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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 (HF(dys)) 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 HF(dys); however, CRT restored both. Phosphatase PP1 induced calcium desensitization in control and CRT-treated cells, while HF(dys) 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 HF(dys) and reactivated by CRT. Mass spectrometry of myofilament proteins from HF(dys) 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 HF(dys), but did not affect control or CRT cells. These data indicate that CRT improves calcium responsiveness of myofilaments following HF(dys) through GSK-3β reactivation, identifying a therapeutic approach to enhancing contractile function

SUBMITTER: Kirk JA

PROVIDER: S-EPMC3871225 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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

Kirk Jonathan A JA Holewinski Ronald J RJ Kooij Viola V Agnetti Giulio G Tunin Richard S RS Witayavanitkul Namthip N de Tombe Pieter P PP Gao Wei Dong WD Van Eyk Jennifer J Kass David A DA

The Journal of clinical investigation 20140101 1

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 tachypac ...[more]

PMID: 24292707

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Project description:Testosterone induces cardiac hypertrophy through a mechanism that involves a concerted crosstalk between cytosolic and nuclear signaling pathways. Nuclear factor of activated T-cells (NFAT) is associated with the promotion of cardiac hypertrophy, glycogen synthase kinase-3β (GSK-3β) is considered to function as a negative regulator, mainly by modulating NFAT activity. However, the role played by calcineurin-NFAT and GSK-3β signaling in testosterone-induced cardiac hypertrophy has remained unknown. Here, we determined that testosterone stimulates cardiac myocyte hypertrophy through NFAT activation and GSK-3β inhibition. Testosterone increased the activity of NFAT-luciferase (NFAT-Luc) in a time- and dose-dependent manner, with the activity peaking after 24 h of stimulation with 100 nM testosterone. NFAT-Luc activity induced by testosterone was blocked by the calcineurin inhibitors FK506 and cyclosporine A and by 11R-VIVIT, a specific peptide inhibitor of NFAT. Conversely, testosterone inhibited GSK-3β activity as determined by increased GSK-3β phosphorylation at Ser9 and β-catenin protein accumulation, and also by reduction in β-catenin phosphorylation at residues Ser33, Ser37, and Thr41. GSK-3β inhibition with 1-azakenpaullone or a GSK-3β-targeting siRNA increased NFAT-Luc activity, whereas overexpression of a constitutively active GSK-3β mutant (GSK-3βS9A) inhibited NFAT-Luc activation mediated by testosterone. Testosterone-induced cardiac myocyte hypertrophy was established by increased cardiac myocyte size and [3H]-leucine incorporation (as a measurement of cellular protein synthesis). Calcineurin-NFAT inhibition abolished and GSK-3β inhibition promoted the hypertrophy stimulated by testosterone. GSK-3β activation by GSK-3βS9A blocked the increase of hypertrophic markers induced by testosterone. Moreover, inhibition of intracellular androgen receptor prevented testosterone-induced NFAT-Luc activation. Collectively, these results suggest that cardiac myocyte hypertrophy induced by testosterone involves a cooperative mechanism that links androgen signaling with the recruitment of NFAT through calcineurin activation and GSK-3β inhibition.

Project description:BackgroundAltered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3β (glycogen synthase kinase 3β) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3β's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance.MethodsInducible cardiomyocyte-specific GSK-3β knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied.ResultsSkinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3β. Agreeing with the localization of its targets, GSK-3β that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3β in neonatal rat ventricular cardiomyocytes. One of GSK-3β's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3β. Last, GSK-3β myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA.ConclusionsWe identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in patients with heart failure, indicating z-disc localized GSK-3β is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

Project description:Oxidative stress is one of the main causes of osteoblast apoptosis induced by post‑menopausal osteoporosis. The authors previously found that metformin can reverse the loss of bone mass in post‑menopausal osteoporosis. The present study aimed to further clarify the effects and mechanisms of action of metformin in post‑menopausal osteoporosis under conditions of oxidative stress. Combined with an in‑depth investigation using the transcriptome database, the association between oxidative stress and mitochondrial dysfunction in post‑menopausal osteoporosis was confirmed. A pre‑osteoblast model of oxidative stress was constructed, and the apoptotic rate following the addition of hydrogen peroxide and metformin was detected using CCK‑8 assay and Annexin V‑FITC/PI staining. Mitochondrial membrane potential was detected using the JC‑1 dye, the intracellular calcium concentration was detected using Fluo‑4 AM, the intracellular reactive oxygen species (ROS) level was observed using DCFH‑DA, and the mitochondrial superoxide level was observed using MitoSOX Red. Bay K8644 was used to increase the level of intracellular calcium. siRNA was used to interfere with the expression of glycogen synthase kinase (GSK)‑3β. Western blot analysis was used to detect the expression of mitochondrial dysfunction‑related proteins. The results revealed that oxidative stress decreased mitochondrial membrane potential and increased intracellular ROS, mitochondrial superoxide and cytoplasmic calcium levels in pre‑osteoblasts; however, metformin improved mitochondrial dysfunction and reversed oxidative stress‑induced injury. Metformin inhibited mitochondrial permeability transition pore opening, suppressed the cytoplasmic calcium influx and reversed pre‑osteoblast apoptosis by promoting GSK‑3β phosphorylation. Moreover, it was found that EGFR was the cell membrane receptor of metformin in pre‑osteoblasts, and the EGFR/GSK‑3β/calcium axis played a key role in metformin reversing the oxidative stress response of pre‑osteoblasts in post‑menopausal osteoporosis. On the whole, these findings provide a pharmacological basis for the use of metformin for the treatment of post‑menopausal osteoporosis.

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

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

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