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Impaired cardiac anti-oxidant activity in diabetes: human and correlative experimental studies

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Abstract

Increased reactive oxygen species (ROS) are traditionally viewed as arising from the metabolic flux of diabetes, although reduction in the activity of anti-oxidant systems has also been implicated. Among the latter is the major thiol reducing thioredoxin system, the activity of which may be diminished by high glucose-induced expression of its endogenous inhibitor, thioredoxin interacting protein (TxnIP). We assessed TxnIP mRNA/protein expression along with thioredoxin activity in human right atrial biopsy specimens from subjects with and without diabetes undergoing coronary artery grafting. In correlative experimental studies, we examined TxnIP expression in both type 1 and type 2 rodent models of diabetic cardiomyopathy. Finally, we used in vitro gene silencing to determine the contribution of changes in TxnIP abundance to the high glucose-induced reduction in thioredoxin activity. In human right atrial biopsies, diabetes was associated with a >30-fold increase in TxnIP gene expression and a 17 % increase in TxnIP protein expression (both p < 0.05). This was associated with a 21 % reduction in thioredoxin activity when compared to human non-diabetic cardiac biopsy samples (all p < 0.05). In correlative animal studies, both type 1 and type 2 diabetic rats demonstrated a significant increase in TxnIP mRNA and reduction in thioredoxin activity when compared to non-diabetic animals (all p < 0.05). This was associated with a significant increase in ROS (p < 0.05 when compared with control). In cultured cardiac myocytes, high glucose increased ROS and TxnIP mRNA expression, in association with a reduction in thioredoxin activity (p < 0.01). These findings were abrogated by TxnIP small interfering RNA (siRNA). Scrambled siRNA had no effect upon ROS or TxnIP expression. High glucose reduces thioredoxin activity and increases ROS via TxnIP overexpression. These findings suggest that impaired thiol reductive capacity, through altered TxnIP expression, contributes to increased ROS in the diabetic heart.

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Abbreviations

ATCC:

American type culture collection

ANOVA:

Analysis of variance

DMEM:

Dulbecco’s modified Eagle’s medium

EDP:

End-diastolic pressure

EDPVR:

The slope of the end-diastolic pressure volume relationship

EDV:

End-diastolic volume

ESP:

End systolic pressure

FBS:

Fetal bovine serum

NADPH:

Nicotinamide adenine dinucleotide phosphate

OCT:

Optimal cutting temperature

PCR:

Polymerase chain reaction

PLSD:

Fishers protected least significant difference

PRSW:

The slope of the preload recruitable stroke work relationship

PV:

Left ventricular pressure–volume loop

RAS:

Renin-angiotensin system

Ren-2:

TGR(mRen-2)27 transgenic (Ren-2) rats

RVU:

Relative volume unit

ROS:

Reactive oxygen species

SiRNA:

Small interfering RNA

STZ:

Streptozotocin

SW:

Stroke work

TRX:

Thioredoxin

TxnIP:

Thioredoxin interacting protein

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Acknowledgments

These studies were supported by a HSF Grant-in-aid #000484, #7201 and a Grant from the J. P Bickell Foundation. Dr. Kim Connelly was supported by a HSF Canada Phase 1 clinician scientist award. Dr. Richard Gilbert is the Canada Research Chair in Diabetes Complications and this study was supported, in part, by the Canada Research Chair Program. Dr. Advani is supported by a Canadian Diabetes Association Clinician Scientist Award. Imaging for this study work was carried out at the BioImaging Facility of St. Michael’s Hospital, Toronto, Canada.

Conflict of interest

Kim A Connelly, Andrew Advani, Suzanne L. Advani, Yuan Zhang, Young M. Kim, Vanessa Shen, Kerri Thai, Darren J. Kelly and Richard E. Gilbert declare that they have no conflict of interest.

Human and Animal Rights

All animal studies were approved by the St Michael’s Animal ethics care committee in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).

Informed Consent

Informed consent was obtained from all patients for being included in the study in accordance with the requirements of the St Michael’s Hospital human research ethics committee. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008.

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Authors and Affiliations

  1. Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, 30 Bond St, 209 Victoria Street, Room 7-052, Toronto, ON, M5B 1W8, Canada

    Kim A. Connelly, Andrew Advani, Suzanne L. Advani, Vanessa Shen, Kerri Thai & Richard E. Gilbert

  2. University of Melbourne Department of Medicine, St. Vincent’s Hospital, Fitzroy, VIC, 3065, Australia

    Yuan Zhang & Darren J. Kelly

  3. Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, USA

    Young M. Kim

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  1. Kim A. Connelly
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  2. Andrew Advani
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  3. Suzanne L. Advani
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  9. Richard E. Gilbert
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Correspondence to Kim A. Connelly.

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Connelly, K.A., Advani, A., Advani, S.L. et al. Impaired cardiac anti-oxidant activity in diabetes: human and correlative experimental studies. Acta Diabetol 51, 771–782 (2014). https://doi.org/10.1007/s00592-014-0608-9

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