Transcriptomic Sequencing Reveals a Biosignature for Myocardial Reductive Stress

levels correlate with biomarkers of early atherosclerosis such as carotid intima media thickness and blood pressure.

3

University of Utah, USA

G6PD-deficient cells are susceptible to OS, which can lead to

Background:

exhaustion/deficiency of GSH. We examined the hypothesis

(Nfe2l2/Nrf2) is a master regulator of antioxidant gene

that G6PD-deficiency decreases GSH and Nitric oxide (NO),

transcription. We recently identified that constitutive

and upregulates cell adhesion molecules (CAMs) which can

activation of Nrf2 (CaNrf2) caused reductive stress (RS) in

lead

and

the myocardium. Here, we investigate how chronic Nrf2

atherosclerosis risk. G6PD deficiency in HUVEC and U937

activation alters the mRNA transcriptome in the hearts of

monocytes was induced by using siRNA or by its

CaNrf2 transgenic mice using an unbiased integrated

to

excess

pharmacological

endothelial

inhibitor

dysfunction

(6-AN).

(ED)

G6PD-deficiency

The

nuclear

factor

erythroid

2

like

2

systems approach and next generation RNA sequencing

exhibited a dose-dependent increase in CAMs (ICAM-1 &

(RNAseq) followed by qRT-PCR methods.

VCAM-1), NADPH Oxidase 4, iNOS, MCP-1 and ROS, and

Methods: Cardiac-specific transgenic mice expressing the

reduction in GSH, eNOS and NO levels (p<0.05) and an

constitutively active form of Nrf2 were generated using a

increase in monocyte adhesion to EC [119.3%]. Similarly,

construct lacking a functional Neh2 domain responsible for

GSH deficiency (induced by siRNA or by its chemical

Keap1 repression. Founder lines expressing the transgene at

inhibitor BSO) caused increased expression of adhesion

low (TGL) and high (TGH) levels were physically separated

molecules and monocyte adhesivity to EC [107.6%]. G6PD-

and distinct colonies were maintained. RNA was isolated

deficient cells showed greater (p<0.05) increase in CAMs

from 6-8 month male non-transgenic (NTG), TGL and TGH

after

hearts (n=3-4/group) and subjected to 50 cycle single read

stimulation with HG [15.13%], TNF-α [18.49%], and MCP-1

sequencing using Illumina’s HiSeq 2000. Reads were aligned

and

monocyte-EC

[62.19%]

adherence

respectively

[10.08%],

compared

with

and

G6PD-normal

using Novoalign and counts were used in DESeq to

stimulated cells. In addition, L-cysteine (LC) treatment (a

determine differential expression. Differentially expressed

precursor of GSH) protects HUVEC and monocytes by

genes (DEGs) in TGL and TGH groups were filtered through

increasing GSH; attenuating ROS, CAMs and monocyte-EC

Ingenuity Pathway Analysis (IPA). A total of 4 genes within

adhesion (p<0.05). These results provide a role of

9 enriched biological processes were validated by qRT-PCR.

G6PD/GSH deficiency in the vascular dysfunction and the

Results: A total of 246 and 1031 DEGs were identified in the

events leading to excess atherosclerosis, and that LC

hearts of TGL and TGH mice relative to NTG littermates.

supplementation can reduce the atherosclerosis risk in

Notably, the expression and validation of the transcripts

G6PD-deficienct conditions.

were gene-dosage dependent and statistically significant

Supported by Malcolm W. Feist Cardiovascular Research

(p<0.05).

Fellowship and Endowed Chair in Diabetes from CCDS,

functions

LSUHSC, Shreveport.

myocardial RS

IPA and

identified canonical in

CaNrf2

overrepresented pathways

biological

associated

mice. Enriched

with

xenobiotic

metabolic signaling, glutathione mediated detoxification, DOI: 10.1016/j.freeradbiomed.2017.10.180

unfolded protein response, and protein ubiquitination was observed. Other signaling pathways identified included: eNOS,

integrin-linked

kinase,

glucocorticoid

receptor,

PI3/AKT, actin cytoskeleton, cardiac hypertrophy, and the endoplasmic reticulum stress response.

168

Conclusion: This mRNA profiling identified a "biosignature" for pro-reductive (TGL) and reductive stress (TGH) that can

Transcriptomic Sequencing Reveals a

predict the onset, rate of progression, and clinical outcome

Biosignature for Myocardial Reductive

of Nrf2-dependent myocardial complications. We anticipate

Stress

that this global sequencing analysis will illuminate the

Justin Quiles1, Madhusudhanan Narasimhan2, Timothy

mediated

Mosbruger3, Gobinath Shanmugam1, David Crossman1, and Namakkal S Rajasekaran1,3 1

University of Alabama at Birmingham, USA

2

Texas Tech University Health Sciences Center, USA

120

undesirable effect of chronic Nrf2 signaling leading to RS pathogenesis

besides

providing

important

guidance for the application of Nrf2 activation-based cytoprotective strategies. DOI: 10.1016/j.freeradbiomed.2017.10.181

SfRBM 2017