Human cardiac tissue in a microperfusion chamber simulating extracorporeal circulation--ischemia and apoptosis studies.
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.
Usta
Engin
E
Department of Thoracic-, Cardiac- and Vascular Surgery, Tübingen University Hospital, Germany. engin.usta@med.uni-tuebingen.de
Renovanz
Mirijam
M
Mustafi
Migdat
M
Ziemer
Gerhard
G
Aebert
Hermann
H
D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000704:Analysis of Variance; D017209:Apoptosis / Q000502:physiology*; D001706:Biopsy; D002118:Calcium / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D000077261:Carvedilol; D002448:Cell Adhesion; D016257:Fura-2 / Q000494:pharmacology; D006324:Heart Arrest, Induced / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques; D066298:In Vitro Techniques; D017202:Myocardial Ischemia / Q000473:pathology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000473:pathology*; D011412:Propanolamines / Q000494:pharmacology; D015427:Reperfusion Injury / Q000473:pathology*
D000319:Adrenergic beta-Antagonists; D002227:Carbazoles; D011412:Propanolamines; D000077261:Carvedilol; D002118:Calcium; D016257:Fura-2