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

Exercise training corrects control of spontaneous calcium waves in hearts from myocardial infarction heart failure rats

Impaired cardiac control of intracellular diastolic Ca<sup>2+</sup> gives rise to arrhythmias. Whereas exercise training corrects abnormal cyclic Ca<sup>2+</sup> handling in heart failure, the effect on diastolic Ca<sup>2+</sup> remains unstudied. Here, we studied the effect of exercise training on the generation and propagation of spontaneous diastolic Ca<sup>2+</sup> waves in failing cardiomyocytes. Post-myocardial infarction heart failure was induced in Sprague–Dawley rats by coronary artery ligation. Echocardiography confirmed left ventricular infarctions of 40 ± 5%, whereas heart failure was indicated by increased left ventricular end-diastolic pressures, decreased contraction-relaxation rates, and pathological hypertrophy. Spontaneous Ca<sup>2+</sup> waves were imaged by laser linescanning confocal microscopy (488 nm excitation/505–530 nm emission) in 2 μM Fluo-3-loaded cardiomyocytes at 37°C and extracellular Ca<sup>2+</sup> of 1.2 and 5.0 mM. These studies showed that spontaneous Ca<sup>2+</sup> wave frequency was higher at 5.0 mM than 1.2 mM extracellular Ca<sup>2+</sup> in all rats, but failing cardiomyocytes generated 50% (P < 0.01) more waves compared to sham-operated controls at Ca<sup>2+</sup> 1.2 and 5.0 mM. Exercise training reduced the frequency of spontaneous waves at both 1.2 and 5.0 mM Ca2+ (P< 0.05), although complete normalization was not achieved. Exercise training also increased the aborted/completed ratio of waves at 1.2 mM Ca<sup>2+</sup> (P < 0.01), but not 5.0 mM. Finally, we repeated these studies after inhibiting the nitric oxide synthase with L-NAME. No differential effects were found; thus, mediation did not involve the nitric oxide synthase. In conclusion, exercise training improved the cardiomyocyte control of diastolic Ca<sup>2+</sup> by reducing the Ca<sup>2+</sup> wave frequency and by improving the ability to abort spontaneous Ca<sup>2+</sup> waves after their generation, but before cell-wide propagation

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

CaMKII augments ROS and Ca2+ entry processes in female cardiac fibroblasts in hyperglycemic and hypertensive conditions

Introduction Heart Failure with preserved Ejection Fraction (HFpEF) affects over 50% of patients with HF, majority of whom are females with conditions such as hypertension, obesity and diabetes. Our in vivo animal models of these conditions show increased fibrosis and oxidized Calcium-calmodulin dependent protein kinase II (CaMKII) activity. At the cellular level, mitochondrial health is known to be implicated in HF, and our in vitro models have signified the impact of female cardiac fibroblasts (CFs) on altered calcium (Ca2+) signalling in cardiac myocytes (CMs), during co-culture. However, knowledge on the characteristics of CFs in HFpEF still remains obscure. Here, we investigated altered Ca2+ signalling, mitochondrial Reactive Oxygen Species (ROS) production, and the therapeutic potential of inhibiting CaMKII activity in CFs, during hyperglycaemia and hypertension. Methods Adult human CFs (Promocell) sourced from both female and male donors were cultured under hyperglycaemic (22 mM Glucose), hypertensive (200nM Angiotensin II) or HFpEF-like (hyperglycaemic plus hypertensive) conditions, in the absence or presence of a CaMKII inhibitor (5µM KN93), for 48 hours. Following pathological conditioning of CFs, cells were loaded with Cal520AM calcium indicator or MitoSOX red mitochondrial superoxide indicator. Live cell fluorescence imaging was utilised to assess Ca2+ activity and mitochondrial ROS production in CFs. Results Female CFs treated under hyperglycaemic conditions showed a greater Endothelin-1-induced Ca2+ transient amplitude (ΔF/F0) relative to control and hypertensive conditioning [Control: 0.184±0.008; Diabetes: 0.305±0.026; Hypertension: 0.208±0.030; HFpEF: 0.241±0.008, n=4passages]. Mitochondrial superoxide (AU) levels were elevated only in female CFs, in both hyperglycaemic and hypertensive conditions [Control: 18.0±2.0; Diabetes: 36.7±3.4; Hypertension: 29.5±4.5; HFpEF: 22.7±2.0, n=4passages]. These alterations in Ca2+ transient amplitude and superoxide production were impeded by the presence of KN93 [Ca2+ Transient Amplitude- Control: 0.197±0.020; Diabetes: 0.167±0.022; Hypertension: 0.199±0.037; HFpEF: 0.205±0.023, Mitochondrial superoxide- Control: 16.6±2.4; Diabetes: 14.7±2.6; Hypertension: 13.3±1.1; HFpEF: 18.1±2.6, [n=4passages]. Conclusions These results indicate CaMKII is important in mitochondrial oxidative stress in female CFs, in hyperglycaemic and hypertensive conditions. Further work is needed to investigate the importance of these processes in the development of fibrosis in HFpEF

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

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

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