Abnormal calcium cycling and cardiac arrhythmias associated with the human Ser96Ala genetic variant of histidine-rich calcium-binding protein

Abstract

Background: A human genetic variant (Ser96Ala) in the sarcoplasmic reticulum (SR) histidine-rich Ca(2+)-binding (HRC) protein has been linked to ventricular arrhythmia and sudden death in dilated cardiomyopathy. However, the precise mechanisms affecting SR function and leading to arrhythmias remain elusive.

Methods and results: We generated transgenic mice with cardiac-specific expression of human Ala96 HRC or Ser96 HRC in the null background to assess function in absence of endogenous protein. Ala96 HRC decreased (25% to 30%) cardiomyocyte contractility and Ca2+ kinetics compared with Ser96 HRC in the absence of any structural or histological abnormalities. Furthermore, the frequency of Ca2+ waves was significantly higher (10-fold), although SR Ca2+ load was reduced (by 27%) in Ala96 HRC cells. The underlying mechanisms involved diminished interaction of Ala96 HRC with triadin, affecting ryanodine receptor (RyR) stability. Indeed, the open probability of RyR, assessed by use of ryanodine binding, was significantly increased. Accordingly, stress conditions (5 Hz plus isoproterenol) induced aftercontractions (65% in Ala96 versus 12% in Ser96) and delayed afterdepolarizations (70% in Ala96 versus 20% in Ser96). The increased SR Ca2+ leak was accompanied by hyperphosphorylation (1.6-fold) of RyR at Ser2814 by calmodulin-dependent protein kinase II. Accordingly, inclusion of the calmodulin-dependent protein kinase II inhibitor KN93 prevented Ser2814 phosphorylation and partially reversed the increases in Ca2+ spark frequency and wave production. Parallel in vivo studies revealed ventricular ectopy on short-term isoproterenol challenge and increased (4-fold) propensity to arrhythmias, including nonsustained ventricular tachycardia, after myocardial infarction in Ala96 HRC mice.

Conclusions: These findings suggest that aberrant SR Ca2+ release and increased susceptibility to delayed afterdepolarizations underlie triggered arrhythmic activity in human Ala96 HRC carriers.

Keywords: arrhythmia; calcium; ryanodine receptor calcium release channel; sarcoplasmic reticulum.

Figure 1.

SR Ca²⁺‐cycling proteins and gross inspection of Ser96 (S96) and Ala96 (A96) HRC hearts. A, Representative blots of Ca²⁺‐cycling protein levels in WT (nontransgenic), Ser96, and Ala96 HRC hearts. B, Quantitative assessment of protein levels in WT (nontransgenic), Ser96, and Ala96 HRC hearts. Values are mean±SEM; N=3 hearts/group. C, Left: hematoxylin‐eosin staining of ventricular sections of Ser96 and Ala96 HRC mice (×100), bars=10 μm. Middle: trichromic Masson stain of ventricular sections of Ser96 and Ala96 HRC (×100), bars=10 μm. Right: representative electron micrographs (EM) from cross sections of the papillary muscles from Ser96 and Ala96 HRC hearts. D, Ventricular cardiomyocytes from Ser96 and Ala96 mice immunostained with human anti‐HRC and mouse anti‐SERCA antibodies. Bars=20 μm. CSQ indicates calsequestrin; GAPDH, glyceraldehydes 3‐phosphate dehydrogenase; HRC, histidine‐rich Ca²⁺‐binding protein; NCX, sodium‐calcium exchanger; PLN, phospholamban; PLN‐16 and PLN‐17, PLN phosphorylation at Ser16 and Thr17 sites; RyR2, ryanodine receptor; SERCA, sarcoplasmic reticulum Ca²⁺ ATPase; SR, sarcoplasmic reticulum; TRI, triadin; WT, wild type.

Figure 2.

Mechanics and Ca²⁺ kinetics of Ser96 (S96) and Ala96 (A96) HRC myocytes and their responses to isoproterenol. A, Representative cell shortening tracings of Ser96 and Ala96 cells before isoproterenol stimulation, field stimulated at 0.5 Hz. B, The fractional shortening (FS) in the absence and presence of 100 nmol/L isoproterenol (ISO). C, Rates of contraction, +dL/dt, in the absence and presence of ISO. D, Rates of relaxation, −dL/dt, in the absence and presence of ISO. E, Representative tracings of Ca²⁺ transients in Ser96 and Ala96 cells before isoproterenol stimulation. F, Ca²⁺ transient amplitude, as indicated by the Fura‐2 ratio (340:380 nm) in the absence and presence of ISO. G, Time to 80% decay of the transient (T80) in the absence and presence of ISO. H, Ca²⁺ transient decline (Tau). For cell mechanics (−ISO): n=44 to 55 cells from 5 hearts/group; twitch Ca²⁺ transients (−ISO): n=40 to 55 cells from 5 hearts/group; cell mechanics (+ISO): n=25 to 35 cells from 4 hearts/group, and Ca²⁺ transients (+ISO): n=38 to 50 cells from 3 hearts/group. Data are mean±SEM of the total number of cells/group. Comparisons were performed by using t test. *P≤0.05 vs Ser96 mice. HRC indicates histidine‐rich Ca²⁺‐binding protein.

Figure 3.

SR Ca²⁺ content and NCX function in Ser96 (S96) and Ala96 (A96) HRC cardiomyocytes. A, Representative tracings of caffeine‐induced Ca²⁺ transients recorded from Fura‐2 AM–loaded, field‐stimulated myocytes at 0.5 Hz. B, Amplitude of caffeine‐induced Ca²⁺ transients showing a decrease in SR Ca²⁺ load in myocytes from Ala96 mice in the absence and presence of ISO. C, Mean data for Ca²⁺ transient decline (Tau) during caffeine‐induced Ca²⁺ transients, recorded in the presence of caffeine. D, Average data for fractional release (ratio of twitch Ca²⁺ transient/caffeine‐induced Ca²⁺ transient). ISO (100 nmol/L). For SR Ca²⁺ load, n=15 to 25 cells for 4 Ser96 hearts; n=25 to 38 cells from 4 Ala96 hearts. Data are represented as mean±SEM of the total number of cells/group, and t test was used to calculate statistical significance. *P<0.05 vs Ser96 mice. AM indicates acetoxymethyl; HRC, histidine‐rich Ca²⁺ binding; ISO, isoproterenol; NCX, sodium‐calcium exchanger; SEM, standard error of the mean; SR, sarcoplasmic reticulum.

Figure 4.

Ca²⁺ sparks and waves in intact Ser96 (S96) and Ala96 (A96) HRC cardiomyocytes. A, Representative line‐scan images of Ca²⁺ sparks acquired in Ser96 and Ala96 cardiomyocytes in the presence of 100 nmol/L isoproterenol. B, Cumulative data on Ca²⁺ spark frequency in the presence of 100 nmol/L isoproterenol (n=55 Ser96 cells from 5 hearts and n=61 Ala96 cells from 5 hearts). *P<0.05, Ser96 vs Ala96 (t test). C, Representative line‐scan images of Ca²⁺ waves acquired in Ser96 and Ala96 cardiomyocytes in the presence of 1 μmol/L isoproterenol and 5 Hz. D, percentage of cells showing waves (n=20 cells for 3 Ser96 hearts; n=20 cells for 3 Ala96 hearts). *P<0.05, Ser96 vs Ala96 (Fisher Exact test). E, SR Ca²⁺ leak measurements in Ser96 and Ala96 HRC myocytes in the presence of 100 nmol/L isoproterenol. SR Ca²⁺ leak was determined as the tertacaine sensitive drop in diastolic Fura‐2 ratio. F, Comparison of average SR Ca²⁺ leak (ratio of twitch Ca²⁺ transient/caffeine‐induced Ca²⁺ transient) G, Bar graph showing quantification of leak/SR load relationship in Ser96 and Ala96 HRC myocytes. Ser96 HRC myocytes, n=22 from 3 hearts; Ala96 HRC myocytes, n=28 from 3 hearts. *P<0.05, Ser96 vs Ala96 (Fisher Exact test). HRC indicates histidine‐rich Ca²⁺ binding; ISO, isoproterenol; SR, sarcoplasmic reticulum.

Figure 5.

Aftercontractions (Acs) and DADs in Ser96 (S96) and Ala96 (A96) HRC cardiomyocytes at 5‐Hz and 1‐μmol/L isoproterenol stimulation. A, Representative traces of Acs in Ser96 and Ala96 cardiomyocytes. B, Percentage of the Ser96 and Ala96 cardiomyocytes that developed Acs (n=55 cells for 5 Ser96 hearts; n=68 cells for 5 Ala96 hearts). C, Representative traces of action potential in Ser96 and Ala96 cells. D, Percentage of the Ser96 and Ala96 cells that showed DADs (n=14 cells for 4 Ser96 hearts; n=15 for 5 Ala96 hearts). Acs (A) and DADs (C) are marked with arrows. Data are mean±SEM of the total number of cells/group. *P<0.05 vs Ser96 (Fisher Exact test was used to calculate statistical significance). DADs indicates delayed afterdepolarizations; HRC, histidine‐rich Ca²⁺ binding; SEM, standard error of the mean.

Figure 6.

Ala96 (A96) HRC reduces binding to triadin. A, The anti‐HRC antibody, coupled to protein G–agarose beads, was used for coimmunoprecipitation of triadin from cardiac homogenates (1 mg total protein) of Ser96 (S96) and Ala96 HRC mice. The precipitates were analyzed by immunoblotting with anti‐HRC and anti‐TRD antibodies, as indicated. C, Coimmunoprecipitation of triadin from HEK293 cells that coexpressed triadin and Ser96 or Ala96 HRC, under high Ca²⁺ (0.1 mmol/L) and low Ca²⁺ (0.1 μmol/L) conditions was also performed using anti‐HRC antibody, coupled to protein G–agarose beads. Top panel: immunoblotting with HRC antibodies; Bottom panel: immunoblotting with triadin antibodies. Immunoprecipitate with anti‐IgG PLUS agarose was used as negative control (A and C). B and D, The summary of interactions is presented. Ala96, N=3; Ser96, N=3. *P<0.05, Ala96 vs Ser96 mice (t test); ^&P<0.05, Ser96 low vs high Ca²⁺ (comparisons were performed by using 1‐way ANOVA). ANOVA indicates analysis of variance; GFP, green fluorescent protein; HRC, histidine‐rich Ca²⁺ binding; IPs, immunoprecipitations; TRD, Triadin.

Figure 7.

CaMKII‐dependent RyR2 phosphorylation and Ca²⁺ sparks/waves in Ser96 (S96) and Ala96 (A96) HRC hearts. A, Representative western blots of protein levels and phosphorylation of RyR2. Total protein homogenates from the left ventricle were used for western blots. Hearts were perfused in the absence or presence of β‐adrenergic agonist (100 nmol/L ISO) and/or CaMKII inhibitor (1 μmol/L KN93) with a modified HEPES‐Tyrode's solution prior to isolation of the left ventricles. RyR2 phosphorylation at sites S2808 and S2814 was determined with phospho‐specific antibodies. B and C, Bar graph showing quantification of ryanodine receptor phosphorylation at S2808 and S2814 sites in the absence (−) or presence of ISO and KN93. Actin was used to verify the amount of loaded samples. Two‐way repeated‐measures ANOVA were used to compare groups. Values are mean±SEM; N=5 hearts/group. *P<0.05 vs Ser96 (−ISO), #P<0.05 vs Ala96 (±ISO). D, Ca²⁺ spark frequency recorded in Ser96 and Ala96 HRC cardiomyocytes in the absence (basal) or presence of ISO (100 nmol/L) and in the absence or presence of CaMKII inhibitor KN93 (1 μmol/L). E, Percentage of increases in Ca²⁺ waves acquired in Ser96 and Ala96 HRC cardiomyocytes in the presence of 1 μmol/L isoproterenol plus 5 Hz and in the absence or presence of CaMKII inhibitor KN93 (1 μmol/L). For Ca²⁺ sparks: n=32 to 35 cells (−ISO) and n=20 to 28 cells (+ISO); and for Ca²⁺ waves: n=24 to 32 cells. A total of 4 to 5 hearts were used/group. Two‐way repeated‐measures ANOVA were used to compare groups. *P<0.05 vs Ser96; ^#P<0.05 vs Ala96. ANOVA indicates analysis of variance; HRC, histidine‐rich Ca²⁺ binding; ISO, isoproterenol; RyR2, ryanodine receptor.

Figure 8.

Ala96 (A96) HRC mice display increased ventricular ectopy after catecholamine challenge. A and B, Representative examples of ECG traces showing premature ventricular complexes (PVCs, #) (A) and nonsustained ventricular tachycardia (NSVT, B) in anesthetized Ala96 mice after intraperitoneal injection of isoproterenol (2 mg/kg). C, A high proportion of mice expressing Ala96 displayed complex forms of ventricular arrhythmias (bigeminy/trigeminy and NSVT), compared with Ser96 (S96) mice, following isoproterenol injection. n=8 Ala96 mice; n=8 Ser96 mice. *P<0.05 vs Ser96, the Mann–Whitney U test was used to compare absolute PVC incidence and Fisher exact test was used to evaluate ventricular tachycardia incidence. ECG indicates electrocardiogram; HRC, histidine‐rich Ca²⁺ binding.

Figure 9.

Increased susceptibility to arrhythmias in Ala96 (A96) HRC mice upon myocardial infarction. A, Typical examples of ECG changes during the course of ischemia (top left panel) and representative ECG traces showing ventricular premature beats (#: VPBs, top right panel) and ventricular tachycardia (VT, bottom panel) in an Ala96 HRC mouse after MI. B, Percentage of Ser96 (S96) and Ala96 HRC mice showing VT within 24 hours after MI as monitored by implanted telemeters (N=6 for Ser96 and N=10 for Ala96 mice; *P<0.05 Ala96 vs Ser96). C, Echocardiographic measurements of ejection fraction, and (D) left ventricular diastolic volume in Ser96 (N=4) and Ala96 (N=4) mice at 12 weeks post‐MI. *P<0.05 baseline vs 12 weeks (t test). E, Representative telemetry traces depicting (#: VPBs) in a Ser96 mouse and non‐sustained ventricular tachycardia (NSVT) in an Ala96 mouse at 12 weeks post MI. F, Bar graph showing incidence of VT in Ser96 and Ala96 mice. VT was observed only in Ala96 mice and in none of the Ser96 mice at 12 weeks after MI. *P<0.05 Ala96 vs Ser96 mice. Fisher exact test was used to evaluate ventricular tachycardia incidence. ECG indicates electrocardiogram; EF, ejection fraction; HRC, histidine‐rich Ca²⁺ binding; I/R, ischemia/reperfusion; LV, left ventricle; MI, myocardial infarction.

Figure 10.

Proposed scheme on the functional interactions between calsequestrin (CSQ), triadin (TRI), histidine‐rich Ca²⁺ binding protein (HRC), and ryanodine receptor (RyR) in the cardiac sarcoplasmic reticulum (SR) membrane. In Ser96 (S96) HRC heart, CSQ interacts with TRI, resulting in deactivation of the RyR channel at low luminal Ca²⁺ concentrations, following SR Ca²⁺ release (A). On refilling the SR with Ca²⁺, CSQ inhibition is relieved as CSQ dissociates from the RyR complex; however, binding of HRC prevents the RyR from becoming leaky (B). In Ala96 (A96) HRC hearts, CSQ interactions are similar to Ser96 HRC at both low and high Ca²⁺ concentrations; however, the interaction of Ala96 HRC with triadin is impaired (C and D), leading to increased RyR2 leak. RyR leakiness is further amplified via phosphorylation by calmodulin‐dependent protein kinase II (D).