Accelerated Development of Pressure Overload-Induced Cardiac Hypertrophy and Dysfunction in an RyR2-R176Q Knockin Mouse Model

In response to chronic hypertension, the heart compensates by hypertrophic growth, which frequently progresses to heart failure. Although intracellular calcium (Ca(2+)) has a central role in hypertrophic signaling pathways, the Ca(2+) source for activating these pathways remains elusive. We hypothesized that pathological sarcoplasmic reticulum Ca(2+) leak through defective cardiac intracellular Ca(2+) release channels/ryanodine receptors (RyR2) accelerates heart failure development by stimulating Ca(2+)-dependent hypertrophic signaling. Mice heterozygous for the gain-of-function mutation R176Q/+ in RyR2 and wild-type mice were subjected to transverse aortic constriction. Cardiac function was significantly lower, and cardiac dimensions were larger at 8 weeks after transverse aortic constriction in R176Q/+ compared with wild-type mice. R176Q/+ mice displayed an enhanced hypertrophic response compared with wild-type mice as assessed by heart weight:body weight ratios and cardiomyocyte cross-sectional areas after transverse aortic constriction. Quantitative PCR revealed increased transcriptional activation of cardiac stress genes in R176Q/+ mice after transverse aortic constriction. Moreover, pressure overload resulted in an increased sarcoplasmic reticulum Ca(2+) leak, associated with higher expression levels of the exon 4 splice form of regulator of calcineurin 1, and a decrease in nuclear factor of activated T-cells phosphorylation in R176Q/+ mice compared with wild-type mice. Taken together, our results suggest that RyR2-dependent sarcoplasmic reticulum Ca(2+) leak activates the prohypertrophic calcineurin/nuclear factor of activated T-cells pathway under conditions of pressure overloa

Ryanodine Receptor Phosphorylation by Calcium/Calmodulin-Dependent Protein Kinase II Promotes Life-Threatening Ventricular Arrhythmias in Mice With Heart Failure

Background Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca(2+) remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results Mice in which the S2814 Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca(2+) release events, resulting in reduced sarcoplasmic reticulum Ca(2+) load on confocal microscopy. These Ca(2+) release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca(2+)/calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. Conclusions Our results suggest that Ca(2+)/calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca(2+) release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failur

Role of RyR2 Phosphorylation at S2814 During Heart Failure Progression

Rationale: Increased activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is thought to promote heart failure (HF) progression. However, the importance of CaMKII phosphorylation of ryanodine receptors (RyR2) in HF development and associated diastolic sarcoplasmic reticulum Ca(2+) leak is unclear. Objective: Determine the role of CaMKII phosphorylation of RyR2 in patients and mice with nonischemic and ischemic forms of HF. Methods and Results: Phosphorylation of the primary CaMKII site S2814 on RyR2 was increased in patients with nonischemic, but not with ischemic, HF. Knock-in mice with an inactivated S2814 phosphorylation site were relatively protected from HF development after transverse aortic constriction compared with wild-type littermates. After transverse aortic constriction, S2814A mice did not exhibit pulmonary congestion and had reduced levels of atrial natriuretic factor. Cardiomyocytes from S2814A mice exhibited significantly lower sarcoplasmic reticulum Ca(2+) leak and improved sarcoplasmic reticulum Ca(2+) loading compared with wild-type mice after transverse aortic constriction. Interestingly, these protective effects on cardiac contractility were not observed in S2814A mice after experimental myocardial infarction. Conclusions: Our results suggest that increased CaMKII phosphorylation of RyR2 plays a role in the development of pathological sarcoplasmic reticulum Ca(2+) leak and HF development in nonischemic forms of HF such as transverse aortic constriction in mic

Reduced glucose-sensitive Ca²⁺ pools and the putative mechanism underlying glucose-metabolism defects in S2814D mice.

<p>(<b>A</b>) Representative [Ca²⁺]_cyt tracings in islets isolated from WT and S2814D mice following stimulation with 5 µM thapsigargin (TG). (<b>B</b>) Quantification of [Ca²⁺]_cyt transient rise per sec in response to 5 µM TG in islets from WT and S2814D mice. Data are represented as average ± SEM. n = 19−24 islets from 3–5 mice per group. **<i>P</i><0.01 versus WT. (<b>C</b>) Mutation S2814D results in reduced glucose-sensitive Ca²⁺ pools in islets from S2814D mice. (1) Due to S2814D mutation, RyR2 leaks Ca²⁺ from intracellular Ca²⁺ stores in pancreatic β cells. (2) Chronic Ca²⁺ leak, in turn, leads to (dotted arrow) a decrease (downward arrow) in the glucose-sensitive Ca²⁺ pool in S2814D mice. (3) This decrease in the Ca²⁺ pool is reflected by a decrease in glucose-stimulated intracellular Ca²⁺ transients, which (4) blunts glucose-stimulated insulin secretion in S2814D mice.</p

Defective glucose-stimulated insulin secretion and Ca²⁺ transient in S2814D mice.

<p>(<b>A</b>) Insulin secretion from 4 sets of 5 islets each (each set from a different mouse, n = 20 islets from 4 mice per group) during 30-minute sequential exposures to glucose (2.8, 11 and 25 mM). Quantification revealed higher basal insulin secretion in islets from S2814D mice, which did not increase in response to glucose stimulation, compared to WT islets. (<b>B</b>) Representative tracings of [Ca²⁺]_cyt in WT and S2814D islets following glucose stimulation at varying concentrations (6, 8, and 10 mM). (<b>C</b>) Representative [Ca²⁺]_cyt tracings following stimulation with 40 mM KCl. (<b>D</b>) Quantification revealed blunted [Ca²⁺]_cyt transient amplitude in response to 10 mM glucose or 40 mM KCl in islets from S2814D mice compared to WT. In addition, frequency of [Ca²⁺]_cyt oscillations (<b>E</b>) and amplitude of [Ca²⁺]_cyt oscillations following initial peak (<b>F</b>) after 10 mM glucose stimulation were also reduced in S2814D islets. n = 6−14 islets from 3 mice per group. Data are represented as average ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 vs. WT; ^#<i>P</i><0.05, ^##<i>P</i><0.01 vs. 2.8 mM glucose.</p

Glucose intolerance and impaired glucose-induced insulin secretion in S2814D mice.

<p>(<b>A, B</b>) Glucose tolerance tests were conducted by injecting glucose (2 g/kg body weight) in WT and S2814D mice after a period of fasting for 6 hours. Quantifaction revealed glucose intolerance in S2814D mice as evidenced by higher blood glucose levels (<b>A</b>) and the lack of an insulin response (<b>B</b>). (<b>C</b>) Insulin tolerance tests were conducted by injecting insulin (0.75 U/kg body weight) after 6 h of fasting. (<b>D</b>) Acute phase insulin secretion in response to glucose (3 g/kg body weight) after 16 hours of fasting revealed a blunted response in S2814D mice. Data are represented as average ± SEM. N = 4−14 mice per group. **<i>P</i><0.01, *<i>P</i><0.05 vs. WT.</p

Increased CaMKII phosphorylation of RyR2 in type 2 diabetes.

<p>(<b>A, B</b>) Immunolabeling of sequential frozen human pancreatic sections from normal donors and type 2 diabetic donors revealed similar total RyR2 levels in β cells, but increased S2814 phosphorylation (pS2814) of RyR2 in diabetic donors. (<b>C, D</b>) Immunolabeling showing similar levels of S2808 phosphorylation (pS2808) of RyR2 in human β cells from normal and diabetic donors. Scale bars, 50 µm. (<b>E</b>) Bar graph showing quantification of S2814 and S2808 phosphorylation, normalized to total RyR2 signal, respectively, in human islets. Quantification revealed increased S2814 phosphorylation in human islets isolated from diabetic donors. n = 17−29 islets from 3 donors per group. ***<i>P</i><0.001 versus healthy donors. (<b>F</b>) Quantifications of RyR2 phosphorylation at S2814 and S2808 normalized to total RyR2 signal, respectively, also demonstrated increased levels of phosphorylated S2814 (pS2814) in high fat diet-fed HFD (type 2 diabetic) mice compared to low fat diet-fed (LFD) mice. Data are represented as average ± SEM. n = 17−29 islets from 3 donors per group, n = 18−22 islets from 3 mice per group. ***<i>P</i><0.001 vs. normal donor, **<i>P</i><0.01 vs. LFD.</p

Detection of CaMKII-mediated phosphorylation on RyR2 and determination of its sensitivity to glucose in INS-1 cells.

<p>(<b>A</b>) Quantification of qRTPCR analyses showing mRNA transcript levels of RyR1, RyR2 and RyR3 in islets from WT mice (represented in arbitrary units (A.U.)). (<b>B</b>) Representative Western blots for RyR2 expression and phosphorylated RyR2-S2814 in lysates of skeletal muscles (SM), heart (Ht), and brain (Br) from WT mice and rat insulinoma INS-1 cells (IS). β-actin was used to normalize protein concentrations. Observation indicated that RyR2 was present and could be phosphorylated in IS cells, comparable to the Ht. (<b>C</b>) Representative Western blots for RyR2 and its phosphorylation at S2814 from immunoprecipitates obtained from IS lysates using anti-RyR2 antibody. The lysates were prepared from INS-1 cells incubated with 2.8 mM (−) glucose or stimulated with 25 mM (+) glucose with and without pre-treatment with 10 µM KN-93. (<b>D</b>) Representative Western blots for CaMKII and its autophosphorylation at T287 from IS lysates generated in the above experiments. (<b>E</b>) Quantification revealed increased S2814 phosphorylation (pS2814) normalized to total RyR2 level upon glucose (25 mM) stimulation. Pre-treatment with 10 µM KN-93 blunted this increase. (<b>F</b>) Quantification also revealed increased autophosphorylation of CaMKII at T287 (pT287) normalized to total CaMKII level upon stimulation with 25 mM glucose but not in the presence of KN-93. Data (N = 3–6 experiments) are represented as average ± SEM. *<i>P</i><0.05, vs. 2.8 mM glucose; ^#<i>P</i><0.05 vs. 25 mM glucose.</p