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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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 μM] was investigated by adding into the perfusate.</p> <p>Results</p> <p>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).</p> <p>Conclusions</p> <p>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.</p> <p>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 β-blocker, carvedilol had even a significantly higher reduction of apoptotis.</p

The challenge to verify ceramide's role of apoptosis induction in human cardiomyocytes - a pilot study

<p>Abstract</p> <p>Background</p> <p>Cardioplegia and reperfusion of the myocardium may be associated with cardiomyocyte apoptosis and subsequent myocardial injury. In order to establish a pharmacological strategy for the prevention of these events, this study aimed to verify the reliability of our human cardiac model and to evaluate the pro-apoptotic properties of the sphingolipid second messenger ceramide and the anti-apoptotic properties of the acid sphingomyelinase inhibitor amitryptiline during simulated cardioplegia and reperfusion ex vivo.</p> <p>Methods</p> <p>Cardiac biopsies were retrieved from the right auricle of patients undergoing elective CABG before induction of cardiopulmonary bypass. Biopsies were exposed to <it>ex vivo </it>conditions of varying periods of cp/rep (30/10, 60/20, 120/40 min). Groups: I (untreated control, n = 10), II (treated control cp/rep, n = 10), III (cp/rep + ceramide, n = 10), IV (cp/rep + amitryptiline, n = 10) and V (cp/rep + ceramide + amitryptiline, n = 10). For detection of apoptosis anti-activated-caspase-3 and PARP-1 cleavage immunostaining were employed.</p> <p>Results</p> <p>In group I the percentage of apoptotic cardiomyocytes was significantly (p < 0.05) low if compared to group II revealing a time-dependent increase. In group III ceramid increased and in group IV amitryptiline inhibited apoptosis significantly (p < 0.05). In contrast in group V, under the influence of ceramide and amitryptiline the induction of apoptosis was partially suppressed.</p> <p>Conclusion</p> <p>Ceramid induces and amitryptiline suppresses apoptosis significantly in our ex vivo setting. This finding warrants further studies aiming to evaluate potential beneficial effects of selective inhibition of apoptosis inducing mediators on the suppression of ischemia/reperfusion injury in clinical settings.</p

Hydrogen Sulfide Attenuated Tumor Necrosis Factor-α-Induced Inflammatory Signaling and Dysfunction in Vascular Endothelial Cells

S donor) on tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) dysfunction.Application of NaHS concentration-dependently suppressed TNF-α-induced mRNA and proteins expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), mRNA expression of P-selectin and E-selectin as well as U937 monocytes adhesion to HUVEC. Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of NaHS. Furthermore, TNF-α-induced NF-κB activation assessed by IκBα degradation and p65 phosphorylation and nuclear translocation and ROS production were diminished in cells subjected to treatment with NaHS.S can exert an anti-inflammatory effect in endothelial cells through a mechanism that involves the up-regulation of HO-1