Han, Young Soo

Cardiac troponin I (cTnI) is a key regulator of cardiomyocyte contraction. Phosphorylation of cTnI at Ser22/23 is mediated by protein kinase A, C, and G, and is associated with reduced Ca 2+ sensitivity of myocardial contraction. In previous in vivo and in vitro studies, we showed that hypothermia/rewarming (rewarming shock) reduces the Ca 2+ sensitivity of myocardial contraction due to cTnI Ser22/23 phosphorylation. Rewarming shock was also found in isolated cardiomyocytes; however, demonstrating a relationship with cTnI phosphorylation is complicated by varying cardiomyocyte viability after isolation. Using a modified Langendorff technique, the cardiomyocyte viability gradually declines over a 4‐h period from ~80% to ~40%. This decline in cell viability is not accounted for in typical Western blot measurements and, therefore, assessments of cTnI phosphorylation are highly subject to misrepresenting actual physiological changes in cTnI phosphorylation. To address this issue, we developed a novel quantitative technique to measure cTnI phosphorylation at Ser22/23 in viable cardiomyocytes using confocal microscopy. We demonstrated that cardiomyocytes with a rod‐shaped morphology and uniform striation patterns display robust transient contractile and cytosolic Ca 2+ responses to electrical stimulation. All of these rod‐shaped cardiomyocytes exclude trypan blue, while a disproportionate fraction of non‐rod‐shaped cardiomyocytes are non‐viable. Separately, using 2D gel electrophoresis, we validated the specificity of an antibody that targets phosphorylated cTnI. Using this antibody, we verified that the extent of cTnI phosphorylation in rod‐shaped cardiomyocytes increased with isoproterenol in a dose dependent fashion. This novel methodology allows a more accurate measurement of cTnI phosphorylation in isolated primary cardiomyocytes that is not affected as cell viability declines over time. Support or Funding Information This publication was made possible by Grant Number T32 HL 105355 from the National Heart, Lung and Blood Institute (NHLBI), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NIH.

Inflammation and pro‐inflammatory cytokines (e.g., TNFα, IL‐13) induce airway smooth muscle (ASM) hyper‐reactivity and remodeling characteristic of asthma. We hypothesized that exposure to TNFα (and IL‐13) increases isometric force generation in porcine ASM via several underlying mechanisms: 1) increasing sensitivity to ACh stimulation; 2) enhancing the cytosolic Ca 2+ ([Ca 2+ ] cyt ) response to ACh; 3) increasing the Ca 2+ sensitivity of force generation; 4) enhancing the Ca 2+ ‐dependent phosphorylation of myosin regulator light chain (rMLC); 5) promoting expression of non‐muscle rMLC; 6) increasing actin and myosin heavy chain (MyHC) content (i.e., the number of contractile units); and/or 7) promoting cytoskeletal remodeling (actin polymerization). Studies were performed on intact ASM strips (~1 mm wide × ~5 mm long) dissected from porcine tracheas obtained from a local abattoir. The ASM strips were placed in PSS with and without TNFα (100 ng/ml) for 24 h. The ACh concentration dependence of force generation ASM was determined (ACh range from 10 nM to 1mM), and the EC50 (i.e., the ACh concentration that produces 50% of maximal ASM force) was determined. In control, untreated ASM, the EC50 for ACh was 2.6 μM compared to 1.3 μM following TNFα treatment. We found that across all ACh concentrations, ASM force increased after TNFα treatment with an ~50% increase in maximum ASM force. The [Ca 2+ ] cyt response induced by the EC50 [ACh] was similar in control and TNFα treated ASM. Thus, with an increase in force and no effect on [Ca 2+ ] cyt , there was an apparent increase in Ca 2+ sensitivity of force after TNFα treatment. However, in both control and TNFα treated ASM, the extent of rMLC phosphorylation induced by ACh stimulation was not affected by TNFα treatment. Importantly, the extent of rMLC phosphorylation at the ACh EC50 was comparable between control and TNFα treatment indicating no change in Ca 2+ sensitivity. The expression of non‐muscle rMLC (determined by 2D gel electrophoresis) was also unaffected by TNFα treatment. Total actin and MyHC concentrations increased by ~45% following TNFα treatment; thus, the number of contractile units contributing to force increased. This was reflected by a marked increase in the Ca 2+ activation of ATP consumption (cross‐bridge cycling) of ASM following TNFα treatment. Cytoskeletal remodeling was evaluated by ACh‐induced actin polymerization (F‐/G‐actin ratio) but was found to be unaffected by TNFa treatment. Together, our results show that two primary mechanisms underlie the marked increase in ASM force generation induced by 24‐h TNFα exposure: 1) an increase in ACh sensitivity, and 2) an increase in actin and MyHC concentrations (number of contractile units). Support or Funding Information Supported by NIH grant HL126451 (GCS) and HL150890 (GCS)

Cytoskeletal remodeling in smooth muscle occurs with activation and involves actin polymerization. Remodeling of the cortical cytoskeleton allows tethering of contractile proteins and force transmission to the extracellular matrix. Smooth muscle contraction involves cross‐bridge cycling and ATP hydrolysis, which are both sensitive to loading. During isometric activation, the rate of ATP hydrolysis is initially high but declines with time reaching a steady‐state, while peak force is maintained. Thus, tension cost, i.e., the rate of ATP hydrolysis per unit of force, decreases over time, reflecting increased efficiency of smooth muscle activation. We hypothesize that the time‐dependent decrease in ATP hydrolysis rate and tension cost is due to cytoskeletal remodeling and an increase in internal loading on cross‐bridges. In permeabilized porcine airway smooth muscle (ASM) strips, isometric force and ATP hydrolysis rate were simultaneously measured after maximum Ca 2+ activation (pCa 4.0). From each animal, two ASM strips were tested: 1) Inhibition of actin polymerization and cytoskeletal remodeling using Cytochalasin‐D (Cyto‐D; treated (1 μM for 30 min); and 2) Untreated controls. Compared to untreated controls, Cyto‐D‐induced inhibition of actin polymerization reduced both peak and steady‐state isometric force by ~40%, while peak and steady‐state ATP hydrolysis rate increased by ~30% and ~20%, respectively. As a result, at peak and steady‐state, tension cost increased by ~100% and ~60%, respectively, following Cyto‐D treatment. These results support our hypothesis that the time‐dependent decrease in ATP hydrolysis rate reflects an increase in internal load on cross‐bridges via cytoskeletal remodeling. Cyto‐D inhibition of cytoskeletal remodeling impairs contractile protein tethering and thus reduces internal loading, resulting in faster cross‐bridge cycling and increased ATP hydrolysis rate. Support or Funding Information Supported by NIH grant HL126451(GCS), HL150890 (GCS) and the Mayo Clinic

Rewarming shock (acute cardiac failure) during resuscitation from profound hypothermia is partially ameliorated by low dose epinephrine (EPI). However, hypothermia‐induced cardiac Ca 2+ overload persists with rewarming. The reactive oxygen species (ROS) scavenger superoxide dismutase (SOD) protects isolated hearts from hypothermia‐induced Ca 2+ overload. In electrically stimulated rat cardiac myocytes loaded with Ca 2+ indicators fluo‐3 (cytosolic) and rhod‐2 (mitochondria) and imaged using confocal microscopy, we investigated the effects of EPI and SOD on cytosolic vs. mitochondrial Ca 2+ during rewarming. Myocytes were cooled from 37 to 15 °C over 1h, maintained at 15 °C for 30min, and rewarmed over 1h to 37 °C. EPI 0.125 μg/ml was administered from 27 °C upwards, and SOD 0.1 mg/ml from 15 °C upwards during rewarming. Control cells did not receive drugs. Other cells received EPI alone. Hypothermia resulted in elevated cytosolic and mitochondrial Ca 2+ . Compared to normothermia, mitochondrial Ca 2+ uptake and release was slowed by hypothermia, and not immediately reversed with rewarming. EPI prolonged mitochondrial Ca 2+ overload during rewarming. With SOD, mitochondrial as well as cytosolic Ca 2+ were comparable to controls during hypothermia and rewarming. These data suggest that during rewarming, mitochondrial Ca 2+ buffering remains impaired, and that ROS may play a role in Ca 2+ overload. Administration of EPI may exacerbate mitochondrial dysfunction, potentially worsening rewarming shock. Supported by Mayo Foundation Clinical Research grants, and NIH grants HL74309 and GM56686.

The mechanisms underlying rewarming shock (cardiac failure during rewarming from profound hypothermia) are not known. Increased myocardial Ca 2+ levels and altered mitochondrial ultrastructure have been reported. We hypothesized that mitochondrial buffering of cytosolic Ca 2+ contributes to cardiac dysfunction. Enzymatically‐dissociated rat cardiac myocytes loaded with Ca 2+ indicators fluo‐3 (cytosolic) and rhod‐2 (mitochondrial) were electrically stimulated (0.5 Hz) and visualized using real time confocal microscopy. Myocytes were cooled from 37 to 15 °C over 1h, maintained at 15 °C for 30min, and rewarmed over 1h back to 37 °C. Hypothermia increased baseline mitochondrial Ca 2+ (134% of normothermic control), which returned to control levels after rewarming. In contrast, rewarming elevated cytosolic Ca 2+ significantly compared to controls (126% vs. 94%). During hypothermia both cytosolic and mitochondrial peak Ca 2+ , time to peak, and time to 50% decay increased by 135%, 150% and 200%, respectively, but returned to control levels during rewarming. These data indicate that during hypothermia, mitochondria buffer cytosolic Ca 2+ , and during rewarming, mitochondrial Ca 2+ is redistributed to the cytosol, leading to continued elevation in cytosolic Ca 2+ . The temporary Ca 2+ overload in mitochondria may further affect subsequent energy production and myocyte function. Supported by Mayo Foundation Clinical Research grants, and NIH grants HL74309 and GM56686.

Rewarming the intact heart after a period of hypothermia is associated with reduced myocardial contractility, decreased Ca2+ sensitivity, and increased cardiac troponin-I (cTnI) phosphorylation. We hypothesized that hypothermia/rewarming (H/R) induces left ventricular (LV) contractile dysfunction due to phosphorylation of cTnI at Ser23/24. To test this hypothesis, the response of wild-type mice ( n = 7) to H/R was compared with transgenic (TG) mice expressing slow skeletal TnI (TG-ssTnI; n = 7) that lacks the Ser23/24 phosphorylation sites. Hypothermia was induced by surface cooling and maintained at 23–25°C for 3 h. Subsequently, the animals were rewarmed to 37°C. LV systolic and diastolic function was assessed using a 1.4 F pressure-volume Millar catheter introduced via the right carotid artery. At baseline conditions, there were no significant differences in LV systolic function between wild-type and TG-ssTnI mice, whereas measurements of diastolic function [isovolumic relaxation constant (τ) and end-diastolic pressure-volume relationship (EDPVR)] were significantly ( P < 0.05) reduced in TG-ssTnI animals. Immediately after rewarming, significant differences between groups were found in cardiac output (CO; wild-type 6.6 ± 0.7 vs. TG-ssTnI 8.8 ± 0.7 mL/min), stroke work (SW; wild-type 796 ± 112 vs. TG-ssTnI 1208 ± 67 mmHg/μL), and the preload recruited stroke work (PRSW; wild-type 38.3 ± 4.9 vs. TG-ssTnI 68.8 ± 8.2 mmHg). However, EDPVR and τ returned to control levels within 1 h in both groups. We conclude that H/R-induced LV systolic dysfunction results from phosphorylation of cTnI at Ser23/24. NEW & NOTEWORTHY Rewarming following a period of accidental hypothermia leads to a form of acute cardiac failure (rewarming shock), which is in part due to reduced sensitivity to Ca2+ activation of myocardial contraction. The results of the present study support the hypothesis that rewarming shock is due to phosphorylation of cardiac troponin I.

New Findings What is the central question of this study? Will discontinued stimulation of isolated cardiomyocytes (asystole) during hypothermia mitigate hypothermia–rewarming‐induced cytosolic Ca2+ overload? What is the main finding and its importance? Mimicking asystole or hypothermic cardiac arrest by discontinued stimulation of cardiomyocytes during hypothermia resulted in normal contractile function after rewarming. This result suggests that asystole during severe hypothermia provides protection from hypothermia–rewarming‐induced contractile dysfunction in cardiomyocytes. AbstractAfter exposure of spontaneously beating hearts or electrically stimulated isolated cardiomyocytes to hypothermia–rewarming (H/R), cardiac dysfunction or alteration in excitation–contraction coupling, respectively, is a consequence. In contrast, hypothermic cardiac arrest, as routinely applied during cardiac surgery, will not impose any hazard to cardiac function after rewarming. We hypothesize that by maintaining asystole during H/R, cardiomyocytes will avoid Ca2+ overload attributable to the transient stimulation‐evoked elevation of [Ca2+]i and thus, H/R‐induced elevation of phosphorylated cardiac troponin I and reduced Ca2+ sensitivity after rewarming. To test this hypothesis, the aim of the study was to determine whether discontinued electrical stimulation (to imitate hypothermic cardiac arrest) versus stimulation during 3 h of H/R prevents disruption of excitation–contraction coupling in our established cardiomyocyte H/R model. Cytosolic Ca2+ and the contractile response (sarcomere length shortening) were measured using an IonOptix system, and the dynamic assessment of Ca2+ sensitivity of contraction was conducted using a phase‐loop plot. Cardiomyocytes were divided into three groups. Group 1 (time‐matched control) was continuously stimulated at 0.5 Hz for 3 h at 35°C. Group 2 was continuously stimulated during H/R at 0.5 Hz, whereas in group 3 stimulation was discontinued during H/R and thus the cells remained quiescent until the resumption of stimulation after rewarming. The results demonstrate that discontinued stimulation of cardiomyocytes during H/R, imitating hypothermic cardiac arrest during cardiac surgery, provides protection against H/R‐induced disruption of excitation–contraction coupling. We suggest that protective effects are caused by preventing the protein kinase A‐induced elevation of phosphorylated cardiac troponin I, which is a key mechanism to reduce myofilament Ca2+ sensitivity of contraction.

Maximum velocity of the actomyosin ATPase reaction ( VmaxATPase) and ATP consumption rate during maximum isometric activation (ATPiso) were determined in human vastus lateralis (VL) muscle fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that the reserve capacity for ATP consumption [1 − (ratio of ATPisoto VmaxATPase)] varies across VL muscle fibers expressing different MHC isoforms. Biopsies were obtained from 12 subjects (10 men and 2 women; age 21–66 yr). A quantitative histochemical procedure was used to measure VmaxATPase. In permeabilized fibers, ATPisowas measured using an NADH-linked fluorometric procedure. The reserve capacity for ATP consumption was lower for fibers coexpressing MHC2Xand MHC2Acompared with fibers singularly expressing MHC2Aand MHCslow(39 vs. 52 and 56%, respectively). Tension cost (ratio of ATPisoto generated force) also varied with fiber type, being highest in fibers coexpressing MHC2Xand MHC2A. We conclude that fiber-type differences in the reserve capacity for ATP consumption and tension cost reflect functional differences such as susceptibility to fatigue.

In airway smooth muscle (ASM), ACh induces propagating intracellular Ca2+concentration ([Ca2+]i) oscillations (5–30 Hz). We hypothesized that, in ASM, coupling of elevations and reductions in [Ca2+]ito force generation and relaxation (excitation-contraction coupling) is slower than ACh-induced [Ca2+]ioscillations, leading to stable force generation. When we used real-time confocal imaging, the delay between elevated [Ca2+]iand contraction in intact porcine ASM cells was found to be ∼450 ms. In β-escin-permeabilized ASM strips, photolytic release of caged Ca2+resulted in force generation after ∼800 ms. When calmodulin (CaM) was added, this delay was shortened to ∼500 ms. In the presence of exogenous CaM and 100 μM Ca2+, photolytic release of caged ATP led to force generation after ∼80 ms. These results indicated significant delays due to CaM mobilization and Ca2+-CaM activation of myosin light chain kinase but much shorter delays introduced by myosin light chain kinase-induced phosphorylation of the regulatory myosin light chain MLC20and cross-bridge recruitment. This was confirmed by prior thiophosphorylation of MLC20, in which force generation occurred ∼50 ms after photolytic release of caged ATP, approximating the delay introduced by cross-bridge recruitment alone. The time required to reach maximum steady-state force was >15 s. Rapid chelation of [Ca2+]iafter photolytic release of caged diazo-2 resulted in relaxation after a delay of ∼1.2 s and 50% reduction in force after ∼57 s. We conclude that in ASM cells agonist-induced [Ca2+]ioscillations are temporally and spatially integrated during excitation-contraction coupling, resulting in stable force production.

Perkins, William J., Young-Soo Han, and Gary C. Sieck.Skeletal muscle force and actomyosin ATPase activity reduced by nitric oxide donor. J. Appl. Physiol.83(4): 1326–1332, 1997.—Nitric oxide (NO) may exert direct effects on actin-myosin cross-bridge cycling by modulating critical thiols on the myosin head. In the present study, the effects of the NO donor sodium nitroprusside (SNP; 100 μM to 10 mM) on mechanical properties and actomyosin adenosinetriphosphatase (ATPase) activity of single permeabilized muscle fibers from the rabbit psoas muscle were determined. The effects of N-ethylmaleimide (NEM; 5–250 μM), a thiol-specific alkylating reagent, on mechanical properties of single fibers were also evaluated. Both NEM (≥25 μM) and SNP (≥1 mM) significantly inhibited isometric force and actomyosin ATPase activity. The unloaded shortening velocity of SNP-treated single fibers was decreased, but to a lesser extent, suggesting that SNP effects on isometric force and actomyosin ATPase were largely due to decreased cross-bridge recruitment. The calcium sensitivity of SNP-treated single fibers was also decreased. The effects of SNP, but not NEM, on force and actomyosin ATPase activity were reversed by treatment with 10 mMdl-dithiothreitol, a thiol-reducing agent. We conclude that the NO donor SNP inhibits contractile function caused by reversible oxidation of contractile protein thiols.