Schaible, Niccole

Abstract Background Regulator of G protein signaling (RGS)2 terminates bronchoconstrictive Gaq signaling, and reductions in RGS2 expression associate with a hypercontractility phenotype. RGS2 promoter variants rs2746071 and rs2746072 are hypofunctional in gene reporter assays, and individuals with severe asthma homozygous for these two RGS2 variants experience higher asthma exacerbation rates. Whether these RGS2 promoter variants directly cause a hypercontractile human airway smooth muscle (HASM) phenotype is unknown. Methods We used the hTERT immortalized HASM cell line, derived from a human non-asthmatic donor. RGS2expression was reduced with siRNA (150nM anti-RGS2 vs. control siRNA) or through CRISPR transformation (Synthego©) to express the RGS2 promoter variants of interest, confirmed by Sanger sequencing. Gene and protein expression were quantified by qPCR and Western blotting. Intracellular Ca ++ flux and oscillation was measured with Fluo-8 (Abcam) with excitation/emission at 490/525 nm. HASM contractility to histamine treatment was measured using Traction Force Microscopy implemented in a 96-well plate format. Results Relative to cell lines with reference genotypes, HASM CRISPR transformed with the two RGS2 promoter variants exhibited a 41.8% (95% CI 28.6%--62.5%; p < 0.0001) reduction in RGS2 relative expression by qPCR, and a 35% (95% CI 8.6%—85.7%; p = 0.024) reduction in protein expression by Western blot. Methacholine-triggered calcium flux was nearly 50% greater for the cell lines with the RGS2 homozygous variants vs. those with reference genotypes (p = 0.043) as was the contractile response to histamine. HASM with reference genotypes exposed to anti-RGS2 siRNA showed the same phenotype, consistent with a causal relationship between the variants and expression (decreased) and receptor function (enhanced). Similarly, HASM CRISPR-transformed with the two RGS2 promoter variants exhibited an up to 3-fold increase in contraction magnitude (represented by strain energy, reported in pJ) in response to histamine >1uM (p < 0.05) relative to cell lines with reference genotypes. Conclusion Two promoter single nucleotide polymorphisms substitutions are sufficient to reduce RGS2 expression and induce a hypercontractile HASM phenotype. This abstract is funded by: AAAAI Bridge Award, NHLBI R21

In previous studies, we found that prolonged hypothermia (2 h or greater) followed by rewarming induces cardiac contractile dysfunction (rewarming shock) mimicking the effects of accidental hypothermia followed by rapid rewarming in trauma patients. We found that rewarming shock was also presented in isolated cardiomyocytes in which reduced contractility was attributed to PKA‐mediated TnI phosphorylation and reduced Ca 2+ sensitivity. During hypothermia rewarming in cardiomyocytes there is increased production of reactive oxidant species (ROS ‐ superoxide anion), which may trigger the intracellular mechanisms underlying reduced contractility. We hypothesized that in cardiomyocytes, hypothermia/rewarming‐induced ROS production is due to altered mitochondrial respiratory function. To test this hypothesis, isolated cardiomyocytes from 6 rats underwent the following hypothermia/rewarming protocol in which they were electrically stimulated every 2 s: experimental temperature was cooled from 35°C to 15°C during the initial 30 min, then maintained at 15°C for 2 h, followed by rewarming to 35°C in the final 30 min. Time‐matched control cardiomyocytes were also electrically stimulated every 2 s and maintained at 35°C for 3 h. After the 3‐h protocol, cardiomyocytes were placed into separate chambers of an Oroboros Oxygraph (high‐resolution respirometry) to measure O 2 consumption rate (OCR) under varying conditions. Initially, basal OCR was measured for 5 min followed by serial exposure to oligomycin (1 μM for 5 min) to block ATP synthase activity, FCCP (0.5 μM repeated 3 times for 5 min each) an H + ionophore to dissipate mitochondrial membrane potential; and antimycin‐A (0.5 μM for 5 min) to block the electron transport chain. This procedure allowed assessment of the key parameters of mitochondrial respiratory function: basal and maximal respiration (after FCCP exposure), ATP production and proton leak (after oligomycin), and reserve respiratory capacity (calculated after antimycin‐A). We found that compared to control cardiomyocytes, hypothermia/rewarming decreased key parameters of mitochondrial function, which suggests that altered mitochondrial function may have contributed to increased ROS production. In addition, decreased OCR following rewarming may contribute to contractile dysfunction by limiting ATP supply in response to metabolic demand, as well as by contributing to a decline in cell viability (e.g., triggering apoptosis). Support or Funding Information 5T32HL105355 (NSS); Norwegian Air Ambulance Foundation (YSH)

Hypothermia/Rewarming (H/R) is poorly tolerated by the myocardium; however, the underlying intracellular basis of H/R‐induced cardiac injury remains elusive. We hypothesized that in cardiomyocytes, H/R leads to contractile dysfunction primarily through reduced Ca 2+ sensitivity of the myofilaments. To test this hypothesis, isolated rat cardiomyocytes (13–15 cells from 6 rats per group) were electrically stimulated to evoke both cytosolic Ca 2+ ([Ca 2+ ] cyto ) and contractile (sarcomere shortening) responses that were simultaneously measured using an IonOptix system. These measurements were subjected to the following temperature protocols: 1) hypothermia (15°C) followed by rewarming (35°C) or 2) time‐matched control (35°C). Contractile dysfunction after H/R was indicated by reduced shortening velocity and reduced extent of shortening of sarcomere length (SL) compared to time‐matched controls. Throughout hypothermia, basal [Ca 2+ ] cyto increased and the duration of the evoked [Ca 2+ ] cyto transients was prolonged. Phase‐loop plots of [Ca 2+ ] cyto versus contraction in cells exposed to H/R indicate that Ca 2+ sensitivity of contraction is reduced. Consistent with decreased Ca 2+ sensitivity, we found that cardiac troponin I (cTnI) phosphorylation (measured by Western blot) increased after H/R. Our results support our overall hypothesis and suggest that reduced Ca 2+ sensitivity and increased cTnI phosphorylation contributes to the altered excitation‐contraction coupling of cardiomyocytes following H/R. The clinical importance of our results brings focus to Ca 2+ sensitivity as a plausible therapeutic target in treating patients exposed to accidental hypothermia. Support or Funding Information NIH Grant 5T32HL10535 (NSS)

Hypothermia/Rewarming (H/R) induces myocardial dysfunction although the underlying intracellular mechanisms remain elusive. We hypothesized that in cardiomyocytes, H/R disrupts intracellular Ca 2+ ([Ca 2+ ] cyt ) homeostasis via an effect on both release and clearance of cytosolic Ca 2+ leading to a prolongation of evoked [Ca 2+ ] cyt transients associated with activation with downstream impact on contractility. To test this hypothesis, isolated rat cardiomyocytes (13–15 cells from 6 rats per group) were loaded with the Ca 2+ indicator, Fura‐2AM (0.5 μM), and exposed to H/R with or without electrical pacing at 0.5 Hz. In both groups, evoked [Ca 2+ ] cyt and contractile (sarcomere length (SL) shortening) responses were simultaneously measured at 35°C before and after hypothermia using an IonOptix system. H/R experiments were conducted as follows: experimental temperature was cooled from 35°C to 15°C in 30 min, maintained at 15°C for 2 h, and then rewarmed back to 35°C in 30 min. Time‐matched controls were implemented for 3 h at 35°C. With electrical pacing throughout hypothermia, basal [Ca 2+ ] cyt increased and the kinetics of the evoked [Ca 2+ ] cyt responses were prolonged. In addition, the extent and velocity of SL shortening was reduced in cardiomyocytes that were paced during hypothermia. In contrast, in cardiomyocytes that were not electrically paced during hypothermia, basal [Ca 2+ ] cyt was maintained and the kinetics of evoked [Ca 2+ ] cyt transients were unaffected. In addition, in the absence of pacing during hypothermia, contractility of cardiomyocytes was unaffected. These results suggest that H/R affects the release and clearance of evoked [Ca 2+ ] cyt transients (e.g., RyR mediated Ca 2+ release and SERCA mediated clearance) such that basal [Ca 2+ ] cyt increases. This effect of H/R on [Ca 2+ ] cyt homeostasis is associated with reduced cardiac contractility.

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)

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)

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.

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.

Hypothermia/rewarming (H/R) is poorly tolerated by the myocardium; however, the underlying intracellular basis of H/R-induced cardiac dysfunction remains elusive. We hypothesized that in cardiomyocytes, H/R disrupts excitation-contraction coupling by reducing myofilament Ca2+sensitivity due to an increase in cardiac troponin I (cTnI) phosphorylation. To test this hypothesis, isolated rat cardiomyocytes (13–15 cells from 6 rats per group) were electrically stimulated to evoke both cytosolic Ca2+([Ca2+]cyto) and contractile (sarcomere shortening) responses that were simultaneously measured using an IonOptix system. Cardiomyocytes were divided into two groups: 1) those exposed to hypothermia (15°C for 2 h) followed by rewarming (35°C; H/R); or 2) time-matched normothermic (35°C) controls (CTL). Contractile dysfunction after H/R was indicated by reduced velocity and extent of sarcomere length (SL) shortening compared with time-matched controls. Throughout hypothermia, basal [Ca2+]cytoincreased and the duration of evoked [Ca2+]cytotransients was prolonged. Phase-loop plots of [Ca2+]cytovs. contraction were shifted rightward in cardiomyocytes during hypothermia compared with CTL, indicating a decrease in Ca2+sensitivity. Using Western blot, we found that H/R increases cTnI phosphorylation. These results support our overall hypothesis and suggest that H/R disrupts excitation-contraction coupling of cardiomyocytes due to increased cTnI phosphorylation and reduced Ca2+sensitivity.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/hypothermiarewarming-disrupts-e-c-coupling/ .