Figures
Abstract
Background
Although high aerobic capacity is associated with effective cardiac function, the effect of aerobic capacity on atrial function, especially in terms of cellular mechanisms, is not known. We aimed to investigate whether rats with low inborn maximal oxygen uptake (VO2 max) had impaired atrial myocyte contractile function when compared to rats with high inborn VO2 max.
Methods and Results
Atrial myocyte function was depressed in Low Capacity Runners (LCR) relative to High Capacity Runners (HCR) which was associated with impaired Ca2+ handling. Fractional shortening was 52% lower at 2 Hz and 60% lower at 5 Hz stimulation while time to 50% relengthening was 43% prolonged and 55% prolonged, respectively. Differences in Ca2+ amplitude and diastolic Ca2+ level were observed at 5 Hz stimulation where Ca2+ amplitude was 70% lower and diastolic Ca2+ level was 11% higher in LCR rats. Prolonged time to 50% Ca2+ decay was associated with reduced sarcoplasmic reticulum (SR) Ca2+ ATPase function in LCR (39%). Na+/Ca2+ exchanger activity was comparable between the groups. Diastolic SR Ca2+ leak was increased by 109%. This could be partly explained by increased ryanodine receptors phosphorylation at the Ca2+-calmodulin-dependent protein kinase-II specific Ser-2814 site in LCR rats. T-tubules were present in 68% of HCR cells whereas only 33% LCR cells had these structures. In HCR, the significantly higher numbers of cells with T-tubules were combined with greater numbers of myocytes where Ca2+ release in the cell occurred simultaneously in central and peripheral regions, giving rise to faster and more spatial homogenous Ca2+-signal onset.
Citation: Johnsen AB, Rolim NPL, Stølen T, Alves M, Sousa MML, Slupphaug G, et al. (2013) Atrial Myocyte Function and Ca2+ Handling Is Associated with Inborn Aerobic Capacity. PLoS ONE 8(10): e76568. https://doi.org/10.1371/journal.pone.0076568
Editor: Xun Ai, Loyola University Chicago, United States of America
Received: April 24, 2013; Accepted: August 26, 2013; Published: October 16, 2013
Copyright: © 2013 Johnsen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the K.G. Jebsen Foundation and the Norwegian Research Council Funding for Outstanding Young investigators (to U.W.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Despite the important contribution of atria to refilling of the ventricles during increased workload such as physical activity, there are limited data on the association between atrial function and aerobic capacity, especially in terms of cellular mechanisms. Recent studies have shown that failure to increase atrial function on exercise impairs compensatory late diastolic filling with increased heart rate. This contributes to genesis of exertional dyspnoea [1], [2]. It is well established that aerobic exercise training improves left ventricular cardiac function with increased cardiac output during systole and faster relaxation during diastole [3]. These functional alterations of the heart are also supported by several studies which indicate a clear association between both training induced and high inborn aerobic capacity with improved left ventricular myocyte function and Ca2+ handling (reviewed in Kemi et al. [4]). How inborn aerobic capacity influences on atrial myocyte function and Ca2+ handling is presently not known.
Here we apply a model of rats with diverging inborn aerobic running capacities [5] to study the association between intrinsic aerobic fitness and atrial myocyte function and Ca2+ handling. Rats with low inborn aerobic running capacity (Low Capacity Runners; LCR rats) have a high-risk cardiovascular profile whereas rats with high inborn aerobic running capacity (High Capacity Runners; HCR rats) developes a healthy athletic profile with improved cardiac function [6]. We hypothesised that LCR rats have impaired atrial myocyte function associated with defective intracellular Ca2+ handling compared to HCR rats.
Methods
Animal Model
LCR and HCR rats were artificially selected and bred over 22 generations on the basis of difference in inborn running capacities between two populations, the LCR and HCR rats. Breeding started from N:NIH stock obtained from the National Institute of Health (USA), as described previously [5], [6].
The Norwegian Council for Animal Research approved the study, which was in accordance with the Guide for the Care and Use of Laboratory Animals by the European Commission Directive 86/609/EEC.
Maximal Oxygen Uptake (VO2 max) Measurement
VO2 max was measured by uphill (25°) treadmill running in a metabolic chamber until exhaustion as previously described [7], [8].
Atrial Myocyte Isolation
Left atria from rats were isolated using a modified mouse model protocol [9]. After removal, hearts were kept in ice-cold cell isolation buffer (130 mM NaCl, 5.4 mM KCL, 0.5 mM MgCl2, 0.33 mM NaH2PO4, 22 mM glucose, 50 µU/ml bovine insulin (I-5500, Sigma), 25 mM HEPESNaOH (pH = 7.4)) with 0.4 mM EGTA and immediately canulated through aorta and retrogradely perfused (7.5 ml/min, 37 C) with isolation buffer containing 0.4 mM EGTA for 2–4 min. Then the hearts were perfused with the enzyme solution containing isolation buffer supplemented with 0.048 mM CaCl2 and 1 mg/ml collagenase (Type II, Worthington, 295 U/mg). From the digested hearts (10–15 minutes perfusion) left atria were removed, cut into 3–5 pieces, and further digested by gentle stirring for 5–10 min in fresh enzyme solution until myocytes appeared. Tissue chunks were then transferred to isolation buffer containing 0.096 mM CaCl2 and 10 mg/ml 0.1% bovine serum albumin (Sigma), cut into as small pieces as possible and mechanically agitated with a pipette. The cell suspension was centrifuged at 40×g for 2 minutes in a 15 mL plastic centrifuge tube, the supernatant was gently removed and the cell pellet was resuspended in 2 ml of isolation buffer with 0.026 mM CaCl2.
Ca2+ Measurements
For intracellular Ca2+ recordings, Ca2+ concentration in the perfusion buffer was increased to 1.8 mM. Fura-2/AM-loaded (20 minutes in 2 µM, Molecular Probes, Eugene, OR) cardiomyocytes were field stimulated by bipolar electrical pulses at 2 Hz and then 5 Hz on an inverted epifluorescence microscope (Nikon TE-2000E, Tokyo). Cell shortening was measured by video-based sarcomere spacing (Ionoptix, Milton, MA) and intracellular Ca2+ concentration was measured by counting 510 nm emissions with a photomultiplier tube (PMTACQ, IonOptix, Milton, MA) after exciting with alternating 340 and 380 nm wavelengths (F340/380 ratio) (Optoscan, Cairn Research, Kent, UK). Quantification of the Sarcoplasmic reticulum (SR) Ca2+ content and rate constant for fractional contribution of Ca2+ removal by SR Ca2+ ATPase (SERCA2a) and Na+/Ca2+ exchanger (NCX) are previously described in Seidler et al. [10].
A method similar to that established by Shannon et al. [11] was used to determine diastolic Ca2+- leak from the SR. To bring the cellular Ca2+-content to a steady state, cardiomyocytes were electrically stimulated at 1 Hz in normal HEPES based 1.8 mM Ca2+-solution for 30–60 seconds. After the last electric stimulus, perfusion was switched to a 0 Na+/0 Ca2+ containing solution and diastolic Ca2+ concentration was measured in quiescent non-stimulated cardiomyocytes (40 seconds) ± Tetracaine (1 mmol/L). The 0 Na+/0 Ca2+ solution prevents the Na+ - Ca2+ exchange, which is the primary Ca2+-influx and efflux mechanism at rest. Tetracaine blocks the Ca2+-leak over the RyR. The quantitative difference between diastolic Ca2+-concentration with and without tetracaine determines leak. After the 40 second period in 0 Na+/0 Ca2+ ± Tetracaine solution, caffeine was added (10 mM) to assess the SR Ca2+-content. Diastolic Ca2+-leak is presented as diastolic [Ca2+]i in relation to total SR Ca2+-content.
Confocal Microscopy
Imaging of T-tubular network and spatiotemporal characteristics of Ca2+ transients were studied using a laser scanning microscope (LSM 5 PASCAL, Zeiss, Jena, Germany) and a Zeiss 63×1.23NA oil-immersion objective. To visualize T-tubular network, quiescent, non-perfused cardiomyocytes loaded with the membrane specific Di-8-ANEPPS dye (10 µM, Molecular Probes) were confocal Z-stack scanned (488 nm excitation and detection at >514 nm). This was performed with pinhole of 1 airy unit and 0.38 micron thick stacks. T-tubule density was analyzed with custom-made applications in IDL 6.0 (ITT Visual, Boulder, CO, USA), by counting pixels stained with the dye relative to the total number of pixels after removing pixels associated with the external cell membrane. To study spatiotemporal characteristics of Ca2+ transients, Fluo-3/AM (10 µM, Molecular Probes) loaded cardiomyocytes were confocal line-scan recorded (488 nm excitation and detection at >514 nm) during steady state stimulation at 1 Hz. Repetitive scanning of a line parallel to the transversal axis of the cell were used to visualize Ca2+ signal. For the Ca2+ synchrony analysis, the transients were divided into 5 equal strips. Time from stimulation to 50% peak Ca2+ release was measured for each strip by the programme LabTalk Origin (OriginLab Corporation, Northhampton, MA) to determine spatial differences in systolic rise time of the Ca2+ transient from the edges to the center of the cardiomyocytes.
Western Blot Analyses
Proteins (100 µg total lysate) from left atrium were heated in LDS loading buffer (Invitrogen) and subjected to electrophoresis on pre-cast 3–8% Tris-acetate denaturing NuPAGE gels (Invitrogen). After separation for 3 hours at 150 V/220 mA and 4°C, gels were incubated in 2× NuPAGE transfer buffer (Invitrogen) contatining 0.02% SDS for 10 minutes. And proteins were electro-transferred onto PVDF membranes (Immobilon-FL, Millipore) at 20 V overnight and 4°C (BioRad, Hercules, CA). The membranes were blocked with Odyssey blocking buffer (LiCOR) prior to incubation with monoclonal anti-ryanodine receptor (RyR2) (1∶5,000; Thermo Fisher Scientific, Waltham, MA), polyclonal anti-pS2809-RyR2 (1∶1,000; Badrilla, Leeds, UK), and monoclonal anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶100,000; Millipore (Chemicon), Temecula, CA) antibodies overnight at 4°C. After incubation with secondary goat anti-mouse IRDye800LT and goat anti-rabbit IRDye680LT secondary antibodies (Li-COR) bands were detected using an Odyssey infrared imaging system (Li-COR, Lincoln, NE). Quantitative analyses were performed with Odyssey v.3.0 software and ImageJ Data Acquisition Software (National Institute of Health, Bethesda, MD).
Statistics
Data are presented as mean ± SD. Student T-test was used to identify statistical differences between the groups. Man-whitney Rank Sum test was used if normality test (Shapiro-Wilk) failed. The Fisher’s Exact test was applied to the categorical data. P<0.05 was considered statistical significant.
Results
Intrinsic Aerobic Capacity and Cardiac Contractility
VO2 max was 24% lower in LCR rats compared to HCR rats (Figure 1, p<0.01).
Data are presented as mean±SD.
Atrial Myocyte Function
Fractional shortening in atrial myocytes from LCR was 52% lower at 2 Hz and 60% lower at 5 Hz stimulation (Figure 2B, p<0.01) compared to that observed in HCR. Diastolic atrial myocyte function, measured as time to 50% re-lengthening was 43% (2 Hz) and 55% (5 Hz) slower in LCR rats (Figure 2C, p<0.01).
A, Exemplary tracings of atrial myocyte function in Low Capacity Runner (LCR)- compared to High Capacity Runner (HCR) rats display a deteriorated viability in LCR rats both at systolic and diastolic properties. B, Fractional shortening was depressed at 2 and 5 Hz stimulation in LCR rats and, C, Time to 50% relaxation was increased LCR rats. n = 5 animals, n = 4−6 cells from each animal. Data are presented as mean±SD.
Ca2+-handling
We found that atrial myocyte Ca2+- handling was significantly impaired in LCR rats compared to HCR rats. Exemplary tracings of Ca2+ transients are shown in figure 3A and 3B. At 2 Hz stimulation the Ca2+-amplitude was similar in both groups, whereas it was 70% lower in atrial myocytes from LCR rats at 5 Hz (Figure 3C, p<0.05). Diastolic Ca2+-levels were also similar between groups when studied at 2 Hz stimulation but significantly elevated in LCR rats at 5 Hz (Figure 3D, p<0.05). In line with the prolonged time to cell relengthening in atrial myocytes from LCR rats, time to 50% Ca2+- decay was significantly longer at both 2 and 5 Hz stimulation when compared to that observed in HCR (Figure 3E, p<0.01).
A, Exemplary recordings of Ca2+-transients in Low Capacity Runner (LCR)- and High Capacity Runner (HCR) rats. B, Exemplary tracings of one single twitch Ca2+ transient at 2 hz (left panel) 5 hz (right panel) with comparison of LCR and HCR (normalized diastolic Ca2+ levels). C, Ca2+-amplitude during systolic contraction of the atrial myocytes. D, Diastolic Ca2+-level measured at end diastole. E, Time to 50% Ca2+-removal during diastole. All Ca2+-recordings are presented as the 340/380 ratio of FURA2/AM. n = 5 animals, n = 4−6 cells from each animal. Data are presented as mean±SD.
Sarcolemmal and SR Ca2+-cycling
Prolonged time to 50% Ca2+-decay was associated with a 39% reduction in Ca2+-removal via SERCA2a in atrial myocytes from LCR rats when compared to HCR (Figure 4A, p<0.01). NCX activity was comparable between the groups (Figure 4B). SR Ca2+-content was not different between LCR and HCR rats (Figure 4C). Measuring Ca2+ in quiescent cardiomyocytes over a prolonged period of time (1 min) with and without tetracaine provides a quantitative assessment of SR (RyR2) Ca2+ leak (Figure 5A). We found that diastolic SR Ca2+ leak over the RyR2 was increased by 109% in LCR compared to HCR (Figure 5B). To analyse mechanisms of increased diastolic SR Ca2+ leak, RyR2 expression and phosphorylation were quantified. We found that RyR2 phosphorylation at the Ca2+-calmodulin-dependent protein kinase-II (CaMKII) specific Ser-2814 site is apparently induced in LCR rats (Figure 5C and 5D). The protein kinase A (PKA) phosphorylation site Serine-2808 was not significantly altered (data not shown).
Total SR Ca2+ content was measured by assessing peak Ca2+ amplitude after rapidly applying Caffeine (10 mM) to the perfusion solution immediately after stopping the electrical stimulation in normal HEPES solution. To quantify the SERCA2a function, a simple model was used based on the following assumptions: SERCA2a transport rate is: Ktwitch – KCaffeine/NCX, where Ktwitch is the Ca2+ removal (F340/380 ratio) during the time period from peak electrical stimulated twitch Ca2+ to 50% Ca2+ decay in normal HEPES solution and the KCaffeine/NCX is the Ca2+ removal (F340/380 ratio) during the time period from peak caffeine induced Ca2+ release to 50% of decay (10 mM Caffeine+HEPES). In presence of caffeine the SERCA is inhibited and the Ca2+ removal in this condition is mainly determined by NCX. A, SR Ca2+ ATPase (SERCA2a) function was significantly lower in Low Capacity (LCR) rats than High Capacity Runner (HCR) rats. B, Na+/Ca2+ exchanger (NCX) function was not different between groups. C, SR Ca2+-content assessed by application of 10 mM of caffeine after electrical 1 Hz stimulation did not reveal any difference LCR and HCR atrial myocytes. n = 5 animals, n = 4−6 cells from each animal. Data are presented as mean±SD. D, Exemplary recordings of twitch Ca2+ transients (red lines) compared to Caffeine transients (black lines). Twitch Ca2+ transients are magnified in respective figures for better evaluation of Ca2+ handling kinetics.
A, Exemplary recordings show the protocol of quantification of SR Ca2+-leak by determination of diastolic Ca2+-levels in quiescent atrial cells with 0 Na+/0 Ca2+ in the external perfusion solution compared to perfusion solution with 0 Na+/0 Ca2++Tetracaine (TET) that inhibits the opening of the ryanodine receptor (RyR2). Recordings were followed by Caffeine (10 mM) induced Ca2+ depletion of the SR to determine SR Ca2+ storage B, Diastolic SR Ca2+-leak was significantly increased in Low Capacity Runner (LCR) rats compared to High Capacity Runner (HCR) rats. n = 5 animals, n = 4−6 cells from each animal. C, Western blot analyses of the ratio between phosphorylated Serine-2814/RyR2 display a significant higher expression in LCR rats (n = 4) compared to HCR rats (n = 3). D, Representative Western blots. Data are presented as mean±SD.
Transverse (T)- tubule and Cell Dimensions
Synchronous activation of Ca2+ -induced Ca2+ release is facilitated by T-tubules that are inward invaginations in the plasma membrane that ensure close proximity of L-type Ca2+ channels and RyRs in the cell interior We determined T-tubule structure in atrial cells stained with the membrane specific dye Di-8-ANNEPS (typical examples in Figure 6A). We found that fewer atrial cells from LCR had T-tubule structures compared with that observed in HCR (33% in LCR (n = 57 cells) versus 68% in HCR rats (n = 37 cells), P<0.01). However, there was no difference in T-tubule density between the two groups in cells presenting T-tubule structure. In agreement with previous studies from larger animals [12], [13], we observed that isolated myocytes with T-tubules was significantly wider than myocytes without T-tubules (Figure 6B).
A, Confocal images of Di-8-Anepps stained atrial myocytes with and without T-tubules for Low Capacity Runner (LCR) and High Capacity Runner (HCR) rats. B, Proportion of cells with and without T-tubules for LCR and HCR rats. Absence of T-tubules in the majority of LCR rats may impair Ca2+ handling. Comparison of cell thickness in cells with and without T-tubules. Data are presented as mean±SD. n = 57 cells for LCR and 37 cells for HCR.
Spatiotemporal Properties of Ca2+ Transients
Two types of Ca2+ transients were observed in atrial myocytes from LCR and HCR, U-shaped and W-shaped (Exemplary tracings are illustrated in Figure 7), as observed in atrial myocytes in previous rat models [12], [13]. The majority of atrial myocytes from LCR displayed mainly an U- shaped Ca2+ transient (84%, n = 19 cells, Figure 8A), where the Ca2+ release initiated at the edges of the cells and then propagated inwards. Such response has been observed in cells devoid of T-tubules [12] and is in line with our finding of low proportion of myocytes with T-tubules in LCR. In contrast, the majority of atrial myocytes from HCR displayed W-shaped Ca2+ transients (56%, n = 16 cells Figure 8A), where the Ca2+ signal initiated at the edges of the cells as well as in the central regions of the cells, giving rise to more complex pattern of transient. LCR had a significant lower proportion of W shaped Ca2+ transients compared to HCR and we observed that time to 50% peak Ca2+ was slower in LCR than HCR (p<0.05, Figure 8B). Analysis of time to 50% peak of Ca2+ transient in U- compared to W-shaped transients revealed that U-shaped transients were slower than W-shaped (p<0.05, from HCR group) and no differences were observed when comparing U- vs. U –shaped transients and W- vs. W shaped transients between groups. This suggests that the slower time to peak in LCR was partly due to high proportion slow U-shaped transients. Further spatiotemporal analysis of U-shaped Ca2+ transient revealed that the central Ca2+ release within the myocytes was significantly slower than the edges (p<0.05, within LCR and HCR group, Figure 8C and 8D). Furthermore, central Ca2+ release in U-shaped Ca2+ transients was significantly slower than the corresponding central Ca2+ release in W-shaped transients (p<0.01, from HCR group).
Cells were labeled with fluo-4 and confocal line scanned transversely. Panels A–D depict the spatiotemporal properties of Ca2+ transient in: A, atrial myocyte with U-shaped Ca2+ signal in in Low Capacity Runner (LCR); B, atrial myocyte with W-shaped Ca2+ signal in LCR; C, atrial myocyte with U-shaped Ca2+ signal in High Capacity Runner (HCR); D, atrial myocyte with W-shaped Ca2+ signal in HCR.
A, Proportion of cells with different Ca2+ response pattern (U- or W-shaped). B, Time to 50% peak Ca2+ release in Low Capacity Runner (LCR) vs. High Capacity Runner (HCR) rats. C and D, Spatial characteristics of time to 50% peak Ca2+ release in U- vs W shaped transients in LCR and HCR. Data are mean±SD. Difference in time to 50% peak Ca2+ release between edges (A and E, x-axis) and center (C, x-axis) in U shaped transient: *p<0.05. Difference in time to 50% peak Ca2+ release between central region of U- and W-shaped transient: †p<0.05. Data are presented as mean±SD. n = 19 cells for LCR and 16 cells for HCR.
Discussion
This is the first study to demonstrate that low inborn aerobic capacity is directly associated with reduced contractile function and impaired Ca2+ handling in atrial myocytes.
Cardiomyocyte Function and Ca2+ Handling
We have previously reported that left ventricular myocytes from LCR rats have impaired systolic and diastolic function relative to HCR rats [6]. Ventricular contractile dysfunction has been strongly associated with altered Ca2+ handling in heart failure [14] and such association has also been reported in atrial myocytes in HF model [15]. This study revealed reduced fractional shortening and prolonged time to diastolic re-lengthening combined with depressed atrial myocyte Ca2+ handling in LCR compared to HCR rats, which confirms that there is an association between aerobic capacity and development of atrial myocyte function. Ca2+ amplitude together with duration of Ca2+ transient are main determinants of cardiac contraction [16]. In this study atrial myocyte Ca2+ amplitude was preserved at 2 Hz in LCR compared to HCR rats, still fractional shortening was depressed in LCR rats, indicating reduced Ca2+ sensitivity. At 5 Hz stimulation there was a significant decrease in Ca2+ amplitude in LCR rats. The observed negative frequency dependent alteration in systolic Ca2+ amplitude in the LCR (illustrated in Figure 3) is important and likely contributes to limited aerobic capacity during increasing workload such as endurance exercise. In our data there are two mechanisms that potentially may cause this negative response in LCR: 1) reduced reuptake of Ca2+ to the SR by SERCA2a and 2) less developed T-tubule structures and reduced initiation sites for Ca2+ activated Ca2+ release.
Earlier studies have shown that reduced SERCA2a function is related to a negative frequency dependent acceleration of Ca2+ removal [17]. When increasing the frequency from 2 Hz to 5 Hz SERCA2a may not have the capacity to cope with the increased demand of rapidly circulating Ca2+ and thereby not able to reload the SR with Ca2+ available between stimulation. Despite this obvious explanation we were unable to detect any significant difference SR Ca2+ content after caffeine-stimulated depletion. The stimulation frequency before caffeine stimulation in our experiments was, however, performed after 1 Hz electrical stimulation, which probably is too low to tax the capacity of SERCA2a. Therefore, despite that the SERCA2a capacity is reduced in LCR already at low frequencies compared to HCR, the capacity may still be adequate to maintain a preserved end-diastolic Ca2+ and SR Ca2+content at this frequency. Our finding of a significantly increased end-diastolic Ca2+ level at 5 Hz stimulation supports a failure of SERCA2a for reuptake of Ca2+ during increased Ca2+ cycling rates which potentially also mediated a reduced SR Ca2+ available for release.
T-tubule system of variable extent has been reported in rat atrial cells [12], [13]. Here we show a greater proportion of cells devoid of any T-tubule system in LCR compared to HCR rats and we suggest that differences in this could be associated with intrinsic aerobic capacity. The high number of U-shaped Ca2+ transients in the myocytes from LCR compared to HCR rats, together with relative low number of atrial myocytes with T-tubules in LCR rats, suggests a lack of central initiation sites for Ca2+ response. The transients showing this spatial profile rises rapidly at the edges of the myocytes and more slowly in the interior, which is in agreement with association between lack of T-tubules and spatiotemporal characteristics of Ca2+ transients demonstrated in atrial cells previously [12], [13], [18]. In cells devoid of T-tubules, the close apposition of L-type Ca2+ channels (LTCCs) and RyRs that is necessary for Ca2+ induced Ca2+ release, occurs only at the cells periphery leading to dyssynchronous Ca2+ release [19]. Similar Ca2+ dynamics has been reported in ventricular myocytes of HF models because of a loss of or reorganization of T-tubules leaving some orphaned RyRs that become physically separated from LTCCs [20], [21]. The average signal of Ca2+ release across the entire spatial dimension of the line scan was faster in HCR rats compared to LCR rats. This may be explained by the relative higher number of W-shaped Ca2+ transients due to more developed T-tubular network in HCR myocytes, which provide central initiation sites for Ca2+ release with faster and more spatial homogenous onset of Ca2+-signal. This is supported by Smyrnias et al. [13] who found cells with W-shaped Ca2+ transients to have significantly faster recovery of systolic Ca2+ amplitude after complete depletion of Ca2+ by caffeine application. At increasing frequencies the functional consequences of delayed central Ca2+ rise in LCR rats will be even more pronounced because of the increased demand of rapid initiation of Ca2+ induced Ca2+ release. Therefore, we suggest an association between the observed differences in spatio-temporal characteristics of Ca2+-signal and the observed differences in atrial myocyte systolic performance due to the fact that slow rise in Ca2+ release may limit synchronous contractile activation, especially at high cardiac frequencies [14].
Increased Diastolic SR Ca2+ Leak
The observation of increased diastolic SR Ca2+ leak in atrial myocytes is interesting since this is the first report showing that low aerobic capacity leads to a cellular substrate that may be more prone to triggering of atrial arrhythmias. Several studies on ventricle cardiomyocytes [22]–[24] and also from patients with atrial fibrillation [25] have shown that increased RyR2 Ca2+ leak from the SR during diastole is a potent trigger for uncontrolled electrical activity that may cause spontaneous contractions and arrhythmias. On this basis several novel Ca2+ release RyR2-stabilizing drugs have been proposed [26]. Phosphorylation of serine −2814 at the RyR2 by CaMKII is a well-documented cause of increased Ca2+ leak [17], [22], [27]. Although further studies including higher number of animals are necessary to elucidate the mechanism involved in the regulation of Ca2+ leak, our data indicates that RyR2 serine-2814 phosphorylation is apparently increased in the LCR rats. Importantly, this suggests a deleterious signaling induced by contrasting for low aerobic capacity.
Conclusions
This study report for the first time that contrasting for low or high aerobic capacity leads to diverse functional and structural remodeling of atrial myocytes. Compared to rats with high aerobic capacity we found that low aerobic capacity in LCR rats was associated with reduced atrial myocyte contractility and diastolic relaxation that were associated with impaired Ca2+-handling. Reduced systolic Ca2+ amplitude in LCR rats was associated with reduced ability to initiate Ca2+ release from the SR that probably is caused by a less developed T-tubule network. Furthermore, low aerobic capacity in LCR rats led to an increased diastolic SR Ca2+ leak over the RyR2, which has been linked to cardiac arrhythmias in several studies on left ventricular myocytes. Our study therefore suggests that low aerobic capacity may lead to negative signaling in atrial myocytes with defective properties of Ca2+ handling that is not only negative for atrial function but also may cause a cellular substrate that is more prone for triggering of atrial arrhythmias. It is likely that the improved cardiomyocyte function and Ca2+ handling associated with high aerobic capacity has a positive effect during increased workload of the atria. It is furthermore tempting to speculate that the positive adaptations in the atrial cellular mechanisms may protect against atrial dysfunction such as atrial fibrillation.
Author Contributions
Conceived and designed the experiments: ABJ GLS UW TS MAH. Performed the experiments: ABJ NPLR MAH UW. Analyzed the data: ABJ MAH NPLR. Contributed reagents/materials/analysis tools: LGK SLB MMLS GS MA. Wrote the paper: ABJ MAH.
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