We generated a knock-in mouse model in which aspartic acid replaces serine at RyR2-2814 (S2814D) to mimic constitutive phosphorylation of RyR2 by CaMKII (Fig. S1
). CaMKII can phosphorylate RyR2 immunoprecipitated from wild type (WT) hearts but not from S2814D mice (). Furthermore, CaMKII inhibition by KN-93 prevents RyR2 phosphorylation by CaMKII in WT hearts, but has no effect in S2814D hearts, indicating that S2814 is the major CaMKII target site of RyR2 (). FKBP12.6 binding to RyR2 can alter RyR2 function, but we found that the S2814D mutation did not alter FKBP12.6 binding to RyR2 in cardiac homogenates (), as in an earlier report 19
. At baseline, cardiac structure and function are similar in young (3-month-old) WT and S2814D mice as determined by echocardiography (; Supplemental Table 1
). Transverse H&E sections from WT and S2814D hearts showed there were no significant differences in right ventricular (RV) wall thickness (WT: 0.58 ± 0.17 mm; S2814D: 0.47 ± 0.14 mm, P
= 0.10), left ventricular (LV) posterior wall thickness (WT: 0.74 ± 0.21 mm; S2814D: 0.74 ± 0.22 mm, P
= 0.91), or LV anteroposterior diameter (WT: 3.51 ± 1.02 mm; S2814D: 3.63 ± 1.05 mm, P
= 0.41) (). Quantitative analysis of Masson Trichrome stainings revealed no differences in the amount of interstitial fibrosis comparing WT (0.06 ± 0.02 % of total surface area) and S2814D mouse hearts (0.07 ± 0.02 %, P
= 0.61) (). Wheat germ agglutinin (WGA) staining and quantification of myocardial cell size revealed no differences in myocyte surface areas in WT (2311 ± 166 μm2
) and S8214D (2315 ± 256 μm2
) mouse hearts (P
= 0.99) (). On Western blots, expression of WT and S2814D heart lysates using antibodies against cardiac ryanodine receptor (RyR2), L-type Ca2+
channel (Cav1.2), Na+
-exchanger (NCX), and Ca2+
/calmodulin-dependent protein kinase II (CaMKII) was not statistically different (Cav1.2, P
= 0.66; NCX, P
= 0.17; CaMKII, P
= 0.61) ().
Echocardiographic parameters of WT and S2814D mice at 3 and 12 months of age.
At the myocyte level, HF and CaMKII overexpression (and activation) enhance SR Ca2+
leak (manifested as increased Ca2+
sparks or waves mediated by RyR2) and can thus serve as the molecular trigger for arrhythmia 26
. To determine how SR Ca2+
leak is altered in young S2814D and S2814A mice, we used confocal microscopy to image local SR Ca2+
release events, or Ca2+
sparks, in permeabilized isolated cardiomyocytes (). At baseline, Ca2+
spark frequency (CaSpF) was significantly increased in S2814D (9.8 ± 0.5 /s/100μm) vs. WT mice (6.4 ± 0.3 /s/100μm; P
< 0.001; ). Activation of endogenous CaMKII significantly increased CaSpF in WT myocytes (9.9 ± 0.5 /s/100μm), but had no additional effect in S2814D cells (which were already at this higher level, 10.2 ± 0.6 /s/100μm). In S2814A myocytes, CaSpF was comparable to WT at baseline both before (5.1 ± 0.1 /s/100μm) and after activation of CaMKII (6.1±0.2/s/100μm). The small rise with CaMKII was entirely attributable to enhanced SR Ca2+
content (), and did not significantly alter Ca2+
spark amplitude, full duration at half-maximum (FDHM), full width at half-maximum (FWHM), or maximum Ca2+
release (Fig. S2
). Inclusion of the specific CaMKII inhibitory peptide AIP (1 μM) prevented CaMKII-dependent activation of CaSpF in WT cells, but did not alter CaSpF in S2814D myocytes (). Combined, these results show that constitutive RyR2-S2814 pseudo-phosphorylation mimics maximal CaMKII activation of WT RyR2 in ventricular myocytes, and that S2814 is the only functionally important CaMKII site with respect to these measurements.
CaMKII phosphorylation induces RyR2 mediated SR calcium leak in permeabilized myocytes
We also measured Ca2+
sparks and SR Ca2+
load in intact ventricular myocytes. Twitch Ca2+
transient amplitude during electrical field stimulation (1 Hz) was similar in WT, S2814D and S2814A cardiomyocytes (P
=0.43; ). However, the time constant of twitch [Ca2+
decline was significantly lower in S2814D (403.8±18 ms, P
< 0.05) compared to WT (274.8±13 ms) and S2814A (238±18 ms). Moreover, SR Ca2+
load (assessed by rapid application of 10 mM caffeine) was 50% lower in S2814D compared to WT or S2814A myocytes (P
= 0.02 ). This is presumably due to the slightly reduced SERCA2a function and higher diastolic SR Ca2+
leak in S2814D myocytes (see above), and also evidenced by significantly higher CaSpF in intact S2814D myocytes vs. WT or S2814A myocytes (Fig. S3A-B
). However, other parameters such as Ca2+
spark amplitude, FDHM, FWHM, and rate of rise were unchanged among the groups (Fig. S3C-F
). Note that CaMKIIδC
transgenic mouse myocytes and rabbit HF myocytes also exhibit enhanced leak and reduced SR Ca2+
load 9, 12
. There was no statistical difference in NCX function - measured as the time constant of [Ca2+
decline during a caffeine-induced Ca2+
transient - among the three mouse groups: WT (τ=1.9±0.2 s), S2814D (τ=2.2±0.6 s) and S2814A (τ=1.7±0.2 s) (P
= 0.54). However, the S2814D mice did exhibit enhanced fractional SR Ca2+
release (ratio of twitch:caffeine-induced Ca2+
transient) compared to WT or S2814A mice (P
<0.001; ). Thus, S2814D mice maintain normal Ca2+
transients (and cardiac function) with a smaller SR Ca2+
load, but a larger fractional release at each contraction. This is consistent with prior work suggesting that CaMKII-dependent RyR2 phosphorylation sensitizes RyR2 to a given Ca2+
current trigger at a given SR Ca2+
. Thus RyR2 phosphorylation at S2814 activates both diastolic and systolic RyR2 activation.
CaMKII phosphorylation induces RyR2 mediated SR calcium leak and reduced SR calcium load in intact myocytes
To directly assess single RyR2 channel opening we extracted microsomes containing RyR2 from young WT and S2814D mouse hearts and reconstituted them in lipid bilayers. RyR2-S2814D channels exhibited much higher open probability (Po 54.3%, [interquartile range, 25.8% to 86.5%]) compared to RyR2 from WT mice (1.0 % [interquartile range, 0.9% to 2.5%], P<0.001; ). It is likely, however, that the relative increase in open probability of S2814D mutant channels will be more modest in vivo, as the frequency of Ca2+ sparks in S2814D myocytes was only increased two-fold. The single channel and whole cell experiments demonstrate that pseudo-phosphorylation of RyR2 at the S2814 CaMKII site increases the open probability of RyR2, resulting in diastolic Ca2+ leak.
We then used young (3–4 month old) S2814D knock-in mice to evaluate the effects of CaMKII mediated RyR2 phosphorylation on arrhythmogenesis in structurally normal hearts. ECG telemeters were implanted in both WT and S2814D mice to allow recording of ambulatory ECG waveforms. S2814D mice had normal heart rhythm at rest, with unaltered electrophysiological parameters such as heart rate (HR), depolarization intervals (PQ, QRS), and repolarization intervals (QTc) (Table S2
). Moreover, the ventricular effective refractory period (VERP) was also unaffected in S2814D mice (Table S3
). However, when challenged with the β-adrenergic agonist isoproterenol (0.5 mg/kg i.p.), S2814D mice exhibited a significantly higher increase in premature ventricular complexes (PVCs) vs. WT (10.0 [interquartile range, 6.0 to 14.0] vs. 3.8 [interquartile range, 1.0 to 4.8]; P
= 0.002), which is indicative of ventricular ectopic arrhythmic activity ().
Constitutive CaMKII phosphorylation of RyR2 leads to ventricular arrhythmias and sudden cardiac death
Given that hearts of S2814D mice were structurally and electrically normal at rest, but exhibited ventricular ectopy upon β-adrenergic stimulation, we considered that the model phenotype might resemble catecholaminergic polymorphic ventricular tachycardia (CVPT). To further test S2814D mice for predisposition to ventricular arrhythmias under more stringent catecholaminergic conditions 5, 28
, we injected caffeine and epinephrine (120 mg/kg and 2 mg/kg i.p., respectively). There was a significantly increased incidence of sustained ventricular tachycardia in S2814D mice (71%) compared to WT mice (13%, P
= 0.04) (). This is consistent with the presence of a pro-arrhythmogenic substrate caused by CaMKII pseudo-phosphorylation of RyR2. One S2814D mouse exhibited persistent ventricular tachycardia following caffeine and epinephrine that deteriorated into bradycardia and then asystole (). Thus, CaMKII-mediated RyR2 phosphorylation promotes in vivo
ventricular arrhythmias, and increases the risk of sudden cardiac death.
In order to further differentiate the role of CaMKII activation of RyR2 from the effects of PKA activation, we sought to examine the role of elevated heart rate on arrhythmogenesis by performing in vivo
intracardiac electrophysiology studies. Programmed electrical stimulation using ventricular burst pacing was performed to compare cardiac susceptibility to ventricular ectopic activity in WT and S2814D mice. Burst pacing evoked sustained ventricular tachycardia in 53% of S2814D mice compared with 6% of WT mice (P
= 0.006; ). Consistent with previous studies 19
, rapid pacing induced CaMKII but not PKA phosphorylation of RyR2; whereas, the S2814D mutation inhibited CaMKII phosphorylation of RyR2 under paced and non-paced conditions (, S4A-B
). In contrast, pacing increased phospholamban (PLN) phosphorylation at the CaMKII site T17 (), but not the PKA site S16 (Fig. S4C-D
) in both S2814D and WT mice, suggesting that enhanced SR Ca2+
loading due to SERCA2a stimulation facilitates VT induction in S2814D mice.
Finally, treatment with the β-adrenergic receptor blocker propranolol (3 mg/kg) did not significantly alter the incidence of ventricular arrhythmia induction (S2814D: 50%; WT: 0%) (). Because sudden changes in heart rate may induce a reflex sympathetic response and change in blood pressure, we also performed control experiments in which the arterial blood pressure was continuously monitored while the right ventricle was paced from 500 to 800 bpm at 100 bpm intervals. The blood pressure at a pacing rate of 800 bpm was not significantly higher (3.1 ± 0.1%; P = 0.88) than at 500 bpm, thus excluding that a reflex sympathetic response due to blood pressure changes is evoked in mice receiving EP studies.
To test whether CaMKII targets other than RyR2 may contribute to the observed arrhythmogenesis, we crossed S2814D mice with AC3I transgenic mice, in which the CaMKII-inhibitory peptide AC3I reduces CaMKII activity in the heart 17
. S2814D:AC3I double mutant mice experienced a slight decline in arrhythmia incidence (33% versus 53% in S2814D), suggesting that CaMKII effects on other targets, such as L-type Ca2+
or PLN might promote SR Ca2+
loading and arrhythmogenesis. However, when these experiments were repeated after injection of β-adrenergic receptor agonist isoproterenol (0.5 mg/kg, i.p.), the incidence of sustained VT was not reduced in S2814D-AC3I mice (66%) (). These results suggest that PKA activation and phosphorylation of Ca2+
handling proteins (e.g. PLN, L-type Ca2+
channel and RyR2) - even in the absence of CaMKII activation - can also enhance SR Ca2+
loading and promote arrhythmias in S2814D mice.
Previously, other groups have demonstrated that transgenic overexpression of CaMKII-δc
induces heart failure in mice 12–15
, and so we sought to define the specific role of CaMKII phosphorylation of RyR2 in progression to heart failure. As mentioned above, S2814D mice had no significant echocardiographic differences compared to WT littermates at 3 months of age (). However, at 12 months of age, S2814D mice demonstrated a significant increase in LV posterior wall diameter and end-diastolic diameter, and a small but significant decrease in ejection fraction (52.4 ± 1.8%) compared to WT mice (56.9 ± 0.7%; P =
0.005) (, , P
CaMKII phosphorylation of RyR2 causes cardiac dilation, loss of contractility, and early death from arrhythmias
Additionally, we performed transverse aortic constriction (TAC) in young (3–4 month old) S2814D and WT mice to evaluate the effects of constitutive CaMKII phosphorylation of RyR2 on the development of heart failure and arrhythmias. At 4 weeks post-TAC, CaMKII phosphorylation of RyR2 was not significantly elevated in WT mice (Fig. S5A-B
). Moreover, phosphorylation of S2808 on RyR2 was not altered in WT and S2814D mice after TAC (Fig. S5C-D
). However, survival was significantly lower for S2814D mice (40%) compared to WT mice (91%; P
= 0.02) 3 weeks following TAC surgery (). To determine whether the difference in survival was caused by arrhythmias, we repeated TAC studies in 5 WT and 5 S2814D mice in which a telemetric ECG transmitter was implanted 1 week prior to TAC. These studies revealed that the 2 S2814D mice in this group that died following TAC experienced episodes of ventricular arrhythmias immediately preceding death, whereas none of the WT mice died within 3 weeks after TAC (). These results implicate CaMKII phosphorylation of RyR2 as an important factor contributing to arrhythmogenesis and sudden death in heart failure.
Next, we assessed whether prevention of CaMKII-mediated phosphorylation of RyR2 at S2814 could ameliorate ventricular arrhythmogenesis in mice with HF. To test this hypothesis, we used knock-in mice in which S2814 of RyR2 is replaced by alanine (S2814A) to genetically inhibit CaMKII phosphorylation of RyR2 5
. Surgical TAC was performed in young (3–4 month old) WT and S2814A mice to induce HF. Following echocardiography at 8 weeks post-TAC, WT and S2814A mice were matched such that on average, both groups exhibited equal levels of cardiac dysfunction (see Table S4
). Similar to patients with heart failure 30
, WT mice subjected to TAC developed an increased propensity toward ventricular arrhythmias. Programmed electrical stimulation revealed that 75% (6 of 8) of WT mice developed non-sustained ventricular tachycardia following overdrive pacing at 8 weeks after TAC (). In contrast, only 14% (1 of 7) of S2814A mice developed non-sustained VT (P = 0.04). Western blots using a phospho-epitope specific antibody revealed increased CaMKII phosphorylation of RyR2 in WT mice following TAC, whereas the S2814 phosphorylation site could not be phosphorylated in S2814A mice as expected (). In contrast, phosphorylation of the PKA site S2808 was not altered in WT and S2814A mice compared to sham-operated controls (). These results suggest that CaMKII phosphorylation of S2814 on RyR2 is an essential signaling event that promotes ventricular arrhythmias in TAC-induced heart failure. Taken together, our data in S2814D and S2814A mice demonstrate that CaMKII phosphorylation of RyR2 at this site is critical for the development of cardiac arrhythmia.