Simulated ischemia does not mimic stop flow ischemia in perfused mouse heart

AGUILAR SÁNCHEZ Y; VALVERDE CA; SALAS M; ESCOBAR AL; MATTIAZZI A

Ciudad Autónoma de Buenos Aires

Congreso; XXII ISHR WORLD CONGRESS 2016; 2016

International Society for Heart Research

When hearts are submitted to an ischemia itsuffers intracellular calcium (Ca2+) handling and mechanical dysfunction,recovering partially during reperfusion. The magnitude of these alterations ishigher with increasing ischemia duration. When studding this pathology, usuallyit requires different cardiac preparations, the aortic retro-perfusion ofintact heart (Langendorff technique) being the most commonly used. Isolatedhearts are normally submitted to a coronary flow cessation (global/regionalischemia). Meanwhile, some studies require utilizing isolated cardiomyocytes, whichdue to its features they have to be submitted to chemically simulated ischemia.Aim: Tocompare mechanical function, cytosolic Ca2+ handling and key Ca2+handling proteins phosphorylation during ischemia (simulated vs. non-simulated).Methods:Hearts from 2-months-old male Balb/c mice were removed and perfused accordingto Langendorff technique perfused at constant flow/temperature. Left ventricle(LV) mechanical activity (LV developed pressure, LVDP, and LV end-diastolicpressure, LVEDP) was assessed with a latex balloon connected to a pressuretransducer. Cytosolic Ca2+ was assessed in Rhod-2-loaded hearts in apulsed-local-field fluorescence microscope. Action potentials were registeredwith the aid of a microelectrode. Hearts were submitted to a 15 min-period ofnormothermic ischemia (chemically simulated, CS, pH 6.2+no glucose+N2;or flow restriction, FR). Phosphorylation was assessed by western blot ofcardiac homogenates from hearts freezed-clamped at different time-points. Results: Duringischemia, CS hearts showed lesser mechanical dysfunction when compared to FRhearts. During reperfusion CS-hearts presented a higher LVDP respect toFR-hearts (48.5±6.2 vs. 11.9±6.6%). LVEDP was significantly higher in CS-hearts(29.0±5.5 vs. 56.6±3.1mmHg in FR-hearts). Additionally, the action potentialamplitude was lower in FR vs. CS-hearts together with lower Ca2+transient amplitude. Phospholamban phosphorylation by CaMKII at early reperfusionin FR-hearts was higher respect to FR-hearts (812±46 vs. 155±26%). No changeswere observed in PLN-Ser16-phosphorylation. Conclusion: Simulatedischemia generates mild alteration of mechanical and calcium handling functionrespect to flow cessation ischemia. These results have to be taken into accountwhen using different cardiac preparations for assessing ischemia/reperfusion.