Search

Cardiac action potential (AP) shape and propagation are regulated by several key dynamic factors such as ion channel recovery and intracellular Ca2+ cycling. Experimental methods for manipulating AP electrical dynamics commonly use ion channel inhibitors that lack spatial and temporal specificity. In this work, we propose an approach based on optogenetics to manipulate cardiac electrical activity employing a light-modulated depolarizing current with intensities that are too low to elicit APs (sub-threshold illumination), but are sufficient to fine-tune AP electrical dynamics. We investigated the effects of sub-threshold illumination in isolated cardiomyocytes and whole hearts by using transgenic mice constitutively expressing a light-gated ion channel (channelrhodopsin-2, ChR2). We find that ChR2-mediated depolarizing current prolongs APs and reduces conduction velocity (CV) in a space-selective and reversible manner. Sub-threshold manipulation also affects the dynamics of cardiac electrical activity, increasing the magnitude of cardiac alternans. We used an optical system that uses real-time feedback control to generate re-entrant circuits with user-defined cycle lengths to explore the role of cardiac alternans in spontaneous termination of ventricular tachycardias (VTs). We demonstrate that VT stability significantly decreases during sub-threshold illumination primarily due to an increase in the amplitude of electrical oscillations, which implies that cardiac alternans may be beneficial in the context of self-termination of VT. [ABSTRACT FROM AUTHOR]

Key points: Although optogenetics has clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies lack the capability to react acutely to ongoing cardiac wave dynamics. Here, we developed an all‐optical platform to monitor and control electrical activity in real‐time. The methodology was applied to restore normal electrical activity after atrioventricular block and to manipulate the intraventricular propagation of the electrical wavefront. The closed‐loop approach was also applied to simulate a re‐entrant circuit across the ventricle. The development of this innovative optical methodology provides the first proof‐of‐concept that a real‐time all‐optical stimulation can control cardiac rhythm in normal and abnormal conditions. Abstract: Optogenetics has provided new insights in cardiovascular research, leading to new methods for cardiac pacing, resynchronization therapy and cardioversion. Although these interventions have clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies do not take into account cardiac wave dynamics in real time. Here, we developed an all‐optical platform complemented by integrated, newly developed software to monitor and control electrical activity in intact mouse hearts. The system combined a wide‐field mesoscope with a digital projector for optogenetic activation. Cardiac functionality could be manipulated either in free‐run mode with submillisecond temporal resolution or in a closed‐loop fashion: a tailored hardware and software platform allowed real‐time intervention capable of reacting within 2 ms. The methodology was applied to restore normal electrical activity after atrioventricular block, by triggering the ventricle in response to optically mapped atrial activity with appropriate timing. Real‐time intraventricular manipulation of the propagating electrical wavefront was also demonstrated, opening the prospect for real‐time resynchronization therapy and cardiac defibrillation. Furthermore, the closed‐loop approach was applied to simulate a re‐entrant circuit across the ventricle demonstrating the capability of our system to manipulate heart conduction with high versatility even in arrhythmogenic conditions. The development of this innovative optical methodology provides the first proof‐of‐concept that a real‐time optically based stimulation can control cardiac rhythm in normal and abnormal conditions, promising a new approach for the investigation of the (patho)physiology of the heart. [ABSTRACT FROM AUTHOR]

Well-coordinated activation of all cardiomyocytes must occur on every heartbeat. At the cell level, a complex network of sarcolemmal invaginations, called the transverse-axial tubular system (TATS), propagates membrane potential changes to the cell core, ensuring synchronous and uniformexcitation-contraction coupling. Although myocardial conduction of excitation has been widely described, the electrical properties of the TATS remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of TATS. Fluorescence recovery after photobleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: A fluorescent dextran inside TATS lumen is photobleached, and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along TATS. The method is first validated in cells where most TATS elements are acutely detached by osmotic shock and then applied to probe TATS electrical conductivity in failing heart cells. We find that acute and pathological tubular remodeling significantly affect TATS electrical conductivity. This may explain the occurrence of defects in action potential propagation at the level of single T-tubules, recently observed in diseased cardiomyocytes. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: None. Cardiac action potential (AP) shape and propagation are regulated by several key dynamic factors such as ions channel recovery and intracellular Ca2+-cycling. Experimental methods for manipulating AP electrical dynamics commonly use ion channel inhibitors that lack spatial and temporal specificity. In this work, we propose a novel approach based on optogenetics to manipulate cardiac electrical activity employing a light-modulated depolarizing current with intensities that are too low to elicit APs (sub-threshold illumination) but are sufficient to fine-tune AP electrical dynamics. We investigated the effects of sub-threshold illumination in isolated cardiomyocytes and whole hearts by using transgenic mice constitutively expressing a light-gated ion channel (channelrhodopsins-2, ChR2). We find that ChR2-mediated depolarizing current prolongs APs and reduces conduction velocity (CV) in a space-selective and reversible manner. Sub-threshold manipulation also affects the dynamics of cardiac electrical activity, increasing the magnitude of cardiac alternans. We use an optical system that uses real-time feedback control to generate re-entrant circuits with user-defined cycle lengths to explore the role that cardiac alternans plays in spontaneous termination of ventricular tachycardias (VTs). We find that VT stability significantly decreases during sub-threshold illumination primarily due to an increase in the amplitude of electrical oscillations, which implies that cardiac alternans may be beneficial in the context of self-termination of VT. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondazione Intesa San Paolo Introduction Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive degeneration of striated muscles; in addition to skeletal muscle impairment, DMD is also characterized by progressive myocardial disfunction. The low translational value of animal models and the low availability of human samples make DMD hard to investigate; induced pluripotent stem cells (iPSCs) represent a novel tool to model this disease, preserving the genetic heritage of the patient, including the pathogenic mutation causing dystrophy. Aim Our aim is to characterize cardiomyocytes differentiated from iPSCs (iPSC-CMs) derived from healthy donors (CTRL) and DMD patients, to identify the pathophysiological mechanisms of DMD-related cardiomyopathy. Materials and Methods Cardiomyocytes are differentiated from IPSCs obtained by reprogramming isolated mononucleated blood cells from healthy donors and DMD patients. IPSC-CMs are cultured until day 60, 75 or 90 post-differentiation after plating on nanostructured substrates with two different stiffness levels: PEG-substrates, with lower rigidity, mimicking healthy extracellular tissue, and DEG-substrates, with greater rigidity, that mimic the presence of myocardial fibrosis. Through imaging techniques, we evaluated calcium handling and action potentials (AP) on DMD and CTRL iPSC-CMs by using specific fluorescent dyes for Ca2+ (CAL630) and membrane voltage (Fluovolt). Cells were stimulated at different pacing rates. Results The calcium transient amplitude of CTRL-iPSC-CMs became larger during maturation. This adaptation did not occur in DMD lines, showing a deficit calcium release due to poor maturation of the sarcoplasmic reticulum (SR). AP duration was shorter in the DMD line at d75 but at d90 we observed no differences when compared with the CTRL line. CTRL iPSC-CMs showed a marked ability to adapt to different substrate stiffnesses. Indeed, the calcium transient amplitude was larger and its kinetics faster when cells were grown on the rigid DEG substrates rather than on PEG plates. In the DMD line, however, no differences were observed between the substrates. Conclusions Our results highlight a scarce ability of DMD iPSC-CM to adapt to different substrate stiffness, resulting in mechanical and electrical impairment, especially in the presence of stiffer substrates. This might explain why cardiac impairment is usually absent in the early stages of DMD, when cardiac structural changes are still absent. However, the electrophysiological and mechanical impairment of DMD hearts may precipitate rapidly when extracellular stiffness starts to increase due to development of cardiac fibrosis. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union's Horizon 2020 research and innovation programme - No 952166 (REPAIR) Regione Toscana - Bando Ricerca Salute 2018 - PERCARE project. Severe remodeling processes may occur in the heart due to both genetic and non-genetic diseases. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction at different orders of magnitude and, eventually, lead to arrhythmias. In this scenario, arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease that involves ventricular dysfunction, arrhythmias, and localized replacement of contractile fibers with fibrofatty scar tissue. Unfortunately, nowadays, predicting the impact of fine structural alterations on the electrical dysfunction in entire organs is challenging, due to the inefficacy of standard imaging methods in performing high-resolution three-dimensional reconstructions in massive tissues. In this work, we developed a new full-optical correlative approach to quantify and integrate the electrical dysfunctions with three-dimensional structural reconstructions of entire hearts, both in controls and in a mouse model of ACM. We combined optical mapping of the action potential propagation (APP) with advances in tissue clearing and light-sheet microscopy techniques. First, we employed an optical platform to map and analyze the APP in Langendorff-perfused hearts. Then, we optimized the SHIELD procedure for the clearing of cardiac tissue, thus converting the previously electrically characterized samples into well-preserved and fully-transparent specimens. A high-throughput light-sheet microscope has been developed following the mesoSPIM project: the conceived microscope allows the reconstruction of the whole mouse heart with a micrometric resolution allowing fine quantification of myocytes alignment and fibrosis deposition across the organ. Finally, we developed a software pipeline that employs high-resolution 3D images to analyze and co-register APP maps with the 3D anatomy, contractile fibers disarray, and fibrosis deposition on each heart. We believe that this promising methodological framework will allow clarifying the involvement of fine structural alterations in the electrical dysfunctions, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): In Silico project Background Hypertrophic cardiomyopathy (HCM) represents the commonest inherited cardiac disease, with a prevalence of 1/500 in the general population. The most devastating consequence of HCM is sudden cardiac death (SCD) due to ventricular fibrillation, particularly common in children and young adults. The positive correlation between the extent of late gadolinium enhancement (LGE, reflecting myocardial fibrosis) and the arrhythmic risk suggests that ventricular arrhythmias are held to originate from the fibrotic regions, by a mechanism of electrical re-entry. However, recent data suggest that enhanced cellular automaticity (i.e.  early- or delayed-afterdepolarizations, EADs or DADs-) may be clinically more relevant in promoting ventricular arrhythmias in patients. Purpose Aiming to better understand the cellular and molecular mechanisms of arrhythmogenesis in HCM and to establish a reliable arrhythmic risk stratification in patients, we performed a translational and retrospective study in 61 HCM patients who underwent surgical myectomy, by combining a clinical follow-up study with in vitro assessments of cellular arrhythmogenicity. Methods We retrospectively studied 61 HCM patients who underwent surgical interventricular-septum myectomy to relieve refractory obstruction-related symptoms. At the time of surgery, fresh ventricular tissue was collected and used to isolate single ventricular cardiomyocytes (CMs), which were used for patch-clamp measurements and Ca2+ imaging experiments to assess the occurrence of EADs and DADs. Patients were followed up for a median time of 8 years and the occurrence of non-sustained ventricular tachycardia (NSVT) or life-threatening arrhythmic events (LAE, including sustained VT and ventricular fibrillation, VF) was monitored. Moreover, data from ECG and cardiac magnetic-resonance studies were collected. Results EADs occurred in CMs from 36% of patients and were associated with prolonged action potential duration while DADs occurred in 24% of patients and correlated with abnormalities of intracellular Ca2+ handling. During follow up, NSVT events occurred in 19/61 patients while LAE occurred in 4/61 patients. Their combined occurrence (37%) strongly correlated with the presence of DADs in cardiomyocytes. Patients with NSVT/LAE were more likely to show specific "pro-arrhythmic" pathological ECG-patterns. Among patients with LGE, the presence of DADs in cells behaved as a necessary pre-requisite for NSVT/LAE, as none of the patients with evidence of fibrosis but negative for DADs had arrhythmic events. Conclusions The presence of pro-arrhythmic changes appears to be necessary for arrhythmia generation in HCM and seems to be related with specific alterations at ECG level, that might be used as clinical arrhythmia predictors in HCM patients. Fibrosis per se is not a major predictor of arrhythmias in HCM but may contribute to generate sustained arrhythmias in the presence of substantial cellular triggers (DADs). [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministero dell'università e della ricerca Electrophysiological remodeling in primary versus secondary cardiac hypertrophy: a study in human cardiomyocytes Introduction Myocardial hypertrophy is an adaptive condition to hemodynamic stress, characterized by the increase in size of the cardiomyocytes and in the cardiac mass. It involves more frequently the left ventricle (LV) and leads to an enhanced risk of arrhythmias; it's characterized by the increase walls thickness and by a large remodelling of the cardiac tissue in terms of fibrosis, impaired calcium handling and ion current balance. The LV hypertrophy could be induced by genetic abnormalities, such as in hypertrophic cardiomyopathy (HCM) or it could be secondary to other pathological conditions, like aortic stenosis (SAO-HT). Purpose In this work we will compare the mechanisms that mainly determine primitive (HCM samples) or secondary (to aortic stenosis: SAO-HT) LV hypertrophy to assess common points and disagreements. Methods Human septal specimens from HCM, SAO-HT and control (CTRL) surgical patients were collected, tissue was in part rapidly frozen, in part processed to isolate responsive single cells or trabeculae. We performed patch clamp experiments to measured action potential duration (APD) and calcium current (ICaL). To study the Ca2+ handling we performed imaging analysis; isolated cardiomyocytes were loaded with a Ca2+ sensitive dye (CAL530) by incubating cells for 30 min, then we evaluate the calcium transients at different stimulation frequencies. Results We evaluated APD, ICaL, and Ca2+ handling in the 3 groups: HCM, SAO-HT and CTRL cardiomyocytes. Both pathological groups show to be depolarized, to have a prolonged APD, diastolic Ca2+ concentration is higher and the kinetics are slower than in CTRL cardiomyocytes. HCM cardiomyocytes has longer APD than SAO-HT and higher diastolic Ca2+ concentration, HCM also shows longer kinetics of the decay phase of the Ca2+ transient compare to SAO-HT. The ICaL is comparable between CTRL and SAO-HT, it is higher in HCM and the inactivation kinetics are slower. Discussion and Conclusion We evaluate the electrophysiological differences among cardiomyocytes derived from a secondary or primary myocardial hypertrophy and from non-hypertrophic myocardia. Alterations in the two groups of pathological samples are similar. Both shows slowed Ca2+ transient kinetics, that slow down the relaxation machinery, and increase diastolic Ca2+ concentration, that could cause arrhythmic events (DAD). The increase in diastolic Ca2+ could also be responsible for the hyperactivation of the CAMK-II that has among its target ICaL channel and the Na2+ channel that are hyperactivated causing the prolongation of APD. The functional alterations observed in the hypertrophic cardiomyocytes could be considered not as disease specific response but a mechanism of adaptation to the hypertrophy and to diastolic dysfunction, those are more serious where the hypertrophy is genetic determined. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): M.I.U.R. PRIN 2017 to E.C and ECRF to L.S. Introduction Ivabradine (Iva), Zatebradine (Zat) and ZD7288 (ZD) are blockers of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, which play definite electrophysiological functions in cardiac pacemakers, working cardiomyocytes, peripheral and central neurons [1]. Among them, Iva is in clinical use as bradycardic agent for uncontrolled angina and heart failure with reduced ejection fraction. Accumulating evidence suggests Iva may have a more complex pharmacological profile, including targets different from HCN channels [2, 3]. Reports on experimental atrial fibrillation, detrusor contraction and bladder overactivity [4] led us to hypothesize that muscarinic receptors (mAChRs) may represent still unexplored targets of Iva and analogues. Present study was designed to identify the effect of Iva on acetylcholine-gated inward-rectifier K+-current (IKACh), the main effector of atrial mAChRs, and to assess whether Iva and analogues interact directly with human atrial and recombinant mAChRs. Methods Patch-clamp recordings were performed on human atrial myocytes (hAMs) isolated from atrial appendages of patients in sinus rhythm. Competition and kinetic radioligand binding assays were performed on hAM preparations and on mAChR(1,2,3) expressed in heterologous systems. Results Patch-clamp recordings on hAMs demonstrated Iva is able to suppress IKAch similarly to atropine, thus suggesting a functional antagonism of Iva on atrial mAChRs.  Competition binding assays on hAM membranes revealed Iva, Zat and ZD bind to mAChRs with affinity values close to those of muscarinic ligands carbachol (Cch) and gallamine (Gal). Competition binding assays on recombinant mAChR(1,2,3), which are functionally relevant in hAMs, showed all blockers are ligands for each single mAChR type with affinity values close to those of CCh and Gal. Finally, using dissociation binding assays on mAChR(2) lacking the allosteric binding site, we showed Iva and Zat behave as allosteric ligands with affinity values similar to Gal. Doking studies confirmed Iva and Zat, but not ZD, have the structural requirements necessary to interact with the allosteric and the orthosteric site of mAChR(2). Conclusions This study demonstrates Iva directly interacts with mAChR expressed in hAMs, showing antimuscarinic activity on IKAch. This property is likely dependent on a double interaction of Iva with the orthosteric and the allosteric site of mAChR(2). Zat, but not ZD, shows similar binding properties compared to Iva. Our results identify a novel pharmacological target for Iva and analogues in human atria, which may have important consequences for the function of the healthy and diseased atrium. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): FAS-Salute 2014, regione Toscana Background Sudden cardiac arrest (SCA) or death (SCD) in young people represents a dramatic event, often leading to severe neurologic outcomes or death. However, the incidence of this phenomenon remains largely unknown, since the recording of cases, and consequently the identification of the underlying causes, requires a multi-professional and disciplinary approach, including genetic counselling. Purpose In this study, we aimed to monitor and investigate SCA and SCD in young people (≤50 years) recorded in the ToRSADE© registry comparing clinical and molecular data. Methods and Results A total of 22 blood samples were analyzed; 14 were collected from dead patients during autopsy and 8 from resuscitated patients after cardiac arrest. Next Generation Sequencing (NGS) analysis revealed 38% of total cases with Likely Pathogenetic (LP) variants associated to Cardiomyopathy (CM) or Channelopathy, and 61% with Variant of Uncertain Significance (VUS). In three cases, NGS confirmed autopsy and histology findings: the p.(Leu466Phe) variant in SCN5A associated with Brugada Syndrome (1a), the p.(Glu173del) in TNNT2 for Hypertrophic CM (1b) and the p. (Asn480Lysfs*20) in PKP2 for Arrhythmogenic Cardiomyopathy (AC) (1c). Conclusions Creation of the ToRSADE© registry allowed implementation of a blood repository for molecular and genetic analysis. Genetic analysis combined with clinical information and post-mortem evaluation constitute a multi-disciplinary approach to juvenile SCD and SCA, while providing medical and genetic assistance to families, public awareness especially among youths and athletes, as well as up-to-date research on the underlying mechanisms of cardiomyopathies. Open in new tab Download slide [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): M.I.U.R. PRIN 2017 Background and purpose Increased inflammatory cytokines, including interleukin 6 (IL-6), are associated to enhanced arrhythmogenic risk, including atrial fibrillation [1]. Moreover, direct effects of cytokines on ion channels are emerging as important mediators of arrhythmogenic remodeling [2]. In line with this, enhanced arrhythmogenesis in COVID-19 patients is hypothesized to be driven by cytokine storms, a well demonstrated condition in this setting [3]. To dissect the underlying mechanisms explaining such an association, we evaluated the proarrhythmogenic alterations of IL-6, assessing the impact on the expression of Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, of the regulatory subunits MiRP1, and on the action potential (AP) profile in HL-1 cardiomyocytes (CMs). In human left atrial samples we studied the relation occurring between the expression levels of IL-6 and of HCN channels. In human induced pluripotent stem cell (hiPSC)-derived CMs we evaluated the acute effects of IL-6 on pacemaker activity. Methods HL-1 CMs exposed to 50 ng/ml IL-6 or vehicle were collected after 0, 0.5, 6, 12, 24 and 48 h to study intracellular signaling, ion channel expression and AP profile. The latter was assessed through a high-throughput system allowing optical detection of APs with optical stimulation. In human atrial samples obtained from patients undergoing surgery, IL-6 and HCN mRNA expression were analyzed by quantitative RT-PCR. The acute effects on pacemaker activity were evaluated in hiPSC-derived CMs exposed to increasing concentrations of IL-6. Results In HL1 CMs IL-6 rapidly induces STAT3 phosphorylation, demonstrating the activation of IL-6 signaling cascade. IL-6 modifies HCN channel transcript and proteins at different time points, evidencing a significant downregulation of HCN4 isoform and significant upregulations of HCN1, HCN2 and MiRP1. In line with this, in human left atrial samples, expression levels of IL-6 were linearly and directly related to HCN1 channel, while they were linearly and inversely related to HCN4. Electrophysiological recordings on HL-1 CMs showed a decreasing trend of AP amplitude and of maximum diastolic potential, while AP durations tended to increase. In hiPSC-derived CMs IL-6 reduces the frequency of AP in a concentration-dependent manner. Conclusions Our data demonstrate that in HL-1 CMs IL-6 activates a STAT3 dependent intracellular signaling that is associated to subsequent variation of HCN channel expression and a concurrent alteration of AP profile. The relation between IL-6 and HCN1,4 expression in human samples suggests a mechanistic link between IL-6 levels and ionic channel targets, including HCN channels. The reduction of AP frequency in hiPSC-derived CMs suggests a direct interaction with ion channels. We hypothesize that these modifications may lay the basis to enhance the propensity of atria to develop arrhythmias in condition of elevate IL-6 levels. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): M.I.U.R. PRIN 2017 Evidence for hydrogen-sulfide as a novel modulator of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels in human atrial cardiomyocytes Background Hydrogen sulfide (H2S) is an endogenous gasotransmitter that controls several physiological and pathological processes in mammalian organs. H2S is synthesized by endogenous enzymes and directly regulates the function of target proteins by sulfhydration of cysteine residues and/or indirect effects mediated by modification of intracellular cyclic nucleotide (cAMP/cGMP) levels. In the cardiovascular system, H2S activates protective responses aimed to counterbalance pathological processes, such as those involved in glucose dysmetabolism, vasal constriction, oxidative stress and cytotoxicity. In cardiac cells, H2S regulates electrogenesis through the modulation of different ion channels, comprising potassium, calcium and TRP channels. No information is available on the effects of H2S on the funny current (If), which is involved in cardiac pacemaking and working myocyte electrogenesis. Purpose The aim of this study was to assess whether H2S modulates the properties of If in human atrial myocytes. If is mediated by Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, which are expressed in human atria and are directly regulated by intracellular cAMP/cGMP levels. Methods Human atrial cardiomyocytes were isolated by enzymatic and mechanical digestion from atrial specimens of patients undergoing cardiac surgery. The acute effects of H2S on If were tested by single-cell patch-clamp recordings using increasing concentrations of NaHS as H2S donor. Results High NaHS concentrations increased If conductance at all potential tested. At physiological potential (-80 mV), relative If conductance changed from 0.33±0.08 to 0.45±0.11 and 0.60±0.11 at 10 and 100 µM concentrations, respectively (p<0.05, n=5-7), while half-maximal activation potential (V½) was shifted from -92.4±0.9 to -83.9±0.7 and -82.4±0.4 mV, respectively (p<0.05, n=5-7). Conversely, low NaHS concentrations reduced If conductance that at -80 mV changed to 0.23±0.07 and 0.25±0.06 at 1 and 5 µM concentrations, respectively (p<0.05, n=5-7). Accordingly, at low NaHS concentration If V½ showed a trend toward less positive potentials. Both high and low concentrations did not modify If kinetics. Conclusions H2S exerts a dual, concentration-dependent effect on If expressed in human atrial cardiomyocytes. This response is similar to that induced by NO, a different gas signaling molecule. The effect of H2S on If properties may be caused by direct S-sulfhydration of HCN proteins and/or to modifications of intracellular cAMP/cGMP levels resulting from phosphodiesterase inhibition by H2S. These findings may open novel perspectives to modulate If function and dysfunction in cardiac pacemaker cells and working myocytes. [ABSTRACT FROM AUTHOR]

A characteristic histological feature of striated muscle cells is the presence of deep invaginations of the plasma membrane (sarcolemma), most commonly referred to as T-tubules or the transverse-axial tubular system (TATS). TATS mediates the rapid spread of the electrical signal (action potential) to the cell core triggering Ca 2+ release from the sarcoplasmic reticulum, ultimately inducing myofilament contraction (excitation–contraction coupling). T-tubules, first described in vertebrate skeletal muscle cells, have also been recognized for a long time in mammalian cardiac ventricular myocytes, with a structure and a function that in recent years have been shown to be far more complex and pivotal for cardiac function than initially thought. Renewed interest in T-tubule function stems from the loss and disorganization of T-tubules found in a number of pathological conditions including human heart failure (HF) and dilated and hypertrophic cardiomyopathies, as well as in animal models of HF, chronic ischemia and atrial fibrillation. Disease-related remodeling of the TATS leads to asynchronous and inhomogeneous Ca 2+-release, due to the presence of orphan ryanodine receptors that have lost their coupling with the dihydropyridine receptors and are either not activated or activated with a delay. Here, we review the physiology of the TATS, focusing first on the relationship between function and structure, and then describing T-tubular remodeling and its reversal in disease settings and following effective therapeutic approaches. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Recent clinical data suggest beneficial effects of ivabradine, a specific heart rate (HR)-lowering drug, in heart failure patients. However, the mechanisms responsible for these effects have not been completely clarified. Thus, we investigated functional/molecular changes in If, the specific target of ivabradine, in the failing atrial and ventricular myocytes where this current is up-regulated as a consequence of maladaptive remodelling. EXPERIMENTAL APPROACH We investigated the effects of ivabradine (IVA; 10 mg·kg−1·day−1 for 90 days) on electrophysiological remodelling in left atrial (LA), left ventricular (LV) and right ventricular (RV) myocytes from post-mycardial infarcted (MI) rats, with sham-operated (sham or sham + IVA) rats as controls. If current was measured by patch-clamp; hyperpolarization-activated cyclic nucleotide-gated (HCN) channel isoforms and microRNA (miRNA-1 and miR-133) expression were evaluated by reverse transcription quantitative PCR. KEY RESULTS Maximal specific conductance of If was increased in MI, versus sham, in LV ( P < 0.01) and LA myocytes ( P < 0.05). Ivabradine reduced HR in both MI and sham rats ( P < 0.05). In MI + IVA, If overexpression was attenuated and HCN4 transcription reduced by 66% and 54% in LV and RV tissue, respectively, versus MI rats (all P < 0.05). miR-1 and miR-133, which modulate post-transcriptional expression of HCN2 and HCN4 genes, were significantly increased in myocytes from MI + IVA. CONCLUSION AND IMPLICATION The beneficial effects of ivabradine may be due to the reversal of electrophysiological cardiac remodelling in post-MI rats by reduction of functional overexpression of HCN channels. This is attributable to transcriptional and post-transcriptional mechanisms. [ABSTRACT FROM AUTHOR]

Background and Purpose: Increased angiotensin II levels and insulin resistance coexist at the early stages of cardiomyopathies. To determine whether angiotensin II increases insulin resistance in cardiomyocytes, we studied the effect of angiotensin II on basal and insulin-stimulated transport rate of energy substrates in immortalized cardiomyocytes (HL-1 cells).Experimental Approach: Glucose and palmitic acid uptakes were measured using [(3)H]2-deoxy-D-glucose and [(14)C]palmitic acid, respectively, in cells exposed or not exposed to angiotensin II (100 nM), angiotensin II plus irbesartan or PD123319, type 1 and 2 receptor antagonists, or PD98059, an inhibitor of ERK1/2 activation. Cell viability, DNA, protein synthesis and surface area were evaluated by the MTT test, [(3)H]thymydine, [(3)H]leucine and morphometric analysis, respectively. Type 1 receptor levels were measured by western blot analysis.Key Results: Basal uptakes of glucose and palmitic acid by HL-1 cells (0.37+/-0.07 and 7.31+/-0.22 pmol per 10(4)cells per min, respectively) were both stimulated by 100 nM insulin (+91 and +64%, respectively). Cells exposed to angiotensin II remained viable and did not show signs of hypertrophy. In these conditions, the basal palmitic acid uptake of the cells increased (11.41+/-0.46 pmol per 10(4) cells per min) and insulin failed to stimulate the uptake of glucose and fatty acids. Changes in the rate of uptake of energy substrates were prevented or significantly reduced by irbesartan or PD98059.Conclusions and Implications: Angiotensin II is a candidate for increasing insulin resistance in cardiomyocytes. Our results suggest a further mechanism for the cardiovascular protection offered by the angiotensin II type 1 receptor blockers. [ABSTRACT FROM AUTHOR]

The article presents a study which evaluates barium exposure to the population in Tuscany, Italy. It states that graphite furnace atomic absorption spectrometry was used in barium determination. It concludes that barium drinking water standards have been developed without studies on human health effects of drinking water intake.

We used intracellular microelectrodes to study the effects of histamine on both normal and abnormal automaticity in sheep Purkinje fibers. Histamine, dimaprit and 4-methylhistamine caused a similar reduction of the action potential duration in driven Purkinje fibers. Histamine (10 M) induced spontaneous activity in previously quiescent preparations more often than did equimolar concentrations of dimaprit and 4-methylhistamine. The effects of histamine on automaticity were enhanced in the presence of barium. In fact histamine, at concentrations which were unable to induce automaticity in normal preparations, induced it in the presence of barium. In Purkinje fibers manifesting barium-induced automatic activity, histamine (10-10 M) significantly increased the average number of spontaneous action potentials and shortened the time of their appearance. In the same range of concentrations, histamine dose-dependently increased the amplitude of the barium-induced oscillatory afterpotentials (OAP), which are considered an electrophysiological manifestation of calcium overload. Histamine (10-10 M) increased the OAP amplitude of strophanthidin-treated Purkinje fibers, eventually inducing triggered extrabeats. All these previously described effects were selectively blocked by cimetidine (10 M). It is concluded that histamine may induce cardiac arrhythmias under conditions of calcium overload and that this effect may be due to induction or enhancement of oscillatory afterpotentials. [ABSTRACT FROM AUTHOR]

Action potential (AP), via the transverse axial tubular system (TATS), synchronously triggers uniform Ca2+-release throughout the cardiomyocyte. Cardiac diseases associated with TATS structural remodeling preclude a uniform Ca2+-release across the myocyte, contributing to contractile dysfunction. Here, we combined the advantage of an ultrafast random access multi-photon (RAMP) microscope with a double staining approach to optically record AP in several TATS elements and, simultaneously, the corresponding local Ca2+-transient.In rat control cardiomyocytes, although AP was uniform between surface sarcolemma (SS) and t-tubules (TT) at steady-state stimulation, we observed a non-negligible beat-to-beat variability of local Ca2+-transient amplitude and kinetics. This variability was significantly reduced applying 0.1 μM Isoproterenol, which increases the open probability of Ca2+-release units. Ca2+ sparks, detected at times throughout the TATS, did not induce any membrane potential variation in the surrounding tubular elements.In a rat model of post-ischemic heart failure (HF), we previously demonstrated that some tubular elements fail to propagate AP (AP-; Sacconi PNAS 2012). Here, we found that those AP- tubules displayed a slower and reduced local Ca2+-transient compared to electrically coupled tubules (AP+). Consistently, in a model of acute detubulation, tubules that do not conduct AP showed a local reduction and delay of Ca2+-transient rise. In addition, variability of Ca2+-transient kinetics was increased in HF. Finally, AP- tubular elements, occasionally exhibited spontaneous depolarisations of various amplitude and kinetics. These events were never accompanied by local Ca2+-release in the absence of pro-arrhythmogenic stimuli, while they could induce local Ca2+-release in the presence of 0.1 μM Isoproterenol. Simultaneous recording of AP and Ca2+-transient allows us to probe the spatio-temporal variability of Ca2+-release, whereas the investigation of Ca2+-transient in HF discloses an unexpected uncoupling between t-tubular depolarization and Ca2+-release in remodelled tubules. This work was supported by the European Union 7th Framework Program (FP7/2007- 2013) under grant agreement n° 284464, 241526, by the Italian Ministry of University and Research (NANOMAX), and by Telethon-Italy (GGP13162). [ABSTRACT FROM AUTHOR]

Introduction: In HCM human tissue, primary functional alterations at the level of the sarcomeres are associated with secondary changes of Ca2+ handling and membrane EP, leading to a pro-arrhythmogenic phenotype and alteration of myocardial relaxation (Circulation. 2013 Feb 5;127(5):575-84). The relative contribution of primary vs secondary alterations is still unknown.Methods: Here we aim to study these changes in intact trabeculae, single cardiomyocytes and skinned preparations from the ventricles of transgenic mouse models aged 6 months carrying HCM-related mutations of cTnT (R92Q, E163R).Results: Compared to WT, R92Q trabeculae showed: (i) preserved peak isometric twitch tension at low inotropic level with reduced contractile reserve; (ii) prolonged relaxation kinetics associated with reduced SERCA function; (iii) faster mechanical restitution, indicating shorter RyR2 refractoriness; (iv) Frequent after-contractions or spontaneous beats during pauses, which increased in response to isoproterenol. Compared to WT, R92Q cardiomyocytes showed: (i) prolonged action potentials due to ionic current remodeling; (ii) reduced amplitude and slower decay rate of Ca2+transients; (iii) elevated diastolic [Ca2+]I and (iv) spontaneous Ca2+ waves and Ca2+transients under isoproterenol.In E163R vs. WT trabeculae and cells, peak isometric tension and Ca2+ transient amplitude were preserved in all conditions. Interestingly, the kinetics of force development and relaxation was prolonged, despite Ca2+ transient kinetics was faster and SERCA function unchanged. E163R myocardium showed faster mechanical restitution and increased spontaneous activity. Further, E163R myofibrils showed a prolongation of the overall relaxation, with incomplete inactivation in the absence of Ca2+. Energy cost of contraction, measured with an enzymatic assay of sarcomeric ATPase activity, as well as myofilaments Ca2+ sensitivity, were increased in E163R vs. WT skinned trabeculae.Conclusions: Primary changes of myofilaments function (increased Ca2+ sensitivity), previously described in R92Q hearts, are associated with a large spectrum of EC-coupling and membrane EP changes, which appear to be a major contributor to the observed mechanical dysfunction and arrhythmogeneicity in this mouse line, resembling advanced human disease. In E163R instead, impairment of myofilament function appear to be the leading element determining mechanical abnormalities. In the absence of major EC-coupling changes, the increased arrhythmogeneicity in E163R myocardium may be a direct consequence of the increased myofilaments Ca2 sensitivity. [ABSTRACT FROM AUTHOR]

Introduction: Ca2+ handling abnormalities are a main determinant of electro-mechanical dysfunction in human hypertrophic cardiomyopathy (HCM), leading to a pro-arrhythmogenic cellular phenotype. The late-Na+ current blocker ranolazine reduced the rate of arrhythmogenic events in HCM human myocardium via shortening of action potentials and reduction of intracellular Na+ and Ca2+, with increased forward NCX function (Circulation. 2013 Feb 5;127(5):575-84).Methods: Here we characterize pro-arrhythmogenic changes in E-C coupling that occur in cardiomyocytes and intact trabeculae from cTnT mutant mouse models of HCM (R92Q and E163R) and test the effects of ranolazine.Results: Compared to wild type (WT), R92Q cardiomyocytes showed (i) prolonged action potentials associated with ionic current remodeling; (ii) lower Ca2+transient amplitude with slower rate of decay; (iii) elevated diastolic [Ca2+]i; (iv) spontaneous Ca2+ waves and premature Ca2+ transients during pauses.Compared to WT, R92Q trabeculae showed (i) prolonged relaxation kinetics associated with reduced SERCA function and (ii) Frequent after-contractions or regular spontaneous beats during stimulation pauses.In E163R vs. WT trabeculae and cells, the kinetics of force development and relaxation was prolonged, despite Ca2+ transient kinetics and SERCA function were not slower. E163R vs. WT myocardium showed increased rate of spontaneous arrhythmogenic activity.In R92Q preparations, ranolazine (10μM): (i) hastened Ca2+ transient kinetics and reduced diastolic Ca2+; (ii) increased forward NCX function and (iii) reduced the rate of spontaneous beats and Ca2+ waves.In E163R trabeculae and cells ranolazine did not affect the kinetics of Ca2+ transients. Nonetheless, the drug prolonged RyR2 refractoriness and reduced the occurrence of spontaneous Ca2+ and contractile activity.None of these effects of ranolazine was observed in WT.Discussion: As in human HCM, the beneficial effects of ranolazine on R92Q myocardium are likely mediated by the consequences of late Na+ current inhibition. In E163R preparations, myocardial arrhythmogeneicity is not accompanied by remodeling of ion currents or changes of SERCA/NCX function, and thus appear to be a direct consequence of increased myofilaments Ca2+ sensitivity or anomalies of RyR2 function. Therefore the beneficial effects of ranolazine on Ca2+ mediated spontaneous activity in E163R myocardium supports the hypothesis that the anti-arrhythmic effect of ranolazine can also be mediated by mechanisms other that INaL inhibition (i.e. reduction of myofilaments Ca2+ sensitivity or RyR2 inhibition). [ABSTRACT FROM PUBLISHER]