Jian Shan

Role of chronic ryanodine receptor phosphorylation in heart failure and β-adrenergic receptor blockade in mice

Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-S2808D+/+ mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-S2808D+/+ mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR Ca2+ store content, and increased diastolic SR Ca2+ leak. After myocardial infarction, RyR2-S2808D+/+ mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR Ca2+ leak and reduced HF progression in WT and RyR2-S2808D+/+ mice. In contrast, β-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-S2808D+/+ mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of β-blocker therapy in HF.

Role of CaMKIIδ phosphorylation of the cardiac ryanodine receptor in the force frequency relationship and heart failure

The force frequency relationship (FFR), first described by Bowditch 139 years ago as the observation that myocardial contractility increases proportionally with increasing heart rate, is an important mediator of enhanced cardiac output during exercise. Individuals with heart failure have defective positive FFR that impairs their cardiac function in response to stress, and the degree of positive FFR deficiency correlates with heart failure progression. We have identified a mechanism for FFR involving heart rate dependent phosphorylation of the major cardiac sarcoplasmic reticulum calcium release channel/ryanodine receptor (RyR2), at Ser2814, by calcium/calmodulin–dependent serine/threonine kinase–δ (CaMKIIδ). Mice engineered with an RyR2-S2814A mutation have RyR2 channels that cannot be phosphorylated by CaMKIIδ, and exhibit a blunted positive FFR. Ex vivo hearts from RyR2-S2814A mice also have blunted positive FFR, and cardiomyocytes isolated from the RyR2-S2814A mice exhibit impaired rate-dependent enhancement of cytosolic calcium levels and fractional shortening. The cardiac RyR2 macromolecular complexes isolated from murine and human failing hearts have reduced CaMKIIδ levels. These data indicate that CaMKIIδ phosphorylation of RyR2 plays an important role in mediating positive FFR in the heart, and that defective regulation of RyR2 by CaMKIIδ-mediated phosphorylation is associated with the loss of positive FFR in failing hearts.

Mitogen-Activated Protein Kinase Inhibitors Improve Heart Function and Prevent Fibrosis in Cardiomyopathy Caused by Mutation in Lamin A/C Gene

Background— Mutations in the lamin A/C gene, LMNA, can cause dilated cardiomyopathy. We have shown abnormal activation of the extracellular signal-regulated kinase (ERK) and the c-jun N-terminal kinase (JNK) branches of the mitogen-activated protein kinase signaling cascade in hearts from LmnaH222P/H222P mice that develop dilated cardiomyopathy. We recently showed that partial inhibition of ERK and JNK signaling before the onset of cardiomyopathy in LmnaH222P/H222P mice prevented the development of left ventricle dilatation and decreased cardiac ejection fraction at a time when they occurred in untreated mice. Methods and Results— To determine whether pharmacological inhibitors of ERK and JNK signaling could be clinically useful to treat cardiomyopathy caused by LMNA mutation, we administered them to LmnaH222P/H222P mice after they developed left ventricular dilatation and decreased ejection fraction. LmnaH222P/H222P mice were treated with ERK and JNK signaling inhibitors from 16 to 20 or, in pilot experiments, 19 to 24 weeks of age. The inhibitors blocked increased expression of RNAs encoding natriuretic peptide precursors and proteins involved in sarcomere architecture that occurred in placebo-treated mice. Echocardiography and histological analysis demonstrated that treatment prevented left ventricular end-systolic dilatation, increased ejection fraction, and decreased myocardial fibrosis. Conclusion— Inhibitors of ERK and JNK signaling could potentially be used to treat humans with cardiomyopathy caused by LMNA mutations.

Pharmacological inhibition of c-Jun N-terminal kinase signaling prevents cardiomyopathy caused by mutation in LMNA gene.

Mutations in LMNA, which encodes A-type nuclear lamins, cause disorders of striated muscle that have as a common feature dilated cardiomyopathy. We have demonstrated an abnormal activation of both the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) branches of the mitogen-activated protein kinase signaling cascade in hearts from Lmna(H222P/H222P) mice that develop dilated cardiomyopathy. We previously showed that pharmacological inhibition of cardiac ERK signaling in these mice delayed the development of left ventricle dilatation and deterioration in ejection fraction. In the present study, we treated Lmna(H222P/H222P) mice with SP600125, an inhibitor of JNK signalling. Systemic treatment with SP600125 inhibited JNK phosphorylation, with no detectable effect on ERK. It also blocked increased expression of RNAs encoding natriuretic peptide precursors and proteins involved in the architecture of the sarcomere that occurred in placebo-treated mice. Furthermore, treatment with SP600125 significantly delayed the development of left ventricular dilatation and prevented decreases in cardiac ejection fraction and fibrosis. These results demonstrate a role for JNK activation in the development of cardiomyopathy caused by LMNA mutations. They further provide proof-of-principle for JNK inhibition as a novel therapeutic option to prevent or delay the cardiomyopathy in humans with mutations in LMNA.

Reply to Eisner et al.: CaMKII phosphorylation of RyR2 increases cardiac contractility

The ryanodine receptor/calcium-release channel (RyR2) on the sarcoplasmic reticulum (SR) is the source of Ca2+ required for myocardial excitation–contraction (EC) coupling. During stress (i.e., exercise), contractility of the cardiac muscle is increased largely because of phosphorylation and activation of key proteins that regulate SR Ca2+ release. These include the voltage-gated calcium channel (Cav1.2) on the plasma membrane through which Ca2+ enters the cardiomyocyte, the sarco/endoplasmic reticulum calcium ATPase (SERCA2a)/phospholamban complex that pumps Ca2+ into the SR, and the RyR2 channel that releases Ca2+ from the SR, all of which are activated by phosphorylation.