Díaz‐Navarro, Rienzi

Ischemia/reperfusion (I/R) injury occurs during acute myocardial infarction and is a frequent cause of contractile failure, morbidity and mortality. There is evidence that reduced cardiac contractility following I/R injury is associated with disruption of excitation‐contraction (E‐C) coupling. We hypothesized that I/R injury alters E‐C coupling in cardiomyocytes by reducing the Ca 2+ sensitivity of contraction. Cardiomyocytes were isolated from adult rat (Sprague Dawley) hearts using a modified Langendorff system. Cardiomyocytes were then loaded with fura‐2 AM (Ca 2+ fluorescent indicator; 1 μM) and electrically stimulated every 2 s (0.5 Hz). Cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) and sarcomere length shortening responses to stimulation were simultaneously measured using an IonOptix System equipped with a hypoxia‐regulated incubator system (Live Cell Instrument). To model I/R, cardiomyocytes were initially exposed to normoxia (21% O 2 ) for 30 min followed by 30 min exposure to 2.5% O 2 and finally 30 min exposure to normoxia. Throughout the I/R protocol, Ca 2+ sensitivity of cardiomyocytes was assessed using a phase loop plot of concurrently measured [Ca 2+ ] cyt and sarcomere length shortening. I/R reduced the extent of sarcomere length shortening, but it had no effect on the amplitude of evoked [Ca 2+ ] cyt transients. Furthermore, I/R reduced Ca 2+ sensitivity reflected by a rightward shift in the phase loop plots. These results support the hypothesis that I/R alters E‐C coupling in cardiomyocytes by reducing the Ca 2+ sensitivity of contraction. Support or Funding Information Supported by Mayo Foundation This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Ca 2+ sensitivity plays a critical role in regulating cardiac contraction, and pathophysiological changes in Ca 2+ sensitivity of cardiac contraction may significantly impact cardiac function. For example, oxidative stress causes myocardial dysfunction, which is due in part to a decrease in Ca 2+ sensitivity. Oxidative stress (e.g., elevated H 2 O 2 ) underlies, at least in part, various conditions of cardiac dysfunction including ischemia‐reperfusion injury, heart failure, hypertension, and atherosclerosis. In cardiac muscle, Ca 2+ sensitivity can be dynamically assessed using a phase loop plot of concurrently measured cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) and sarcomere length shortening (Spurgeon et al J Physiol 447:83–102, 1992; Han et al Am J Physiol 298:890–897, 2010; Schaible et al Am J Physiol 310:1533–40, 2016). We hypothesized that oxidative stress reversibly reduces Ca 2+ sensitivity of cardiomyocytes within minutes. Cardiomyocytes were isolated from adult rat hearts (Sprague Dawley) using a modified Langendorff system. Cardiomyocytes were loaded with Fura‐2 AM (Ca 2+ fluorescent indicator; 1 mM) and electrically stimulated every 2 s. [Ca 2+ ] cyt and sarcomere shortening responses to stimulation were simultaneously measured using an IonOptix System. After stabilization for 30 min at 35°C, cardiomyocytes were exposed to PSS for 10 min, then to H 2 O 2 (200 μM) for 10 min and finally to either PSS washout or DTT (1 mM)‐induced reduction for 10 min. H 2 O 2 ‐induced oxidative stress increased basal [Ca 2+ ] cyt and the amplitude of the evoked [Ca 2+ ] cyt response. H 2 O 2 ‐induced oxidative stress also reduced the extent of sarcomere shortening and slowed the kinetics of shortening/relaxation. As a result, H 2 O 2 ‐induced oxidative stress reduced Ca 2+ sensitivity reflected by a rightward shift in the phase loop plots. Interestingly, with both PSS washout and DTT‐induced reduction Ca 2+ sensitivity was further reduced. These results support the hypothesis that H 2 O 2 ‐induced oxidative stress reduces Ca 2+ sensitivity of cardiomyocytes, but this effect was not immediately reversed, but indeed worsened by PSS washout or by DTT‐induced reduction. Together, these results provide insight into the dynamic effects of oxidative stress on Ca 2+ sensitivity of cardiomyocytes that may be reflected under pathological conditions. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .