Phenotypic screen quantifying differential regulation of cardiac myocyte hypertrophy identifies CITED4 regulation of myocyte elongation

Abstract

Cardiac hypertrophy is controlled by a highly connected signaling network with many effectors of cardiac myocyte size. Quantification of the contribution of individual pathways to specific changes in shape and transcript abundance is needed to better understand hypertrophy signaling and to improve heart failure therapies. We stimulated cardiac myocytes with 15 hypertrophic agonists and quantitatively characterized differential regulation of 5 shape features using high-throughput microscopy and transcript levels of 12 genes using qPCR. Transcripts measured were associated with phenotypes including fibrosis, cell death, contractility, proliferation, angiogenesis, inflammation, and the fetal cardiac gene program. While hypertrophy pathways are highly connected, the agonist screen revealed distinct hypertrophy phenotypic signatures for the 15 receptor agonists. We then used k-means clustering of inputs and outputs to identify a network map linking input modules to output modules. Five modules were identified within inputs and outputs with many maladaptive outputs grouping together in one module: Bax, C/EBPβ, Serca2a, TNFα, and CTGF. Subsequently, we identified mechanisms underlying two correlations revealed in the agonist screen: correlation between regulators of fibrosis and cell death signaling (CTGF and Bax mRNA) caused by AngII; and myocyte proliferation (CITED4 mRNA) and elongation caused by Nrg1. Follow-up experiments revealed positive regulation of Bax mRNA level by CTGF and an incoherent feedforward loop linking Nrg1, CITED4 and elongation. With this agonist screen, we identified the most influential inputs in the cardiac hypertrophy signaling network for a variety of features related to pathological and protective hypertrophy signaling and shared regulation among cardiac myocyte phenotypes.

Computational modeling of cardiac fibroblasts and fibrosis

“…Computational models can help identify the mechanisms by which these drugs suppress fibrosis and can identify therapeutic strategies that could be more targeted and efficacious against cardiac fibrosis. Computational models of cardiomyocyte signaling have successfully been used to predict which signaling nodes are most important for cardiomyocyte hypertrophy in vitro[105,106] or how phenomena such as compartmentalization affect hypertrophy[107,108]. Markov models are also useful for determining the effect of ion channel mutations on electrical conduction[109].…”

Size matters: Finding growth pathways that protect the heart

“…For example, the authors confirmed that CT1 increases the length-width ratios of cells, in contrast to the pattern seen with phenylephrine, which they use as a pathological stimulus. In fact, cardiomyocyte elongation is seen in pathological hypertrophy and notable physiological growth pathways (e.g., neuregulin-1 and CITED4) inhibit elongation [6]. Moreover, phenylephrine itself likely also activates physiological growth pathways given the role of α-adrenergic signaling in postnatal cardiac growth [7].…”

Metabolic regulation of myocardial adaptation to exercise.

“…Exercise training downregulated Cebpb and upregulated Cited4, an observation which is consistent with previous findings showing necessary involvement of these transcriptional mediators in regulating physiologic growth. 45,46,[217][218][219] Moreover, the expression of Nfatc2, the deletion of which ameliorates pathological hypertrophy and heart failure but does not prevent physiological hypertrophy, 231 was markedly lower in hearts from exercise-adapted mice (Fig. 11F).…”

“…Expression of a dominantnegative form of PFK2 (Glyco Lo ) in the heart decreased glycolytic rate and was sufficient to modulate the Cebpb/Cited4 transcriptional program that regulates physiologic cardiac growth. 45,46,219 Mice in which myocardial glycolytic rate is constitutively high also showed slight hypertrophy, but these hearts did not engage the physiologic growth gene program. The corollary of these findings is that exercise-induced changes in phosphofructokinase activity are necessary to activate transcriptional programs dictating cardiac growth and hypertrophy.…”

“…27,218 For example, C/EBPβ and CITED4 are known to be critical for regulating the transcriptional programs that instigate cardiac growth 45,46,219 ; however, the mechanisms by which such transcriptional programs are activated with exercise are unclear. In tissues such as skeletal muscle, adaptive changes in gene expression are thought to be initiated by the repetitive bouts of metabolic stress triggered by strenuous physical activity.…”