CRISPR-Cas9–Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin—Brief Report

Lyu, Qing; Dhagia, Vidhi; Han, Yu; Guo, Bing; Wines-Samuelson, Mary; Christie, Christine K.; Yin, Qin; Slivano, Orazio J.; Herring, Paul; Long, Xiaochun; Gupte, Sachin A.; Miano, Joseph M.

doi:10.1161/atvbaha.118.311171

Cited by 27 publications

(22 citation statements)

References 38 publications

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“…It has been shown that the specific transcriptional regulation of these contractile genes in VSMCs is dependent on complex interactions of multiple cis ‐elements and their trans ‐binding factors . Among these factors, myocardin, an extremely potent SRF co‐activator, has been shown to selectively induce expressions of all CArG‐dependent VSMC marker genes . Our study herein provided evidences that two TRIM family members, TRIM28 and TRIM27, are novel regulators of SRF and myocardin in human VSMCs.…”

Section: Discussionsupporting

confidence: 54%

TRIM28 and TRIM27 are required for expressions of PDGFRβ and contractile phenotypic genes by vascular smooth muscle cells

et al. 2020

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Vascular smooth muscle cells (VSMCs) in the normal arterial media continually express contractile phenotypic markers which are reduced dramatically in response to injury. Tripartite motif-containing proteins are a family of scaffold proteins shown to regulate gene silencing, cell growth, and differentiation. We here investigated the biological role of tripartite motif-containing 28 (TRIM28) and tripartite motif-containing 27 (TRIM27) in VSMCs. We observed that siRNA-mediated knockdown of TRIM28 and TRIM27 inhibited platelet-derived growth factor (PDGF)-induced migration in human VSMCs. Both TRIM28 and TRIM27 can regulate serum response element activity and were required for maintaining the contractile gene expression in human VSMCs. At the same time, TRIM28 and TRIM27 knockdown reduced the expression of PDGF receptor-β (PDGFRβ) and the phosphorylation of its downstream signaling components. Immunoprecipitation showed that TRIM28 formed complexes with TRIM27 through its N-terminal RING-B boxes-Coiled-Coil domain.Furthermore, TRIM28 and TRIM27 were shown to be upregulated and mediate the VSMC contractile marker gene and PDGFRβ expression in differentiating human bone marrow mesenchymal stem cells. In conclusion, we identified that TRIM28 and TRIM27 cooperatively maintain the endogenous expression of PDGFRβ and contractile phenotype of human VSMCs.

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Section: Discussionsupporting

confidence: 54%

TRIM28 and TRIM27 are required for expressions of PDGFRβ and contractile phenotypic genes by vascular smooth muscle cells

et al. 2020

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“…While this manuscript is in review, Lyu et al, reported a size variability for MYOCD protein. Further studies using the antibodies reported by Lyu et al will further confirm our findings 24 .…”

Section: Acknowledgement and Funding Sourcessupporting

confidence: 83%

Myocardin ablation in a cardiac-renal rat model

Mittal

Rana

Sharma

et al. 2018

Preprint

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34Cardiorenal syndrome is defined by primary heart failure conditions influencing or 35 leading to renal injury or dysfunction. Dilated cardiomyopathy (DCM) is a major co-36 existing form of heart failure (HF) with renal diseases. Myocardin (MYOCD), a 37 cardiac-specific co-activator of serum response factor (SRF), is increased in DCM 38 mice and patient cardiac tissues and plays a crucial role in the pathophysiology of 39 DCM. Inhibiting the increased MYOCD has shown to be partially rescuing the DCM 40 mice phenotype. However, expression levels of MYOCD in the cardiac tissues of the 41 cardiorenal syndromic patients and the beneficial effect of inhibiting MYOCD in a 42 cardiorenal syndrome model remains to be explored. 43Here, we analyzed the expression levels of MYOCD in the DCM patients with and 44 without renal diseases. We also explored, whether cardiac specific silencing of 45 MYOCD expression could ameliorate the cardiac remodeling and improve cardiac 46 function in a renal artery ligated rat model (RAL). We observed an increase in 47 homing peptide conjugated MYOCD siRNA resulted in attenuation of cardiac 50 hypertrophy, fibrosis and restoration of the left ventricular functions. 51Our data suggest hyper-activation of MYOCD in the pathogenesis of the cardiorenal 52 failure cases. Also, MYOCD silencing showed beneficial effects by rescuing cardiac 53 hypertrophy, fibrosis, size and function in a cardiorenal rat model. 54 55Introduction 56 DCM is a major cause of HF, 1 accounting for nearly 1/3rd of total cases. Many of 57 these patients subsequently display kidney dysfunction or injury leading to 58 cardiorenal syndrome. More than half of the heart failure patients show renal 59 diseases. Co-existence of cardiac and renal dysfunction in the patients increases the 60 mortality significantly compared to cardiac or renal disease alone patients. 61Various molecular pathways including Renin-angiotensin-aldosterone system (RAAS) 62 are shown to be influencing the cardiorenal syndrome. Notably, circulating Ang II (an 63 essential component of RAAS) affects cardiac function by, increasing systemic 64 arteriolar vasoconstriction, vascular resistance, and cardiac afterload through AT1 65 receptor-mediated endothelial dysfunction 2 . 66Ang II has been shown to induce MYOCD under hypoxic condition 3 . MYOCD is a 67 cardiac-specific transcriptional co-activator present in cardiomyocytes and smooth 68 muscle cells. MYOCD is involved in heart development and cardiomyocyte 69 differentiation 4,5 . Also, MYOCD is required for maintenance of structural integrity, 70 cardiomyocyte survival, and heart function 5-7 . Besides, cardiomyocytes and smooth 71 muscle cells MYOCD expression has been detected in fibroblasts 8 . MYOCD has 72 been shown to promote fibroblast to myofibroblast differentiation and to inhibit cell 73 proliferation 9 . Forced expression of MYOCD in fibroblasts induces cardio-myogenic 74 4 properties alone 8 and/or in combination with other factors 10 . Transforming growth 75 factor (TGF-β) was shown to induc...

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“…Additionally, infection of hMA rings with a Lv-ccMYOCD induced knock-down of myocardin ( Figure 6C), together with a pronounced vessel remodeling in 0% FBS ( Figure 6D). Due to the concerns regarding specificity of myocardin antibodies 35 , we also explored expression of other myocardin target genes (MYHII and SM22), which were also decreased. (Supplemental Figure VIII).…”

Section: Resultsmentioning

confidence: 99%

Myocardin-Dependent Kv1.5 Channel Expression Prevents Phenotypic Modulation of Human Vessels in Organ Culture

Arévalo-Martínez

Cidad

García-Mateo

et al. 2019

ATVB

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Objective: We have previously described that changes in the expression of Kv channels associate to phenotypic modulation (PM), so that Kv1.3 /Kv1.5 ratio is a landmark of vascular smooth muscle cells (VSMCs) phenotype. Moreover, we demonstrated that the Kv1.3 functional expression is relevant for PM in several types of vascular lesions. Here, we explore the efficacy of Kv1.3 inhibition for the prevention of remodeling in human vessels, and the mechanisms linking the switch in Kv1.3 /Kv1.5 ratio to PM. Approach and Results: Vascular remodeling was explored using organ culture and primary cultures of VSMCs obtained from human vessels. We studied the effects of Kv1.3 inhibition on serum-induced remodeling, as well as the impact of viral vectormediated overexpression of Kv channels or myocardin knock-down. Kv1.3 blockade prevented remodeling by inhibiting proliferation, migration and extracellular matrix (ECM) secretion. PM activated Kv1.3 via downregulation of Kv1.5. Hence, both Kv1.3 blockers and Kv1.5 overexpression inhibited remodeling in a non-additive fashion. Finally, myocardin knock-down induced vessel remodeling and Kv1.5 downregulation and myocardin overexpression increased Kv1.5, while Kv1.5 overexpression inhibited PM without changing myocardin expression. Conclusions: We demonstrate that Kv1.5 channel gene is a myocardin-regulated, VSMCs contractile marker. Kv1.5 downregulation upon PM leaves Kv1.3 as the dominant Kv1 channel expressed in dedifferentiated cells. We demonstrated that the inhibition of Kv1.3 channel function with selective blockers or by preventing Kv1.5 downregulation can represent an effective, novel strategy for the prevention of intimal hyperplasia and restenosis of the human vessels used for coronary angioplasty procedures.

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CRISPR-Cas9–Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin—Brief Report

Cited by 27 publications

References 38 publications

TRIM28 and TRIM27 are required for expressions of PDGFRβ and contractile phenotypic genes by vascular smooth muscle cells

TRIM28 and TRIM27 are required for expressions of PDGFRβ and contractile phenotypic genes by vascular smooth muscle cells

Myocardin ablation in a cardiac-renal rat model

Myocardin-Dependent Kv1.5 Channel Expression Prevents Phenotypic Modulation of Human Vessels in Organ Culture

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