Osorio, Sara

A transient elevation of cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) triggers a force response in excitation‐contraction coupling. The Ca 2+ sensitivity of muscle reflects the relationship between [Ca 2+ ] cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca 2+ ] cyt at which 50% force is generated (ED50). In smooth muscle, Ca 2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca 2+ ] cyt . However, this conventional measurement overlooks the dynamic nature of Ca 2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca 2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca 2+ ] cyt and force and determined their temporal dependence using a phase loop plot. We examined the phase loop plot assessment of Ca 2+ sensitivity in ASM under the condition in which the conditional assessment indicated an increase ( Calyculin A treatment) in Ca 2+ sensitivity . Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips were loaded with Fura‐2 AM (Ca 2+ fluorescent indicator) and stimulated using 1 mM ACh. Isometric force and [Ca 2+ ] cyt responses were simultaneously measured using a Guth Muscle Research System. ASM strips were exposed to Calyculin A (phosphatase inhibitor; 100 nM for 10 min), which was previously reported to alter static Ca 2+ sensitivity. Phase loop plots were calculated from simultaneous [Ca 2+ ] cyt and force responses. The extent of rMLC phosphorylation was determined at different time periods after ACh stimulation using Phos‐tag SDS‐PAGE (SuperSep Phos‐tag; Wako Pure Chemical Industries, Ltd.) and Western blotting. Calyculin A increased ACh‐induced isometric force in ASM strips, but it had no effect on the amplitude of evoked [Ca 2+ ] cyt transients. These results suggested that Calyculin A increased static Ca 2+ sensitivity. However, the phase loop plots showed no significant shift in dynamic Ca 2+ sensitivity with Calyculin A. These results indicate that the phase loop plot provides a valuable tool to assess dynamic Ca 2+ sensitivity of isometric force generation in ASM, which better reflects the complex process of excitation‐contraction coupling in smooth muscle. These results further indicate that dynamic Ca 2+ sensitivity of force generation in ASM is independent of rMLC phosphorylation. Support or Funding Information Supported by NIH grant HL126451 (GCS) This abstract is from the Experimental Biology 2018 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 .