In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Summary

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

Abstract

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

Introduction

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery^1,2. During nonpulsatile low flow perfusion and periods of total circulatory arrest, damage involving all types of heart cells still occurs. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, therapeutic hypothermia (TH) affords substantial protection against an expected ischemic insult in patients undergoing cardiac surgery^3,4. TH is defined as a core body tempera....

Protocol

Information regarding commercial reagents and instruments are listed in the Table of Materials.

The AC16 human cardiomyocyte cell line was derived from the fusion of primary cells from adult ventricular heart tissue with SV40-transformed human fibroblasts¹⁷, which were purchased from BLUEFBIO (Shanghai, China). The cell line develops many biochemical and morphological features characteristic of cardiomyocytes. In addition, the cell line is widely used to evaluate myocardial damage and myocardial function in vitro^18,19.

Results

The effect of OGD exposure on the viability of HCMs was determined by CCK-8 assay. Compared with that observed in the control group, cell viability was significantly decreased in a time-dependent manner (Figure 2A). The apoptosis rates of HCMs at different times after reperfusion showed a specific trend, where from 0 to 16 h, the apoptosis rates gradually increased and reached the maximum rate at the 16 h time point (Figure 2B). As OGD for 12 h reduced cell acti.......

Discussion

The complexities of intact animals, including the interactions between different types of cells, often prevent detailed studies of specific components of I/R injury. Therefore, it is necessary to establish an in vitro cell model that can accurately reflect the molecular changes after ischemia in vivo. Research on OGD models has been previously reported^13,22, and many sophisticated methods have been established^23,

Materials

Name	Company	Catalog Number	Comments
Annexin V-FITC cell apoptosis detection kit	Bio-Technology,China	C1062M
Cardiac myocyte growth supplement	Sciencell,USA	6252
Caspase 3 activity assay kit	Bio-Technology,China	C1115
Caspase 8 activity assay kit	Bio-Technology,China	C1151
DMEM, no glucose	Gibco,USA	11966025
Dulbecco's modified eagle medium	Gibco,USA	11960044
Fetal bovine serum	Gibco,USA	16140071
Flow cytometry	CytoFLEX,USA	B49007AF
Human myocardial cells	BLUEFBIO,China	BFN60808678
Mitochondrial membrane potential assay kit with JC-1	Bio-Technology,China	C2006
Penicillin/Streptomycin solution	Gibco,USA	10378016
Reactive oxygen species assay kit	Bio-Technology,China	S0033S
Three-gas incubator	Memmert,Germany	ICO50
Trypsin-EDTA (0.25%)	Gibco,USA	25200056