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

The Subcellular Distribution of Ryanodine Receptors and L-Type Ca2+ Channels Modulates Ca2+-Transient Properties and Spontaneous Ca2+-Release Events in Atrial Cardiomyocytes

Spontaneous Ca2+-release events (SCaEs) from the sarcoplasmic reticulum play crucial roles in the initiation of cardiac arrhythmias by promoting triggered activity. However, the subcellular determinants of these SCaEs remain incompletely understood. Structural differences between atrial and ventricular cardiomyocytes, e.g., regarding the density of T-tubular membrane invaginations, may influence cardiomyocyte Ca2+-handling and the distribution of cardiac ryanodine receptors (RyR2) has recently been shown to undergo remodeling in atrial fibrillation. These data suggest that the subcellular distribution of Ca2+-handling proteins influences proarrhythmic Ca2+-handling abnormalities. Here, we employ computational modeling to provide an in-depth analysis of the impact of variations in subcellular RyR2 and L-type Ca2+-channel distributions on Ca2+-transient properties and SCaEs in a human atrial cardiomyocyte model. We incorporate experimentally observed RyR2 expression patterns and various configurations of axial tubules in a previously published model of the human atrial cardiomyocyte. We identify an increased SCaE incidence for larger heterogeneity in RyR2 expression, in which SCaEs preferentially arise from regions of high local RyR2 expression. Furthermore, we show that the propagation of Ca2+ waves is modulated by the distance between RyR2 bands, as well as the presence of experimentally observed RyR2 clusters between bands near the lateral membranes. We also show that incorporation of axial tubules in various amounts and locations reduces Ca2+-transient time to peak. Furthermore, selective hyperphosphorylation of RyR2 around axial tubules increases the number of spontaneous waves. Finally, we present a novel model of the human atrial cardiomyocyte with physiological RyR2 and L-type Ca2+-channel distributions that reproduces experimentally observed Ca2+-handling properties. Taken together, these results significantly enhance our understanding of the structure-function relationship in cardiomyocytes, identifying that RyR2 and L-type Ca2+-channel distributions have a major impact on systolic Ca2+ transients and SCaEs

End-diastolic force pre-activates cardiomyocytes and determines contractile force: role of titin and calcium

Abstract: Titin functions as a molecular spring, and cardiomyocytes are able, through splicing, to control the length of titin. We hypothesized that together with diastolic [Ca2+], titin-based stretch pre-activates cardiomyocytes during diastole and is a major determinant of force production in the subsequent contraction. Through this mechanism titin would play an important role in active force development and length-dependent activation. Mutations in the splicing factor RNA binding motif protein 20 (RBM20) result in expression of large, highly compliant titin isoforms. We measured single cardiomyocyte work loops that mimic the cardiac cycle in wild-type (WT) and heterozygous (HET) RBM20-deficient rats. In addition, we studied the role of diastolic [Ca2+] in membrane-permeabilized WT and HET cardiomyocytes. Intact cardiomyocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT) at low pacing frequencies, but this difference disappeared at high pacing frequencies. Length-dependent activation (force–sarcomere length relationship) was blunted in HET cardiomyocytes, but the force–end-diastolic force relationship was not different between HET and WT cardiomyocytes. To delineate the effects of diastolic [Ca2+] and titin pre-activation on force generation, measurements were performed in detergent-permeabilized cardiomyocytes. Cardiac twitches were simulated by transiently exposing permeabilized cardiomyocytes to 2 µm Ca2+. Increasing diastolic [Ca2+] from 1 to 80 nm increased force development twofold in WT. Higher diastolic [Ca2+] was needed in HET. These findings are consistent with our hypothesis that pre-activation increases active force development. Highly compliant titin allows cells to function at higher diastolic [Ca2+]

Cellular contribution to left and right atrial dysfunction in chronic arterial hypertension in pigs

Aims: Atrial contractile dysfunction contributes to worse prognosis in hypertensive heart disease (HHD), but the role of cardiomyocyte dysfunction in atrial remodelling in HHD is not well understood. We investigated and compared cellular mechanisms of left (LA) and right atrial (RA) contractile dysfunction in pigs with HHD. Methods and results: In vivo electrophysiological and magnetic resonance imaging studies were performed in control and pigs treated with 11-deoxycorticosterone acetate (DOCA)/high-salt/glucose diet (12 weeks) to induce HHD. HHD leads to significant atrial remodelling and loss of contractile function in LA and a similar trend in RA (magnetic resonance imaging). Atrial remodelling was associated with a higher inducibility of atrial fibrillation but unrelated to changes in atrial refractory period or fibrosis (histology). Reduced atrial function in DOCA pigs was related to reduced contraction amplitude of isolated LA (already at baseline) and RA myocytes (at higher frequencies) due to reduced intracellular Ca release (Fura 2-AM, field stimulation). However, Ca regulation differed in LA and RA cardiomyocytes: LA cardiomyocytes showed reduced sarcoplasmic reticulum (SR) [Ca], whereas in RA, SR [Ca] was unchanged and SR Ca2+-ATPase activity was increased. Sodium-calcium exchanger (NCX) activity was not significantly altered. We used ORM-10103 (3 mu M), a specific NCX inhibitor to improve Ca availability in LA and RA cardiomyocytes from DOCA pigs. Partial inhibition of NCX increased Ca2+ transient amplitude and SR Ca in LA, but not RA cells. Conclusions: In this large animal model of HHD, atrial remodelling in sinus rhythm in vivo was related to differential LA and RA cardiomyocyte dysfunction and Ca signalling. Selective acute inhibition of NCX improved Ca release in diseased LA cardiomyocytes, suggesting a potential therapeutic approach to improve atrial inotropy in HHD

A two dimensional electromechanical model of a cardiomyocyte to assess intra-cellular regional mechanical heterogeneities

Experimental studies on isolated cardiomyocytes from different animal species and human hearts have demonstrated that there are regional differences in the Ca2+ release, Ca2+ decay and sarcomere deformation. Local deformation heterogeneities can occur due to a combination of factors: regional/local differences in Ca2+ release and/or re-uptake, intra-cellular material properties, sarcomere proteins and distribution of the intracellular organelles. To investigate the possible causes of these heterogeneities, we developed a two-dimensional finite-element electromechanical model of a cardiomyocyte that takes into account the experimentally measured local deformation and cytosolic [Ca2+] to locally define the different variables of the constitutive equations describing the electro/mechanical behaviour of the cell. Then, the model was individualised to three different rat cardiac cells. The local [Ca2+] transients were used to define the [Ca2+]-dependent activation functions. The cell-specific local Young’s moduli were estimated by solving an inverse problem, minimizing the error between the measured and simulated local deformations along the longitudinal axis of the cell. We found that heterogeneities in the deformation during contraction were determined mainly by the local elasticity rather than the local amount of Ca2+, while in the relaxation phase deformation was mainly influenced by Ca2+ re-uptake. Our electromechanical model was able to successfully estimate the local elasticity along the longitudinal direction in three different cells. In conclusion, our proposed model seems to be a good approximation to assess the heterogeneous intracellular mechanical properties to help in the understanding of the underlying mechanisms of cardiomyocyte dysfunction.This study was partly supported by grants from Ministerio de Economia y Competitividad (ref. SAF2012-37196, TIN2014-52923-R); the Instituto de Salud Carlos III (ref. PI11/01709, PI14/00226) integrado en el Plan Nacional de I+D+I y cofinanciado por el ISCIII-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER) “Otra manera de hacer Europa”; the EU FP7 for research, technological development and demonstration under grant agreement VP2HF (n° 611823); The Cerebra Foundation for the Brain Injured Child (Carmarthen, Wales, UK); Obra Social “la Caixa” (Barcelona, Spain); Fundació Mutua Madrileña; Fundació Agrupació Mutua (Spain) and AGAUR 2014 SGR grant n° 928 (Barcelona, Spain). P.G.C. was supported by the Programa de Ayudas Predoctorales de Formación en investigación en Salud (FI12/00362) from the Instituto Carlos III, Spain. P.G.C wants to acknowledge to Boehringer Ingelhiem Fonds for the travel grant to do her research stay at LaBS group in Politecnico di Milano

A two dimensional electromechanical model of a cardiomyocyte to assess intra-cellular regional mechanical heterogeneities.

Experimental studies on isolated cardiomyocytes from different animal species and human hearts have demonstrated that there are regional differences in the Ca2+ release, Ca2+ decay and sarcomere deformation. Local deformation heterogeneities can occur due to a combination of factors: regional/local differences in Ca2+ release and/or re-uptake, intra-cellular material properties, sarcomere proteins and distribution of the intracellular organelles. To investigate the possible causes of these heterogeneities, we developed a twodimensional finite-element electromechanical model of a cardiomyocyte that takes into account the experimentally measured local deformation and cytosolic [Ca2+] to locally define the different variables of the constitutive equations describing the electro/mechanical behaviour of the cell. Then, the model was individualised to three different rat cardiac cells. The local [Ca2+] transients were used to define the [Ca2+]-dependent activation functions. The cell-specific local Young's moduli were estimated by solving an inverse problem, minimizing the error between the measured and simulated local deformations along the longitudinal axis of the cell. We found that heterogeneities in the deformation during contraction were determined mainly by the local elasticity rather than the local amount of Ca2+, while in the relaxation phase deformation was mainly influenced by Ca2+ re-uptake. Our electromechanical model was able to successfully estimate the local elasticity along the longitudinal direction in three different cells. In conclusion, our proposed model seems to be a good approximation to assess the heterogeneous intracellular mechanical properties to help in the understanding of the underlying mechanisms of cardiomyocyte dysfunction

Different images recorded during the cardiomyocyte electrical stimulation experiments, with a pacing rate of 1Hz.

<p>A: Transmitted light image of the whole cell. The blue arrow corresponds to the line-scan where the images acquisition was performed. B: Line-scan transmitted light image. The red box indicates a region within the cell with zero displacement. C: Confocal FM4-64 image where the T-Tubule and sarcolemma are visible. D: Confocal Fluo-4 image corresponding to cytosolic [<i>Ca</i>²⁺]. The vertical axis corresponds to the line-scan (blue arrow) and the horizontal one to the time. The line-scan images resolution is 3.2 ⋅ 10⁻³ <i>s</i> × 0.28<i>μm</i>.</p

Experimental measured local and global [<i>Ca</i>²⁺] transients and dynamic vs. steady-state force and [<i>Ca</i>²⁺] relationship.

<p>A: Experimental measured local [<i>Ca</i>²⁺] transients normalised to basal fluorescence (<i>F</i>₀) at different positions along the longitudinal axis of the cell (Long. pos). B: Global experimental measured (solid line) and fitted (dash line) with the two exponential functions (<i>Z</i>(<i>t</i>) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182915#pone.0182915.e008" target="_blank">Eq 4</a>) [<i>Ca</i>²⁺] transients. C: Individual time course of cytosolic [<i>Ca</i>²⁺] and active stress (<i>S</i>_<i>act</i>). D: Phase-plane plot relating force to <i>Ca</i>²⁺ for both heterogeneous and homogeneous <i>Ca</i>²⁺ activation. The dynamic behaviour for a single contraction is compared with the steady-state relation.</p

Synthetic data generated for validating the inverse problem procedure and results of the proposed framework validation in presence of gaussian noise.

<p>A: Local [<i>Ca</i>²⁺] transients. B: Active stress <i>S</i>_<i>act</i>(<i>t</i>) at different longitudinal positions (Long. pos) of the synthetic cell. C: Undeformed (grey) and deformed mesh of the synthetic cell at maximum contraction time frame. Colormap indicates the simulated longitudinal strain. D: Original (black solid line) and simulated strains along the longitudinal axis of the cell at maximum contraction time frame after the optimisation process with 0% (*), 5% (□), 10% (◇) and 15% (∘) of noise. E: Original (black solid line) and estimated local Young’s moduli along the longitudinal axis (line-scan) of the cell with 0% (*), 5% (□), 10% (◇) and 15% (∘) of noise.</p

Results of the cell-specific simulations performed for three different cardiomyocytes, for both [<i>Ca</i>²⁺]-activation (Act.) scenarios: Homogeneous (homo.) and heterogeneous (hetero.)

<p>Results of the cell-specific simulations performed for three different cardiomyocytes, for both [<i>Ca</i>²⁺]-activation (Act.) scenarios: Homogeneous (homo.) and heterogeneous (hetero.)</p