Phosphorylation of the ryanodine receptor mediates the cardiac fight or flight response in mice
Jian Shan, … , Peter J. Mohler, Andrew R. Marks
Jian Shan, … , Peter J. Mohler, Andrew R. Marks
Published November 22, 2010
Citation Information: J Clin Invest. 2010;120(12):4388-4398. https://doi.org/10.1172/JCI32726.
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Research Article

Phosphorylation of the ryanodine receptor mediates the cardiac fight or flight response in mice

Abstract

During the classic “fight-or-flight” stress response, sympathetic nervous system activation leads to catecholamine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium (Ca2+). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-S2808A+/+ mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte Ca2+ transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-S2808A+/+ mice. The blunted cardiac response to catecholamines in RyR2-S2808A+/+ mice resulted in impaired exercise capacity. RyR2-S2808A+/+ mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation.

Authors

Jian Shan, Alexander Kushnir, Matthew J. Betzenhauser, Steven Reiken, Jingdong Li, Stephan E. Lehnart, Nicolas Lindegger, Marco Mongillo, Peter J. Mohler, Andrew R. Marks

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Figure 7

Chronic β-adrenergic stimulation causes cardiac dysfunction and remodeling of the RyR2 channel complex.

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Chronic β-adrenergic stimulation causes cardiac dysfunction and remodeli...
(A) Echocardiography of left ventricular EF (A) and LVESD (B) in WT mice and RyR2-S2808A+/+ mice during chronic Iso treatment (n = 6 in both groups; mean ± SEM; *P < 0.05 versus WT; #P < 0.05 versus WT baseline). (C) Equivalent amounts of RyR2 were immunoprecipitated with RyR2-specific antibody followed by immunoblotting for relative PKA phosphorylation of RyR2 at Ser2808, oxidation (DNP), and of calstabin2 bound to RyR2. Immunoblotting of PLN- and PKA-phosphorylated PLN was performed on whole heart lysates from WT and RyR2-S2808A+/+ mice. (D and E) Quantification summaries. Mice were continuously treated with Iso for 56 days at a dose of 30 mg/kg/d (WT, n = 3; RyR2-S2808A+/+, n = 3; mean ± SD; *P < 0.05). (F) Aliquots of cardiac lysates from WT mice treated with Iso for 1 or 28 days were incubated at 37°C before the addition of phosphatase inhibitors at the indicated times (top) to stop the RyR2 dephosphorylation reaction. (G) Quantification of RyR2 PKA phosphorylation from F (*P < 0.05). (H) Cardiac RyR2 single-channel activity measured from microsomes from Iso-treated WT and (I) RyR2-S2808A+/+ mice. Channel openings are upward, “C” indicates closed state of the channel, and dotted lines indicate levels of partial openings or subconductance states. Top dash indicates the fully open 4 pA level. The top tracings are condensed time scale (5 s) and the bottom tracings are expanded time scale (500 ms). Amplitude histograms are shown for each channel. To, average open time; Tc, average closed time. (J) Summary of WT (n = 6 channels from 3 mice) and RyR2-S2808A+/+ (n = 8 channels from 3 mice) Po. *P < 0.05.