Calsequestrin 2 (CASQ2) mutations increase expression of calreticulin and ryanodine receptors, causing catecholaminergic polymorphic ventricular tachycardia

Abstract

Catecholamine-induced polymorphic ventricular tachycardia (CPVT) is a familial disorder caused by cardiac ryanodine receptor type 2 (RyR2) or calsequestrin 2 (CASQ2) gene mutations. To define how CASQ2 mutations cause CPVT, we produced and studied mice carrying a human D307H missense mutation (CASQ(307/307)) or a CASQ2-null mutation (CASQ(DeltaE9/DeltaE9)). Both CASQ2 mutations caused identical consequences. Young mutant mice had structurally normal hearts but stress-induced ventricular arrhythmias; aging produced cardiac hypertrophy and reduced contractile function. Mutant myocytes had reduced CASQ2 and increased calreticulin and RyR2 (with normal phosphorylated proportions) but unchanged calstabin levels, as well as reduced total sarcoplasmic reticulum (SR) Ca(2+), prolonged Ca(2+) release, and delayed Ca(2+) reuptake. Stress further diminished Ca(2+) transients, elevated cytosolic Ca(2+), and triggered frequent, spontaneous SR Ca(2+) release. Treatment with Mg(2+), a RyR2 inhibitor, normalized myocyte Ca(2+) cycling and decreased CPVT in mutant mice, indicating RyR2 dysfunction was critical to mutant CASQ2 pathophysiology. We conclude that CPVT-causing CASQ2 missense mutations function as null alleles. In the absence of CASQ2, calreticulin, a fetal Ca(2+)-binding protein normally downregulated at birth, remains a prominent SR component. Adaptive changes to CASQ2 deficiency (increased posttranscriptional expression of calreticulin and RyR2) maintained electrical-mechanical coupling, but increased RyR2 leakiness, a paradoxical response further exacerbated by stress. The central role of RyR2 dysfunction in CASQ2 deficiency unifies the pathophysiologic mechanism underlying CPVT due to RyR2 or CASQ2 mutations and suggests a therapeutic approach for these inherited cardiac arrhythmias.

Figure 1. Introduction of missense mutation D307H and exon 9 deletion into the mouse CASQ2 gene.

(A) The mouse CASQ2 gene is encoded in 11 exons spread over 70 kb. The WT (+), D307H knock-in (307) and exon 9–deletion KO (ΔE9) alleles are contained on a 15-kb XmaI restriction fragment, which encodes exons 9–11. (B) The genotypes of WT (+/+), D307H-knockin heterozygous (307/+) and homozygous (307/307), and exon 9–deletion KO heterozygous (ΔE9/+) and homozygous (ΔE9/ΔE9) mice were determined by size characterization of PCR-amplified DNA fragments. The WT and Neo-excised alleles of D307H-knockin mice were amplified using primers 307F and 307R, which yielded 140-bp and 250-bp fragments, respectively (lanes 1–3). The WT and ΔE9 alleles were amplified with primers F1, F2, and R, producing 250-bp and 400-bp fragments, respectively (lanes 4–6). The D307H allele was amplified by PCR primers 307FB and 307RB, which yielded a 750-bp fragment. After BamHI digestion, the D307H allele produced 500-bp and 250-bp bands (lanes 7–9). M, molecular marker.

Figure 2. Assessment of RNA and proteins in CASQ2-deficient hearts.

(A) Cardiac mRNA levels of CASQ2, CASQ1, and CRT in age-matched (n = 3) WT (+/+), heterozygous (CASQ^ΔE9/+; CASQ^307/+), and homozygous (CASQ^ΔE9/ΔE9; CASQ^307/307) mice. Note low and comparable CASQ1 RNA levels and normal, comparable CASQ2 and CRT RNA levels in WT and CASQ^307/307 mice. In contrast, CASQ2 RNA levels are very low in CASQ^ΔE9/ΔE9 mice. GAPDH RNA was used for normalization. (B) Representative Western blots show dramatic reduction in CASQ2 protein expression in hearts from CASQ^ΔE9/ΔE9 and CASQ^307/307 mice and increased cardiac CRT protein levels (n = 3). (C) Densitometry of Western blots (n = 3) shows significantly increased CRT levels in CASQ^ΔE9/ΔE9 (white bar) and CASQ^307/307 (gray bar) versus WT (black bar) hearts. (D) Western blot analyses of triadin, SERCA2, sorcin, calstabin, PLN monomers (M) and pentamers (P), phosphorylated PLN (residues Ser16 and Thr17) monomers and pentamers, and sarcalumenin showed unchanged levels in CASQ2-deficient hearts (n = 3). (E) Western blots of both dephosphorylated (RyR2-dep-Ser2809) and phosphorylated (RyR2-p-Ser2809) RyR2 in CASQ2-deficient and WT hearts. (F and G) Densitometry of Western blots (n = 3, normalized to Coomassie blue–stained proteins) showed that both phosphorylated and dephosphorylated RyR2 were significantly increased (WT versus CASQ^ΔE9/ΔE9 or CASQ^307/307) and ratios (H) of phosphorylated RyR2 (RyR2-p-Ser2809) and nonphosphorylated (RyR2-dep-Ser2809) were unchanged.

Figure 3. Cardiac morphology and histology of CASQ2-deficient hearts.

(A) Gross cardiac anatomy of adult WT (+/+) and CASQ^307/307 mice (n = 4) demonstrated enlarged atria (arrows) and ventricles in mutant hearts. (B) Low magnification of paraffin-embedded ventricular transverse heart sections from 35-week-old mice studied with Masson trichrome stain. No fibrosis was observed in either WT (+/+) or homozygous CASQ^307/307 mice (n = 4). (B). Scale bars, 1 mm.

Figure 4. Representative ventricular arrhythmias recorded in CASQ2-deficient mice.

(A) Ventricular bigeminy in a resting CASQ^ΔE9/ΔE9 mouse. (B) Dimorphic ventricular couplets induced by exercise in a CASQ^307/307 mouse. (C) Telemetry recording of CASQ^307/307 mouse showed baseline sinus rhythm (left), but after isoproterenol administration, multiple polymorphic VPBs and supraventricular tachycardia (SVT) occurred, which deteriorated to sustained polymorphic VT with bidirectional pattern. (D) Sinus arrest with nodal escape during EP study in CASQ^{ΔE9/ ΔE9} mouse. V, ventricular beat; S, sinus beat; N, nodal beat; P, p wave. Scale bar: 0.2 second.

Figure 5. Functional analyses and SR Ca²⁺ transients of CASQ2-deficient myocytes.

In Ca²⁺ Tyrode solution (A) and in epinephrine 5.5 μM Tyrode solution (B), electrical stimulation (60 Hz) produced Ca²⁺ transients (left paired panels) and sarcomere shortening (right paired panels) of WT and CASQ2-deficient myocytes (M, CASQ^ΔE9/ΔE9 and CASQ^307/307). Traces represent the average of 15 contraction-relaxation cycles from 1 representative cell of each genotype. Scale bars: 0.2 second. (C) Representative traces of caffeine-induced (10 mM) Ca²⁺ transients in myocytes. Scale bars: 1 second. Graphs denote pooled data from 8 WT (black) and 11 mutant (white) cells. (D) Representative traces from CASQ^307/307 myocyte during constant epinephrine infusion for 5 minutes with electrical pacing at 1 Hz (blue ticks). Note elevation of diastolic Ca²⁺ levels (diamond), reduction of transient peak height, and development of Ca²⁺ oscillation after 4 minutes of epinephrine infusion (right side of the panel). (E) Representative traces from another CASQ^307/307 myocyte in Tyrode solution during the fifth minute of epinephrine infusion show multiple events of SCR and Ca²⁺ oscillations. Arrows, paced electrical stimulus, 1 Hz.

Figure 6. A model for CPVT based on abnormal SR Ca²⁺ homeostasis in CASQ2-deficient myocytes.

(A) Under resting conditions, SR Ca²⁺ (blue circles) is plentiful and buffered by CASQ2 (blue rectangles) in WT myocyte. Ca²⁺ influx via L-type Ca²⁺ channel (left side) causes Ca²⁺-induced Ca²⁺ release (CICR) through cardiac RyR2 channels (purple). Reuptake of cytosolic Ca²⁺ (black arrows) occurs via the SERCA/PLN complex (orange). When SR Ca²⁺ concentration falls (right half), CASQ2 binds to the RyR2 channel, closing the channel (x), thus preventing Ca²⁺ leak (right side arrow). (B) In CASQ2-deficient myocytes, CRT (brown symbols) replaces CASQ2. Total SR Ca²⁺ is lower than in WT myocytes due to lower Ca²⁺-binding capacity by CRT (50% of CASQ2) and diastolic Ca²⁺ leak (red dashed arrows) through abundant RyR2 channels and inadequate calstabin. SERCA2-mediated Ca²⁺ reuptake may also be impaired (dashed black arrows) due to increased free Ca²⁺ gradient given lower Ca²⁺-binding capacity of CRT. Total SR Ca²⁺ transients and cytosolic Ca²⁺ are near normal. (C) Catecholaminergic stress applied to CASQ2-deficient myocytes phosphorylates RyR2 channels and PLN, dissociating calstabin from RyR2. Excessive RyR2 activity causes extensive diastolic Ca²⁺ leak, further depletes SR Ca²⁺, reduces SR Ca²⁺ transients, and increases cytosolic Ca²⁺ levels. Excess cytosolic Ca²⁺, effluxed via the Na⁺/Ca²⁺ exchanger, increases Na⁺ entry into myocyte, which increases the probability of delayed after-polarization and cardiac arrhythmia. Toxicity of Ca²⁺ overload may increase myocyte death.