Functional consequences of stably expressing a mutant calsequestrin (CASQ2^D307H) in the CASQ2 null background

Abstract

The role of calsequestrin (CASQ2) in cardiac sarcoplasmic reticulum (SR) calcium (Ca²⁺) transport has gained significant attention since point mutations in CASQ2 were reported to cause ventricular arrhythmia. In the present study, we have critically evaluated the functional consequences of expressing the CASQ2^D307H mutant protein in the CASQ2 null mouse. We recently reported that the mutant CASQ2^D307H protein can be stably expressed in CASQ2 null hearts, and it targets appropriately to the junctional SR (Kalyanasundaram A, Bal NC, Franzini-Armstrong C, Knollmann BC, Periasamy M. J Biol Chem 285: 3076–3083, 2010). In this study, we found that introduction of CASQ2^D307H protein in the CASQ2 null background partially restored triadin 1 levels, which were decreased in the CASQ2 null mice. Despite twofold expression (relative to wild-type CASQ2), the mutant protein failed to increase SR Ca²⁺ load. We also found that the Ca²⁺ transient decays slower in the CASQ2 null and CASQ2^D307H cells. CASQ2^D307H myocytes, when rhythmically paced and challenged with isoproterenol, exhibit spontaneous Ca²⁺ waves similar to CASQ2 null myocytes; however, the stability of Ca²⁺ cycling was increased in the CASQ2^D307H myocytes. In the presence of isoproterenol, Ca²⁺-transient amplitude in CASQ2^D307H myocytes was significantly decreased, possibly indicating an inherent defect in Ca²⁺ buffering capacity and release from the mutant CASQ2 at high Ca²⁺ concentrations. We also observed polymorphic ventricular tachycardia in the CASQ2^D307H mice, although lesser than in the CASQ2 null mice. These data suggest that CASQ2^D307H point mutation may affect Ca²⁺ buffering capacity and Ca²⁺ release. We propose that poor interaction between CASQ2^D307H and triadin 1 could affect ryanodine receptor 2 stability, thereby increasing susceptibility to delayed afterdepolarizations and triggered arrhythmic activity.

calsequestrin (casq2), an ∼55-kDa protein located in the lumen of the sarcoplasmic reticulum (SR) plays an important role in regulating calcium (Ca²⁺) concentration within the cardiomyocyte (1, 13, 20). CASQ2 has a COOH-terminal sequence of highly negative amino acids that facilitates sequestration of a large number of Ca²⁺ ions (26). Ca²⁺ binding is further enhanced due to the protein's elaborate polymerization into long, interlocked twines that localize at the junctional SR (jSR) (7, 22). CASQ2 is also shown to interact with triadic proteins triadin 1 (TRD1) (18) and junctin (JCN) (15), along with the Ca²⁺ release channel, ryanodine receptor 2 (RyR2), via very specific protein-protein interactions (9). Due to its Ca²⁺ binding property, CASQ2 functions as a Ca²⁺ buffer to trap Ca²⁺ during the influx of high Ca²⁺ via the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a) pump (9, 29). This buffering maintains the free Ca²⁺ concentration within the SR lumen below the level conducive for RyR2 activation, thereby preventing extrasystolic Ca²⁺ release (29). These known functions of CASQ2 have established the protein as a key constituent of Ca²⁺ homeostasis in excitation-contraction (E-C) coupling.

Recently, Knollmann et al. (17) developed a CASQ2 null mouse model, and these mice live and reproduce well. They do not display any obvious abnormal alterations to E-C coupling at rest; however, when stressed with adrenergic triggers, such as isoproterenol infusion, cardiomyocytes from CASQ2 null mice displayed a dramatic susceptibility to abnormal, extrasystolic Ca²⁺ release, which led to incidences of delayed afterdepolarizations (DADs), which can cause deleterious ventricular arrhythmias (17). This finding emphasizes the importance of CASQ2 expression in regulating E-C coupling in the cardiac muscle, especially under conditions of adrenergic stress.

Our laboratory has been studying the functional consequence of a point mutation D307H in domain III of CASQ2 (4, 16). This mutation is of particular interest, since it disrupts a charge cluster of negative amino acids involved in Ca²⁺ buffering and is also linked to the human disease catecholaminergic polymorphic ventricular tachycardia (CPVT) (5, 19, 24, 31). We have shown that the mutant protein can be stably expressed under the control of the α-myosin heavy chain promoter in a wild-type (WT) background (4). However, in this mouse model, both WT and mutant protein were present, and the higher levels of mutant CASQ2^D307H could interfere with WT CASQ2 function in a dominant-negative manner, probably by interfering with CASQ2's orderly polymerization sequence. To better understand the role of the mutant protein, our laboratory recently developed a new mouse model in which the mutant protein is solely expressed in the null background (16) and showed that the mutant protein can be stably expressed in the CASQ2 null myocardium and targeted correctly to the jSR. In addition, to our surprise, we found that critical alterations in the CASQ2 null myocardium, such as the extensive ultrastructural modifications to the jSR, were partially restored by the expression of this functionally deficit mutant form of CASQ2. This finding was very interesting, since it suggested that expression of CASQ2 not only contributes to Ca²⁺ homeostasis by virtue of its Ca²⁺ binding properties, but also serves additional roles to maintain SR architecture.

In the present study, we have critically evaluated the functional consequences of expressing the CASQ2^D307H mutant protein in the CASQ2 null mouse.

MATERIAL AND METHODS

Mouse model.

We have used both CASQ2 null mice (17) and a mouse model that expresses CASQ2^D307H protein in the CASQ2 null background (16) for this study. The partial characterization of the mouse model (16) that expresses CASQ2^D307H protein in the CASQ2 null background was recently reported. This investigation conforms to the guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85–23, revised 1996), and protocol was approved by the Institutional Animal Care and Use Committee. All experiments were done on 10- to 12-wk-old male, littermate-controlled mice, unless otherwise specified.

Protein analysis.

Expression of SR proteins SERCA2a (Zymed), phospholamban (PLB, Zymed), TRD1 (Santa Cruz), CASQ2 (Thermo Scientific), L-type Ca²⁺ channel (DHPR2α, Thermo Scientific), RyR2 (Thermo Scientific), phosphorylated PLB (serine 16 and threonine 17; Badrilla), and GAPDH (Cell Signaling) were determined by quantitative Western blotting techniques (4) using whole heart homogenates from WT, CASQ2^D307H, and CASQ2 null (n = 6). Protein bands were scanned, and integrated densities were measured using ImageJ Data Acquisition Software (NIH, Bethesda, MD). Protein-signal densities were normalized to the corresponding GAPDH signal densities.

Mouse cardiomyocyte isolation and electrophysiological recordings.

Ventricular myocytes were isolated from CASQ2^D307H, CASQ2 null, and WT hearts by enzyme digestion, as before (4). Transmembrane ionic currents were recorded by whole cell patch-clamp experiments, as described previously (4). Experiments were performed either in patch-clamped or in permeabilized myocytes at room temperature (21 to 23°C) Whole cell patch-clamp recordings of transmembrane currents were performed using an Axopatch 200B amplifier (Axon Instruments) and pClamp-9 software. External solution contained (in mmol/l) 140 NaCl, 5.4 KCl, 1 CaCl₂, 0.5 MgCl₂, 10 HEPES, and 5.6 glucose (pH 7.3). Micropipettes made from borosilicate glass (Sutter Instrument; 1–3 MΩ resistance) were filled with a solution that contained (in mmol/l) 120 potassium-aspartate, 20 KCl, 3 Na₂ATP, 3.5 MgCl₂, 4 NaCl, 5 HEPES, 0.05 fluo 3, and pCa 7 (pH 7.3). For Ca²⁺ current (I_Ca) measurements, K⁺ in pipette solution was replaced to Cs⁺. Voltage pulses were applied from a holding potential of −50 mV at 1-min intervals. Rapid applications of caffeine (10 mmol/l) were used to measure SR Ca²⁺ content. Peak amplitude of the caffeine-induced Ca²⁺ transients and the integration of the Na⁺/Ca²⁺ exchange current were used to calculate the amount of Ca²⁺ released.

Confocal Ca²⁺ measurements.

Intracellular Ca²⁺ imaging was performed as described in Ref. 4 using an Olympus Fluoview 1000 and Nikon A1 laser scanning confocal microscopes equipped with a 60 × 1.4 numerical aperture oil objectives. Fluo 3 was excited by 488-nm argon-ion laser and the fluorescence acquired at wavelengths >500 nm in the line-scan mode at the rate of 2–7 ms per scan.

Ca²⁺ cycling stability measurements.

Myocytes were incubated with 10 μM fluo 3-AM for 30 min. Twenty to thirty minutes were allowed for diesterification. Stability of Ca²⁺ signaling was studied in the presence of 100 nM isoproterenol and 2 mM Ca²⁺ in the external solution. Cells were field stimulated at 2 Hz for 20 s. Fluo 3 fluorescence was recorded during stimulation and for 20 s after the stimulation was stopped. Most cells displayed spontaneous Ca²⁺ waves (SCW) within the 20-s rest period. However, SCWs were not observed during this period in 7/22, 1/26, and 3/25 of WT, CASQ2^D307H, and CASQ2 null myocytes, respectively. For the rest of the myocytes, time between SCW initiation and peak Ca²⁺ transient evoked by last stimulus was measured and considered as an indicator of Ca²⁺ cycling stability. In addition, myocytes from the three genotypes were field stimulated at 0.5 Hz in the presence of 100 nM isoproterenol and 2 mM Ca²⁺ in the external solution, and percentage of cells displaying diastolic Ca²⁺ waves was taken as an indicator of susceptibility to Ca²⁺-dependent arrhythmia.

ECG monitoring and induction of arrhythmia.

Electrocardiographic (ECG) recordings were performed in CASQ2^D307H, CASQ2 null, and WT mice (n = 8), before and after catecholaminergic challenge, as previously described (4). Briefly, continuous ECG recordings were obtained from mice anesthetized with isoflurane, at minimum effective concentration (1–1.5%), and placed on a heating pad to maintain normothermia. ECGs were recorded using a physiological data acquisition system (MP 100, Biopac Systems) with a sampling rate of 2 kHz for 60 min. After baseline recording (10 min), each mouse received four doses of isoproterenol (1.5 mg/kg ip) in 10-min intervals, the first two of which were combined with caffeine (120 mg/kg ip). All of the ECGs were analyzed using Acknowledge software, by performing both a qualitative and quantitative assessment of ECGs in a blinded fashion. Leads 1 and 3 were analyzed for determination of heart rate and detection of irregular beats. Arrhythmias were classified as simple or complex, with isolated ventricular ectopic beats classified as simple. Complex ventricular arrhythmias included the following: couplets, bigeminy, trigeminy, parasystole, and nonsustained ventricular tachycardia.

Statistical analysis.

All experiments were done blinded to the genotype. Cross tabulations with χ², one-way and two-way analysis of variance, Student's T-tests, and two-sample Kolmogorov-Smirnov tests were performed as appropriate. Unless otherwise indicated, results are expressed as means ± SD.

RESULTS

Expression of CASQ2^D307H mutant protein (twofold) in the CASQ2 null background does not cause cardiac pathology.

In the present study, we critically examined structure and function of the myocardium in CASQ2 null, CASQ2^D307H, and WT littermates up to 1 yr. There were no statistically significant differences between the three experimental groups in survival rates (>1 yr), litter sizes, and survival of litters. There were no incidences of sudden death in both CASQ2 null and CASQ2^D307H mice at any age (3–12 mo) throughout the duration of this study. Heart weight-to-body weight ratios (Fig. 1A) and heart weight-to-tibia length ratios (data not shown) were not statistically different between the three groups. In addition, histological sections from 1-yr-old hearts analyzed for gross abnormalities did not reveal any significant evidence of cardiac hypertrophy and/or fibrosis (Fig. 1B).

Fig. 1.

Expression of calsequestrin mutant protein (CASQ2^D307H) does not induce cardiac hypertrophy in the CASQ2 null heart. A: heart weight-to-body weight ratios (HW/BW) were not significantly different in the CASQ2 null and CASQ2^D307H hearts compared with wild-type (WT) values. Values are means ± SD. B: histological analyses were done on cardiac sections (1-yr-old hearts were used) from the three groups stained with hematoxylin-eosin (H&E) and Masson-trichrome. Both techniques showed neither structural alterations nor fibrosis in the hearts (n = 3).

CASQ2^D307H protein expression partially rescues TRD1 levels in the CASQ2 null background.

Our laboratory has recently reported that the mutant CASQ2^D307H can be stably expressed and appropriately targeted to the jSR in the CASQ2 null mouse heart (16). In this study, we determined if the approximately twofold expression of the mutant protein affects the expression of other SR proteins, specifically TRD1, a jSR protein known to interact with CASQ2. Quantification and correction for loading variations showed that TRD1 protein levels that were decreased to ∼60% (P < 0.05) in the CASQ2 null hearts were restored to ∼75% of WT level (Fig. 2). This decrease was evident in both the glycosylated (gly) and unglycosylated (ungly) forms of TRD1 in the CASQ2 null (gly-60% and ungly-59%; P < 0.05) and CASQ2^D307H (gly-64% and ungly-78%) hearts. These data indicate that the expression of the mutant CASQ2^D307H protein can partially restore the expression of its interacting partner TRD1.

Fig. 2.

CASQ2^D307H expression partially rescues triadin 1 (TRD1) level in the CASQ2 null background. Top: immunoblot analysis of TRD1 in WT, CASQ2 null, and CASQ2^D307H hearts. Twenty micrograms of whole heart homogenates were separated by SDS-PAGE on 8% gels and probed with a suitable antibody to detect TRD1 [both glycosylated (Gly), 40 kDa, and unglycosylated (unGly), 36 kDa, bands shown] (n = 6), repeated three times. Band densities of GAPDH were used to normalize those of TRD1 and quantified with Image J software. Bottom: summarized data of relative percentages of TRD1 expression compared with WT levels in the three genotypes (*P < 0.05). Values are means ± SD.

CASQ2^D307H expression does not affect I_ca-induced Ca²⁺ transients or SR Ca²⁺ content, but improves the rate of Ca²⁺ transient decay.

Györke et al. (10) have shown that CASQ2 acting as a Ca²⁺ buffer affects the RyR2 activity via regulation of free SR Ca²⁺. Since we expressed CASQ2^D307H that has reduced Ca²⁺ buffering properties, we studied the Ca²⁺ signaling parameters, especially Ca²⁺ release from the SR.

The effects of mutant CASQ2^D307H expression compared with CASQ2 null and WT CASQ2 on intracellular Ca²⁺ handling in fluo 3 loaded patch-clamped myocytes are illustrated in Fig. 3. There are no apparent changes in the peak amplitude of the I_ca between the three experimental groups (n = 12 control, 7 CASQ2 null, and 7 CASQ2^D307H myocytes), indicating that the trigger for Ca²⁺ release is unaltered in the three genotypes (Fig. 3A, left, bottom trace, and 3B). In addition, no statistically significant alteration was found in the amplitude of Ca²⁺ transients triggered by membrane potential depolarization steps from −50 to 0 mV among the three groups (Fig. 3A, left, top trace). However, the Ca²⁺ transient time in milliseconds for 50% decay, half relaxation time (Ca²⁺), was significantly longer in the CASQ2 null and CASQ2^D307H myocytes compared with WT cells (WT: 278 ± 16 ms; CASQ2 null: 484 ± 26 ms; P < 0.05; and CASQ2^D307H: 314 ± 18 ms; Fig. 3A, right). Thus, compared with the WT and CASQ2^D307H myocytes, Ca²⁺ transient decay time was significantly prolonged in the CASQ2 null myocytes, suggesting that Ca²⁺ reuptake from the cytosol could be impaired.

Fig. 3.

Expression of mutant CASQ2^D307H does not affect sarcoplasmic reticulum (SR) Ca²⁺ load. A, left: representative recordings of Ca²⁺ current (I_Ca; bottom traces) and calcium transients (top traces) evoked by membrane potential depolarization steps from −50 to 0 mV. *P < 0.05. Right: summarized data of half relaxation time (RT₅₀) values of the Ca²⁺ transient decay. B, left: I_Ca measurements in ventricular myocytes, isolated from WT (solid lines), CASQ2 null (dotted lines), and CASQ2^D307H hearts (dashed lines). Right: summarized data of the Na⁺/Ca²⁺ exchange current (I_NCX) in the three groups. C, left: profiles of confocal images of Ca²⁺ load measured as amplitude of caffeine (10 mM)-induced Ca²⁺ release. Right: summarized data of caffeine-induced Ca²⁺ release from the three groups. F/F₀, florescence ratio. Values are means ± SD.

To determine the Ca²⁺ storing capacity of the SR following the expression of CASQ2^D307H, we measured caffeine-induced Ca²⁺ transients in cardiomyocytes expressing CASQ2^D307H and in CASQ2 null and WT myocytes [n = 6 (WT), 5 (CASQ2 null), and 5 (CASQ2^D307H) myocytes]. Changes in fluo 3 fluorescence were used to determine the amount of Ca²⁺ released after 10 mmol/l caffeine application. We did not find a significant difference in the amplitude of caffeine-induced Ca²⁺ release between the WT and CASQ2^D307H myocytes (Fig. 3C). In addition, there were no changes among the three groups in the Na⁺/Ca²⁺ exchanger current recordings (Fig. 3B, right).

To understand the underlying mechanism that causes the slow decay of the Ca²⁺ transient, we next determined the expression of SERCA2a protein and its regulator PLB (Fig. 4). We found no alterations in the expression of SERCA2a protein (Fig. 4A); however, PLB expression was significantly increased in the CASQ2 null hearts (∼20% above WT levels, P < 0.05) and only by ∼7% in the CASQ2^D307H hearts (Fig. 4B). Moreover, phosphorylation of PLB, which relieves the inhibition of SERCA2a pump, was also decreased at both Ser16 and Thr17 sites in the two groups [CASQ2 null 76% and 75%; CASQ2^D307H 65% (P < 0.05) and 45% (P < 0.05)], respectively (Fig. 4B).

Fig. 4.

Expression of SR Ca²⁺ handling proteins are unaffected by the introduction of CASQ2^D307H. A: immunoblot analyses of SR calcium handling proteins and ion channels in CASQ2^D307H, CASQ2 null, and WT hearts (n = 6). Five to twenty micrograms of whole heart homogenates were separated by SDS-PAGE on 8–10% gels [sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a), CASQ2, DHPR2α, and GAPDH] and ryanodine receptor 2 (RyR2) (5%) and probed with suitable antibodies. Experiments were repeated three times. Expressions of the above-tested proteins were not statistically different between CASQ2^D307H, CASQ2 null, and WT mouse hearts. B: immunoblot analyses of phospholamban (PLB) total protein expression, phosphorylation status at serine 16 and threonine 17 sites (n = 6), and summarized data of the relative percentages of TRD1 expression compared with WT levels (straight line) in the three genotypes (*P < 0.05). Values are means ± SD.

CASQ2^D307H myocytes show increased stability of Ca²⁺ cycling when challenged with a β-adrenergic agonist.

To assess the effects of the mutant protein on Ca²⁺ cycling following adrenergic stimulation, we examined Ca²⁺ transients in single ventricular myocytes that were field stimulated at 2 Hz in the presence of 100 nM isoproterenol. The time delay between the end of field stimulation and SCW initiation was used as a measure of Ca²⁺ cycling stability: shorter time delay to SCW, lesser Ca²⁺ cyling stability. We found a significant decrease in SCW time delay (P < 0.05) in both CASQ2^D307H and CASQ2 null cells compared with the WT (Fig. 5, A and B; Table 1). Compared with CASQ2 null, CASQ2^D307H myocytes displayed longer time delay to SCW, suggesting increased Ca²⁺ cycling stability. When paced at 0.5 Hz, myocytes from both CASQ2^D307H and CASQ2 null groups showed significant increase in incidence of diastolic SCW relative to WT myocytes (Fig. 5C, Table 1). Interestingly, in the presence of isoproterenol, the Ca²⁺ transient amplitude was significantly lesser in the CASQ2^D307H myocytes (1.8 ± 0.3, P < 0.05) than in the WT (3.7 ± 0.2) and CASQ2 null (2.9 ± 0.2, P < 0.05) myocytes.

Fig. 5.

CASQ2^D307H cardiomyocytes show increased incidences of spontaneous calcium waves (SCW) and altered Ca²⁺ cycling stability. A: representative line-scan images and temporal profiles of fluo 3 fluorescence recorded in WT, CASQ2^D307H, and CASQ2 null myocytes stimulated at 2 Hz. Top: field-stimulation protocol. Timing of electrical stimulations is indicated by upward deflections. Only last 10 out of 40 stimuli are shown. B: distributions of time delay between the end of field stimulation and SCW initiation recorded in WT, CASQ2^D307H, and CASQ2 null myocytes, as shown in A. C: representative line-scan images and temporal profiles of fluo 3 fluorescence recorded in WT, CASQ2^D307H, and CASQ2 null myocytes stimulated at 0.5 Hz. Top: field-stimulation protocol. Experiments were performed in the presence of 100 nM isoproterenol and 2 mM Ca²⁺ in the external solution.

Table 1.

Properties of Ca²⁺ signaling in WT, CASQ2^D307H, and CASQ2 null myocytes recorded in the presence of isoproterenol (100 nM)

Parameter	WT	CASQ2D307H	CASQ2 Null
SCW delay at 2 Hz, s	6.3 ± 1.2 (22)	3.0 ± 0.7* (26)	2.8 ± 0.7* (25)
SCW incidence at 0.5 Hz, %	9 (2 of 20)	33 (8 of 24)	35 (8 of 23)
Ca2+ transient amplitude (ΔF/F0) at 0.5 Hz	3.7 ± 0.2 (20)	1.8 ± 0.3* (19)	2.9 ± 0.2*† (18)
Ca2+ transient decay (τ) at 0.5 Hz, ms	85 ± 5 (20)	62 ± 4* (19)	80 ± 5† (16)

Values are mean ± SE (no. of cells studied are in parentheses). WT, wild type; CASQ2, calsequestrin; SCW, spontaneous Ca²⁺ waves; ΔF/F₀, change in fluorescence ratio. P < 0.05 vs.

^*control and

^†CASQ2^D307H (ANOVA, Bonferroni test).

The CASQ2^D307H mutant mice display incidences of ventricular arrhythmia.

We recorded surface ECGs on mildly anesthetized mice from the three groups after catecholaminergic challenge using isoproterenol. CASQ2^D307H mice developed simple and complex ventricular arrhythmias after catecholaminergic challenge. However, the number of simple and complex ventricular arrhythmias observed in CASQ2^D307H mice was significantly decreased by 13- and 5-fold, respectively, compared with that in CASQ2 null mice (P < 0.001, Fig. 6). The CASQ2 null mice developed significantly more junctional arrhythmias (i.e., junctional ectopic beats and junctional tachycardia) compared with the other two groups (cumulative number of observations: 3,036 in CASQ2 null vs. 174 in CASQ2^D307H and 11 in WT mice, P < 0.001, Fig. 6), as well as simple and complex ventricular arrhythmias, including nonsustained ventricular tachycardia, compared with WT mice (P < 0.001, Fig. 6).

Fig. 6.

CASQ2 ^D307H mice are susceptible to triggered ventricular arrhythmia. Ventricular arrhythmias were recorded in mildly anesthetized CASQ2^D307H, CASQ2 null, and WT mice following isoproterenol challenge. A cumulative number of simple and complex ventricular arrhythmias occurring in vivo in CASQ2 null (n = 8), CASQ2^D307H (n = 8), and WT mice (n = 8) are shown. Simple ventricular arrhythmias included single isolated ventricular ectopic beats. Complex ventricular arrhythmias included couplet, bigeminy, trigeminy, and nonsustained ventricular tachycardia (NSVT). P < 0.001, CASQ2 null vs. CASQ2^D307H mice, and P < 0.001, CASQ2^D307H vs. WT mice. A trace of NSVT from a CASQ2 null heart is shown below the graph. S, sinus beat; P, compensatory pause; *, intermittent premature ventricular ectopic beats.

DISCUSSION

Ca²⁺ release from cardiac SR via the RyR2 is regulated by both cytosolic and luminal Ca²⁺ concentrations. The precise termination of Ca²⁺ release and maintenance of RyR2 refractoriness is dependent on both luminal Ca²⁺ concentration and RyR2 modulation by jSR proteins. Among them, CASQ2 is the major Ca²⁺ binding protein that serves as a Ca²⁺ sensor and modulator of RyR2 (CASQ2 stabilizes both intra-SR Ca²⁺ concentration and RyR2 activity) during the myocyte's Ca²⁺ cycle. CASQ2, together with its triadic/junctional partners TRD1 and JCN (8–10, 37), synergistically operate as a single multiprotein complex to regulate Ca²⁺ release via RyR2, and recent studies have confirmed that loss of one partner not only affects the expression of one or more of the other components, but, more importantly, can destabilize the structural and functional integrity of the complex (3, 17, 35) and contribute to triggered arrhythmias.

CASQ2's Ca²⁺ buffering function modulates Ca²⁺ release at high SR Ca²⁺ load and can also affect SERCA2a-mediated Ca²⁺ uptake.

In this present study, we took advantage of both the CASQ2 null and a mutant CASQ2^D307H mouse model to critically evaluate the importance of CASQ2. CASQ2 is long believed to be the major Ca²⁺ buffering protein within the SR lumen. Hence it is logical to expect that SR Ca²⁺ load is a direct consequence of both CASQ2 levels and/or function. In fact, overexpression of WT CASQ2 can significantly increase SR load (14, 25). However, the CASQ2 null mouse contradicted this tenet by presenting an unaltered Ca²⁺ load wherein the compensatory increase in SR volume in these mice could have normalized Ca²⁺ load, despite the absence of CASQ2 to buffer Ca²⁺. Given these previous reports, it is very intriguing that we did not find the SR Ca²⁺ load to be different in the CASQ2^D307H myocytes, even though we found a twofold increase in the mutant protein. We reasoned that this finding could indicate a decreased capacity to buffer Ca²⁺, thereby reducing the amount of Ca²⁺ that can be released. Previous studies (12) have shown that the D307H mutation, which occurs in domain III of the molecule, can affect its ability to buffer Ca²⁺. In addition, we tested the hypothesis that CASQ2's pivotal role as a buffer would become apparent only at higher SR Ca²⁺ concentrations (when challenged with isoproterenol), and, in its absence, Ca²⁺ release would be decreased. Although at rest CASQ2^D307H myocytes show unaltered Ca²⁺ transient peak amplitude, isoproterenol treatment indeed decreased the Ca²⁺ transient amplitude significantly than in the CASQ2 null myocytes compared with the WT. It is interesting to note that the CASQ2 null cells also show decreased peak amplitudes in response to isoproterenol, which infers that, in the absence of CASQ2, the dynamics of Ca²⁺ release during catecholamine stress are altered. Despite expressing higher levels of CASQ2^D307H, we found only a slight improvement in the Ca²⁺ release efficiency, which indicates an inherent defect in the mutant CASQ2^D307H at high Ca²⁺ concentrations.

Incidentally, we found that the Ca²⁺ transient decays significantly slower in the CASQ2 null myocytes compared with that of WT cells, and that this deficit is partially restored in the CASQ2^D307H cells. Since the Ca²⁺ transient decay phase is predominantly due to Ca²⁺ reuptake by SERCA2a (23), the data may suggest that the loss of Ca²⁺ buffering could negatively impact SERCA2a pump activity. Interestingly, we found no apparent changes in SERCA2a expression, but rather only in the expression of its regulator, PLB. PLB was significantly increased in the CASQ2 null hearts, which could negatively impact SERCA2a pumping function; in addition, we also found decreases in phosphorylation both at the PKA (Ser16) and CamKII (Thr17) sites, which could further decrease pump activity. Although we did not find a statistically significant increase in PLB levels in the CASQ2^D307H hearts, the PKA and CamKII sites were less phosphorylated (65%, P < 0.05, and 45%, P < 0.05, of WT, respectively), which could contribute to the decreased pump activity. Taken together, these findings show, for the first time, that alterations in CASQ2 protein expression and or Ca²⁺ store can impact PLB expression, although the two proteins are not known to directly interact [unlike the jSR complex that CASQ2 forms with RyR2, TRD1, and JCN (8)].

CASQ2 and TRD1 interaction is essential for jSR architecture.

TRD1 has been shown to interact with CASQ2 via its luminal domain, and that this interaction anchors CASQ2 to the junctional face membrane (8, 37). More importantly, it has been suggested that the clustering of CASQ2 at the jSR mediated by the interaction between CASQ2 and TRD1/JCN (30, 36) dictates the specific architecture of the jSR cisternae (6). The recent knockout models for both TRD1 and CASQ2 have indeed shown that the loss of one protein affects the other and is paralleled by significant architectural changes to the jSR (3).

In our laboratory's previous report, we showed that the mutant CASQ2^D307H expression was able to partially restore the ultrastructure of jSR cisternae in the CASQ2 null hearts (16). CASQ2 is required within the SR lumen to facilitate the tight zippering effect of TRD1 and JCN, which results in the narrow configuration of the jSR cisternae (17). The proper targeting and localization of the mutant CASQ2^D307H could have indirectly contributed to the restoration of the widened jSR cisternae observed in the absence of CASQ2. More interestingly, we found that the TRD1 levels were increased in the D307H hearts to ∼75% from the ∼60% observed in the CASQ2 null hearts, indicating that CASQ2 expression has a direct effect on the expression of TRD1 via synthesis and/or stability. Since mRNA levels of TRD1 were unchanged in the CASQ2 null hearts (17), we hypothesized that decreased levels of TRD1 occurs posttranslationally. In keeping with this possibility, we found decreases in both the glycosylated and unglycosylated forms of TRD1 in the CASQ2 null (gly-60% and ungly-59%; P < 0.05) and CASQ2^D307H (gly-64% and ungly-78%) hearts relative to WT hearts. Recent studies have shown that glycosylation is an important posttranslational modification that can regulate TRD1 expression and trafficking (21). Unglycosylated TRD1 is more prone to active degradation relative to the more stable glycosylated version (21). Interestingly, we found a significant decrease only in the unglycosylated form of TRD1 in CASQ2 null hearts, which prompted us to suggest that CASQ2 could influence glycosylation of TRD1, but the exact mechanism is unknown, since TRD1 levels could be influenced by synthesis, trafficking, localization, and protein-protein interaction at the jSR. On the other hand, the unglycosylated TRD1 level was relatively increased in the CASQ2^D307H hearts (to 78% of WT levels), indicating that CASQ2 expression can positively regulate TRD1 expression.

Introduction of CASQ2^D307H in CASQ2 null hearts modifies arrhythmic susceptibility.

Although mutations in both CASQ2 and RyR2 are associated with ventricular arrhythmia in humans, complete loss or decrease of any one component of the complex is equally proarrhythmic (2–3, 17, 24, 27). CASQ2 null mice, when adrenergically stressed, show enhanced fractional SR Ca²⁺ release, Ca²⁺ leak, and arrhythmias (17). Similarly, loss of TRD1 increases susceptibility to triggered arrhythmia (2). These knockout mouse models have shown that the integrity of the triadic protein complex is critical to the proper modulation of RyR2 activity.

In this study, we found that the predisposition to proarrhythmic SCWs is decreased in CASQ2^D307H myocytes compared with the CASQ2 null myocytes. Knollmann et al. (17) have demonstrated that altered RyR2 channel activity and/or leak is the main defect behind the CPVT phenotype in the CASQ2 null mice. This finding was further confirmed by blocking RyR2 open state with Flecainide, which, despite absence of CASQ2, completely abolished triggered Ca²⁺ waves in the CASQ2 null myocytes and CPVT in the CASQ2 null mice and in CPVT human patients (11, 32). Our results suggest that the partial restoration of TRD1 expression, a key component of the Ca²⁺ release complex, along with jSR ultrastructure (16) in the mutant cells, could have contributed to stabilization of Ca²⁺ cycling and decreased the incidence of triggered arrhythmias.

Partial buffering by CASQ2^D307H could also have additive effects in modifying arrhythmia susceptibility by decreasing the free luminal Ca²⁺ within the SR compared with the complete lack of CASQ2. A recent report shows that loss of SERCA pump expression increases probability for SCWs under β-adrenergic stimulation, emphasizing the role of Ca²⁺ reuptake in SCW induction, in addition to RyR2 phosphorylation (28). Our data showing increased PLB and decreased phosphorylation in the CASQ2 null hearts suggest that a decrease in SERCA pump activity could contribute to increased propensity for triggered arrhythmias.

Taken together, our data suggest that alterations in CASQ2-mediated Ca²⁺ buffering and its effect on Ca²⁺ release via the RyR2 is apparent, specifically under high Ca²⁺ concentrations. Loss of tight regulation of the RyR2 [by CASQ2 (2, 17) and/or other modifications (33–34)], causing “Ca²⁺ leakiness” of the SR, has been postulated as a prominent proarrhythmic mechanism. In addition, our study reveals other multifaceted mechanisms that occur, along with the loss of CASQ2, emphasizing the need to consider the entire SR as a cooperative unit that regulates Ca²⁺ cycling. Studies focused on understanding the role of posttranslational modifications affecting key SR proteins and their mutual regulatory roles will shed more light on the unique mechanisms involved in maintaining cardiac Ca²⁺ homeostasis and may open new avenues to manipulate Ca²⁺ cycling and signaling for therapeutic purposes.

GRANTS

This work was supported by a Predoctoral Fellowship from the American Heart Association (AHA) (to A. Kalyanasundaram); US National Heart, Lung, and Blood Institute (Grants HL88635 and HL71670 to B. C. Knollmann, HL 080551 and HL 64140 to M. Periasamy), and the AHA Established Investigator Award (0840071N to B. C. Knollmann).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: A.K. and M.P. conception and design of research; A.K., S.V.-K., A.B., V.A.L., and H.S.H. performed experiments; A.K., S.V.-K., A.B., V.A.L., and H.S.H. analyzed data; A.K. and M.P. interpreted results of experiments; A.K., S.V.-K., A.B., and V.A.L. prepared figures; A.K. and M.P. drafted manuscript; A.K., B.C.K., S.G., and M.P. edited and revised manuscript; A.K. and M.P. approved final version of manuscript.