. 2023 Mar 15;2(3):251-267.

doi: 10.1038/s44161-022-00199-2.

Live-cell photo-activated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes

Yufeng Hou^#¹, Martin Laasmaa^#¹, Jia Li¹, Xin Shen¹, Ornella Manfra¹, Einar S Norden^{1

2}, Christopher Le¹, Lili Zhang^{1

2}, Ivar Sjaastad^{1

2}, Peter P Jones³, Christian Soeller⁴, William E Louch^{1

2}

Affiliations

¹ Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, NO-0424 Oslo, Norway.
² K.G. Jebsen Centre for Cardiac Research, University of Oslo, Oslo Norway.
³ Department of Physiology, School of Biomedical Sciences and HeartOtago, University of Otago, Dunedin, New Zealand.
⁴ Department of Physiology, University of Bern, Bern, Switzerland.

^# Contributed equally.

PMID: 38803363
PMCID: PMC7616007
DOI: 10.1038/s44161-022-00199-2

Live-cell photo-activated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes

Yufeng Hou et al. Nat Cardiovasc Res. 2023.

. 2023 Mar 15;2(3):251-267.

doi: 10.1038/s44161-022-00199-2.

Authors

Affiliations

¹ Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, NO-0424 Oslo, Norway.
² K.G. Jebsen Centre for Cardiac Research, University of Oslo, Oslo Norway.
³ Department of Physiology, School of Biomedical Sciences and HeartOtago, University of Otago, Dunedin, New Zealand.
⁴ Department of Physiology, University of Bern, Bern, Switzerland.

^# Contributed equally.

PMID: 38803363
PMCID: PMC7616007
DOI: 10.1038/s44161-022-00199-2

Abstract

Ca²⁺ sparks constitute the fundamental units of Ca²⁺ release in cardiomyocytes. Here we investigate how ryanodine receptors (RyRs) collectively generate these events by employing a transgenic mouse with a photo-activated label on RyR2. This allowed correlative imaging of RyR localization, by super-resolution Photo-Activated Localization Microscopy, and Ca²⁺ sparks, by high-speed imaging. Two populations of Ca²⁺ sparks were observed: stationary events and "travelling" events that spread between neighbouring RyR clusters. Travelling sparks exhibited up to 8 distinct releases, sourced from local or distal junctional sarcoplasmic reticulum. Quantitative analyses showed that sparks may be triggered by any number of RyRs within a cluster, and that acute β-adrenergic stimulation augments intra-cluster RyR recruitment to generate larger events. In contrast, RyR "dispersion" during heart failure facilitates the generation of travelling sparks. Thus, RyRs cooperatively generate Ca²⁺ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca²⁺ release.

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Conflict of interest statement

Competing Interests Statement The authors declare no competing interests.

Figures

**Extended Data Figure 1. Knock-in strategy and validation.**
(a) Schematic detailing the insertion plan using homologous recombination and neomysin cassette (provided by Cyagen, Santa Clara, CA, USA). (b) PCR analyses were employed to genotype mouse progeny, based on the presence of the WT (0) RyR allele (281 base pairs) or PA-Tag RFP knock in transgene (TG, 413 base pairs). Randomly-selected mice examined in this example blot include those expressing the homozygous TG, which were used in experiments (animals #26 and 27), a heterozygous animal (#28), and an animal null for the TG (#29). TG gene expression was validated in all experimental animals.

**Extended Data Figure 2. Validation of normal animal growth and cardiomyocyte Ca²⁺ homeostasis in transgenic animals.**
(a) Animal body and heart weights were similar in knock-in (TG) and wild-type (WT) animals. n = 29 animals; 5 male WT, 6 female WT, 7 male TG, 10 female TG. (b) Ca²⁺ transients were measured in isolated cardiomyocytes using confocal line-scan imaging during 1 Hz field stimulation. Representative line-scans are show in the upper panels, with spatially-averaged Ca²⁺ transients below. (c) Mean measurements of Ca²⁺ transient parameters were similar in TG and WT cardiomyocytes. RT50 = time to half decline. TTP = time to peak. n_recordings: control = 42, TG = 39.

**Extended Data Figure 3. RyR cluster size measurements and resolution obtained in dSTORM and PALM imaging experiments.**
(a) Distribution of RyR cluster size measurements from dSTORM imaging at the cell surface. (b) Control experiments performed with secondary antibody alone indicated non-specific labeling, artefactually manifested as small RyR clusters. (c) Distribution of RyR cluster size measurements by PALM imaging at the cell surface. In comparison with dSTORM results, proportionally fewer small RyR clusters were identified by PALM imaging, as issues with non-specificity of antibody labeling were avoided. (d) Resolution obtained for dSTORM and PALM imaging, as estimated by Fourier ring correlation. Data are mean ± standard error. n = 10 PALM images, 7 dSTORM images.

**Extended Data Figure 4. Representative examples illustrating steps in image processing.**
(a) Raw fluorescence image. (b) Pixel-wise extrapolated background. (c) Background-subtracted image, with a putative Ca²⁺ spark indicated. (d) Temporally-averaged data for initial coarse identification of Ca²⁺ spark. (e) Extracted spark time course, with frames separated by 20 ms. Raw and diffusion-subtracted outputs are presented, blurred by a 2 pixel Gaussian filter to reduce false detections. (f) Averaged background revealing SR structure. (g) PALM RyR image used to align the signal with SR structure, accounting for chromatic shift. This alignment enabled direct correlation of confirmed Ca²⁺ sparks (h) to RyR positions (i, merged image). The described analysis steps were performed for all recordings of Ca²⁺ sparks and RyRs. ~80% of Ca²⁺ release events were successfully aligned with RyRs, i.e. situated within 300 nm of a cluster (see Fig. 3c).

**Extended Data Figure 5. Modeling approaches to examine RyR collaboration during Ca²⁺ sparks.**
(a) Left panel: schematic of a model employed for calculating average Ca²⁺ efflux from SR during a spark. 100 Ca²⁺ spark simulations are presented for a cluster of 10 RyRs, with the number of open RyRs and Ca²⁺ release rate shown in the middle and right panels (the average flux shown in red). As illustrated in (b), the average Ca²⁺ efflux was used in a reaction-diffusion model. An example of a simulated Ca²⁺ spark is shown at the right. The top row shows changes in Ca²⁺ fluorescence at the focal plane (z=0.0). The middle and bottom rows show the same spark’s line-scans at different focal planes. The white contours from inside-out indicate 1, 0.75, 0.5, 0.25, and 0.1x maximum fluorescence. (c) Dependence of spark characteristics on the focal plane, indicating that out-of-focus events present with smaller amplitudes and broader geometries. FWHM values were used as a metric to exclude out-of-focus sparks in experimental recordings, using a cut-off value of 0.8 μm. (d) Simulated spark amplitudes from different cluster sizes were fitted with a Michaelis-Menten type equation to estimate the maximal number of simultaneously open RyRs for measured sparks (see orange arrows). (e) The simulations were repeated using different parameter values for Ca²⁺ buffers (see Supplementary Table 2). The uncertainty (gray fill) is based on sensitivity analysis of the spark detection algorithm (see Methods).

**Extended Data Figure 6. Sensitivity analysis of the spark fitting algorithm.**
(a) Spark fitting performance is illustrated for two synthetic example sparks (left panels). Sparks were degraded by Poisson and Gaussian noise, blurred, and then fitted by a 2D elliptical Gaussian function as described in Supplementary Method 5. Black and red contours (from outside in) in the right panel correspond to 10%, 34.1% (one SD), 50%, and 75% of the amplitude of the ground truth and fitted result, respectively. (b) Fitting error (δ) indicating deviation from ground truth, based on 2000 simulations at fixed F₀ and spark amplitude (40 and 1.0, respectively). Measurements are presented for (from left to right), angle of rotation (θ), x and y positions of the centroid (x₀ and y₀), spark amplitude, and spark width in x and y dimensions (σ_x and σ_y). (c), (d) show changes in average deviance (squares) and SD (shaded) of the fitting error over a range of F₀ and amplitudes, respectively. Each data point is derived from 2000 simulations.

**Extended Data Figure 7. Quantitative analysis of Ca²⁺ release sites, assuming looser RyR packing.**
Data are presented as in Figure 4, but with estimates of RyR number based on packing density reported from DNA-PAINT studies. We note that this is a scalar adjustment, and thus proportional differences between groups remained similar. (a) Nearest RyR cluster sizes for single-release (blue) and multi-release sparks (orange), compared to the overall distribution of RyR cluster sizes (pink). (b) Mean estimates of RyR configurations underlying Ca²⁺ sparks. Please refer to Extended Data Fig. 9 for evidence that there was no apparent density shift in RyR packing for single- and multi-release sparks. Data are presented as mean ± SEM. Differences between groups were tested with two-tailed linear mixed models with Tukey posthoc correction for multiple comparisons. *** = P < 0.001. P values: cluster size: 4.57x10^-5, local RyR density: 4.3x10^-4, local cluster count: 0.1748. For n values, see Supplemental Table 3, middle column. (c) Mathematical modeling prediction of maximal spark amplitude based on the underlying RyR cluster arrangement, with experimental data superimposed. (d) Estimated number of peak open RyRs for each recorded Ca²⁺ spark. (e) Estimated RyR open probability.

**Extended Data Figure 8. Spark characteristics in wild-type cells.**
In cardiomyocytes isolated from wild-type mice, ISO treatment increased spark rate within the cell interior (a), but not the proportion of multi-release events (b) or the displacement of the spark centroid during its time course (radius of gyration, c). These findings closely paralleled observations made in knock-in cardiomyocytes (Fig. 7). Data are presented as mean ± SEM. Differences between groups were tested with two-tailed linear mixed models with Tukey posthoc correction for multiple comparisons. *** = P < 0.001. P values: a: 0.0538; b: 0.287; c: Ctl vs Iso 0.8425, Ctl single vs Ctl multi <0.0001, Iso single vs Iso true: <0.0001. For spark rate and multi-release proportion measurements, n = control: 3 animals, 35 cells; iso: 3 animals, 34 cells. For radius of gyration measurements, n = control_{single release}: 3 animals, 13 cells, 45 sparks; control_{multi release}: 3 animals, 9 cells, 15 sparks; iso_{single release}: 3 animals, 21 cells, 82 sparks; iso_{multi release}: 3 animals, 17 cells, 47 sparks.

**Extended Data Figure 9. RyR organization in sham and failing myocytes assessed by PALM imaging.**
RyR cluster density **(a)** and size **(b)**, as well as overall RyR density measurements **(c)** were similar in sham and post-infarction heart failure (HF) myocytes, when assessed by PALM imaging. Data are presented as mean ± SEM. Differences between groups were tested with two-tailed linear mixed models with Tukey posthoc correction for multiple comparisons. n values: sham = 32 cells, 4 animals; HF = 34 cells, 3 animals.

**Extended Data Figure 10. RyR fluorescent event counts are correlated with segmented RyR area.**
In preliminary analysis, we tallied RyR blinking events as a possible alternative approach to quantifying RyR number. We noted an approximately linear relationship with the segmented area of the thresholded RyR signal. Importantly, very similar relationships were observed for RyR arrangements underlying single and multi-release Ca²⁺ sparks, suggesting that initiation of these distinct types of events is not dependent on differential RyR packing. Correlations were performed using least squares fitting with a linear function. For n values, please refer to Supplemental Table 3, middle column.

**Figure 1. Demonstration of live-cell PALM imaging in RFP-RyR cardiomyocytes.**
(a) Effects of photoactivation. Minimal fluorescence was observed at 543 nm at baseline (left). Photoactivation was achieved using brief (30 s) low intensity illumination with a 405 nm confocal laser, resulting in a robust fluorescence signal increase within the illuminated band across the center of the cell. An intensity plot measured along the dotted orange line illustrates regional RyR photoactivation (right). (b) Super-resolution RyR images obtained by fixed-cell surface dSTORM (left) and live-cell TIRF-PALM (middle). dSTORM and PALM measurements showed similar RyR organization within the cell interior, as indicated by measurements of cluster sizes (right). Data are presented as mean ± standard error of the mean (SEM). Difference between groups was tested with two-tailed linear mixed models nested by cell and animal levels. n_dSTORM = 3 animals, 20 cells; n_PALM = 3 animals, 20 cells.

**Figure 2. Single- and multiple-release Ca²⁺ sparks.**
TIRF/ imaging of cells loaded with Cal520-AM revealed two types of Ca²⁺ sparks. (a) Single-release Ca²⁺ spark. Representative raw recordings of a spark time course are presented above diffusion-subtracted images. The centroid of the Ca²⁺ signal for each frame is indicated by a red dot, with the surrounding white circle estimating localization uncertainty within the 300 nm diffraction limit. Contours indicate fitting dimensions corresponding to 5 levels between the 70^th signal percentile and peak spark amplitude. The overall spark time course is indicated in the lower panel, with new Ca²⁺ released since the previous frame indicated as an orange bar, and the duration of spark detection shown in gray. 0 ms on the time axis is set as the point of earliest spark detection (>2 SD above background). (b) Multiple-release Ca²⁺ spark. In a subset of recorded sparks, multiple releases were observed, separated by at least one frame where no Ca²⁺ release occurred. (c) Comparison of properties of single- and multi-release sparks. From left to right: radius of gyration (as a measure of centroid movement), amplitude, time to peak (TTP), full duration half maximum (FDHM), and diffusion-subtracted (DS) ΔF/F₀. Data are presented as mean ± SEM. Differences between groups were tested with two-tailed linear mixed models with Tukey posthoc correction for multiple comparisons. Significance levels: * = P < 0.05, ** = P < 0.01, *** = P < 0.001. P values: radius of gyration: 1.11x10^-5, DS amplitude: 0.0601, amplitude: 0.0395, TTP: <2x10^-16, FDHM: 7.5x10^-15. For n values, see Source Data. (d) Simulation of 1D line-scans reveals mischaracterization of multi-release sparks. Slices were made along various axes in the vertical (V) or horizontal (H) orientation across a region with multiple releases (2D image shows pixel-wise maximal intensity during the spark). Corresponding line-scans and measured spark time courses are illustrated.

**Figure 3. Correlation of single- and multiple-release Ca²⁺ sparks to RyR clusters.**
**(a)** Super-resolution PALM imaging of RyRs at the cell surface (left) with enlargement of the indicated region and adaptive thresholding. Correlative Ca²⁺ imaging revealed super-imposition of a Ca²⁺ spark centroid (red dot, uncertainty = white circle) to an RyR cluster (right, upper panels). Diffusion-subtraction analyses revealed only a single Ca²⁺ release pulse at 4 ms, and no further release at 8 ms (lower right panels). **(b)** For multiple-release Ca²⁺ sparks, diffusion subtraction improved mapping to RyR cluster origins, and revealed “travelling” of Ca²⁺ release between adjacent clusters. In the presented example, distinct release events occurred at 4 ms and 8 ms, which summated to generate a large Ca²⁺ spark. **(c)** Histogram showing distance from the centroid of each diffusion-subtracted release event to the nearest RyR cluster. Dotted line indicates the expected localization accuracy of 300 nm, used as a cutoff for subsequent correlation analysis. n = 4 animals, 11 cells, 359 sparks, 608 frames. **(d)** Left: visualization of the jSR using background Cal520 fluorescence (red), together with RyR positions (white) and Ca²⁺ release events (white crosses). Multiple-release sparks were observed to remain within the same jSR (middle) or travel to distant jSR (right). Experiments in **a, b**, and d were repeated independently in cells from 4 hearts with similar results.

**Figure 4. Quantitative analysis of Ca²⁺ release sites.**
**(a)** Measurements of nearest RyR cluster sizes revealed that single-release sparks (blue) generally mapped to smaller clusters than multiple-release sparks (orange). Kernel density estimates (kde) are shown as black lines. The overall distribution of RyR cluster size measurements is illustrated in pink. **(b)** Multi-release sparks were tracked to regions of the cell with larger nearest clusters (left) and higher RyR density (weighted RyR measurements based on proximity, center). The mean number of local RyR clusters was similar for the two types of sparks (right). Data are presented as mean ± SEM. Differences between groups were tested with two-tailed linear mixed models with Tukey posthoc correction for multiple comparisons. *** = P < 0.001. P values: cluster size: 4.57x10^-5, local RyR density: 4.3x10^-4, local cluster count: 0.1748. For n values, see Source Data. **(c)** Proposed schematic showing that multi-release sparks are most likely to be generated at sites with large and/or tightly packed RyR clusters with short nearest-neighbour distances (NND). **(d)** Mathematical modeling predicted the maximal spark amplitude based on the underlying RyR cluster size, assuming all channels open (left). 4 models were employed (shaded region = 2 S.D. of uncertainty), and experimental measurements of spark magnitude and nearest RyR cluster sizes were superimposed. Comparison was made with correlation of sparks to local RyR “superclusters” (middle, ie. RyRs in clusters within 100 nm) and weighted RyR density (right). **(e)** Using the model, the number of peak open RyRs was estimated for each recorded Ca²⁺ spark, and compared with the size of the underlying RyR cluster, supercluster, or weighted RyR density. **(f)** Estimated maximal RyR open probability for each spark, as a function of contributing RyR arrangement.

**Figure 5. Mapping of Ca²⁺ sparks origins during β-adrenergic stimulation.**
**(a)** Correlative Ca²⁺ and RyR imaging in control and isoproterenol-treated cardiomyocytes, with spark locations marked with yellow crosses. Despite markedly increased spark frequency during isoproterenol stimulation (b), the majority of Ca²⁺ sparks continued to be superimposed on RyR clusters (79% within 300 nm for control, vs 83% in isoproterenol). Data are presented as mean ± SEM. Differences between groups were tested with two-tailed linear mixed models with Tukey posthoc correction for multiple comparisons. * = P < 0.05. P value for spark rate: 0.0291. For n values, see Source Data. Representative diffusion-subtracted Ca²⁺ sparks during control **(c)** and isoproterenol treatment **(d)** are illustrated with Ca²⁺-RyR mapping (left), with spark centroids indicated by red dots and localization uncertainly illustrated by surrounding white circles. Overall spark time-courses are shown at right. Both example sparks are single-release events. Diffusion-subtracted Ca²⁺ release (blue bars) was larger during isoproterenol, resulting in larger amplitude Ca²⁺ sparks. The dashed line indicates the time point for the Ca²⁺-RyR overlay, and the duration of spark detection is shown in gray.

**Figure 6. β-adrenergic stimulation increases RyR recruitment during Ca²⁺ sparks.**
**(a)** Isoproterenol treatment did not alter the proportion of total sparks which exhibit multiple releases, or their travel distance **(b)**. However, the amplitude of each diffusion-subtracted Ca²⁺ release event was larger during isoproterenol **(c)**, resulting in larger overall Ca²⁺ sparks **(d)**, without increasing spark time to peak **(e)** or duration **(f)**. Data are presented as mean ± SEM. Differences between groups were tested with two-tailed linear mixed models with Tukey posthoc correction for multiple comparisons. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Please refer to Source Data for exact P values and n values. **(g)** Experimentally-measured spark amplitudes and corresponding weighted RyR counts were plotted, together with the theoretical maximal spark amplitude curve (see also Fig. 4D). Isoproterenol produced an upward shift, as sparks skewing toward larger amplitudes **(h)**. Using the mathematical model to calibrate the number of open RyRs during each spark revealed a similar skewing towards larger values during isoproterenol treatment **(i)**. TTP = time to peak, FDHM = full-duration half maximum.

**Figure 7. Regulation of Ca²⁺ release at internal sites.**
**(a)** Using HILO imaging, correlative Ca²⁺ and RyR imaging was performed within the cell interior. As observed on the cell surface (see previous figures), multi-release Ca²⁺ sparks at internal sites exhibited greater movement than single-release events (b), and tended to be larger **(c)**, with slower kinetics **(d, e)**. ISO treatment markedly increased spark rate **(f)**, but not the proportional occurrence of multi-release sparks (see main text) or RyR cluster sizes (g, estimated by tight RyR packing within thresholded 2D area). Scale bars in a = 800 nm. TTP = time to peak, FDHM = full-duration half maximum. Data are presented as mean ± SEM. Differences between groups were tested with two-tailed linear mixed models with Tukey posthoc correction for multiple comparisons. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Please refer to Source Data for exact P values and n values.

**Figure 8. RyR cluster dispersion during heart failure promotes multi-release, travelling sparks.**
**(a)** Correlative imaging of Ca²⁺ and RyRs within the interior of cardiomyocytes isolated from mice with post-infarction heart failure (HF). Comparison was made with sham-operated controls (Sham). **(b)** Failing cells exhibited an increased proportion of multi-release sparks. As in Sham, multi-release sparks observed in failing cells travelled further, and were larger and slower than single-release events. dSTORM imaging revealed marked “dispersion” of RyRs in HF **(c)**, resulting in the appearance of smaller but more numerous clusters, and no change in overall RyR density **(d)**. Scale bars in a and c = 1.6 μm in zoom-outs, 750 nm in enlargements. TTP = time to peak. Data in b and d are presented as mean ± SEM. Differences between groups were tested with two-tailed linear mixed models with Tukey posthoc correction for multiple comparisons. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Please refer to Source Data for exact P values and n values.

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Live-cell photo-activated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes

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Live-cell photo-activated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes

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