Defective domain-domain interactions within the ryanodine receptor as a critical cause of diastolic Ca2+ leak in failing hearts

Abstract

Aims: A domain peptide (DP) matching the Gly(2460)-Pro(2495) region of the cardiac type-2 ryanodine receptor (RyR2), DPc10, is known to mimic channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), owing to its interference in a normal interaction of the N-terminal (1-600) and central (2000-2500) domains (viz. domain unzipping). Using DPc10 and two other DPs harboring different mutation sites, we investigated the underlying mechanism of abnormal Ca(2+) cycling in failing hearts.

Methods and results: Sarcoplasmic reticulum (SR) vesicles and cardiomyocytes were isolated from dog left ventricular muscles for Ca(2+) leak and spark assays. The RyR2 moiety of the SR was fluorescently labelled with methylcoumarin acetate (MCA) using DPs corresponding to the 163-195 and 4090-4123 regions of RyR2 (DP163-195 and DP4090-4123, respectively) as site-directed carriers. Both DPs mediated a specific MCA fluorescence labelling of RyR2. Addition of either DP to the MCA-labelled SR induced domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. Both SR Ca(2+) leak and Ca(2+) spark frequency (SpF) were markedly increased in failing cardiomyocytes. Upon introduction of DP163-195 or DP4090-4123 into normal SR or cardiomyocytes, both Ca(2+) leak and SpF increased to the levels comparable with those of failing myocytes. K201 (JTV519) suppressed all of the effects induced by DP163-195 (domain unzipping and increased Ca(2+) leak and SpF) or those in failing cardiomyocytes, but did not suppress the effects induced by DP4090-4123.

Conclusion: Defective inter-domain interaction between N-terminal and central domains induces diastolic Ca(2+) leak, leading to heart failure and lethal arrhythmia. Mutation at the C-terminal region seen in CPVT does not seem to communicate with the aforementioned N-terminal and central inter-domain interaction, although spontaneous Ca(2+) leak is similarly induced.

Figure 1

(A) Concentration-dependent effects of DP163–195 and DP4090–4123 on Ca²⁺ leak from normal sarcoplasmic reticulum. Representative time courses of Ca²⁺ leak following Ca²⁺ uptake (top figure) and summarized data (bottom figure). *P < 0.01 vs. baseline. (B) Effects of DPc10, DP163–195, and DP4090–4123 on dissociation of FKBP12.6 from RyR2. Before immunoprecipitation of RyR, sarcoplasmic reticulum vesicles were mixed with DP163–195 or DP4090–4123 for 30 min and then centrifuged, followed by western blotting. (C) Effect of K201 (0.3 µmol/L) on the DP163–195-induced or DP4090–4123-induced Ca²⁺ leak in normal sarcoplasmic reticulum. (D) Effect of K201 (0.3 µmol/L) on spontaneous Ca²⁺ leak seen in failing sarcoplasmic reticulum, in the presence of DP163–195 (30 µmol/L) or DP4090–4123 (30 µmol/L).

Figure 2

Site-directed fluorescence labelling of RyR2 with methylcoumarin acetate. Site-specific methylcoumarin acetate fluorescence labelling was performed using either DPc10, DP163–195, or DP4090–4123 as a site-directing carrier. No methylcoumarin acetate fluorescence was seen when corresponding DPmut was used (left figure). An excess concentration of each unlabelled peptide (10 mmol/L) also inhibited each domain peptide-mediated methylcoumarin acetate labelling (right figure).

Figure 3

Fluorescence quenching analysis of domain unzipping (upper figures). A fluorescent probe methylcoumarin acetate was attached to the RyR2 of normal and failing sarcoplasmic reticulum in a site-specific manner using either DP163–195 (A) or DP4090–4123 (B) as a carrier. Then, the accessibility of the RyR2-bound MCA to a macromolecular fluorescence quencher BSA-QSY conjugate, the Stern–Volmer fluorescence quenching constant (i.e. the slope of the Fo/F vs. [BSA-QSY] plot), was determined as a measure of the degree of domain unzipping. Statistical comparison of the slope of each plot, which is equivalent to the Stern–Volmer quenching constant (K_Q) (bottom figures). DP163–195 increased the accessibility of the protein-bound MCA (carrier: DP163–195) to the quencher, as shown by an increase of the slope of the plot. K201 reversed the effect of DP163–195 in normal SR and decreased an elevated level of quencher accessibility in failing SR. DP4090–4123 also increased the accessibility of the bound MCA (carrier: DP4090–4123) to the quencher in normal SR, but in this case K201 was without effect.

Figure 3

Fluorescence quenching analysis of domain unzipping (upper figures). A fluorescent probe methylcoumarin acetate was attached to the RyR2 of normal and failing sarcoplasmic reticulum in a site-specific manner using either DP163–195 (A) or DP4090–4123 (B) as a carrier. Then, the accessibility of the RyR2-bound MCA to a macromolecular fluorescence quencher BSA-QSY conjugate, the Stern–Volmer fluorescence quenching constant (i.e. the slope of the Fo/F vs. [BSA-QSY] plot), was determined as a measure of the degree of domain unzipping. Statistical comparison of the slope of each plot, which is equivalent to the Stern–Volmer quenching constant (K_Q) (bottom figures). DP163–195 increased the accessibility of the protein-bound MCA (carrier: DP163–195) to the quencher, as shown by an increase of the slope of the plot. K201 reversed the effect of DP163–195 in normal SR and decreased an elevated level of quencher accessibility in failing SR. DP4090–4123 also increased the accessibility of the bound MCA (carrier: DP4090–4123) to the quencher in normal SR, but in this case K201 was without effect.

Figure 4

(A) Delivery of DP163–195 or DP4090–4123, fluorescently labelled with Alexa Fluor 488 (Molecular Probes, OR), into the isolated cardiomyocytes. Confocal microscopy clearly detects the fluorescence signal (shown as green) of DP163–195 or DP4090–4123 in the cardiomyocytes. Cell surface membrane was fluorescently labelled as red by wheat germ agglutinin-Alexa Fluor 633 conjugate (Molecular Probes, OR). (B) Spontaneous Ca²⁺ sparks in DP163–195- or DP4090–4123-incorporated normal and failing cardiomyocytes at either 2 or 5 mmol/L extra-cellular [Ca²⁺]. (C) Effect of K201 on spontaneous Ca²⁺ sparks in DP163–195- or DP4090–4123-incorporated normal and failing cardiomyocytes at 5 mmol/L extra-cellular [Ca²⁺]. Summarized data are shown at the right side. FWHM, full width at half maximum; FDHM, full duration at half maximum.

Figure 4

(A) Delivery of DP163–195 or DP4090–4123, fluorescently labelled with Alexa Fluor 488 (Molecular Probes, OR), into the isolated cardiomyocytes. Confocal microscopy clearly detects the fluorescence signal (shown as green) of DP163–195 or DP4090–4123 in the cardiomyocytes. Cell surface membrane was fluorescently labelled as red by wheat germ agglutinin-Alexa Fluor 633 conjugate (Molecular Probes, OR). (B) Spontaneous Ca²⁺ sparks in DP163–195- or DP4090–4123-incorporated normal and failing cardiomyocytes at either 2 or 5 mmol/L extra-cellular [Ca²⁺]. (C) Effect of K201 on spontaneous Ca²⁺ sparks in DP163–195- or DP4090–4123-incorporated normal and failing cardiomyocytes at 5 mmol/L extra-cellular [Ca²⁺]. Summarized data are shown at the right side. FWHM, full width at half maximum; FDHM, full duration at half maximum.

Figure 5

Model illustrating how defects in the inter-domain interactions within RyR2 cause channel dysfunctions (diastolic Ca²⁺ leak) in heart failure and ARVC2/CPVT. N, N-terminal domain (1–600); C, central domain (2000–2500); I, I-domain; IP, IP-domain (putative partner domain of I-domain; unidentified).