Human cardiac tissue in a microperfusion chamber simulating extracorporeal circulation--ischemia and apoptosis studies

Abstract

Background: After coronary artery bypass grafting ischemia/reperfusion injury inducing cardiomyocyte apoptosis may occur. This surgery-related inflammatory reaction appears to be of extreme complexity with regard to its molecular, cellular and tissue mechanisms and many studies have been performed on animal models. However, finding retrieved from animal studies were only partially confirmed in humans. To investigate this phenomenon and to evaluate possible therapies in vitro, adequate human cardiomyocyte models are required. We established a tissue model of human cardiomyocytes preserving the complex tissue environment. To our knowledge human cardiac tissue has not been investigated in an experimental setup mimicking extracorporeal circulation just in accordance to clinical routine, yet.

Methods: Cardiac biopsies were retrieved from the right auricle of patients undergoing elective coronary artery bypass grafting before cardiopulmonary bypass. The extracorporeal circulation was simulated by submitting the biopsies to varied conditions simulating cardioplegia (cp) and reperfusion (rep) in a microperfusion chamber. Cp/rep time sets were 20/7, 40/13 and 60/20 min. For analyses of the calcium homoeostasis the fluorescent calcium ion indicator FURA-2 and for apoptosis detection PARP-1 cleavage immunostaining were employed. Further the anti-apoptotic effect of carvedilol [10 microM] was investigated by adding into the perfusate.

Results: Viable cardiomyocytes presented an intact calcium homoeostasis under physiologic conditions. Following cardioplegia and reperfusion a time-dependent elevation of cytosolic calcium as a sign of disarrangement of the calcium homoeostasis occurred. PARP-1 cleavage also showed a time-dependence whereas reperfusion had the highest impact on apoptosis. Cardioplegia and carvedilol could reduce apoptosis significantly, lowering it between 60-70% (p < 0.05).

Conclusions: Our human cardiac preparation served as a reliable cellular model tool to study apoptosis in vitro. Decisively cardiac tissue from the right auricle can be easily obtained at nearly every cardiac operation avoiding biopsying of the myocardium or even experiments on animals.The apoptotic damage induced by the ischemia/reperfusion stimulus could be significantly reduced by the cold crystalloid cardioplegia. The additional treatment of cardiomyocytes with a non-selective beta-blocker, carvedilol had even a significantly higher reduction of apoptotis.

Figure 1

Microperfusion chamber. A: Top and B: Base Plexiglas components. Inlet pipe (1), Plexiglas block (2), rhomboid shaped chamber (3), cardiac tissue (4), nylon net (5) and outlet pipe (6). The top and base Plexiglas components were fastened with 4 screws.

Figure 2

Calcium homoeostasis was intact in the cardioplegia group. In the control group (ischemia without cardioplegia) final calcium ratio values were significantly (p < 0.05) elevated. Ratio values are plotted as mean ± SEM of n = 5 experiments. Cp: cardioplegia for 5 min.

Figure 3

Calcium homoeostasis was intact in the cardioplegia group. In the control group (ischemia without cardioplegia) final calcium ratio values were significantly (p < 0.05) elevated. Ratio values are plotted as mean ± SEM of n = 5 experiments. Cp: cardioplegia for 5 min.

Figure 4

Calcium homoeostasis was intact in the cardioplegia group. In the control group (ischemia without cardioplegia) final calcium ratio values were significantly (p < 0.05) elevated. Ratio values are plotted as mean ± SEM of n = 5 experiments. Cp: cardioplegia for 5 min.

Figure 5

Representative grayscaled fluorescent images of cardiomyocytes treated with cardioplegia and reperfusion (control group, first column) versus the same treatment plus the addition of carvedilol (second column). After DAPI counterstaining the greater nuclei of cardiomyocytes allow their distinction from fibroblasts with smaller nuclei. In PARP-1 cleavage positive, apoptotic cardiomyocytes nuclei feature an intensive granular fluorescence intensity (arrows). The exemplary images represent one experiment with the according cardioplegia and reperfusion time set noted on the left side. During the cryosection procedure artifacts presenting as nuclei conglomerates could not be avoided; these were excluded from analyses.

Figure 6

Antiapoptotic effect of cardioplegia versus ischemia. The x-axis represents time in min and the y-axis the number of PARP-1 cleavage positive, apoptotic cardiomyocytes. There is a time-dependent significant (p < 0.05) increase of apoptotic cardiomyocytes after reperfusion. Further there is a significant (p < 0.05) reduction of apoptosis in cardiomyocytes treated with cardioplegia versus ischemia. One column represents data of n = 5 experiments, noted as mean ± SEM. Stars mark the significances between the compared columns. The mean total cardiomyocyte number in 3 analyzed central areas on the cryosections was 300 ± 25 (mean ± SEM).