AN INTEGRATED HEART-ON-A-CHIP NAM RESOLVES DISTINCT CLINICAL PHENOTYPES OF MYOCARDIAL ISCHEMIA-REPERFUSION INJURY

Background: Reperfusion is required for myocardial salvage yet itself injures the heart, and cardioprotective interventions that reduce infarct size experimentally have repeatedly failed to improve outcomes in patients. These interventions are evaluated against viability or infarct size alone, whereas ischemia-reperfusion (I/R) injury produces structural, biochemical, and contractile dysfunction in the same tissue. No in vitro new approach methodology (NAM), however, resolves all three within one system. Methods: A NAM was created within a heart-on-a-chip platform, which was constructed by culturing neonatal rat ventricular myocytes (NRVMs) on muscular thin films. Cardiomyocytes were subjected to normoxia, 2 hours of ischemia, and ischemia followed by 2 hours of reperfusion. Sarcomeric architecture was quantified by immunofluorescence, cardiomyocyte injury by LIVE/DEAD staining and cardiac troponin I release into conditioned media, while contractile stress and spontaneous beat frequency were recorded on the same films before and after each condition. Results: Under ischemia, z-line density decreased while architectural metrics were preserved, indicating cell loss rather than sarcomere disorganization. Furthermore, viability decreased, troponin elevated, and every contractile stress component declined, while spontaneous beat frequency was unchanged. Reperfusion fractured this uniform depression. Cell death and troponin release increased further, yet structure was unchanged from ischemia, so every functional change occurred on a depleted but architecturally intact cardiomyocyte population. Systolic stress recovered partially, whereas diastolic stress exceeded baseline, leaving active stress suppressed despite oxygen recovery. Beat frequency increased with pronounced variability. Conclusions: This heart-on-a-chip NAM resolved structural, biochemical, and contractile injury that global indices conflate, distinguishing ischemic from reperfusion pathophysiology. Interestingly, individual readouts corresponded to distinct clinical syndromes of ischemic injury, positioning this platform for future testing of cardioprotective candidates against the full injury phenotype.