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Automated imaging reveals a concentration dependent delay in reversibility of cardiac myocyte hypertrophy.

ABSTRACT: Cardiac hypertrophy is controlled by a dense signaling network with many pathways associated with cardiac myocyte growth. New large scale methodology is required to quantitatively characterize the pathways that distinguish reversible forms of hypertrophy from irreversible forms that lead to heart failure. Our automated image acquisition method records 5×5 mosaic images of fluorescent protein-labeled cardiac myocytes within each well of a 96-well plate using an automated stage and focus. Post-processing algorithms automatically identify cell edges, quantify cell phenotypes, and track cells. We uniquely applied our imaging platform to study hypertrophy reversibility in a scalable cell model. Cell area changes after washout of a dose response to the ?-adrenergic receptor (?AR) agonist phenylephrine (PE) showed that hypertrophy reverses at low but not high levels of ?-adrenergic signaling: a reversibility delay. Perturbations with specialized ?AR antagonists, a mathematical model, and live imaging of ?AR localization identify the mechanism for this reversibility delay: ligand trapping with internalized PE acting on intracellular ?AR's.

SUBMITTER: Ryall KA

PROVIDER: S-EPMC3389167 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Automated imaging reveals a concentration dependent delay in reversibility of cardiac myocyte hypertrophy.

Ryall Karen A KA Saucerman Jeffrey J JJ

Journal of molecular and cellular cardiology 20120508 2

Cardiac hypertrophy is controlled by a dense signaling network with many pathways associated with cardiac myocyte growth. New large scale methodology is required to quantitatively characterize the pathways that distinguish reversible forms of hypertrophy from irreversible forms that lead to heart failure. Our automated image acquisition method records 5×5 mosaic images of fluorescent protein-labeled cardiac myocytes within each well of a 96-well plate using an automated stage and focus. Post-pro ...[more]

PMID: 22575844

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Project description:RationaleStudies to dissect the role of calcineurin in pathological cardiac remodeling have relied heavily on murine models, in which genetic gain- and loss-of-function manipulations are initiated at or before birth. However, the great majority of clinical cardiac pathology occurs in adults. Yet nothing is known about the effects of calcineurin when its activation commences in adulthood. Furthermore, despite the fact that ventricular hypertrophy is a well-established risk factor for heart failure, the relative pace and progression of these 2 major phenotypic features of heart disease are unknown. Finally, even though therapeutic interventions in adults are designed to slow, arrest, or reverse disease pathogenesis, little is known about the capacity for spontaneous reversibility of calcineurin-dependent pathological remodeling.ObjectiveWe set out to address these 3 questions by studying mice engineered to harbor in cardiomyocytes a constitutively active calcineurin transgene driven by a tetracycline-responsive promoter element.Methods and resultsExpression of the mutant calcineurin transgene was initiated for variable lengths of time to determine the natural history of disease pathogenesis, and to determine when, if ever, these events are reversible. Activation of the calcineurin transgene in adult mice triggered rapid and robust cardiac growth with features characteristic of pathological hypertrophy. Concentric hypertrophy preceded the development of systolic dysfunction, fetal gene activation, fibrosis, and clinical heart failure. Furthermore, cardiac hypertrophy reversed spontaneously when calcineurin activity was turned off, and expression of fetal genes reverted to baseline. Fibrosis, a prominent feature of pathological cardiac remodeling, manifested partial reversibility.ConclusionsTogether, these data establish and define the deleterious effects of calcineurin signaling in the adult heart and reveal that calcineurin-dependent hypertrophy with concentric geometry precedes systolic dysfunction and heart failure. Furthermore, these findings demonstrate that during much of the disease process, calcineurin-dependent remodeling remains reversible.

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-3? (GSK-3?) is considered to function as a negative regulator, mainly by modulating NFAT activity. However, the role played by calcineurin-NFAT and GSK-3? signaling in testosterone-induced cardiac hypertrophy has remained unknown. Here, we determined that testosterone stimulates cardiac myocyte hypertrophy through NFAT activation and GSK-3? 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-3? activity as determined by increased GSK-3? phosphorylation at Ser9 and ?-catenin protein accumulation, and also by reduction in ?-catenin phosphorylation at residues Ser33, Ser37, and Thr41. GSK-3? inhibition with 1-azakenpaullone or a GSK-3?-targeting siRNA increased NFAT-Luc activity, whereas overexpression of a constitutively active GSK-3? mutant (GSK-3?S9A) 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-3? inhibition promoted the hypertrophy stimulated by testosterone. GSK-3? activation by GSK-3?S9A 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-3? inhibition.

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Automated imaging reveals a concentration dependent delay in reversibility of cardiac myocyte hypertrophy.

Publications

Automated imaging reveals a concentration dependent delay in reversibility of cardiac myocyte hypertrophy.

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