Effects of Electrical and Structural Remodeling on Atrial Fibrillation Maintenance: A Simulation Study

Atrial fibrillation, a common cardiac arrhythmia, often progresses unfavourably: in patients with long-term atrial fibrillation, fibrillatory episodes are typically of increased duration and frequency of occurrence relative to healthy controls. This is due to electrical, structural, and contractile remodeling processes. We investigated mechanisms of how electrical and structural remodeling contribute to perpetuation of simulated atrial fibrillation, using a mathematical model of the human atrial action potential incorporated into an anatomically realistic three-dimensional structural model of the human atria. Electrical and structural remodeling both shortened the atrial wavelength - electrical remodeling primarily through a decrease in action potential duration, while structural remodeling primarily slowed conduction. The decrease in wavelength correlates with an increase in the average duration of atrial fibrillation/flutter episodes. The dependence of reentry duration on wavelength was the same for electrical vs. structural remodeling. However, the dynamics during atrial reentry varied between electrical, structural, and combined electrical and structural remodeling in several ways, including: (i) with structural remodeling there were more occurrences of fragmented wavefronts and hence more filaments than during electrical remodeling; (ii) dominant waves anchored around different anatomical obstacles in electrical vs. structural remodeling; (iii) dominant waves were often not anchored in combined electrical and structural remodeling. We conclude that, in simulated atrial fibrillation, the wavelength dependence of reentry duration is similar for electrical and structural remodeling, despite major differences in overall dynamics, including maximal number of filaments, wave fragmentation, restitution properties, and whether dominant waves are anchored to anatomical obstacles or spiralling freely

Rare missense variants in Tropomyosin-4 (TPM4) are associated with platelet dysfunction, cytoskeletal defects, and excessive bleeding

Background: A significant challenge is faced for the genetic diagnosis of inherited platelet disorders in which candidate genetic variants can be found in more than 100 bleeding, thrombotic, and platelet disorder genes, especially within families in which there are both normal and low platelet counts. Genetic variants of unknown clinical significance (VUS) are found in a significant proportion of such patients in which functional studies are required to prove pathogenicity. Objective: To identify the genetic cause in patients with a suspected platelet disorder and subsequently perform a detailed functional analysis of the candidate genetic variants found. Methods: Genetic and functional studies were undertaken in three patients in two unrelated families with a suspected platelet disorder and excessive bleeding. A targeted gene panel of previously known bleeding and platelet genes was used to identify plausible genetic variants. Deep platelet phenotyping was performed using platelet spreading analysis, transmission electron microscopy, immunofluorescence, and platelet function testing using lumiaggregometry and flow cytometry. Results: We report rare conserved missense variants (p.R182C and p.A183V) in TPM4 encoding tromomyosin-4 in 3 patients. Deep platelet phenotyping studies revealed similar platelet function defects across the 3 patients including reduced platelet secretion, and aggregation and spreading defects suggesting that TPM4 missense variants impact platelet function and show a disordered pattern of tropomyosin staining. Conclusions: Genetic and functional TPM4 defects are reported making TPM4 a diagnostic grade tier 1 gene and highlights the importance of including TPM4 in diagnostic genetic screening for patients with significant bleeding and undiagnosed platelet disorders, particularly for those with a normal platelet count

Basal and stimulated angiotensin II-induced cytosolic free calcium in adult rat cardiomyocytes and fibroblasts after volume overload

This study investigates basal and angiotensin II (Ang II)–induced [Ca2+]i concentrations in cells from hearts of rats that have undergone cardiac hypertrophy due to volume overload. [Ca2+]i measurements assessed by digital imaging using fura 2 methodology were performed on isolated ventricular cardiomyocytes and fibroblasts from adult rat hearts with a 4-week aortocaval shunt. Long-term aortocaval shunt induced a significant increase in atrial (72%) and ventricular (41%) weights and a large elevation in plasma atrial natriuretic peptide-(1-98) concentration (160%). For adult cardiomyocytes [Ca2+]i measurements are reported as diastolic (average of the lowest points) and systolic intracellular Ca2+ values (average of the maximum points corresponding to the diastolic points) over a 30-second time interval. Basal diastolic [Ca2+]i (99±4.1 nmol/L for experimental cells versus 90±4.8 for control cells) was not altered, whereas basal systolic [Ca2+]i was significantly greater in ventricular cardiomyocytes from overload hearts (155±2.3 versus 129±4.4 nmol/L for control cells, P<.05). Ang II increased intracellular Ca2+ spike frequency in a concentration-dependent manner in cardiomyocytes from control and overload myocardium. Basal and Ang II–induced intracellular Ca2+ spike frequencies were not modified in cardiomyocytes from hypertrophied hearts. Basal [Ca2+]i in ventricular fibroblasts from overload myocardium was significantly increased (128±5.1 nmol/L for fibroblasts from hypertrophied hearts versus 104±3.5 for control cells, P<.05). Ang II–induced [Ca2+]i was lower in fibroblasts from overload myocardium (P<.05). In conclusion, alterations of intracellular calcium homeostasis in the two predominant cardiac cell types involved in myocardial growth and fibrosis, cardiomyocytes and fibroblasts, respectively, may contribute to the physiopathology of heart failure in adult rats. Ang II signaling through the intracellular calcium transduction pathway in a cell-specific manner may play an important role in cardiac hypertrophy

Intracellular calcium2+ modulation by angiotensin II and endothelin-1 in cardiomyocytes and fibroblasts from hypertrophied hearts of spontaneously hypertensive rats

The vasoactive peptides angiotensin II (Ang II) and endothelin-1 (ET-1) have been implicated in cardiac hypertrophy. This study investigates Ang II and ET-1 effects on intracellular free calcium concentration and the receptor subtype through which agonist-induced calcium responses are mediated in isolated cardiomyocytes and fibroblasts from hypertrophied hearts of spontaneously hypertensive rats (SHR). We measured intracellular free calcium concentration by fura 2 methodology and determined receptor status by radioligand binding assays. Ang II (10-12 to 10-7 mol/L) had no effect on cardiomyocyte calcium levels in control Wistar-Kyoto rats but significantly increased (P < .01) intracellular free calcium concentration in a dose-dependent manner in cardiomyocytes from SHR. Ang II total and specific binding were increased (P < .05) in SHR cardiomyocytes. Calcium responses elicited by 10-7 to 10-5 mol/L Ang II were significantly reduced (P < .01) in SHR fibroblasts despite no significant change in Ang II receptor density. The angiotensin type 1 receptor blocker losartan (1 micro mol/L) blocked Ang II-stimulated calcium transients, whereas the angiotensin type 2 receptor blocker PD 123319 had no effect. ET-1- and sarafotoxin S6c-induced calcium responses in cardiomyocytes and fibroblasts were not different between hypertensive and control groups. In conclusion, Ang II and ET-I elicit distinct and differential responses in a cell-specific manner in cardiomyocytes and fibroblasts from hypertrophied hearts of SHR. Whereas Ang II-mediated effects, which are elicited via angiotensin type 1 receptors, are detectable in cardiomyocytes from SHR, responses to Ang II are blunted in fibroblasts from SHR, and ET-1-related actions are similar in cells from both rat groups. Stimulation of cardiomyocytes by Ang II in hypertrophied hearts associated with pressure overload in genetic hypertension suggests that Ang II could modulate the function of cardiomyocytes of SHR but not those of Wistar-Kyoto rats, whereas cardiac actions of ET-1 do not change with the development of hypertension

Endothelin-1 and angiotensin II receptors in cells from rat hypertrophied heart: receptor regulation and intracellular Ca2+ modulation

This study investigates the cellular localization and regulation of endothelin-1 (ET-1) and angiotensin II (Ang II) receptors and the effects of ET-1 and Ang II on [Ca2+]i in cardiac hypertrophy due to volume overload in the rat. Radioligand binding assays and [Ca2+]i measurements by fura 2 methodology were performed on isolated ventricular cardiomyocytes and fibroblasts from the heart of rats with a 4-week aortocaval shunt. In the hypertrophied myocardium, ET-1 and Ang II concentrations were unchanged in ventricles. Ventricular ET-1 receptors had a cell-specific distribution: >90% of ET receptors in cardiomyocytes are of the ETA subtype, whereas fibroblasts had a nearly equal proportion of the ETA and ETB subtypes. ET-1 receptor densities, affinities, and ET-1–induced [Ca2+]i were not significantly different from control in both ventricular cell types from hypertrophied myocardium. Ang II specific binding was very low on isolated ventricular cardiomyocytes, suggesting few receptors in control conditions. However, [Ca2+]i responses induced by Ang II at concentrations >10−8 mol/L were detectable and were significantly higher in hypertrophied cardiomyocytes. Ang II receptor density (exclusively AT1) on fibroblasts was significantly reduced (42 970±3330 versus 73 870±7940 sites per cell for control cells, P<.01), but AT1 receptor affinity was unchanged after volume overload. The maximum increase in [Ca2+]i evoked by 10−6 to 10−4 mol/L Ang II was significantly lower in fibroblasts from overloaded hearts. In conclusion, ET-1 receptor proportion is cell specific, with cardiomyocytes possessing predominantly the ETA subtype and fibroblasts possessing both ETA and ETB receptors. Plasma and cardiac ET-1 concentrations and ET-1 receptor regulation on both ventricular cell types are not altered in cardiac volume overload, suggesting that cardiac ET-1 may not play a significant role in this model. Cardiac hypertrophy induced a significant downregulation of AT1 receptors on fibroblasts, whereas total binding and [Ca2+]i sensitivity to Ang II were significantly enhanced in hypertrophied cardiomyocytes. This suggests that cardiac Ang II may be involved in the pathophysiology of the cardiac hypertrophy of volume overload

Altered cardiac endothelin receptors and protein kinase C in deoxycorticosterone-salt hypertensive rats

J. Fareh, R. M. Touyz, E. L. Schiffrin and G. Thibault. Altered Cardiac Endothelin Receptors and Protein Kinase C in Deoxycorticosterone-Salt Hypertensive Rats. Journal of Molecular and Cellular Cardiology (2000) 32, 665–676. The aim of the present study was to assess the status of ET-1 receptor subtypes (ETAand ETB) in ventricular myocytes and fibroblasts and to determine the role of PKC-dependent pathways in ET-1-stimulated cardiac cells in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Systolic blood pressure and relative heart to body weight were significantly increased in DOCA-salt rats. In unilaterally nephrectomized (Uni-Nx) control rats, more than 90% of cardiomyocyte ET receptors were of the ETAsubtype, whereas in fibroblasts ETAand ETBreceptors were present in a 1:3 ratio. In DOCA-salt rats, the density of the ETAreceptor subtype was reduced by 31% in cardiomyocytes and in cardiac fibroblasts only ETBreceptor density was decreased by 29%. Affinity was unchanged. The relative expression of immunoreactive PKC α, γ and ϵ was significantly increased, whereas PKC δ was not altered in cardiac extracts of DOCA-salt rats. In cardiac fibroblasts from DOCA-salt rats PKCδ was significantly increased and PKC ϵ was not translocated after ET-1 stimulation. The hearts of DOCA-salt hypertensive rats are thus characterized by: (1) decreased density of cardiomyocyte ETAreceptors and fibroblast ETBreceptors; (2) cell-specific enhanced expression of some PKC isoenzymes (α, γ,δ and ϵ); and (3) unresponsiveness of PKC ϵ to translocate in the presence of ET-1. Together with alterations of ET-1-induced Ca2+handling in cardiac myocytes and fibroblasts, which we previously reported, results from the present study indicate a marked modification of the cardiac ET-1 system of DOCA-salt hypertensive rats

Cardiac type-1 angiotensin II receptor status in deoxycorticosterone acetate salt hypertension in rats

The regulation of angiotensin II (Ang II) receptors and Ang II–induced modulation of intracellular Ca2+ concentration in cardiac cells from hearts of experimentally induced hypertensive deoxycorticosterone acetate (DOCA)–salt and control unilaterally nephrectomized (Uni-Nx) Sprague-Dawley rats was assessed. Ang II receptor density and intracellular Ca2+ concentration measurements were examined in adult ventricular myocytes and fibroblasts by radioligand binding assay and digital imaging using fura 2 methodology, respectively. Four-week DOCA-salt treatment induced hypertension associated with cardiac hypertrophy. Ang II binding studies demonstrated that adult ventricular myocytes and fibroblasts possess mainly the AT1 subtype receptor. Moreover, DOCA-salt hypertension was associated with a 1.8-fold increase in Ang II–specific binding compared with myocytes from Uni-Nx control rats. Intracellular Ca2+ responses induced by increasing Ang II concentrations (10−12 to 10−4 mol/L) were significantly enhanced in cardiomyocytes from DOCA-salt rats. The effects of Ang II on intracellular Ca2+ spike frequency were unaltered in cardiomyocytes from DOCA-salt–hypertensive rats. The density of AT1 subtype receptors was not modified in ventricular fibroblasts after DOCA-salt treatment. Ang II increased intracellular Ca2+ concentration similarly in ventricular fibroblasts from normal and hypertensive rats. In conclusion, DOCA-salt hypertension is characterized by an increased AT1 receptor density and intracellular calcium responses in ventricular myocytes, whereas in ventricular fibroblasts the AT1 receptor status is unaltered. These findings report for the first time the cardiac cell–specific implication of Ang II and the intracellular calcium signaling pathway stimulated by the AT1 receptor in cardiac hypertrophy in DOCA-salt–hypertensive rats

Endothelin-1 signaling is altered in cardiac cells from deoxycorticosterone acetate-salt hypertensive rats

The cardiac cellular effects of endothelin-1 (ET-1) on intracellular free Ca2+ concentration ([Ca2+]i) were investigated in deoxycorticosterone acetate (DOCA)-salt rats with severe cardiac hypertrophy. [Ca2+]i was measured by fura-2 methodology in ventricular cardiomyocytes and fibroblasts of DOCA-salt hypertensive and control unilaterally nephrectomized rats (Uni-Nx). Blood pressure and heart weight were increased (p < 0.01) in DOCA-salt rats compared to control rats. ET-1 (10-12-10-6 M) increased [Ca2+]i in a dose-dependent manner in both cell types from control and hypertensive rats. However, ET-1-induced [Ca2+]i responses were significantly attenuated (p < 0.01) in cardiomyocytes and fibroblasts of DOCA-salt rats. Sarafotoxin S6c (S6c) increased [Ca2+]i in fibroblasts but not in cardiomyocytes. In conclusion, ET-1 dose-dependently increased[Ca2+]i in cardiomyocytes (primarily via ETA receptors) and fibroblasts (via ETA and ETB receptors). Cardiac cell ET-1 signaling pathways are blunted in DOCA-salt hypertensive rats. ET-1 may not play a critical role in the pathophysiology of the severe concentric cardiac hypertrophy present in DOCA-salt hypertensive rats

Modulation of Ca₂₊ transients in neonatal and adult rat cardiomyocytes by endothelin-1

Endothelin-1 (ET-1) may exert inotropic and chronotropic effects in cardiac muscle by modulating intracellular Ca2+. This study assesses effects of ET-1 on intracellular free Ca2+ concentration ([Ca2+ ];) in neonatal and adult rat cardiomyocytes. [Ca2+ ]i was measured by Fura 2 methodology. ET receptor subtypes were determined by binding studies. ET-1 increased neonatal and adult [Ca2+ ]; and spike frequency in a dose-dependent manner. It decreased [Ca2+ ]; amplitude in neonatal cardiomyocytes but had no effect on [Ca2+ ]i amplitude in adult cells. The ETA receptor antagonist BQ-123 reduced ET-1-induced responses and the ETB receptor agonist IRL-1620 increased [Ca2+ ];. ET-1 binding studies demonstrated 85% displacement by BQ-123 and 15% by the ETB receptor agonist sarafotoxin S6c, suggesting a predominance of ETA receptors. These data demonstrate that in neonatal and adult cardiomyocytes ET-1 has stimulatory effects on [Ca2+ ]; which are mediated predominantly via ETA receptors. Therefore, ET-1 may influence cardiac development and function