphorylation. In summary, RV myocytes have a lower TATS organisation than LV myocytes probably related to the lower workload of the RV chamber. Consequently, RV myocytes present several differences with LV myocytes, including changes in the Ca2+ handling or a more uniform distribution of LTCC on the membrane. Diseases induce reduction of TATS and Ca2+ mishandling in both chambers. Due to the intrinsic differences of RV versus LV myocytes, the RV could be more prone to pathological events in early stages of the diseases, which should be investigated further

Modelling pathological effects in intracellular calcium dynamics leading to atrial fibrillation

The heart beating is produced by the synchronization of the cardiac cells' contraction. A dysregulation in this mechanism may produce episodes of abnormal heart contraction. The origin of these abnormalities often lies at the subcellular level where calcium is the most important ion that controls the cell contraction. The regulation of calcium concentration is determined by the ryanodine receptors (RyR), the calcium channels that connect the cytosol and the sarcoplasmic reticulum. RyRs open and close stochastically with calcium-dependent rates. The fundamental calcium release event is known as calcium spark, which refers to a local release of calcium through one or more RyRs. Thus, a deep knowledge on both the spatio-temporal characteristics of the calcium patterns and the role of the RyRs is crucial to understand the transition between healthy to unhealthy cells. The aim of this Thesis has been to figure out these changes at the submicron scale, which may induce the transition to Atrial Fibrillation (AF) in advanced stages. To address this issue, I have developed, and validated, a subcellular mathematical model of an atrial myocyte which includes the electro-physiological currents as well as the fundamental intracellular structures. The high resolution of the model has allowed me to study the spatio-temporal calcium features that arise from both the cell stimulation and the resting conditions. Simulations show the relevance of the assembly of RyRs into clusters, leading to the formation of macro-sparks for heterogeneous distributions. These macro-sparks may produce ectopic beats under pathophysiological conditions. The incorporation of RyR-modulators into the model produces a nonuniform spatial distribution of calcium sparks, a situation observed during AF. In this sense, calsequestrin (CSQ) has emerged as a key calcium buffer that modifies the calcium handling. The lack of CSQ produces an increase in the spark frequency and, during calcium overload, it also promotes the appearance of global calcium oscillations. Finally, I have also characterized the effect of detubulation, a common issue in cells with AF and heart failure. Thus, the present work represents a step forward in the understanding of the mechanisms leading to AF, with the development of computational models that, in the future, can be used to complement in vitro or in vivo studies, helping find therapeutic targets for this disease

25 years of basic and translational science in EP Europace: novel insights into arrhythmia mechanisms and therapeutic strategies.

In the last 25 years, EP Europace has published more than 300 basic and translational science articles covering different arrhythmia types (ranging from atrial fibrillation to ventricular tachyarrhythmias), different diseases predisposing to arrhythmia formation (such as genetic arrhythmia disorders and heart failure), and different interventional and pharmacological anti-arrhythmic treatment strategies (ranging from pacing and defibrillation to different ablation approaches and novel drug-therapies). These studies have been conducted in cellular models, small and large animal models, and in the last couple of years increasingly in silico using computational approaches. In sum, these articles have contributed substantially to our pathophysiological understanding of arrhythmia mechanisms and treatment options; many of which have made their way into clinical applications. This review discusses a representative selection of EP Europace manuscripts covering the topics of pacing and ablation, atrial fibrillation, heart failure and pro-arrhythmic ventricular remodelling, ion channel (dys)function and pharmacology, inherited arrhythmia syndromes, and arrhythmogenic cardiomyopathies, highlighting some of the advances of the past 25 years. Given the increasingly recognized complexity and multidisciplinary nature of arrhythmogenesis and continued technological developments, basic and translational electrophysiological research is key advancing the field. EP Europace aims to further increase its contribution to the discovery of arrhythmia mechanisms and the implementation of mechanism-based precision therapy approaches in arrhythmia management

Arrhythmogenic Mechanisms in Heart Failure: Linking β-Adrenergic Stimulation, Stretch, and Calcium

Heart failure (HF) is associated with elevated sympathetic tone and mechanical load. Both systems activate signaling transduction pathways that increase cardiac output, but eventually become part of the disease process itself leading to further worsening of cardiac function. These alterations can adversely contribute to electrical instability, at least in part due to the modulation of Ca2+ handling at the level of the single cardiac myocyte. The major aim of this review is to provide a definitive overview of the links and cross talk between β-adrenergic stimulation, mechanical load, and arrhythmogenesis in the setting of HF. We will initially review the role of Ca2+ in the induction of both early and delayed afterdepolarizations, the role that β-adrenergic stimulation plays in the initiation of these and how the propensity for these may be altered in HF. We will then go onto reviewing the current data with regards to the link between mechanical load and afterdepolarizations, the associated mechano-sensitivity of the ryanodine receptor and other stretch activated channels that may be associated with HF-associated arrhythmias. Furthermore, we will discuss how alterations in local Ca2+ microdomains during the remodeling process associated the HF may contribute to the increased disposition for β-adrenergic or stretch induced arrhythmogenic triggers. Finally, the potential mechanisms linking β-adrenergic stimulation and mechanical stretch will be clarified, with the aim of finding common modalities of arrhythmogenesis that could be targeted by novel therapeutic agents in the setting of HF

Translational potential of human embryonic and induced pluripotent stem cells for myocardial repair: Insights from experimental models

Heart diseases have been a major cause of death worldwide, including developed countries. Indeed, loss of non-regenerative, terminally differentiated cardiomyocytes (CMs) due to aging or diseases is irreversible. Current therapeutic regimes are palliative in nature, and in the case of end-stage heart failure, transplantation remains the last resort. However, this option is significantly hampered by a severe shortage of donor cells and organs. Human embryonic stem cells (hESCs) can self-renew while maintaining their pluripotency to differentiate into all cell types. More recently, direct reprogramming of adult somatic cells to become pluripotent hES-like cells (a.k.a. induced pluripotent stem cells or iPSCs) has been achieved. The availability of hESCs and iPSCs, and their successful differentiation into genuine human heart cells have enabled researchers to gain novel insights into the early development of the human heart as well as to pursue the revolutionary paradigm of heart regeneration. Here we review our current knowledge of hESC-/iPSC-derived CMs in the context of two fundamental operating principles of CMs (i.e. electrophysiology and Ca2+-handling), the resultant limitations and potential solutions in relation to their translation into clinical (bioartificial pacemaker, myocardial repair) and other applications (e.g. as models for human heart disease and cardiotoxicity screening). © Schattauer 2010.published_or_final_versio

Modelling the interaction between induced pluripotent stem cells derived cardiomyocytes patches and the recipient hearts

Cardiovascular diseases are the main cause of death worldwide. The single biggest killer is represented by ischemic heart disease. Myocardial infarction causes the formation of non-conductive and non-contractile, scar-like tissue in the heart, which can hamper the heart's physiological function and cause pathologies ranging from arrhythmias to heart failure. The heart can not recover the tissue lost due to myocardial infarction due to the myocardium's limited ability to regenerate. The only available treatment is heart transpalant, which is limited by the number of donors and can elicit an adverse response from the recipients immune system. Recently, regenerative medicine has been proposed as an alternative approach to help post-myocardial infarction hearts recover their functionality. Among the various techniques, the application of cardiac patches of engineered heart tissue in combination with electroactive materials constitutes a promising technology. However, many challenges need to be faced in the development of this treatment. One of the main concerns is represented by the immature phenotype of the stem cells-derived cardiomyocytes used to fabricate the engineered heart tissue. Their electrophysiological differences with respect to the host myocardium may contribute to an increased arrhythmia risk. A large number of animal experiments are needed to optimize the patches' characteristics and to better understand the implications of the electrical interaction between patches and host myocardium. In this Thesis we leveraged cardiac computational modelling to simulate \emph{in silico} electrical propagation in scarred heart tissue in the presence of a patch of engineered heart tissue and conductive polymer engrafted at the epicardium. This work is composed by two studies. In the first study we designed a tissue model with simplified geometry and used machine learning and global sensitivity analysis techniques to identify engineered heart tissue patch design variables that are important for restoring physiological electrophysiology in the host myocardium. Additionally, we showed how engineered heart tissue properties could be tuned to restore physiological activation while reducing arrhythmic risk. In the second study we moved to more realistic geometries and we devised a way to manipulate ventricle meshes obtained from magnetic resonance images to apply \emph{in silico} engineered heart tissue epicardial patches. We then investigated how patches with different conduction velocity and action potential duration influence the host ventricle electrophysiology. Specifically, we showed that appropriately located patches can reduce the predisposition to anatomical isthmus mediated re-entry and that patches with a physiological action potential duration and higher conduction velocity were most effective in reducing this risk. We also demonstrated that patches with conduction velocity and action potential duration typical of immature stem cells-derived cardiomyocytes were associated with the onset of sustained functional re-entry in an ischemic cardiomyopathy model with a large transmural scar. Finally, we demonstrated that patches electrically coupled to host myocardium reduce the likelihood of propagation of focal ectopic impulses. This Thesis demonstrates how computational modelling can be successfully applied to the field of regenerative medicine and constitutes the first step towards the creation of patient-specific models for developing and testing patches for cardiac regeneration.Open Acces