Expression level of cardiac ryanodine receptors dictates properties of Ca²⁺-induced Ca²⁺ release

Abstract

The type 2 ryanodine receptor (RyR2) is the major Ca²⁺ release channel required for Ca²⁺-induced Ca²⁺ release (CICR) and cardiac excitation-contraction coupling. The cluster organization of RyR2 at the dyad is critical for efficient CICR. Despite its central role in cardiac Ca²⁺ signaling, the mechanisms that control CICR are not fully understood. As a single RyR2 Ca²⁺ flux dictates local CICR that underlies Ca²⁺ sparks, RyR2 density in a cluster, and therefore the distance between RyR2s, should have a profound impact on local CICR. Here, we studied the effect of the RyR2 expression level ([RyR2]) on CICR activation, termination, and amplitude. The endoplasmic reticulum (ER)-targeted Ca²⁺ sensor RCEPIA-1er was used to directly measure the ER [Ca²⁺] (Ca²⁺]_ER) in the T-Rex-293 the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) stable cell line expressing human RyR2. Cells coexpressing RyR2 and SERCA2a produced periodic [Ca²⁺]_ER depletions in the form of spontaneous Ca²⁺ waves due to propagating CICR. For each studied cell, the [Ca²⁺]_ER at which Ca²⁺ waves are activated and terminated was analyzed as a function of [RyR2]. CICR parameters, such as [Ca²⁺]_ER activation, termination, and amplitude, were inversely proportional to [RyR2] at low-intermediate levels. Increasing the sensitivity of RyR2 to cytosolic Ca²⁺ lowered the [Ca²⁺]_ER at which CICR is activated and terminated. Decreasing the sensitivity of RyR2 to cytosolic Ca²⁺ had the opposite effect on CICR. These results suggest that RyR2 density in the release cluster should have a significant impact on local CICR activation and termination. Since SR Ca²⁺ load is evenly distributed throughout the SR network, clusters with higher RyR2 density would have a higher probability of initiating spontaneous CICR.

Why it matters

Contraction of heart’s muscle cells is activated by calcium ions (Ca²⁺) that are released from an intracellular store via Ca²⁺ channels called ryanodine receptors (RyRs). Defects in RyR regulation lead to a variety of heart diseases, including life-threatening arrhythmias. Therefore, understanding the mechanisms of RyR regulation is of significant clinical importance. The current study aims to extend our knowledge of how RyR abundance within a cell affects intracellular Ca²⁺ release. By directly measuring Ca²⁺ within cells, we found that cells with higher RyR density produce more Ca²⁺ release than cells with low RyR density. These results suggest that cells with more abundant RyR distribution have a higher chance of producing uncontrolled pro-arrhythmogenic Ca²⁺ release events.

Introduction

In adult ventricular myocytes, synchronized activation of the type 2 ryanodine receptors (RyR2s) during an action potential (AP) produces a global cytosolic Ca²⁺ transient that initiates myocardial contraction (1). RyR2 activity is predominantly controlled by cytosolic [Ca²⁺] ([Ca²⁺]_Cyt) via a mechanism called Ca²⁺-induced Ca²⁺ release (CICR) (2). CICR occurs at specialized cellular microdomains described as dyads. At these domains, L-type Ca²⁺ channels in t-tubules of the surface membrane come into close contact with RyR2 clusters in the sarcoplasmic reticulum (SR) (3,4). During an AP, a relatively small inward Ca²⁺ current via L-type Ca²⁺ channels activates a massive SR Ca²⁺ release via RyR2-mediated CICR. The narrow space of the dyadic cleft ensures a high fidelity of local CICR during AP. Thus, the cardiac dyad represents the ideal subcellular structural element to induce robust SR Ca²⁺ release and myocardial contraction during systole. Individual RyR2s can also open spontaneously during diastole, generating SR Ca²⁺ leak (5). The majority of this Ca²⁺ leak occurs as individual openings of RyRs without triggering inter-cluster CICR or Ca²⁺ sparks (6). During adrenergic receptor stimulation and SR Ca²⁺ overload, however, diastolic SR Ca²⁺ leak can occur in the form of local Ca²⁺ sparks (7) and propagating Ca²⁺ waves (8,9). Such spontaneous CICR can cause diastolic as well as systolic myocardial dysfunction in different pathological conditions (10,11).

Several high-resolution optical studies revealed that in cardiac myocytes, RyR2 clusters vary significantly in size and shape (3,12). The majority of RyR2 clusters are assembled from several smaller subclusters, with different densities. Since the amplitude and duration of Ca²⁺ flux during spontaneous RyR2 openings dictate local [Ca²⁺]_Cyt in the dyadic cleft and therefore inter-cluster CICR activation (13,14), denser RyR2 clusters should have a higher chance to trigger spontaneous CICR than diffused clusters at the same SR Ca²⁺ load. However, a correlation between the density of RyR2 and CICR has never been explored. In this study, we take advantage of a newly developed approach that allows direct measurement of the endoplasmic reticulum (ER) [Ca²⁺] ([Ca²⁺]_ER) with the ER-targeted Ca²⁺ sensor. By analyzing CICR in T-Rex-293 SERCA2a stable cells with different RyR2 expression levels (i.e., RyR2 density), we found that the major CICR parameters are inversely proportional to the RyR2 expression level, suggesting that clusters with higher RyR2 density should have a higher probability of triggering spontaneous CICR.

Materials and methods

cDNA vectors

The human GFP-RyR2 cDNA vector was provided by Dr. Christopher George (University of Cardiff, UK). GFP was fused to the N-terminal domain of RyR2. The vector encoding human SERCA2a cDNA was provided by Dr. David Thomas (University of Minnesota, Minneapolis, MN, USA). The pCMV R-CEPIA1er DNA vector was obtained from Addgene (Watertown, MA, USA).

Generation of SERCA2a stable line

A stable inducible Flp-In T-Rex-293 cell line expressing SERCA2a was generated using the Flp-In T-REx Core Kit (Invitrogen, Carlsbad, CA, USA) as described before (15). Briefly, Flp-In T-REx-293 cells were cotransfected with the pOG44 vector encoding the Flp recombinase and the expression vector pcDNA5/FRT/TO containing the SERCA2a cDNA. 48 h after transfection, the growth medium was replaced with a selection medium containing 100 μg/mL hygromycin. The hygromycin-resistant cell foci were selected and grown. Stable line cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 100 units/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum at 5% CO₂ and 37°C. Expression of SERCA2a in the stable cell line was verified by western blot analysis 48 h after the induction of recombinant pump expression with 1 μg/mL tetracycline.

Experimental protocol

An experimental protocol as described in (15) was used here to study ER Ca²⁺ dynamics. T-Rex-293 SERCA2a stable cells at 60%–80% confluency were cotransfected with plasmids containing the cDNA of GFP-RyR2 and R-CEPIA1er. Experiments were conducted 48 h after transfection to obtain the optimal level of recombinant protein expression. The surface membrane was permeabilized with saponin (0.005%). Experiments were conducted after washing out saponin with an experimental solution (in mM unless noted otherwise): 100 K-aspartate, 15 KCl, 5 KH₂PO₄, 5 MgATP, 0.35 EGTA, 0.22 CaCl₂, 0.75 MgCl₂, 10 HEPES, 2% dextran (molecular weight: 40,000), and pH 7.2. Free [Ca²⁺] and [Mg²⁺] of this solution were 200 nM and 1 mM, respectively.

Confocal microscopy

Expression of recombinant proteins and changes in the luminal ER [Ca²⁺] ([Ca]_ER) were measured with laser scanning confocal microscopy (Radiance 2000 MP, Bio-Rad, Watford, UK) equipped with a 40× oil-immersion objective lens (numerical aperture = 1.3). To verify and quantify the expression of RyR2, GFP was excited with the 488 nm line of the argon laser, and the signal was collected at >515 nm. Two-dimensional images were collected at a speed of 6 ms/line. [Ca²⁺]_ER was recorded as changes in fluorescence intensity of the genetically encoded ER-targeted Ca²⁺ sensor R-CEPIA1er (15). R-CEPIA1er was excited with a 514 nm line of the argon laser, and the signal was collected at >560 nm. Line scan images were collected at a speed of 10 ms/line. The R-CEPIA1er signal (F) was converted to [Ca²⁺]_ER by the following formula: [Ca²⁺]_SE = K_d × [(F − F_min)/(F_max − F)]. F_max was recorded in 5 mM Ca²⁺ and 5 μM ionomycin, and F_min was recorded after ER Ca²⁺ depletion with 5 mM caffeine. The K_D (Ca²⁺ dissociation constant) was 564 μM (16). SERCA-mediated Ca²⁺ uptake was calculated as the first derivative of [Ca²⁺]_ER refilling (d[Ca²⁺]_ER/dt) after RyR2 inhibition with ruthenium red (15 μM) and tetracaine (1 mM). RyR2-independent Ca²⁺ leak was analyzed as the first derivative of [Ca²⁺]_ER decline (d[Ca²⁺]_ER/dt) after simultaneous inhibition of RyR2 and SERCA. ER Ca²⁺ uptake and Ca²⁺ leak rates were plotted as a function of [Ca²⁺]_ER to analyze the maximum ER Ca²⁺ uptake rate and maximum ER Ca²⁺ load. All two-dimensional images and line scan measurements for [Ca²⁺]_SR were analyzed with ImageJ software (NIH, Bethesda, MD, USA).

Statistics

Data are presented as mean ± standard error of n measurements. Statistical comparisons between groups were performed with the Student’s t-test for unpaired data sets. Differences were considered statistically significant at p < 0.05. Statistical analysis and graphical representation of averaged data was carried out on OriginPro7.5 software (OriginLab, Northampton, MA, USA).

Results

Cell model to study cardiac CICR

In this study, we used an established cell model system to assess how an expression level of the cardiac RyR2 affects ER Ca²⁺ regulation under well-controlled experimental conditions (15). To have comparable ER Ca²⁺ uptake rates in all studied cells, the inducible SERCA2a stable cell line (T-Rex-293 cells) was employed in these experiments. The cells were cotransfected with the low-affinity ER-targeted Ca²⁺ sensor R-CEPIA1er and human GFP-RyR2 (Fig. 1 A). The plasma membrane was permeabilized to control the cytosolic composition, including [Ca²⁺], [Mg²⁺], and [ATP]. The cells expressing RyR2 produced periodic [Ca²⁺]_ER depletion in the form of Ca²⁺ waves due to spontaneous CICR followed by ER Ca²⁺ reuptake (Fig. 1 B). Hence, the coexpression of RyR2 and SERCA2a creates a Ca²⁺ release/uptake machinery that generates “cardiac-like” CICR events in our cell model system. For each studied cell, the following CICR parameters were analyzed: the [Ca²⁺]_ER at which Ca²⁺ waves are initiated, CICR activation and the [Ca²⁺]_ER at which Ca²⁺ waves are stopped, and CICR termination. The difference between CICR activation and termination represents CICR amplitude (Fig. 1 B). The RyR2 expression level was estimated from the GFP fluorescence intensity and expressed as a.u. At the end of each experiment, the R-CEPIA1er signal was calibrated with ionomycin in the presence of 0 or 5 mM [Ca²⁺] to convert the CEPIA1er fluorescence to [Ca²⁺]_ER.

Figure 1.

Effect of RyR2 expression level on spontaneous CICR. (A) Representative confocal images of HEK293 cells with different GFP-RyR2 expression levels. RyR2 expression levels were measured as the GFP fluorescence and expressed as a.u. (B) Representative recordings of Ca²⁺ waves from corresponding T-Rex-293 SERCA2a stable cells with different RyR2 expression levels. Ca²⁺ waves were recorded as changes of [Ca²⁺]_ER measured with the low-affinity ER-targeted Ca²⁺ sensor R-CEPIA1er. CICR properties—Ca²⁺ wave activation and termination—are labeled with gray dashed lines and gray arrows. The difference between Ca²⁺ wave activation and termination represents Ca²⁺ wave amplitude.

Correlation between the RyR2 expression level and CICR

We analyzed a correlation between the RyR2 expression level and the aforementioned CICR properties. For each individual cell studied under similar conditions described in Fig. 1, CICR activation, termination, and amplitude were plotted against the RyR2 level. Fig. 1 illustrates examples of spontaneous CICR from four cells with different GFP-RyR2 expression levels (presented as a.u. of the GFP signal). From 75 studied cells, the experimental points were binned and averaged within five different groups based on the GFP signal to analyze the effect of RyR2 expression level on CICR (Fig. 2). These analyses revealed that all CICR parameters were inversely proportional to the RyR2 expression level, with a particularly steep dependence of CICR from the RyR2 expression at the low-intermedium levels. As the distance between neighboring RyR2s decreases with increasing RyR2 expression levels, the cells with high RyR2 levels have a higher chance of generating CICR even at low ER Ca²⁺ loads.

Figure 2.

Relationships between RyR2 expression levels and CICR parameters. [Ca²⁺]_ER thresholds for CICR activation, termination, and wave amplitudes are shown as functions of GFP-RyR2 expression levels in T-Rex-293 SERCA2a stable cells. Each dot represents recordings of CICR parameters from an individual cell (n = 75 cells). The lower graphs represent corresponding data binned into five groups (with 50 a.u. size) according to GFP-RyR2 expression levels.

Effect of changes sensitivity of RyR2 to cytosolic Ca²⁺ on CICR

Next, we studied how changes in RyR2 sensitivity to cytosolic Ca²⁺ affects the properties of CICR. Increasing the sensitivity of RyR2 to cytosolic Ca²⁺ with the RyR2 agonist caffeine (0.1 mM) lowered the [Ca²⁺]_ER at which CICR is activated and terminated. It also decreased CICR amplitude but increased CICR frequency (Fig. 3 A). Decreasing the sensitivity of RyR2 to cytosolic Ca²⁺ with Mg²⁺ (8 mM) had the opposite effect on CICR. It increased the CICR activation and termination [Ca²⁺]_ER threshold (Fig. 3 A). Relative changes of CICR properties during RyR2 activation or inhibition are illustrated in Fig. 3 B. These results demonstrate that an increase in RyR2 sensitivity to cytosolic Ca²⁺ enhances CICR at the same RyR2 expression level, whereas a decrease in RyR2 sensitivity to cytosolic Ca²⁺ has an opposite effect on CICR.

Figure 3.

Effect of caffeine and Mg²⁺ on spontaneous CICR. (A) Representative recordings of Ca²⁺ waves in T-Rex-293 SERCA2a stable cells treated with 0.1 M caffeine (top) or 8 mM Mg²⁺ (bottom). (B) Corresponding relative effect of caffeine (n = 12 cells) or Mg²⁺ (n = 9 cells) on CICR parameters (Act, activation; Term, termination; Amp, amplitude; ∗ statistically significant at p < 0.05).

Discussion

In this study, we performed Ca²⁺ imaging in an expressing cell system to study the effect of the RyR2 expression level on properties of CICR under well-controlled experimental conditions. We have shown previously that expression of human cardiac RyR2 in T-Rex-293 SERCA2a stable cells produced periodic Ca²⁺ waves sensitive to RyR agonists, RyR antagonists, and oxidative stress (15,17,18). Thus, coexpression of RyR2 and SERCA2a creates a Ca²⁺ release-uptake system that reproduces the main aspects of cardiac CICR (Fig. 1). Since [Ca²⁺]_ER was measured directly with the Ca²⁺ sensor RCEPIA-1er, we could selectively separate RyR2-mediated Ca²⁺ release from other components of intracellular Ca²⁺ signaling. To estimate the RyR2 expression level, the GFP-RyR2 fluorescence was measured in each studied cell. As RyR2 expression levels varied significantly between cells, we assessed how the major CICR parameters (i.e., activation, termination, and amplitude) depended on the RyR2 expression level. We found that the major CICR parameters were inversely proportional to the RyR2 expression level, particularly at low-intermediate points (Fig. 2). Such a relationship suggests that decreasing the distances between neighboring RyR2s while increasing the RyR2 expression level enhances CICR even at low SR Ca²⁺ loads. It appears that at high RyR2 density, there is higher chance that single RyR2 openings can increase local [Ca²⁺]_Cyt high enough to activate neighboring channels and trigger propagating CICR (Fig. 4). Moreover, at high RyR2 density, a smaller amplitude of ER Ca²⁺ release is sufficient to maintain CICR until it terminates at almost completely depleted [Ca²⁺]_ER. At low RyR2 expression levels, however, single RyR2 openings need to produce larger local [Ca²⁺]_Cyt to activate more remote neighboring channels (Fig. 4). This can be achieved only at high ER Ca²⁺ loads when the ER Ca²⁺ gradient is high and a single RyR2 Ca²⁺ flux is large. At low RyR2 density, a higher amplitude of ER Ca²⁺ release is required to maintain CICR, which is terminated before reaching complete [Ca²⁺]_ER depletion. These observations nicely correlate with a recently published study of ventricular myocytes isolated from heterozygous RyR2 knockout hearts (19). Although no drastic changes were observed on the whole-heart level, RyR2 knockout myocytes exhibited an alteration of RyR2 clusters, with more densely arranged packing. Such structural changes led to local CICR with smaller amplitude and duration, similar to what we reported in our study. We also studied how changes in RyR2’s sensitivity to [Ca²⁺]_Cyt affects properties of CICR. It has been shown that caffeine increases RyR2 activity by enhancing its sensitivity to [Ca²⁺]_Cyt, whereas Mg²⁺ decreases RyR2 activity by competing with Ca²⁺ for the cytosolic Ca²⁺ binding site (20,21,22). We found that increasing RyR2’s sensitivity to cytosolic Ca²⁺ lowered the [Ca²⁺]_ER at which CICR is activated and terminated, whereas decreasing RyR2’s sensitivity to cytosolic Ca²⁺ had an opposite effect on CICR (Fig. 3). These findings are in good agreement with previously published studies that described mechanisms of pernicious attrition (23) and induction decay (24). These mechanisms suggest that a single RyR2 Ca²⁺ flux plays a critical role in local CICR initiation and termination.

Figure 4.

Schematic diagram of the RyR2 expression level effect on CICR parameters. High expression levels of RyR2 result in smaller distances between channels, causing early wave activation and late termination. Low expression levels of RyR2 are associated with larger interchannel distances that cause late CICR activation and early termination.

The limitation of this study is that it was conducted in a heterologous experimental cell system that is lacking several important regulators of RyR2 and CICR in cardiomyocytes. On the cytosolic side, RyR2 interacts with calmodulin and FK-506 binding protein 12.6. It has been shown that FK-506 binding protein 12.6 can affect the RyR2 by stabilizing the interaction between the channel subunits (25), whereas calmodulin inhibits the channel via Ca²⁺-dependent mechanisms (26). Moreover, it has been suggested that two SR-membrane proteins, junctin and triadin, are crucial for RyR2 to sense [Ca²⁺]_SR via interactions with calsequestrin (27). All these mechanisms can additionally contribute to the regulation of CICR activation and termination in cardiomyocytes.

In conclusion, the results of this study suggest that a density of RyR2 within the release cluster should have a significant impact on local CICR activation and termination. Since the SR Ca²⁺ load is evenly distributed throughout the SR network, clusters with higher RyR2 density would have a higher probability of triggering spontaneous Ca²⁺ sparks and Ca²⁺ waves.

Data and code availability

The datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.

Acknowledgments

This work was supported by the National Institutes of Health grant R01HL151990 (to A.V.Z.). The vector encoding the human RyR2 cDNA fused to GFP at the N-terminal domain was kindly provided by Dr. Christopher George (University of Cardiff, UK). The vector encoding human SERCA2a cDNA was kindly provided by Dr. David Thomas (University of Minnesota, USA).

Author contributions

R.N. and A.V.Z. conceived and supervised the study. R.N., E.B., and A.V.Z. designed the experiments. R.N. and E.B. performed the experiments and analysis. R.N. and A.V.Z. wrote the manuscript. All authors read and approved the manuscript version to be published.

Declaration of interests

The authors declare no competing interests.

Editor: Howard Young.