Isoproterenol Promotes Rapid Ryanodine Receptor Movement to Bridging Integrator 1 (BIN1)-Organized Dyads

Abstract

Background: The key pathophysiology of human acquired heart failure is impaired calcium transient, which is initiated at dyads consisting of ryanodine receptors (RyRs) at sarcoplasmic reticulum apposing CaV1.2 channels at t-tubules. Sympathetic tone regulates myocardial calcium transients through β-adrenergic receptor (β-AR)-mediated phosphorylation of dyadic proteins. Phosphorylated RyRs (P-RyR) have increased calcium sensitivity and open probability, amplifying calcium transient at a cost of receptor instability. Given that bridging integrator 1 (BIN1) organizes t-tubule microfolds and facilitates CaV1.2 delivery, we explored whether β-AR-regulated RyRs are also affected by BIN1.

Methods and results: Isolated adult mouse hearts or cardiomyocytes were perfused for 5 minutes with the β-AR agonist isoproterenol (1 µmol/L) or the blockers CGP+ICI (baseline). Using biochemistry and superresolution fluorescent imaging, we identified that BIN1 clusters P-RyR and CaV1.2. Acute β-AR activation increases coimmunoprecipitation between P-RyR and cardiac spliced BIN1+13+17 (with exons 13 and 17). Isoproterenol redistributes BIN1 to t-tubules, recruiting P-RyRs and improving the calcium transient. In cardiac-specific Bin1 heterozygote mice, isoproterenol fails to concentrate BIN1 to t-tubules, impairing P-RyR recruitment. The resultant accumulation of uncoupled P-RyRs increases the incidence of spontaneous calcium release. In human hearts with end-stage ischemic cardiomyopathy, we find that BIN1 is also 50% reduced, with diminished P-RyR association with BIN1.

Conclusions: On β-AR activation, reorganization of BIN1-induced microdomains recruits P-RyR into dyads, increasing the calcium transient while preserving electric stability. When BIN1 is reduced as in human acquired heart failure, acute stress impairs microdomain formation, limiting contractility and promoting arrhythmias.

Keywords: BIN1 protein; heart failure; membrane microdomain; myocytes cardiac; receptors, adrenergic, beta; ryanodine receptor 2, mouse.

Figure 1

Phosphorylation of RyR is increased in Bin1 HT hearts, particularly after acute β-AR stimulation. Western blot of tRyR (A), BIN1 (B) and P2808-RyR (C) in whole heart lysates from WT and Bin1 HT hearts at basal tone with endogenous β-AR auto-activity blocked with antagonists (ICI+CGP) or with acute β-AR activation after 5 min 1 µmol/L isoproterenol (ISO) treatment. *** indicates p<0.001 between genotypes; ††† indicates p<0.001 after ISO.

Figure 2

P2808-RyR is enriched at BIN1-organized dyadic microdomains. (A) 2D-STORM images image of P2808-RyR and BIN1 in adult mouse cardiomyocytes. Cartoon identifies the imaged area relative to the whole cardiomyocyte with surface attached to a glass coverslip. Scale bar: 500 nm. (B) 3D-STORM images of P2808-RyR with BIN1 (left two panels) or BIN1 with Ca_V1.2 (right two panels) in adult mouse cardiomyocytes. (C) 3D-STORM microdomain rendering of P2808-RyR /BIN1/Ca_V1.2 at dyads. (D) P2808-RyR co-immunoprecipitates with BIN1+13+17. IP: mouse anti BIN1 exon 13; IB: rabbit anti P2808-RyR and BIN1-SH3.

Figure 3

BIN1 recruits P2808-RyR to dyad upon β-AR stimulation. Representative confocal images of BIN1 (top) and P2808-RyR (bottom) labeling in WT (A) and Bin1 HT (B) cardiomyocytes at baseline (ICI+CGP) and after 5 min treatment with 1 µmol/L ISO. Scale bar: 10 µm. Fluorescent intensity profiles of boxed areas are included at the bottom for BIN1 (green) and P2808-RyR (red). (C) Average peak t-tubule intensity of BIN1 and P2808-RyR from the boxed areas in (A–B) after ISO treatment in WT and Bin1 HT myocytes. (D) Correlation between P2808-RyR intensity at jSR and t-tubule BIN1 intensity in WT and Bin1 HT cardiomyocytes. Blue lines define 75% quartile of both R2808-RyR and BIN1 signals in baseline WT cardiomyocytes. * indicates p<0.05 between genotypes; †† indicates p<0.01 after ISO.

Figure 4

Alteration in the intracellular distribution of tRyR upon β-AR stimulation. (A). Representative confocal images of tRyR (bottom) labeling in WT (left) and Bin1 HT (right) cardiomyocytes at baseline (ICI+CGP) and after 5 min treatment with 1 µmol/L ISO. Scale bar: 10 µm. (B). Average peak t-tubule intensity of tRyR from the boxed areas in (A) after ISO treatment in WT and Bin1 HT myocytes. ** indicates p<0.01 between genotypes; ††† indicates p<0.001 after ISO. Gene × treatment interaction term was identified as marginal significant (p=0.045) and remained in the final model.

Figure 5

Isoproterenol (ISO) induced spontaneous calcium release (SCR) is increased in Bin1 HT cardiomyocytes. (A) Representative calcium transients from WT and Bin1 HT myocytes with field stimulation at 1 Hz in the presence and absence of ISO. # indicates SCR. (B) Peak amplitude of calcium transient in both WT and Bin1 HT cardiomyocytes with or without ISO. (C) The incidence of SCR is higher in Bin1 HT cells, particularly after ISO treatment. *, **, *** indicate p<0.05, p<0.01, or p<0.001 between genotypes; ††† indicates p<0.001 after ISO.

Figure 6

Cartoon of the proposed model of BIN1 dependent recruitment of P2808-RyR to dyads during acute stress.

Figure 7

BIN1 is reduced in human acquired heart failure, resulting in decreased association with P2808-RyR. (A) Western blot of BIN1 in heart lysates from patients with either non-failing hearts or hearts with end-stage ischemic cardiomyopathy. (B) P2808-RyR co-immunoprecipitates with BIN1+13+17. IP: anti BIN1 exon 13; IB: rabbit anti P2808-RyR and BIN1-SH3. * indicates p<0.05.