Cardiac resynchronization sensitizes the sarcomere to calcium by reactivating GSK-3β
Jonathan A. Kirk, Ronald J. Holewinski, Viola Kooij, Giulio Agnetti, Richard S. Tunin, Namthip Witayavanitkul, Pieter P. de Tombe, Wei Dong Gao, Jennifer Van Eyk, David A. Kass
Jonathan A. Kirk, Ronald J. Holewinski, Viola Kooij, Giulio Agnetti, Richard S. Tunin, Namthip Witayavanitkul, Pieter P. de Tombe, Wei Dong Gao, Jennifer Van Eyk, David A. Kass
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Research Article Cardiology

Cardiac resynchronization sensitizes the sarcomere to calcium by reactivating GSK-3β

Abstract

Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function also increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance myofilament calcium responsiveness. To test this hypothesis, we examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony (HFdys) or CRT. Myofilament force-calcium relationships were measured in skinned trabeculae and/or myocytes. Compared with control, maximal calcium-activated force and calcium sensitivity declined globally in HFdys; however, CRT restored both. Phosphatase PP1 induced calcium desensitization in control and CRT-treated cells, while HFdys cells were unaffected, implying that CRT enhances myofilament phosphorylation. Proteomics revealed phosphorylation sites on Z-disk and M-band proteins, which were predicted to be targets of glycogen synthase kinase-3β (GSK-3β). We found that GSK-3β was deactivated in HFdys and reactivated by CRT. Mass spectrometry of myofilament proteins from HFdys animals incubated with GSK-3β confirmed GSK-3β–dependent phosphorylation at many of the same sites observed with CRT. GSK-3β restored calcium sensitivity in HFdys, but did not affect control or CRT cells. These data indicate that CRT improves calcium responsiveness of myofilaments following HFdys through GSK-3β reactivation, identifying a therapeutic approach to enhancing contractile function.

Authors

Jonathan A. Kirk, Ronald J. Holewinski, Viola Kooij, Giulio Agnetti, Richard S. Tunin, Namthip Witayavanitkul, Pieter P. de Tombe, Wei Dong Gao, Jennifer Van Eyk, David A. Kass

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Figure 5

Calcium sensitization is mediated by phosphorylation, but not at the known targets.

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Calcium sensitization is mediated by phosphorylation, but not at the kno...
(A) Representative force-calcium curves for controls, HFdys, and CRT before protein phosphatase-1 (PP1) and after treatment. (B) While there was no change in Fmax, PP1 caused a desensitization in the controls (n = 6) and CRT (n = 5) groups, but had no effect on HFdys (n = 5). (C) Top: phos-Tag gel blotted for TnI. Bottom: there was an increase in the unphosphorylated form and a decrease in the biphosphorylated form of TnI in the HFdys and CRT groups (n = 3), indicating overall dephosphorylation of TnI in HF. 0P, unphosphorylated; 1P, phosphorylation state 1; 2P, phosphorylation state 2. (D) Top: Western blot with antibodies against 3 phosphorylation sites on MyBPC. Bottom: both HFdys and CRT showed decreased phosphorylation at S273 and S282 (n = 3). (E) Top: Western blot against S15 phosphorylation on MLC2 and phospho-serine motif sites on MLC2. Bottom: HFdys (n = 4) and CRT (n = 4) were decreased from controls (n = 3), but no different from each other at S15, and there was no change in overall serine phosphorylation. (F) Top: phospho-motif antibodies for serine and threonine sites and total TnT. Bottom: there was very little phosphorylation of TnT in the 3 groups, with no differences among them (n = 3). *P < 0.05 vs. control by 1-way ANOVA.