Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy

Huang, Weei‐Yuarn; Aramburu, J.; Douglas, Pamela S.; Izumo, Seigo

doi:10.1038/74914

Cited by 228 publications

(194 citation statements)

References 6 publications

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“…Cellular stress responses have also been reported in cultured cells stably expressing GFP (Zhang et al, 2003), and eGFP expression has also been reported to increase production of superoxide and hydrogen peroxide (Ganini et al, 2017). Adverse effects have also been observed in transgenic GFP animals (Devgan et al, 2004;Huang et al, 2000;Mawhinney and Staveley, 2011). Therefore, despite GFP tagging of proteins driving huge advances in our understanding of biological processes, GFP is not as inert as previously assumed.…”

Section: Introductionmentioning

confidence: 90%

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

Norris

Yang

Krycer

et al. 2017

Journal of Cell Science

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Akt is a key node in a range of signal transduction cascades and play a critical role in diseases such as cancer and diabetes. Fluorescentlytagged Akt reporters have been used to discern Akt localisation, yet it has not been clear how well these tools recapitulate the behaviour of endogenous Akt proteins. Here, we observed that fusion of eGFP to Akt2 impaired both its insulin-stimulated plasma membrane recruitment and its phosphorylation. Endogenous-like responses were restored by replacing eGFP with TagRFP-T. The improved response magnitude and sensitivity afforded by TagRFP-T-Akt2 over eGFP-Akt2 enabled monitoring of signalling outcomes in single cells at physiological doses of insulin with subcellular resolution and revealed two previously unreported features of Akt biology. In 3T3-L1 adipocytes, stimulation with insulin resulted in recruitment of Akt2 to the plasma membrane in a polarised fashion. Additionally, we observed oscillations in plasma membrane localised Akt2 in the presence of insulin with a consistent periodicity of 2 min. Our studies highlight the importance of fluorophore choice when generating reporter constructs and shed light on new Akt signalling responses that may encode complex signalling information.This article has an associated First Person interview with the first author of the paper.

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Section: Introductionmentioning

confidence: 90%

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

Norris

Yang

Krycer

et al. 2017

Journal of Cell Science

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“…By injecting a construct of mouse cardiac-myosin heavy chain promoter fused with GFP, Huang et al (2000) generated transgenic mice lines that expressed GFP in the heart; however, those mice suffered from severely dilated cardiomyopathy. The zebrafish is an excellent animal model for studying cardiovascular development in vertebrates (Fishman and Stainier, 1994;Fishman and Chien, 1997;Alexander et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

Germ‐line transmission of a myocardium‐specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish

Huang

Hsiao

et al. 2003

Developmental Dynamics

476

433

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In response to the lack of a transgenic line of zebrafish labeled with heart-specific fluorescence in vivo to serve as a research model, we cloned a 1.6-kb polymerase chain reaction (PCR) -product containing the upstream sequence (؊870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene, (cmlc2). A germ-line transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the green fluorescent protein (GFP) gene with ends consisting of inverted terminal repeats of an adeno-associated virus. Green fluorescence was intensively and specifically expressed in the myocardial cells located both around the heart chambers and the atrioventricular canal. Neither the epicardium nor the endocardium showed fluorescent signals. The GFP expression in the transgenic line faithfully recapitulated with the spatial and temporal expression of the endogenous cmlc2. Promoter analysis showed that the fragment consisting of nucleotides from ؊210 to 34 (؊210/34) was sufficient to drive heart-specific expression, with a ؊210/؊73 motif as a basal promoter and a ؊210/؊174 motif as an element involved in suppressing ectopic (nonheart) expression. Interestingly, a germ-line of zebrafish whose GFP appeared ectopically in all muscle types (heart, skeletal, and smooth) was generated by injecting the fragment including a single nucleotide mutation from G to A at ؊119, evidence that A at ؊119 combined with neighboring nucleotides to create a consensus sequence for binding myocyte-specific enhancer factor-2. Developmental Dynamics 228:30 -40, 2003.

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“…84 In contrast, a recent study by Beggah et al 85 reported that expression of MR antisense, and thus knockdown of the MR in cardiac myocytes, produced severe cardiac hypertrophy and fibrosis that was made worse with spironolactone treatment. It has been suggested that this response is not a specific effect of loss of MR signaling but was in large part attributable to the overexpression of a foreign protein in the myocytes, given that very similar heart failure and pathology was seen in cardiac myocyte overexpression of green fluorescent protein 86 or an inflammatory response to the use of antisense. It is also important also to note that these results are inconsistent with the heterozygous MR knockout mice, which have no cardiac phenotype, 9 and to the 11␤HSD2 cardiac-expressing mice, 65 in which an appropriate response to spironolactone is observed.…”

Section: Fuller and Youngmentioning

confidence: 99%

Mechanisms of Mineralocorticoid Action

Fuller¹,

Young²

2005

Hypertension

277

169

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Abstract-Sodium transport in epithelial tissues is regulated by the physiological mineralocorticoid aldosterone. The response to aldosterone is mediated by the mineralocorticoid receptor (MR), for which the crystal structure of the ligand-binding domain has recently been established. The classical mode of action for this receptor involves the regulation of gene transcription. Several genes have now been shown to be regulated by aldosterone in epithelial tissues. Of these, the best characterized is serum-and glucocorticoid-regulated kinase, which increases sodium influx through the epithelial sodium channel. Turnover of these channels in the cell membrane is mediated by Nedd4 -2, a ubiquitin protein ligase; serum-and glucocorticoid-regulated kinase interacts with and phosphorylates Nedd4 -2, thereby rendering it unable to bind the sodium channels. Key Words: aldosterone Ⅲ sodium channels Ⅲ fibrosis T he isolation of aldosterone just over 50 years ago established it as the primary physiological mineralocorticoid. 1 Sodium transport, and hence salt balance, is regulated by a number of mechanisms; however, aldosterone has a critical role. Aldosterone exerts its effects on the distal nephron (and colon) as the last point of sodium reabsorption; it is thus the final arbiter. 2,3 The importance of aldosterone in the maintenance of sodium homeostasis is seen in a series of monogenetic causes of hypertension, 2 in Conn's syndrome, and in disorders in which mineralocorticoid action is compromised with consequent, life-threatening salt losses. 3 Although other pathophysiological consequences of aldosterone excess were identified by Selye 4 over 60 years ago, their significance has only recently been appreciated. Evidence from the Randomized ALdactone Evaluation Study (RALES) and EPlerenone neuroHormonal Efficacy and SUrvival Study (EPHESUS) trials of beneficial effects of mineralocorticoid antagonist therapy on morbidity and mortality in cardiac failure has focused attention beyond the kidney to effects of mineralocorticoids more broadly in the cardiovascular system. 5 Although an understanding of the molecular basis of aldosterone action has tended to lag behind advances in the biology of other steroid hormones, the last decade has seen significant advances toward an understanding of the mechanisms of mineralocorticoid action. The primary mediator of the response to aldosterone is the mineralocorticoid receptor (MR), the ligand-binding domain (LBD) of which has been recently crystallized. 6 -8 Although the MR primarily acts as a transcription factor, recent evidence suggests that it may also mediate nongenomic (or nonnuclear) activation of second messenger pathways. In addition, there is a growing body of evidence that some actions of aldosterone may involve a receptor other than the MR. The cellular and molecular mediators, including proteins induced by aldosterone, have been characterized in sodium transporting epithelia; however, the critical molecular events in the vasculature remain to be determined. In this brief r...

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Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy

Cited by 228 publications

References 6 publications

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

Germ‐line transmission of a myocardium‐specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish

Mechanisms of Mineralocorticoid Action

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