Spontaneous Ca2+ waves in rabbit cardiac myocytes: A modelling study

Propagating intracellular Ca2+ waves in cardiac myocytes occur as a consequence of the overloaded state of the sarcoplasmic reticulum (SR). To examine these events in detail, ventricular cardiomyocytes were isolated from rabbit hearts and permeabilised with beta-escin. Cytosolic Ca2+ signals were monitored using Fluo-5F (10muM) in combination with laser-scanning confocal microscopy. Through careful calibration of the intracellular Ca2+ signals and construction of analysis programs, the fluxes which underlie the Ca2+ wave were derived and subsequently incorporated into a mathematical model. The decline in cytosolic Ca2+ subsequent to rapid application of caffeine was used to quantify cellular Ca2+ diffusional loss (diffusional constant = 31.2+/-0.9 s-1). binding to cellular proteins was then calculated and the sum of the free Ca2+, bound Ca2+ and Ca2+ lost by diffusion was used as the integral of the Ca2+ flux across the SR. The first derivative of this was taken as the trans-SR flux rate. From the analysis of these signals it was apparent that the released from the SR during a wave was not significantly different from that released on application of lOmM caffeine (0.149 +/-0.10 mM vs. 0.154+/-0.10 mM)). This information, coupled with values of intra-SR buffering allowed calculation of intra-SR [Ca2+]. This in turn allowed the trans SR [Ca2+] gradient to be estimated and the subsequent calculation of RyR and SERCA mediated Ca2+ flux . These measurements were used to derive parameters for construction of a 3- compartment model of Ca2+ flux using existing models of Ca2+ buffering, SERCA activity and leak. Three experimental interventions were used to study changes in Ca2+ wave properties and assess the effectiveness of the model in predicting wave frequency, minimum and maximum [Ca2+]. These were: (i) changing extracellular Ca2+, (ii) inhibiting the RyR using tetracaine and (iii) inhibiting SERCA using 2',5'-di(tert- butyl)-l ,4-benzohydroquinone (TBQ). As cytosolic Ca2+ was increased from 300 to 900nM, so frequency and systolic Ca2+ were shown to increase nonlinearly, whilst diastolic [Ca2+] increased linearly. Calculated SR release threshold was found not to change. SERCA Vmax and KD both increased, with Vmax rising from 160 to 380 muMs-1 and KD rising from 239+/-48 to 354+/-18nM as extracellular [Ca2+] was increased from 300nM to 900nM. The calculated peak permeability of RyR mediated flux also increased from 41.1 +/-6.5 to 61.2+/-3.6 s-1 over this range. These changes, when included in the model, subsequently provided acceptable predictions of experimental results. Tetracaine caused frequency of the Ca2+ waves to decrease from 0.59+/-0.03 Hz to 0.35+/-0.02 Hz, systolic [Ca2+] to increase from 2.06+/-0.11 muM to 3.16+/-0.24 muM and diastolic [Ca2+] to decrease from 185+/-9 nM to 157+/-10 nM. Flux analysis indicated that these changes were associated with an increase in the SR release threshold from 1.16+/-0.04 mM to 1.58+/-0.08 mM (n=6). Implementation of this threshold change in the computational model predicted a decrease in Ca2+ wave frequency to a similar value to that observed experimentally. The increased systolic [Ca2+] was comparable to but greater than that observed experimentally. In contrast, the model predicted diastolic [Ca2+] to increase while a decrease diastolic was observed experimentally. Application of the SERCA inhibitor TBQ (1muM) decreased SR Ca2+ content, the amplitude and frequency of Ca2+ waves. Analysis of the underlying fluxes suggested that TBQ caused a 43% reduction in SERCA Vmax, with no significant change in KD. Analysis also suggested that this reduction in Vmax was accompanied by a 25% reduction in SR release threshold. While the reduced SERCA Vmax is consistent with TBQ's known action on SERCA, the effect on Ca2+ wave amplitude was unexpected and cannot be easily explained with the current Ca2+ wave model

Calcium signalling in cardiac fibroblasts and myocytes in an in vitro model of HFpEF

Heart Failure with preserved Ejection Fraction [HFpEF] is a pandemic associated with hypertension and diabetes amongst other co-morbidities1. It has been established that fibrosis plays a major role in this form of heart failure2. However, the functional mechanisms of cardiac fibroblasts (CFs) in HFpEF and more importantly, how these cells interact with cardiomyocytes (CMs) and impact calcium (Ca2+) signalling is limited. This limitations majorly due to a lack of physiologically relevant cellular models

Altered regulation of cardiac calcium handling proteins in an in vivo rat model of angiotensin-induced hypertension

Hypertension is a major comorbidity in patients with heart failure with preserved ejection fraction (HFpEF), which remains an increasing global challenge. Cardiac remodelling and dysfunction as well as altered calcium (Ca2+) homeostasis are all characteristics of this disease1,2. However, existing models and evidence on the mechanisms driving this form of cardiomyopathy remain contradictory

Ryanodine receptor cluster fragmentation and redistribution in persistent atrial fibrillation enhance calcium release

In atrial fibrillation (AF), abnormalities in Ca(2+) release contribute to arrhythmia generation and contractile dysfunction. We explore whether RyR cluster ultrastructure is altered and is associated with functional abnormalities in AF.status: publishe

Hyperactive ryanodine receptors in human heart failure and ischaemic cardiomyopathy reside outside of couplons

Aims In ventricular myocytes from humans and large mammals, the transverse and axial tubular system (TATS) network is less extensive than in rodents with consequently a greater proportion of ryanodine receptors (RyRs) not coupled to this membrane system. TATS remodelling in heart failure (HF) and after myocardial infarction (MI) increases the fraction of non-coupled RyRs. Here we investigate whether this remodelling alters the activity of coupled and non-coupled RyR sub-populations through changes in local signalling. We study myocytes from patients with end-stage HF, compared with non-failing (non-HF), and myocytes from pigs with MI and reduced left ventricular (LV) function, compared with sham intervention (SHAM).Methods and resultsSingle LV myocytes for functional studies were isolated according to standard protocols. Immunofluorescent staining visualized organization of TATS and RyRs. Ca2+ was measured by confocal imaging (fluo-4 as indicator) and using whole-cell patch-clamp (37°C). Spontaneous Ca2+ release events, Ca2+ sparks, as a readout for RyR activity were recorded during a 15 s period following conditioning stimulation at 2 Hz. Sparks were assigned to cell regions categorized as coupled or non-coupled sites according to a previously developed method. Human HF myocytes had more non-coupled sites and these had more spontaneous activity than in non-HF. Hyperactivity of these non-coupled RyRs was reduced by Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition. Myocytes from MI pigs had similar changes compared with SHAM controls as seen in human HF myocytes. As well as by CaMKII inhibition, in MI, the increased activity of non-coupled sites was inhibited by mitochondrial reactive oxygen species (mito-ROS) scavenging. Under adrenergic stimulation, Ca2+ waves were more frequent and originated at non-coupled sites, generating larger Na+/Ca2+ exchange currents in MI than in SHAM. Inhibition of CaMKII or mito-ROS scavenging reduced spontaneous Ca2+ waves, and improved excitation–contraction coupling.ConclusionsIn HF and after MI, RyR microdomain re-organization enhances spontaneous Ca2+ release at non-coupled sites in a manner dependent on CaMKII activation and mito-ROS production. This specific modulation generates a substrate for arrhythmia that appears to be responsive to selective pharmacologic modulation

Effects of acute and chronic cobalt treatment on adult rat cardiomyocyte calcium handling

Patients with cobalt-chromium hip arthroplasties display increased cobalt levels in circulation. Elevated cobalt has been linked to cardiomyopathy, yet the mechanisms underlying cobalt-induced pathology remain unknown. Here, we have examined the effects of acute ( Cardiomyocytes were isolated enzymatically from adult male rats (n=9) and treated acutely for 5min, 1h and 24h with 1, 10 and 100µM CoCl2. After loading with Cal520 AM, calcium transients were measured during electrical pacing at 1Hz and sparks were recorded using confocal microscopy. Chronic cobalt effects were measured in cardiac preparations taken from adult male rats (n=8) injected daily with CoCl2(1mg/kg) for 28 days. A concentration and time-dependent decrease in Ca2+ transient amplitude was evident in cobalt-treated cells compared to controls, with 10μM CoCl2 inducing an amplitude reduction of 15.1±5.4% and 30.0±7.2% at 5min and 1h respectively and 100µM resulting in 25.6±0.05% and 58.8±0.09% reduction at 5min and 1h. Spark frequency was increased with 100µM cobalt relative to control, resulting in 55.4±0.9% and 76.8±1.4% increase at 5min and 1h. RyR2 expression in chronic cobalt-treated ventricular tissue was similar to controls, suggesting RyR2 post-translational modification may account for altered spark frequency. This study demonstrates reduced Ca2+ release and increased RyR2 activation in cardiomyocytes treated acutely with CoCl2. Indications suggest RyR2 phosphorylation may be involved in the negative inotropic effects observed

3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes

Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z‐axis and obscured detection by 2D techniques. Non‐overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single ‘rogue’ RyR. Internal CRUs were more tightly packed along z‐lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t‐tubules, which enabled quantification of dyadic and non‐dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes

Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density

Rationale: In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI). Objective: To characterize RyR functional properties in relation to TT proximity, at baseline and after MI. Methods: Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category. Results: In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI. Conclusion: TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves