Han, Young Soo

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 .

Rewarming shock following accidental hypothermia is a potentially fatal scenario involving cardiac dysfunction. Previously our lab demonstrated that rewarming shock results in a decrease in Ca 2+ sensitivity of contraction in cardiac muscle due to an increase in cTnI phosphorylation. In this study, we hypothesized that rewarming shock initiates a cascade of intracellular events including reactive oxygen species (ROS) formation, endoplasmic reticulum (ER) stress, and mitochondrial disruption. Isolated cardiomyocytes from Sprague‐Dawley rats were exposed to a hypothermia/rewarming protocol (cooled for 1 h, held at 17 o C for 30 min, rewarmed for 1 h) that results in contractile dysfunction indicated by an increase in cytosolic Ca 2+ and reduced cell shortening measured using an IonOptix system. Rewarming shock increased ROS levels measured by an HPLC‐based dihydroethidium assay and by MitoSox fluorescence. Hypothermia/rewarming was also associated with ER stress indicated by increased expression of ER stress markers (Grp78, PERK, and CHOP) measured by Western blot. Finally, rewarming shock was associated with mitochondrial disruption evidenced by mitochondrial membrane depolarization (decreased in TMRM fluorescence) and by an increase in cytosolic cytochrome c (measured by Western blot). Our results support our overall hypothesis and suggest that these ER and mitochondrial intracellular events may underlie myocardial contractile dysfunction following hypothermia/rewarming. Supported by NIH Grant 5T32HL105355‐02 (GCS)

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 .

Recovery from accidental hypothermia followed by rewarming (H/R) is often unsuccessful due to heart failure. Although the causes for H/R‐induced heart failure remain elusive, studies at the cellular level suggest alterations in both cytoplasmic and mitochondrial Ca 2+ regulation. We hypothesize that H/R is associated with mitochondrial Ca 2+ overload, which leads to: 1) excessive reactive oxygen species (ROS) generation, 2) mitochondrial membrane depolarization, and 3) mitochondrial cytochrome c release. To test this hypothesis, isolated cardiomyocytes from adult male Sprague‐Dawley rats were subjected to an in vitro H/R protocol (1 h cooling to 15 o C held for 30 min followed by 1 h rewarming to 37 o C). Cytoplasmic (CM‐H 2 DCFDA fluorescence) and mitochondrial (MitoSOX fluorescence) ROS generation increased during cooling and remained elevated throughout the H/R protocol. Mitochondrial membrane potential (TMRM fluorescence) depolarized during rewarming suggesting mitochondrial permeability transition pore opening. After rewarming, cytochrome c content (Western blot) in cytoplasmic to mitochondrial fractions increased indicating release. These results suggest that Ca 2+ overload associated with H/R induces ROS generation leading to mitochondrial disruption. Grant Funding Source : Supported by NIH Grant 5T32HL105355‐02 (GCS)