Advancing preclinical research with human living myocardial slices as a model of ischemia–reperfusion injury

Background Ischemia-reperfusion injury (IR) is a leading cause of cardiac dysfunction following restoration of blood flow to the heart after acute myocardial infarction or cardiac surgery. Despite extensive research, effective therapies are lacking, particularly due to limitations in existing preclinical models. Living myocardial slices (LMS) preserve the multicellular architecture and contractile function of human myocardium, offering a novel and physiologically relevant platform to model IR injury ex vivo. Purpose To establish an LMS-based model of myocardial IR, enabling assessment of functional, structural, and molecular responses to IR. Methods LMS were prepared from ventricular tissue of end-stage heart failure patients undergoing transplantation. Following adaptation for 3-5 days, slices underwent two IR protocols: 5 or 8 hours of ischemia, achieved by stopping the rocking that ensures oxygenation, followed by 20 hours of reperfusion. To increase workload, the electrical stimulation frequency was raised from 0.5 to 1.5 Hz during ischemia, also in control slices. Throughout the protocol, contractile performance was continuously assessed, including contraction force, time to peak (TTP), time to relaxation (TTR), and contraction kinetics (±dF/dt). After completion of the experimental protocol, myocardial slices were harvested for assessment of mitochondrial respiratory capacity using high-resolution respirometry. Extracellular mitochondrial DNA was also measured as a marker of tissue injury (Fig. 1). Results Ischemia impaired contractile performance, which partially recovered during reperfusion but remained reduced overall. This impairment tended to be more pronounced following prolonged ischemia (50±8% vs. 36±8%) (Fig. 2A-C). Contraction kinetics showed a similar trend, with both the maximal rates of contraction (+dF/dt: 55 ± 7% vs. 42 ± 9%) and relaxation (−dF/dt: 61 ± 9% vs. 42 ± 9%) slightly declining with increasing ischemic duration (Fig. 2G-H). TTP and TTR both shortened during ischemia and returned to baseline following reperfusion (Fig. 2E-F). At the end of reperfusion, TTR remained elevated after prolonged ischemia (TTP: 90±5% vs. 101±10%; TTR: 90±6% vs. 134±16%, p<0.001) (Fig. 2F). Mitochondrial respiratory capacity remained unchanged across the different IR protocols and relative to controls (Fig. 2I). Extracellular mitochondrial DNA increased markedly at the end of reperfusion compared to baseline, with levels slightly higher after prolonged ischemia (570±104% vs. 784±287%), suggesting enhanced tissue injury (Fig. 2J-L). Conclusion This study presents an ex vivo model of myocardial IR using LMS, with initial data suggesting sensitivity of contractile function to ischemia duration. This platform serves as a translational tool to study IR and therapeutic strategies. Future perspectives: The effect of IR on structural remodeling and expression of apoptotic markers is currently ongoing.