Author response: Prolonged β-adrenergic stimulation disperses ryanodine receptor clusters in cardiomyocytes and has implications for heart failure
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that ‘dispersion’ of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged β-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hr triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of β-adrenergic stimulation over the longer term and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA, which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology. Editor's evaluation This article is of fundamental interest to our understanding of heart failure and the cardiac contraction process. It applies super-resolution imaging and functional calcium imaging to healthy and failing cardiac cells and combines these with quantitative modeling of the resulting data. The outcome bearing on receptor distribution is a good example of exploiting quantitative super-resolved data in combination with other techniques to gain real insight into a biological problem. https://doi.org/10.7554/eLife.77725.sa0 Decision letter Reviews on Sciety eLife's review process Introduction In cardiomyocytes, the processes underlying initiation of contraction are well described, at least at the macroscale. Sarcolemmal depolarization triggers an influx of Ca2+ through voltage-gated L-type Ca2+ channels (LTCCs) within t-tubules, which in turn elicits a much larger release of Ca2+ into the cytosol via ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR). This process of Ca2+-induced Ca2+ release (CICR) depends on the precise organization of LTCCs and RyRs within dyadic junctions of the two membranes, with LTCCs located in nanoscale apposition to RyRs across the dyadic cleft (Bers, 2001). Intrinsically, RyRs are organized into discrete clusters, and these arrangements are thought to be critical in defining channel function. Indeed, RyR clusters located within close proximity are proposed to cooperatively operate as Ca2+ release units (CRUs) to generate Ca2+ sparks (Baddeley et al., 2009; Sobie et al., 2006), the elementary units of SR Ca2+ release (Cheng et al., 1993). Recent advances in high-resolution imaging techniques such as 3D dSTORM (Shen et al., 2019), DNA-PAINT (Jayasinghe et al., 2018), and electron tomography Asghari et al., 2014 have created an opportunity to understand these dyadic arrangements in unprecedented detail. Contractile dysfunction and arrhythmogenesis are hallmarks of heart failure (HF), and considerable data have linked these phenomena to t-tubule disruption in this condition (Fowler et al., 2020; Louch et al., 2006; Orchard et al., 2013). Recently, we reported that pathological changes occur also on the other side of the dyad, as we observed ‘dispersion’ of RyR clusters in failing cardiomyocytes. This RyR rearrangement specifically included fragmentation of RyR groupings into more numerous, smaller clusters, without any change in overall channel number (Kolstad et al., 2018). RyR dispersion was critically linked to low-fidelity spark generation. Furthermore, when Ca2+ release was successfully triggered by a CRU, propagation of Ca2+ between multiple clusters generated sparks with slow kinetics, and the overall Ca2+ transient was desychronized (Kolstad et al., 2018). Similar fragmentation of CRUs has been reported in a sheep model of persistent atrial fibrillation (Macquaide et al., 2015) and in monocrotaline-induced right ventricular failure in rats (Sheard et al., 2019). Thus, accumulating evidence indicates that RyR mislocalization, and ensuing dysfunction, is a key contributor to pathophysiology. What drives changes in RyR organization in diseased cardiomyocytes? Previous work has indicated that nanoscale RyR positioning is fine-tuned by phosphorylation of the channels, at least acutely (Asghari et al., 2014; Asghari et al., 2020), and that both Ca2+/calmodulin-dependent protein kinase II (CaMKII)- and protein kinase A (PKA)-dependent phosphorylation of RyR disrupt its function during HF (Ling et al., 2009; Marx et al., 2000; Zhang et al., 2003). We presently hypothesized that these two phenomena are intimately linked. We demonstrate that prolonged β-adrenergic stimulation, as is well known to occur in HF, promotes RyR dispersion via both CaMKII- and PKA-dependent phosphorylation of the channel. This RyR reorganization and sensitization are associated with a time-dependent increase in Ca2+ leak, slowing of Ca2+ spark kinetics, and reduced Ca2+ transient magnitude. In healthy cells, these actions appear to be aimed at gradually countering the stimulatory effects of increased RyR phosphorylation and Ca2+ sensitivity. Indeed, we observed that sufficiently dispersed RyRs inhibit the development of Ca2+ waves that underlie arrhythmia. Conversely, blocking hyperphosphorylation of RyRs reverses channel dispersion in HF cells and associated impairment of Ca2+ release, providing new insight into the protective mechanisms of β-blockade in this disease. Results Prolonged β-adrenergic receptor (β-AR) activation causes RyR cluster dispersion Using 3D dSTORM imaging, we investigated the effects of long-term phosphorylation on RyR organization in cardiomyocytes. We specifically hypothesized that phosphorylation would fragment or ‘disperse’ RyR clusters in a time-dependent manner. Numerically, such breaking apart of RyR groupings is expected to be reflected by a reduction in the average number of RyRs contained in each cluster, as illustrated in (Figure 1—figure supplement 1). This change would be accompanied by a concomitant increase in the total number of clusters as there are now more numerous, smaller clusters in a given 3D space. If marked RyR cluster dispersion occurs, the total number of RyRs contained in a CRU may also be reduced as clusters are no longer located in close enough proximity to be grouped in functional release units (edge-to-edge distances ≤ 100 nm; Figure 1—figure supplement 1). Notably, if RyR dispersion occurs without the addition or loss of channels, then the total number of RyRs detected by dSTORM imaging should remain unchanged. We compared RyR arrangements in normal rat ventricular cardiomyocytes to those treated with isoproterenol (100 nM) for varying lengths of time (10, 30, or 60 min). For the control group, cells were kept for 60 min following isolation prior to fixation for imaging. 3D dSTORM reconstructions revealed progressive RyR cluster dispersion as the duration of isoproterenol application increased (Figure 1a). Indeed, following a brief, 10 min bout of isoproterenol exposure, we detected a slight, but non-significant, change in measured cluster parameters. However, after 30 min of isoproterenol treatment, RyR cluster size significantly decreased (Figure 1b), while cluster density increased (Figure 1d). By 1 hr of β-AR activation, continued RyR rearrangement was sufficient to reduce CRU size as fewer RyRs remained within clusters located ≤ 100 nm from their neighbors (Figure 1c, see enlargements in Figure 1a for representative images of CRUs). Since overall RyR density was unchanged by isoproterenol treatment (Figure 1e), the detected differences in RyR organization did not result from channel degradation, but rather indicate progressive dispersion of RyR clusters into more numerous, smaller groupings. Figure 1 with 3 supplements see all Download asset Open asset Prolonged β-adrenergic receptor (β-AR) activation disperses ryanodine receptor (RyR) clusters. (a) Representative reconstructions of interior RyR clusters based on 3D dSTORM in isolated rat cardiomyocytes (clusters were cropped from a region measuring 5 x 8 x 0.6 μm). Control conditions are compared with isoproterenol treatment (100 nM) of varying duration (10, 30, and 60 min). The insets depict single Ca2+ release units (CRUs), which encompass RyR clusters with edge-to-edge distances ≤ 100 nm. (b–d) Quantification of the dSTORM data revealed progressive dispersion of RyR clusters during isoproterenol treatment, as indicated by measurements of RyR cluster size, CRU size, and cluster density. (e) RyR density was unchanged (control: ncells = 50, nhearts = 6; Iso 10: ncells = 21, nhearts = 3; Iso 30: ncells = 43, nhearts = 4; Iso 60: ncells = 37, nhearts = 5). (f) Correlative imaging of the t-tubular network (confocal microscopy) and RyRs (dSTORM) was employed to create 3D reconstructions of dyadic and non-dyadic CRUs (t-tubules: blue; RyRs: orange). Scale bars: 250 nm. (g) Similar proportions of clusters were characterized as dyadic under control conditions and following 60 min isoproterenol. (h) Cluster size measurements indicated that dispersion during isoproterenol occurred exclusively within dyads (control: ncells = 20, nhearts = 3; Iso 60: ncells = 21, nhearts = 3). The bar charts present mean measurements ± SEM, with superimposed data points representing averaged values from each cardiomyocyte. Statistical significance (p<0.05) between groups is indicated by a comparison bar. We next performed 3D correlative imaging of RyRs and the t-tubular network to distinguish between dyadic and non-dyadic (‘orphaned’) RyRs (Figure 1f). Based on these analyses, the majority of RyR clusters were classified as dyadic, and this proportion was observed to be similar under control and isoproterenol treatment conditions (83 ± 1.6% and 84 ± 1.0%, respectively; Figure 1g). Importantly, RyR cluster dispersion was found to be restricted to RyRs within dyads (control: 9.0 ± 0.5 vs. 60 min isoproterenol: 6.7 ± 0.4 RyRs/cluster, Figure 1h). It should be noted that the above analyses were performed on RyR clusters within the cell interior. Interestingly, the organization of surface RyR clusters associated with the cardiomyocyte sarcolemma was largely unaffected by β-AR activation save for a slight increase (p=0.092) in cluster size following 10 min isoproterenol treatment (Figure 1—figure supplement 2), suggesting that RyR dispersion is a phenomenon restricted to internal, dyadic RyRs. We thus focus only on interior RyR clusters for the remainder of the study. CaMKII activation mediates isoproterenol-induced RyR cluster dispersion In cardiomyocytes, it is well established that a substantial portion of β-AR signaling is mediated via CaMKII (Grimm and Brown, 2010) and includes CaMKII-mediated phosphorylation of RyRs (Ferrero et al., 2007; Grimm et al., 2015). Indeed, using flow cytometry analysis, we observed increased RyR phosphorylation at the CaMKII site ser-2814 that was particularly prominent during early stages of isoproterenol treatment (Figure 2—figure supplement 1a–c). We therefore hypothesized that isoproterenol-induced cluster dispersion is, at least in part, driven by the actions of CaMKII. To this end, we first tested the effects of the CaMKII competitive inhibitor autocamtide-2-related inhibitory peptide (AIP, 2 µM) following an initial hour-long isoproterenol incubation (i.e., 1 hr isoproterenol followed by 1 hr isoproterenol + AIP). We found that the addition of AIP markedly reversed cluster dispersion (Figure 2a). This was reflected by a significant increase in both RyR cluster size (Figure 2d) and CRU size (Figure 2e), as well as a significant reduction in RyR cluster density (Figure 2f). In control experiments, the longer 2 hr treatment period with isoproterenol alone did not alter RyR configuration beyond values observed at the 1 hr time point (Figure 2—figure supplement 2a and b). Direct activation of CaMKII through the Epac2 pathway using 8-CPT-cAMP (10 µM) (Pereira et al., 2013) also consistently reproduced the RyR cluster dispersion observed during isoproterenol stimulation (Figure 2b, quantification in Figure 2d–f). RyR dispersion was again reversed by the addition of AIP post-treatment in these cells. To further corroborate our results, we indirectly stimulated CaMKII activity by applying a low concentration of caffeine (0.5 mM) to cardiomyocytes to increase spontaneous Ca2+ release from the SR (Øyehaug et al., 2013). We found that caffeine application gave rise to significant reductions in both RyR cluster and CRU size, as well as a significant increase in cluster density (Figure 2c, quantification in Figure 2d–f). Again, these changes were reversed when AIP was administered. Figure 2 with 2 supplements see all Download asset Open asset Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation promotes ryanodine receptor (RyR) cluster dispersion. (a–c) Representative Ca2+ release units (CRUs) imaged in cardiomyocytes treated with isoproterenol (100 nM), 8-CPT (10 µM), or caffeine (0.5 mM) for 1 hr (left panels) or with inclusion of the CaMKII inhibitor AIP (2 µM) for an additional hour (right panels). Scale bar: 100 nm. (d– f) Induced RyR cluster dispersion was reversed by CaMKII inhibition, as indicated by measurements of RyR cluster size, CRU size, and cluster density (control: ncells = 50, nhearts = 6; Iso: ncells = 37, nhearts = 5; Iso + AIP: ncells = 37, nhearts = 5; 8-CPT: ncells = 48, nhearts = 5; 8-CPT + AIP: ncells = 52, nhearts = 5; caffeine: ncells = 42, nhearts = 4; caffeine + AIP: ncells = 35, nhearts = 3). (g–i) Representative images of RyR organization in cardiomyocytes from wildtype (WT) mice and transgenic mice with constitutively activated (S2814D) or genetically ablated (S2814A) phosphorylation at S2814. Clusters were cropped from a region measuring 5 x 9 x 0.6 μm. Images and quantified experimental data (j–l) are presented under baseline conditions and following 60 min isoproterenol stimulation (WT: ncells = 25, nhearts = 2; WT + Iso: ncells = 24, nhearts = 2; S2814D: ncells = 19, nhearts = 2; S2814D + Iso: ncells = 18, nhearts = 2; S2814A: ncells = 17, nhearts = 2; S2814A + Iso: ncells = 19, nhearts = 2). The bar charts present mean measurements ± SEM, with superimposed data points representing averaged values from each cardiomyocyte. Statistical significance (p<0.05) between groups is indicated by a comparison bar. While these findings suggest that isoproterenol-induced cluster dispersion is dependent on CaMKII, it remained unclear whether these effects can be directly attributed to its phosphorylation of RyRs. To address this issue, we examined RyR cluster characteristics in cardiomyocytes isolated from transgenic mice in which the CaMKII phosphorylation site on RyR is either constitutively active (S2814D) or genetically ablated (S2814A). We found that the RyR arrangement in the phosphomimetic S2814D mutant cells at baseline resembled the fragmented RyR organization observed in wildtype (WT) animals after isoproterenol treatment (Figure 2g and h). Indeed, cluster size (Figure 2j), CRU size (Figure 2k), and cluster density (Figure 2l) were all similar in WT + Iso and S2814D cardiomyocytes, and significantly different from untreated WT cells. Furthermore, application of isoproterenol to S2814D cardiomyocytes did not lead to significant additional RyR dispersion, and only tendencies toward smaller cluster and CRU sizes (Figure 2h and j–l). In contrast, cardiomyocytes from non-phosphorylatable S2814A mutant mice exhibited similar RyR organization as in WT mice at baseline (Figure 2i). Administering isoproterenol to this group promoted a significant reduction in cluster size (Figure 2j) and increased cluster density (Figure 2l). These findings mirror observations in rat cardiomyocytes, where CaMKII inhibition did not fully reverse RyR dispersion in several experiments (Figure 2d–f). Taken together, these results support that CaMKII-dependent RyR phosphorylation during prolonged β-AR stimulation is a key driver for cluster dispersion, but that there are likely additional contributing mechanisms. PKA-dependent phosphorylation also contributes to RyR cluster dispersion during β-AR activation Following β-AR stimulation, increased cyclic AMP levels lead to PKA-dependent phosphorylation of the RyR at ser-2808 (Marx et al., 2000; Figure 2—figure supplement 1d). Based on the above results, we hypothesized that in parallel to CaMKII-mediated RyR phosphorylation, activation of PKA following isoproterenol application also contributes to RyR cluster dispersion. Indeed, β-AR-induced cluster dispersion was reversed by the addition of the PKA inhibitor H89 (Figure 3a), as reflected by significantly increased cluster size (Figure 3c) and CRU size (Figure 3d), and reduced cluster density (Figure 3e). As in experiments investigating the effects of CaMKII inhibition (Figure 2), it should be noted that PKA inhibition did not fully restore the RyR configuration at the CRU level. Indeed, simultaneous treatment of cardiomyocytes with AIP and H89 resulted in further reversal of the effects of isoproterenol on CRU size, beyond those observed with either agent alone (Figure 3—figure supplement 1a–c). These observations are consistent with summative roles of PKA and CaMKII activation in promoting RyR dispersion. Figure 3 with 1 supplement see all Download asset Open asset Protein kinase A (PKA)-dependent phosphorylation also promotes ryanodine receptor (RyR) cluster fragmentation during β-adrenergic receptor (β-AR) activation. (a) Representative Ca2+ release units (CRUs) indicate that PKA inhibition (H89, 10 µM) reverses isoproterenol-induced cluster dispersion. (b) Direct activation of PKA using 6MB-cAMP (100 µM) can also induce cluster dispersion and is similarly reversed when followed by H89 application. (c–e) Data comparing cluster size, CRU size, and cluster density between groups (control: ncells = 26, nhearts = 3; Iso: ncells = 37, nhearts = 5; Iso + H89: ncells = 30, nhearts = 4; 6MB: ncells = 26, nhearts = 3; 6MB + H89: ncells = 44, nhearts = 4). (f) Representative CRUs indicate that 6MB-cAMP treatment effectively disperses RyR clusters in S2814A cardiomyocytes. (g) PKA inhibition also reverses isoproterenol-induced RyR dispersion in S2814A cells. Quantified experimental data are shown in (h–j). (WT: ncells = 49, nhearts = 5; S2814A: ncells = 51, nhearts = 5; S2814A + 6MB: ncells = 36, nhearts = 3; S2814A + Iso: ncells = 17, nhearts = 2; S2814A + Iso + H89: ncells = 38, nhearts = 3). The bar charts present mean measurements ± SEM, with superimposed data points representing averaged values from each cardiomyocyte. Statistical significance (p<0.05) between groups is indicated by a comparison bar. Scale bars: 100 nm. To further validate an involvement of PKA, we quantified RyR arrangement following application of the selective PKA activator 6MB-cAMP (6MB, 100 µM) (Christensen et al., 2003; Szabo-Fresnais et al., 2010). As illustrated in Figure 3b, 6MB-cAMP treatment prompted RyR dispersion comparable to that elicited by isoproterenol. These changes were again reversible by co-incubating 6MB with H89 following an initial treatment period with 6MB alone (Figure 3c–e). Importantly, 6MB induced RyR dispersion in S2814A cardiomyocytes (Figure 3f and h–j), which was comparable to that observed in WT. Similarly, dispersion observed in S2814A cardiomyocytes treated with isoproterenol was reversed by the addition of H89 (Figure 3g–j). These observations support that 6MB and H89 treatments exerted their effects on RyR configuration via modulation of PKA activity, without contribution from the CaMKII phosphorylation site on the RyR. Taken together, these data indicate that prolonged PKA-dependent phosphorylation of RyRs is sufficient to drive RyR cluster fragmentation, but that both PKA and CaMKII concertedly drive this process during β-AR stimulation. β-Stimulation-induced RyR dispersion alters Ca2+ sparks, transients, and waves We next assessed the functional consequences of RyR dispersion. We examined spontaneous Ca2+ sparks since these events occur almost exclusively at dyads (Louch et al., 2013), where RyR arrangements appear to be sensitive to β-AR stimulation (Figure 1f). Previous work has shown that acute β-AR activation increases the frequency of spontaneous Ca2+ sparks, while diverse effects on spark geometry are reported (Laasmaa et al., 2019; Tanaka et al., 1997; Viatchenko-Karpinski and Györke, 2001). We hypothesized, however, that RyR dispersion during prolonged β-AR stimulation would slow spark kinetics and increase Ca2+ leak, as released Ca2+ propagates between multiple clusters. Representative Ca2+ spark recordings from control and isoproterenol-treated cells are presented in Figure 4a and b, respectively, with corresponding spark time courses presented in the right panels. We observed that while spark frequency was augmented throughout the isoproterenol treatment period (Figure 4c), there was indeed a progressive slowing of both spark rise time (Figure 4d) and duration (Figure 4e). Increased overall spark geometry (Figure 4f and g) during prolonged isoproterenol treatment was linked to progressively augmented Ca2+ leak, as calculated based on spark mass and frequency (Figure 4h). Importantly, reversal of RyR dispersion by co-incubation of cells with either AIP or H89 after the initial isoproterenol treatment also reversed slowing of spark kinetics and isoproterenol-induced Ca2+ (Figure and h). A toward effects was noted when AIP and H89 treatments were (Figure 3—figure supplement In control experiments, a 2 hr treatment period with isoproterenol alone the total treatment time in AIP and H89 did not alter Ca2+ spark beyond values observed at the 1 hr time point (Figure 2—figure supplement and Taken together, these data indicate during prolonged β-AR stimulation, RyR dispersion induced by PKA and CaMKII activity is linked to slowing of Ca2+ spark kinetics and increased Ca2+ Figure with 1 supplement see all Download asset Open asset of ryanodine receptor (RyR) dispersion on Ca2+ homeostasis are reversed by Ca2+/calmodulin-dependent protein kinase II (CaMKII) or protein kinase A (PKA) inhibition. Representative of Ca2+ sparks of control and isoproterenol cardiomyocytes by imaging. The time for each spark is shown on with indicated time to and duration at data indicate that spark and slowing of kinetics during isoproterenol were all reversed by CaMKII or PKA inhibition (control: = ncells = 38, nhearts = 3; Iso 10: = ncells = 44, nhearts = 3; Iso 30: = ncells = 44, nhearts = 3; Iso 60: = ncells = nhearts = 3; Iso 60 + AIP: = ncells = nhearts = 5; Iso 60 + H89: = ncells = nhearts = 4). Representative images the in a of Ca2+ transients, followed by a to Ca2+ generation. of the Ca2+ are shown on right to differences in Ca2+ release (j–l) Data initial increases in Ca2+ transient and were reversed with continued to while overall release kinetics slowed (control: ncells = 38, nhearts = 3; Iso 10: ncells = 35, nhearts = 3; Iso 30: ncells = 36, nhearts = 3; Iso 60: ncells = nhearts = 3). Ca2+ increased during early time points following isoproterenol treatment, but then reversed (control: ncells = 43, nhearts = 3; Iso 10: ncells = 44, nhearts = 3; Iso 30: ncells = nhearts = 3; Iso 60: ncells = 44, nhearts = 3). The bar charts present mean measurements ± SEM, with superimposed data points representing averaged values from each cardiomyocyte. Statistical significance (p<0.05) between groups is indicated by a comparison bar. in RyR localization and function during prolonged β-AR stimulation were linked to in Ca2+ (Figure As isoproterenol treatment acutely increased Ca2+ transient however, this reversed with continued (Figure This was by changes in SR Ca2+ (Figure supplement which was also increased by activity during isoproterenol exposure, but then to as RyR Ca2+ progressively Similar changes were observed in the of Ca2+ release across the Ca2+ release to be more at early stages of isoproterenol treatment (Figure in with work that SR Ca2+ and RyR sensitization more Ca2+ et al., et al., 2013). However, after prolonged β-AR stimulation, RyR slowed Ca2+ spark kinetics, and declining SR was to a reversal of the of Ca2+ release observed at time points (Figure and a slowing of the of the transient (Figure The above data are consistent with the that RyR dispersion gradually the stimulatory effects of β-AR stimulation on in a protective manner. To further this we investigated the for spontaneous Ca2+ waves during isoproterenol treatment (Figure Indeed, while we observed an expected initial increase in Ca2+ following isoproterenol this reversed after 60 min as frequency was reduced by (Figure Thus, sufficient dispersion of RyRs and of suggesting a protective role or RyR rearrangement during prolonged β-AR stimulation. RyR cluster dispersion CRU in a model To support the above correlative data RyR arrangement and Ca2+ we employed a model of Ca2+ release in the To this end, we spark employing observed CRU We then the spark model with these to the resulting We also examined the of release events is to be detected in While to this can SR Using images of RyRs and (confocal we generated CRU that were representative of the of RyR dispersion observed in cells (Figure 1). For each a of spark was performed with and without the effects of β-AR stimulation on RyR and SR included (Figure model are in 2 and Figure 5 with 2 supplements see all Download asset Open asset modeling changes in ryanodine receptor (RyR) configuration and function during β-adrenergic stimulation. (a) 3D from correlative imaging of the t-tubular network (confocal microscopy) and RyRs of 10 10 10 nm and a 1 1 1 The were to the of cluster dispersion in imaging 1 for t-tubular are illustrated with superimposed RyR with indicated RyR channel and cleft by the RyRs. The were to the of RyR dispersion observed in experiments, from to fragmented Ca2+ release units (CRUs) and (b) In spark were performed in each geometry with baseline model and were with the effects of β-adrenergic receptor (β-AR) stimulation sensitization and increased sarcoplasmic reticulum The time is shown for each The to the release as an spark a or a spark The of release into spark and events was to spark fidelity and the of that is (i.e., in each f) and time to measurements for sparks
Source: eLife
Language: English
Subfield: Cardiology and Cardiovascular Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4293044779