Valencia, Sara Osorio

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 .

In cardiomyocytes, the endoplasmic reticulum (ER) plays a dynamic role in excitation‐contraction coupling by regulating cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ). Under pathological conditions the ER accumulates unfolded proteins triggering a homeostatic ER stress response to minimize and limit protein unfolding (via chaperone proteins) and accumulation (by decreasing synthesis and increasing degradation). The interactions between ER stress, [Ca 2+ ] cyt regulation and downstream contractile responses remain unknown, although such interactions may underlie the etiology of various cardiovascular diseases. We hypothesized that in cardiomyocytes ER stress alters the regulation of [Ca 2+ ] cyt and reduces cardiac contractility by decreasing myofilament Ca 2+ sensitivity. In isolated rat cardiomyocytes, ER stress was induced by tunicamycin, a naturally occurring antibiotic that induces ER stress by inhibiting N‐linked glycosylation. In one set of experiments, we used Western blotting techniques to evaluate the effects of ER stress on phosphorylation of cTnI at Ser23/24. In a second set of experiments, we evaluated the impact of tunicamycin on excitation‐contraction coupling by simultaneously measuring [Ca 2+ ] cyt (using Fura‐2 AM ratiometric imaging) and contractile (sarcomere length (SL) shortening) responses to electrical field stimulation using an IonOptix system. Tunicamycin induced an increase in phosphorylation of cTnI by 3–6 h. The evoked [Ca 2+ ] cyt transient was relatively unaffected by tunicamycin while the contractile response was markedly reduced. This resulted in a rightward shift in the phase loop relationship between [Ca 2+ ] cyt and SL shortening indicating reduced myofilament Ca 2+ sensitivity. We conclude that ER stress may underlie contractile dysfunction in a variety of cardiac diseases. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .