Recent spikes in interactions between humans and sharks in the New York Bight have sparked widespread reporting of possible causalities, many of which lack empirical support. Here we comment on the current state of knowledge regarding shark biology and management in New York waters emphasizing that the possible drivers of increased human-shark interactions are confounded by a lack of historical monitoring data. We outline several key research avenues that should be considered to ensure the safe and sustainable coexistence of humans, sharks, and their prey, in an era of accelerated environmental change.
Increasing cardiomyocyte contraction during myocardial stretch serves as the basis for the Frank-Starling mechanism in the heart. However, it remains unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres. We investigated sarcomere contractile synchrony and how intersarcomere dynamics contribute to increasing contractility during cell lengthening.
In cardiomyocytes, invaginations of the sarcolemmal membrane called t-tubules are critically important for triggering contraction by excitation-contraction (EC) coupling. These structures form functional junctions with the sarcoplasmic reticulum (SR), and thereby enable close contact between L-type Ca 2+ channels (LTCCs) and Ryanodine Receptors (RyRs). This arrangement in turn ensures efficient triggering of Ca 2+ release, and contraction. While new data indicate that t-tubules are capable of exhibiting compensatory remodeling, they are also widely reported to be structurally and functionally compromised during disease, resulting in disrupted Ca 2+ homeostasis, impaired systolic and/or diastolic function, and arrhythmogenesis. This review summarizes these findings, while highlighting an emerging appreciation of the distinct roles of t-tubules in the pathophysiology of heart failure with reduced and preserved ejection fraction (HFrEF and HFpEF). In this context, we review current understanding of the processes underlying t-tubule growth, maintenance, and degradation, underscoring the involvement of a variety of regulatory proteins, including junctophilin-2 (JPH2), amphiphysin-2 (BIN1), caveolin-3 (Cav3), and newer candidate proteins. Upstream regulation of t-tubule structure/function by cardiac workload and specifically ventricular wall stress is also discussed, alongside perspectives for novel strategies which may therapeutically target these mechanisms.
Invaginations of the cellular membrane called t-tubules are essential for maintaining efficient excitation–contraction coupling in ventricular cardiomyocytes. Disruption of t-tubule structure during heart failure has been linked to dyssynchronous, slowed Ca2+ release and reduced power of the heartbeat. The underlying mechanism is, however, unknown. We presently investigated whether elevated ventricular wall stress triggers remodelling of t-tubule structure and function. MRI and blood pressure measurements were employed to examine regional wall stress across the left ventricle of sham-operated and failing, post-infarction rat hearts. In failing hearts, elevated left ventricular diastolic pressure and ventricular dilation resulted in markedly increased wall stress, particularly in the thin-walled region proximal to the infarct. High wall stress in this proximal zone was associated with reduced expression of the dyadic anchor junctophilin-2 and disrupted cardiomyocyte t-tubular structure. Indeed, local wall stress measurements predicted t-tubule density across sham and failing hearts. Elevated wall stress and disrupted cardiomyocyte structure in the proximal zone were also associated with desynchronized Ca2+ release in cardiomyocytes and markedly reduced local contractility in vivo. A causative role of wall stress in promoting t-tubule remodelling was established by applying stretch to papillary muscles ex vivo under culture conditions. Loads comparable to wall stress levels observed in vivo in the proximal zone reduced expression of junctophilin-2 and promoted t-tubule loss. Elevated wall stress reduces junctophilin-2 expression and disrupts t-tubule integrity, Ca2+ release, and contractile function. These findings provide new insight into the role of wall stress in promoting heart failure progression.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Reduced cardiac contractility during heart failure (HF) is linked to impaired Ca2+ release from Ryanodine Receptors (RyRs). We investigated whether this deficit can be traced to nanoscale RyR reorganization. Using super-resolution imaging, we observed dispersion of RyR clusters in cardiomyocytes from post-infarction HF rats, resulting in more numerous, smaller clusters. Functional groupings of RyR clusters which produce Ca2+ sparks (Ca2+ release units, CRUs) also became less solid. An increased fraction of small CRUs in HF was linked to augmented 'silent' Ca2+ leak, not visible as sparks. Larger multi-cluster CRUs common in HF also exhibited low fidelity spark generation. When successfully triggered, sparks in failing cells displayed slow kinetics as Ca2+ spread across dispersed CRUs. During the action potential, these slow sparks protracted and desynchronized the overall Ca2+ transient. Thus, nanoscale RyR reorganization during HF augments Ca2+ leak and slows Ca2+ release kinetics, leading to weakened contraction in this disease. https://doi.org/10.7554/eLife.39427.001 eLife digest The muscle cells of the heart coordinate how they contract and relax in order to produce the heartbeat. During heart failure, these cells become less able to contract. As a result the heart becomes inefficient, pumping less blood around the body. For the cardiac muscle cells to contract, the levels of calcium ions in the cells needs to rapidly increase. In failing hearts, these increases in calcium ion levels are smaller, slower and less well coordinated. It was not known what causes these changes, making it difficult to treat heart failure. Calcium ions are released in cardiac muscle cells through protein channels called ryanodine receptors. These receptors form clusters that allow them to synchronize when they open and close. Could the reorganization of ryanodine receptors account for the problems seen in failing hearts? To investigate, Kolstad et al. examined rat hearts using a technique called super-resolution microscopy. This showed that the clusters of ryanodine receptors break apart during heart failure to form smaller clusters. Further experiments showed that calcium ions 'leak' from these smaller clusters, reducing the amount of calcium that can be released into cardiac muscle cells during each heartbeat. Released calcium also spreads between the dispersed clusters, resulting in a slower rise of the calcium levels in the cells. Both changes contribute to weakened contractions of cells in failing hearts. Therefore, heart failure can be traced back to very small rearrangements of the ryanodine receptors. This understanding will help researchers as they investigate new ways to treat heart failure. https://doi.org/10.7554/eLife.39427.002 Introduction The basic processes for cardiac excitation-contraction coupling are well described. Depolarization of the sarcolemma triggers the opening of voltage-gated L-Type Ca2+ channels (LTCCs), and the resulting Ca2+ influx elicits additional Ca2+ release via Ryanodine Receptors (RyRs) in the sarcoplasmic reticulum (SR). This process of Ca2+-induced Ca2+ release leads to a sharp increase in cytosolic Ca2+ concentration which initiates cardiomyocyte contraction. In ventricular myocytes, Ca2+ release is tightly controlled by the arrangement of LTCCs and RyRs in dyads, with LTCCs present in t-tubules juxtaposed from RyRs across a narrow 12–15 nm dyadic cleft (Bers, 2001). The RyRs themselves are organized into clusters; an arrangement that couples their gating, promoting synchronized opening and closing of neighbouring channels (Marx et al., 2001; Sobie et al., 2006). Recent data have indicated that neighbouring clusters of RyRs can also act concertedly if the Ca2+ diffusion distance between them is sufficiently short (Macquaide et al., 2015). Referred to as 'superclusters' or Ca2+ Release Units (CRUs), these functional arrangements of RyR clusters generate Ca2+ sparks, the fundamental units of SR Ca2+ release in cardiomyocytes (Cheng et al., 1993). Ca2+ sparks are not only elicited by LTCC opening, but also occur spontaneously during diastole, where spark frequency and geometry can be measured to assess CRU function. While Ca2+ sparks are an important source of RyR-mediated Ca2+ leak from the SR, 'silent' or 'non-spark' events also occur, and involve the opening of a subset of RyRs within a CRU; so-called 'quarky' release (Brochet et al., 2011). Impaired cardiomyocyte Ca2+ homeostasis is believed to importantly contribute to reduced cardiac contractility and arrhythmogenesis in heart failure (HF). SR Ca2+ release is reduced and slowed in this condition, and these changes have been linked to altered dyadic structure (Louch et al., 2010). We and others have observed marked remodeling of the t-tubular system in failing cardiomyocytes, while RyRs remain predominantly distributed along z-lines (Song et al., 2006; Louch et al., 2006; Heinzel et al., 2008). Thus, the coupling between LTCCs and RyRs is disrupted, with 'orphaned' CRUs exhibiting delayed Ca2+ release only after trigger Ca2+ diffuses from intact dyads. However, abnormal gaps occurring between t-tubules only account for a fraction of the overall de-synchronization of Ca2+ release in HF (Louch et al., 2006; Øyehaug et al., 2013). This suggests that other alterations might also occur, perhaps at the nanometer scale of CRU organization, which hinder efficient triggering of Ca2+ release. CRU reorganization could in principle contribute to increased Ca2+ leak, including silent leak, which is a hallmark of heart failure (Zima et al., 2010; Walker et al., 2014). Exaggerated Ca2+ leak in failing cells has been linked to reduced SR Ca2+ content and depressed contractile function, elevation of resting Ca2+ levels and impaired relaxation, pro-arrhythmic early and delayed afterdepolarizations, and energetic inefficiency as Ca2+ is redundantly cycled (Bers, 2014). Thus, a detailed understanding of CRU structure and function in failing cells is critical. The advent of super-resolution microscopy techniques has markedly improved our ability to visualize and quantify CRU organization (Baddeley et al., 2009; Macquaide et al., 2015; Jayasinghe et al., 2018). However, these techniques have not previously been employed to examine RyR configuration in HF. Using direct stochastic optical reconstruction microscopy (dSTORM), we presently report that CRUs become dispersed in failing myocytes, as RyR clusters are broken apart. With the aid of mathematical modeling, we directly link these changes in CRU structure to experimentally measured increases in RyR leak and slowed SR Ca2+ release, identifying a novel mechanism underlying pathological remodeling of Ca2+ homeostasis in this disease. Results dSTORM imaging reveals dispersion of CRUs in failing myocytes Imaging was performed on isolated, fixed cardiomyocytes with antibody labelling of RyR2. Using diffraction-limited confocal imaging (resolution ≈250 nm) and Structured Illumination Microscopy (SIM, resolution ≈120 nm), the localization of RyRs along z-Lines was clearly apparent, but organization of RyRs within CRUs was not discernable (Figure 1A). With dSTORM imaging, spatial resolution was markedly improved (mean localization precision = 21 ± 3 nm) enabling detailed CRU geometry to be assessed. For analysis of RyR cluster and CRU configuration, acquired raw images were fitted to a 30 × 30 nm grid, corresponding to the quatrefoil structure of the RyR protein (Baddeley et al., 2009). Thresholding was then performed to create binary images (Figure 1B), enabling quantification of RyR clusters, with an RyR counted as present if >half the area of a 30 nm square was above threshold. RyR clusters were defined by occupied, neighbouring grid positions, and CRUs were delineated by collecting neighbouring RyR clusters located within 150 nm (Macquaide et al., 2015) (red boundaries in Figure 1B) or 100 nm (Baddeley et al., 2009; Hou et al., 2015) (Figure 2—source data 1). RyR organization was compared in cardiomyocytes from rats with post-infarction HF and cells from Sham-operated controls. Overall RyR expression was similar in Sham and HF, as evidenced by Western blotting of ventricular homogenates (Figure 2— figure supplement 1), and equivalent RyR labeling density in cardiomyocytes (41.9 ± 1.4 RyR/µm, 40.4 ± 1.3 RyR/µm in Sham, HF respectively). In both groups, RyR staining showed a predominantly transverse, striated pattern (Figure 2A). However, despite rather similar organization of RyRs at the macroscale, nanoscale dSTORM imaging revealed fragmentation of RyR clusters in failing cardiomyocytes (see insets in Figure 2A). Cluster breakup resulted in a reduction in the number of RyRs per cluster, and a greater proportion of small clusters in HF (Figure 2B). The overall number of clusters increased accordingly in failing cells (Figure 2D), and inter-cluster distance was reduced (Figure 2E; see Figure 2—source data 1 for mean data across animals). Consistent with fragmentation of clusters into smaller adjacent groupings, the number of clusters contained in a CRU increased in HF (Figure 2F), although the number of RyRs per CRU decreased (Figure 2C) since RyR clusters were markedly reduced in size. Convex hull analysis (see methods) revealed a consequent decrease in CRU solidity in HF (Figure 2G). Thus, RyR reorganization in failing cells resulted in CRUs with a more sparse, dispersed configuration of smaller sub-clusters. Figure 1 Download asset Open asset dSTORM imaging enables quantification of RyR localization within Ca2+ release units (CRUs). RyR imaging was performed with antibody labelling of isolated and fixed rat ventricular cardiomyocytes. (A). Imaging of RyRs with confocal microscopy (left panel) or Structured Illumination Microscopy (SIM, centre panel) revealed a predominantly striated pattern of RyR localization across cells, but individual CRUs could not be discerned (magnified regions in lower panels). dSTORM imaging provided markedly improved spatial resolution enabling identification of RyR clusters (scale bars = 5 µm). (B). Quantification of RyR localization was performed by fitting raw images to a 30 × 30 nm grid (Baddeley et al., 2009), and performing thresholding to create binary images; an RyR was counted as present if > half the area of a 30 nm square was suprathreshold. CRUs were defined as collections of RyR clusters with an edge-to-edge distance < 150 nm (Macquaide et al., 2015) (red boundaries) or < 100 nm (Baddeley et al., 2009; Hou et al., 2015). (Scale bar = 2 µm). https://doi.org/10.7554/eLife.39427.003 Figure 2 with 1 supplement see all Download asset Open asset RyRs are dispersed in failing cardiomyocytes. Alterations in nanoscale RyR organization were examined in cardiomyocytes from rats with post-infarction heart failure (HF). Representative images show that macroscale organization of RyRs was similar in HF and Sham-operated controls (A), upper panels). However, nanoscale examination revealed that RyR clusters were broken apart in HF. For the magnified regions in (A), conversion from raw dSTORM to binary images is shown in the middle and lower panels (saturation levels indicated by high-low look-up table). Mean measurements showed fewer RyRs per cluster in failing cells, with an increased fraction of small clusters (B). Dispersion of RyR clusters into smaller fragments resulted in an increased overall number of clusters (D), reduced inter-cluster distances (E) and inclusion of more clusters in each CRU (F). Overall CRU composition became less solid in failing cells ((G), assessed by convex-hull analysis), as the average CRU contained fewer RyRs (C). See Figure 2—source data 1 for analysis of 100 nm vs 150 nm CRU inclusion criterion (ncells = 46, 50 in Sham, HF; *=P < 0.05 vs Sham). https://doi.org/10.7554/eLife.39427.004 Figure 2—source data 1 Quantification of RyR organization using 150 vs 100 nm CRU inclusion criteria. RyR cluster and CRU data were compared in Sham and HF myocytes, with data cumulated across cells or animals. Two CRU definitions were also compared, with maximum edge-to-edge distances of 150 nm or 100 nm. Significant differences within data cumulated across cells were determined by t-test, while data cumulated across animals were tested with linear mixed effects models (Lindstrom and Bates, 1988). https://doi.org/10.7554/eLife.39427.006 Download elife-39427-fig2-data1-v3.pdf RyR dispersion in HF augments 'silent' RyR Ca2+ leak We examined the functional implications of altered nanoscale organization of RyRs, first hypothesizing that RyR dispersion would augment SR Ca2+ leak in failing cardiomyocytes. Total Ca2+ leak was assessed in SR microsomes obtained from the left ventricle of Sham and failing hearts. Following initiation of microsomal Ca2+ uptake by addition of ATP, SERCA function was halted by thapsigargin treatment to reveal RyR-mediated Ca2+ leak (Figure 3A). The Ca2+ leak rate was markedly higher in HF compared to Sham (inset in Figure 3A, mean data in Figure 3C). In agreement with previous work (reviewed in Bers, 2006), we additionally observed significantly slowed SR Ca2+ uptake in HF (Figure 3B) and lower Ca2+ content (Figure 3D). Ca2+ spark-mediated RyR leak was assessed by confocal linescan imaging of freshly-isolated cardiomyocytes (Figure 3E). While the average Ca2+ release per spark (spark mass) was significantly increased in HF cells, this effect was offset by a tendency toward lower spark frequency (Figure 3F,G). Indeed, overall spark-mediated Ca2+ leak was similar in HF and Sham cells (Figure 3H). Since we observed an increase in total RyR leak in HF, these results are consistent with augmented 'silent', non-spark mediated leak in failing cells. Figure 3 Download asset Open asset Failing cardiomyocytes exhibit increased 'silent' RyR leak. Total RyR-mediated Ca2+ leak was assessed in SR microsomes using fura-2 fluorescence (A). Vesicular Ca2+ uptake was initiated by addition of ATP, and halted by addition of thapsigargin. SR Ca2+ leak was estimated as the thapsigargin-induced rate of rise of [Ca2+], normalized to releasable SR content (rise in [Ca2+] induced by the RyR opener 4-chloro-m-cresol, CMC). While the rate of SR Ca2+ uptake was reduced in HF relative to Sham (B), total RyR leak was increased (C) even with a slight reduction in the releasable Ca2+ store (D). (n = 8 from 3 Sham hearts, 7 from 3 HF hearts; *=P < 0.05 vs Sham). To assess whether elevated SR leak in failing cells could be attributed to Ca2+ sparks, line-scan confocal imaging of resting cardiomyocytes was employed (E). Ca2+ spark mass was increased in HF relative to Sham (F), due to augmented spark geometry (Figure 5B). However, since spark frequency tended to be reduced (G), overall Ca2+ spark-mediated leak was similar in Sham and HF (H). These results are consistent with increased 'silent' non-spark-mediated SR leak in HF cardiomyocytes. (FWHM = full width at half maximum, FDHM = full duration at half maximum; ncells = 43 in Sham, 50 in HF; *=P < 0.05 vs Sham). https://doi.org/10.7554/eLife.39427.007 To investigate whether increased silent Ca2+ leak could be linked to RyR dispersion, we employed a mathematical model of the dyad (illustrated schematically in Figure 4—figure supplement 1A) that enabled simulation of Ca2+ sparks with varied placement of RyRs within the CRU. We first incorporated small idealized CRUs containing as few as 4 RyRs (Figure 4A), as our dSTORM imaging indicated that HF cells contain an increased fraction of small CRUs (Figure 2C). During repeated simulations, a single RyR was opened at a random position within the CRU, and subsequent triggered RyR openings were allowed to proceed stochastically. Simulated Ca2+ release events with amplitudes ΔF/F0 ≥0.4 were defined as sparks, based on the detection threshold determined experimentally (see methods). Ca2+ release from the smallest CRUs was never detected, but a progressively greater proportion of events yielded visible sparks as the number of RyRs in these idealized dyad geometries was increased (Figure 4A). These results support the assertion that an increased fraction of small CRUs in HF promotes undetectable, silent Ca2+ leak. Figure 4 with 1 supplement see all Download asset Open asset CRU dispersion provides the structural basis for silent RyR leak in HF. A mathematical model of the dyad was employed to examine the effects of CRU dispersion on Ca2+ sparks and non-spark mediated RyR leak. (A). As dSTORM imaging indicated an increased fraction of small CRUs in HF (Figure 2C), small idealized CRUs were initially modelled with as few as 4 RyRs. Simulated Ca2+ sparks (300 consecutive simulations) were never detected for the smallest CRUs, based on an experimentally determined spark detection threshold of ΔF/F0 = 0.4. Higher probability of visible spark generation (fidelity) was observed for larger CRUs. (B). Real CRU geometries obtained by dSTORM imaging were employed to simulate sparks from larger dyads. Four configurations were modelled with varying numbers of constituent RyR clusters, but similar total RyR number (≈55). While the single-cluster CRU exhibited high Ca2+ spark fidelity, lower probability of spark generation was observed in dispersed, multi-cluster CRUs (fidelity indicated by colour scale). These data support that CRU rearrangement during HF promotes silent RyR leak, due to an increased fraction of both small CRUs as well as larger CRUs with dispersed, irregular configurations. https://doi.org/10.7554/eLife.39427.008 Figure 4—source data 1 Morphological characteristics for the 4 dSTORM-generated geometries. https://doi.org/10.7554/eLife.39427.010 Download elife-39427-fig4-data1-v3.pdf We next examined whether dispersion of clusters in larger more realistic CRUs could similarly contribute to increased silent Ca2+ leak in failing cells. To this end we incorporated real CRU geometries obtained by dSTORM imaging into the model (Figure 4B). Four CRUs were selected containing roughly the same number of RyRs, but with different numbers of RyR clusters (1, 3, 7 or 10 clusters). As in the simulations described above for idealized CRU geometries, a single, randomly chosen RyR was opened in each simulation, to determine the likelihood that such triggering would result in a detectable Ca2+ spark. While relatively high fidelity spark generation was observed for the single-cluster CRU, Ca2+ release was more rarely observed to propagate between clusters, and spark fidelity was significantly lower in multi-cluster CRUs (Figure 4B). This reduced efficiency of Ca2+ spark triggering in dispersed CRUs partly resulted from greater Ca2+ diffusion distance between neighbouring clusters, as demonstrated by progressively increasing the distance between RyR clusters in an idealized dyad (Figure 4—figure supplement 1B). Furthermore, released Ca2+ is less efficiently confined in the dyadic space when the junctional SR has a more distributed and irregular shape. This latter point was demonstrated in the model by altering the amount of junctional SR surrounding the CRU; increasing junctional SR 'padding' increased spark fidelity in both idealized dyads (Figure 4—figure supplement 1B) and dSTORM-based geometries (Figure 4—figure supplement 1C). In summary, these results indicate that nanoscale reorganization of RyRs in HF promotes non-spark-mediated SR Ca2+ leak by two mechanisms: (1) by creating smaller CRUs which produce Ca2+ release events below the detection limit, and (2) by creating more distributed CRU configurations in which multiple RyR clusters are less likely to co-operatively generate sparks. CRU dispersion in HF causes slowing of Ca2+ sparks We next hypothesized that CRU dispersion would slow cardiomyocyte Ca2+ release; a hallmark of HF. Representative confocal recordings of Ca2+ sparks and their temporal profiles are shown in Figure 5A. Spark kinetics in Sham cells generally exhibited rapid rising and declining phases. While some sparks also showed fast kinetics in HF cells, others were markedly slow to rise and decay (Figure 5A). Indeed, measurements of spark rise time and duration exhibited broader distributions and were, on the average, prolonged in HF compared to Sham (Figure 5B). To investigate whether CRU dispersion in HF could underlie slowing of Ca2+ spark kinetics, we again employed our mathematical model with dSTORM-based CRU configurations. During the simulations, the time to opening of each RyR was registered, and the time course of the overall Ca2+ spark determined. Representative simulations show that RyR opening times were delayed in the dispersed, multi-cluster CRUs compared to the solid, single-cluster CRU (Figure 6A). Simulations of Ca2+ spark time courses further showed that the delayed RyR openings in multi-cluster CRUs resulted in more variable kinetics and overall slowing of spark rise time (Figure 6B, mean data Figure 6C), reproducing experimental observations. Of note, although CRUs were observed to contain fewer RyRs in HF than Sham (Figure 2C), simply reducing the RyR number to an equivalent degree in the mathematical model did not markedly alter Ca2+ spark kinetics (Figure 6—source data 1), further confirming a key role of CRU fragmentation in failing cells. Figure 5 Download asset Open asset Ca2+ spark kinetics are slowed in HF. (A) Representative line-scan images of Ca2+ sparks in Sham and HF, selected from the cell-wide scans presented in Figure 3E. Temporal profiles (right panels) show that spark kinetics were generally tightly constrained in Sham, with low values for both time to peak (TTP) and duration (full duration at half maximum, FDHM). Although many sparks were also brief in HF cells, a subset of sparks exhibited slowed kinetics. (B) Distributions of measurements for TTP and FDHM were right-shifted in HF, and mean values were significantly increased. Spark magnitudes tended to be larger in HF than Sham. (nsparks = 130, 100 from 75, 72 cells in Sham, HF; *=P < 0.05 vs Sham). https://doi.org/10.7554/eLife.39427.011 Figure 6 with 1 supplement see all Download asset Open asset RyR dispersion during HF results in slowing of Ca2+ sparks. To examine whether altered CRU morphology could slow Ca2+ spark kinetics in HF, spark profiles were simulated for a variety of dSTORM-derived RyR configurations. (A) Sparks were triggered by opening a single RyR (circled) which was randomly placed in consecutive simulations (example RyR opening trajectories are shown in the upper panels, with a family of spark time-courses illustrated below). Time to opening was registered for each RyR in the CRU, and the resultant time course of the Ca2+ spark was plotted until the final RyR closure, at which point the simulation was stopped for computational efficiency. Opening times were similar for individual RyRs within a solid, single cluster CRU, and the overall temporal profile of elicited sparks showed rapid kinetics which were rather consistent between consecutive simulations. By contrast, delayed and variable opening times were observed for individual RyRs in multi-cluster CRUs. This resulted in variable and slowed Ca2+ spark kinetics with these CRU configurations, as indicated by temporal spark profiles (A), a right-shifted distribution of time-to-peak measurements (B) and mean data (C). (*=P < 0.05 vs single-cluster CRU). https://doi.org/10.7554/eLife.39427.012 Figure 6—source data 1 CRU size has little effect on Ca2+spark characteristics in the reported range. Clusters smaller than 15 – 20 RyR exhibit early spark termination due to stochastic attrition, whereas larger clusters are more capable of supporting regenerative release, for which spark termination becomes relatively consistent in time and dependent on depletion of the releasable Ca2+ store. The simulations were started with all RyRs in the open state and allowed to proceed stochastically. For each RyR number, 50 simulations were performed. The plot shows the mean time from the start of the simulation until all RyRs close. Error bars are standard deviations. https://doi.org/10.7554/eLife.39427.014 Download elife-39427-fig6-data1-v3.pdf Slow Ca2+ sparks promote slowing and de-synchronization of the Ca2+ transient Finally, we examined the consequences of increased variability in Ca2+ spark kinetics for the Ca2+ transient in failing cells. We observed that field-stimulated Ca2+ transients were significantly slower to rise in HF than Sham (Figure 7A–C). This slowing of Ca2+ release was associated with marked de-synchronization of the Ca2+ transient, which we quantified by measuring the variability in time to reach half-maximal fluorescence (TTF50) across the cell (see lower panels in Figure 7A). This 'dyssynchrony index' (Louch et al., 2006) was significantly increased in HF compared to Sham, with a strongly right-shifted distribution of values (Figure 7D). T-tubule disruption in failing cells (Figure 7—figure supplement 1) has been previously established in this model of HF (Frisk et al., 2016), and is a recognized cause of Ca2+ release dyssynchrony in this disease (Song et al., 2006; Louch et al., 2006; Heinzel et al., 2008). We examined whether alterations in Ca2+ spark kinetics also promote dyssynchrony, by examining local Ca2+ transients within narrow, 2 µm regions of the line scan. These regions were centered at the locations of spontaneous Ca2+ sparks observed when electrical pacing was halted. We specifically distinguished between locations with 'slow' sparks, defined by a rise time >13 ms (ie. 1 S.D. > mean rise time in Sham), and remaining 'fast' sparks. By this definition, 24% of sparks in HF cells were defined as slow, while only 13% of Sham sparks fit this definition. Representative examples of such sparks and their temporal profiles are shown in Figure 7E, with corresponding positions along the line scan indicated in Figure 7A. Local transients from slow spark locations in HF exhibited markedly slower rise times than those from fast spark locations in both HF and Sham (Figure 7F). The association between slow sparks and slow local transients was also apparent in 'heat map' plots (Figure 7G). These results show that by protracting Ca2+ sparks, CRU dispersion during HF slows and desynchronizes the overall Ca2+ transient. Figure 7 with 1 supplement see all Download asset Open asset Slow Ca2+ sparks promote slow, desynchronized Ca2+ transients in HF. (A) Representative confocal linescan images of Ca2+ transients in field-stimulated cells (stimulus illustrated as a horizontal line). The overall Ca2+ transient was slowed in HF compared to Sham, as indicated by plots of spatially-averaged Ca2+ transients ((A), right panel), and measurements of half rise time (TTF50, (B)) and time to peak (C). Slowed Ca2+ transient kinetics included de-synchronization of Ca2+ release across HF cells, as indicated by profiles of local TTF50 (lower panels in A). The standard deviation of these values, defined as the dyssynchrony index (Louch et al., 2006), showed a right-shifted distribution in HF compared to Sham (D). To examine the relationship between slowed Ca2+ spark kinetics and de-synchronized Ca2+ transients in HF, local Ca2+ transients were examined within 2 µm regions of the linescan centered at the location of recorded sparks. Paired representative recordings of sparks and local Ca2+ transients are shown in (E and F), respectively, corresponding to indicated positions in A) (vertical arrows). Local Ca2+ release at 'slow' spark locations (rise time > 13 ms) was protracted during the action potential, in comparison with local transients with 'fast' sparks in both HF and Sham (F). This association is demonstrated by clustering of locations with slow Ca2+ spark and local transient kinetics in 'heat maps' (G), and links slowing of Ca2+ release kinetics at the level of the single CRU and whole cell. (Ca2+ transients: ncells = 43 in Sham, 57 in HF; nfast sparks= 18 in Sham, 19 in HF; nslow sparks= 18 in HF; *=P < 0.05 vs Sham). https://doi.org/10.7554/eLife.39427.015 Discussion In the present study, we have employed dSTORM imaging to reveal key changes in CRU morphology during heart failure. We specifically observed marked dispersion of RyRs, which resulted in a shift towards smaller RyR clusters and CRUs. Remaining larger CRUs became less solid, with more fragmented configurations. Experiments and mathematical modeling linked these changes in RyR arrangement to two central aspects of impaired Ca2+ homeostasis in failing cells: increased 'silent' RyR leak and slowing of Ca2+ release, which are believed causative for reduced contractility in this condition. In contrast to lower resolution optical imaging techniques such as confocal and SIM microscopy, the dSTORM technique allows quantification of RyR organization within CRUs. We presently employed this technique with a grid-based quantification method for RyR counting, as previously developed by the Soeller group (Baddeley et al., 2009; Hou et al., 2015). Using i
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