Korean Circ J.  2015 May;45(3):234-241. 10.4070/kcj.2015.45.3.234.

Glucose-Insulin-Potassium Solution Protects Ventricular Myocytes of Neonatal Rat in an In Vitro Coverslip Ischemia/Reperfusion Model

• Chun WJ
• Nah DY
• Bae JH
• Chung JW
• Lee H
• Moon IS

• KMID: 2389131
• DOI: http://doi.org/10.4070/kcj.2015.45.3.234

Abstract

BACKGROUND AND OBJECTIVES
The benefit of high glucose-insulin-potassium (GIK) solution in clinical applications is controversial. We established a neonatal rat ventricular myocyte (NRVM) in vitro coverslip ischemia/reperfusion (I/R) model and investigated the effects of GIK solution on suppressing reactive oxygen species (ROS) and upregulating O-GlcNacylation, which protects cells from ischemic injury.
MATERIALS AND METHODS
NRVMs were isolated from postnatal day 3-4 Sprague-Dawley rat pups and grown in Dulbecco's modified Eagle's medium containing high glucose (4.5 g/L), fetal bovine serum, and penicillin/streptomycin. The effects of the GIK solution on ROS production, apoptosis, and expression of O-GlcNAc and O-GlcNAc transferase (OGT) were investigated in the coverslip I/R model.
RESULTS
Covering the 24-well culture plates for 3 hr with 12 mm diameter coverslips resulted in the appropriate ischemic shock. Glucose and insulin synergistically reduced ROS production, protected NRVM dose-dependently from apoptosis, and altered O-GlcNAc and OGT expression.
CONCLUSION
The high GIK solution protected NRVM from I/R injury in vitro by reducing ROS and altering O-GlcNacylation.

Keyword

Myocytes, cardiac; Glucose-insulin-potassium cardioplegic solution; GlcNAc; O-GlcNac transferase

MeSH Terms

Animals
Apoptosis
Glucose
Insulin
Muscle Cells*
Myocytes, Cardiac
Rats*
Rats, Sprague-Dawley
Reactive Oxygen Species
Shock
Transferases
Glucose
Insulin
Reactive Oxygen Species
Transferases

Figure

• 

  Fig. 1 Cultured neonatal rat ventricular myocytes (NRVMs) and experimental scheme. (A) NRVM were grown and phase-contrast images were taken on day 3 in vitro (DIV 3) (a) and on day 7 in vitro (DIV 7) (b). (B) Experimental scheme. The cells were grown in a high glucose medium. After the cells reached confluence on DIV 7, ischemia was induced by placing a coverslip directly on the bottom of the well for the indicated time periods. The medium was changed with fresh containing various alterations immediately before removing the coverslip. Reperfusion started when the coverslip was removed. Scale bar, 20 µm. Glu: glucose.

• 

  Fig. 2 Effects of glucose and insulin on cell viability. Cells were covered for 3 hr. After perfusion with the indicated medium for 30 min, the cells were stained with Annexin-V (AV) and propidium iodide (PI). (A-D) The phase-contrast (phase; upper left), AV-stained, PI-stained, and merged images (upper right) of the cells in the core region are shown. (E) Only AV(+) and AV(+)/PI(+) cells were counted and expressed as % of total cells. *p<0.01; **p<0.001. Glu: glucose, I/R: ischemia/reperfusion. Unit: glucose: g/L, insulin: 0.5 U/mL.

• 

  Fig. 3 Effects of glucose and insulin on suppressing reactive oxygen species (ROS) and O,N-acetylglucosamine (O-GlcNAc) and GlcNAc transferase (OGT) expression. The 24-well culture plate ischemia/reperfusion (I/R) model (covered for 3 hr and reperfusion for 30 min) was used. (A) ROS intensity and GlcNAc and OGT expression images are shown with the typical phase-contrast images (Phase) in the fields of ischemic core, penumbra, and periphery of the covered area in the presence or absence of insulin at the indicated glucose concentrations. (B) ROS, O-GlcNAc, and OGT expression signal intensities were measured in the core region using the NIH Image J program and expressed in arbitrary units. *p<0.01. Glu: glucose.

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