Citations

In excitation‐contraction coupling, a transient elevation of cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) triggers a force response. 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. In smooth muscle, Ca 2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca 2+ ] cyt . However, this 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 hypothesized that [Ca 2+ ] cyt /force phase loop plot in ASM would be altered by conditions in which the extent of regulatory myosin light chain (rMLC) phosphorylation was affected. 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 μM ACh. Isometric force and [Ca 2+ ] cyt responses were simultaneously measured using a Guth Muscle Research System. In the first set of experiments we examined the effect of Y27623, a Rho‐kinase inhibitor, and calyculin A, a protein phosphatase inhibitor that affected Ca 2+ ‐induced rMLC phosphorylation. In a second set of experiments, we examined the effect of cytochalasin D (Cyto‐D), an inhibitor of actin polymerization, which reduces force independent of Ca 2+ ‐induced rMLC phosphorylation. As expected, Y27623 decreased ACh‐induced isometric force but had no effect on evoked [Ca 2+ ] cyt transients. Cyto‐D also decreased force with no effects on evoked [Ca 2+ ] cyt . By comparison, calyculin A increased force with no effect on [Ca 2+ ] cyt . Although these results suggest changes in Ca 2+ sensitivity, the phase loop plots showed no significant shift indicative of changes in dynamic Ca 2+ sensitivity. These results do not support our hypothesis that dynamic Ca 2+ sensitivity in ASM is modulated by the extent of rMLC phosphorylation. Under these experimental conditions, it appears that isometric force generation in ASM is not entirely dependent on [Ca 2+ ] cyt . Support or Funding Information Supported by NIH grant HL126451 (GCS)

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.

There is evidence that the anabolic response to exercise is blunted in skeletal muscle in older adults. Older adults also exhibit reduced mitochondrial content and lower oxidative capacity. Since mitochondria provide chemical energy for muscle protein synthesis, it is possible that age‐related changes to mitochondrial physiology may contribute to anabolic resistance. We evaluated this possibility by measuring mitochondrial function and anabolic responses to exercise in 12 young (19–34 yrs) and 12 older (67–83 yrs) adults. Serial vastus lateralis biopsies were collected during an infusion of 13 C 6 phenylalanine to measure muscle protein synthesis at baseline and 16 hours after a single bout of unaccustomed resistance exercise (unilateral knee extension, 8 sets, 10 repetitions at 70% of 1‐repetition maximum). Real‐time PCR and RNA sequencing were used to evaluate the transcriptional response to acute exercise in muscle tissue. Respiratory capacity of isolated mitochondria was measured by high‐resolution respirometry. Mitochondrial respiratory capacity was significantly lower in older adults (4.67±0.47 pmol/s/ug) compared to young (6.60±0.70 pmol/s/ug, p=0.024). Postabsorptive protein synthesis rates were similar in young and old for mixed muscle (Y: 0.067±0.005 %/hr, O: 0.104±0.030 %/hr, p=0.13), mitochondrial (Y: 0.076±0.014 %/hr, O: 0.089±0.017 %/hr, p=0.58), and myofibrillar fractions (Y: 0.073±0.005 %/hr, O: 0.072±0.015 %/hr, p=0.94). Exercise increased protein synthesis similarly in young and old for mixed muscle (Fold changes Y: 1.45±0.16, O: 1.46±0.18, p=0.98), mitochondrial (Y: 0.90±0.28, O: 1.74±0.41, p=0.11), and myofibrillar (Y: 1.35±0.13, O: 1.37±0.18, p=0.93). These data demonstrate older adults have lower mitochondrial respiratory capacity yet exhibit similar anabolic responses to resistance exercise compared to young. Protein synthesis does not appear limited by the decreased mitochondrial capacity with age.

Recent research focuses on the cerebrovascular response to vasodilatory stimuli as an index of cerebrovascular health; however, vasoconstrictor responses in the brain are likely equally important. We previously have shown that cerebral vasoconstrictor responses to hypocapnia are not affected by aging; however, this was evaluated in primarily sedentary adults. Therefore, we hypothesized that middle cerebral artery velocity (MCAv) vasoconstrictor responses to hypocapnia are dependent on age and habitual physical activity. We evaluated 13 young physically active (YA; age = 30±5 y; VO 2 max = 45±8 mL/kg/min), 6 young sedentary (YS; age = 29±4 y; VO 2 max = 26±3 mL/kg/min), 10 older physically active (OA; age= 61±6 y; VO 2 max =35±7 mL/kg/min) and 8 older sedentary (OS; age = 65±8 y; VO 2 max 23±2 mL/kg/min) adults during the recovery from inhaled 6% CO 2 to room air. Mean arterial pressure (MAP) end tidal CO 2 (ETCO 2 ) and MCAv were recorded throughout. Using a breath‐by breath analysis, cerebrovascular conductance index (CVCi=MCAv/MAP) reactivity was calculated as a slope of CVCi and ETCO 2 and used to quantify vasoconstrictor responses. CVCi reactivity was higher in YA adults (slope=2.74±0.9 AU) compared with both YS (slope=2.20±1.1 AU) and older groups (OA: slope=2.08±0.5 AU; OS: slope=2.07±0.7 AU; p<0.05 for all). There were no significant differences in CVCi reactivity between YS and either OA or OS (p=0.94, p=0.99 respectively). Additionally, CVCi negatively correlated with age (r=−0.38, p<0.05). Our results indicate that aging attenuates the cerebral vasoconstrictor response to hypocapnia in older adults in comparison to physically‐active young adults, but not compared to sedentary young controls. A sedentary lifestyle resulted in attenuated vasoconstrictor responses in the young sedentary group, as their responses were not different than the older adults. It remains unclear how habitual physical activity preserves the cerebral vasoconstrictor response to hypocapnia in young people, and if a structured exercise training intervention would impact this response in older adults. Support or Funding Information NIH HL118154

Background Individuals with sleep apnea experience repeated intermittent hypoxic exposures during sleep. Previous work in sleep apnea showed patients have an increased heart rate (HR), corrected QT interval (QTc), QTc dispersion and P‐wave maximum duration and dispersion (max‐min P‐wave duration). Such changes have been linked to increased risk of cardiovascular disease and sudden cardiac death. Unfortunately, the mechanisms contributing to these changes are not completely understood. Given that individuals with sleep apnea experience repeated intermittent hypoxic exposures during sleep, we tested the hypothesis that exposure to intermittent hypoxia (IH) in young healthy adults would result in similar ECG changes to those seen in patients with sleep apnea. Methods To test this hypothesis, we completed ECG analysis of 7 healthy humans (6M/1F, Age=29±5 years, BMI=24±4kg/m 2 ) without sleep apnea before and after 3‐hours of exposure to IH. Subjects were exposed to hypoxic gas (0.05 FiO2) for 25‐seconds separated by 2‐minutes of room air breathing. This resulted in 25 hypoxic events per hour in which oxygen saturation was significantly reduced (Normoxia: 97%, Hypoxia: 89%, p<0.01). ECG analysis was completed in Lab Chart 7 software and consisted of measurement of intervals (RR, PR, QT), durations (P, QRS), and amplitudes (P, Q, R, S, T) during 10 minutes of normoxic quiet rest before and after IH exposure. QTc was calculated using Bazett's formula (QT duration/√HR). Dispersion of QTc and P waves were calculated as max duration ‐min duration. Results HR was significantly increased following IH (60±8 vs. 66±8 beats/min, baseline vs. post‐IH, respectively, p<0.05). There was no significant change in P‐wave maximum duration or dispersion following IH. QT interval was significantly decreased and QTc was significantly increased following IH (QT interval: 377±13 vs. 368±8ms, QTc: 377±22 vs 384±22ms, baseline vs. post‐IH, p<0.05 for both). QTc dispersion was not significantly different from baseline following IH. Conclusions We found that 3 hours of experimentally‐induced IH in young healthy individuals resulted in increased HR, decreased QT interval, and increased QTc. These data suggest that IH exposure experienced by patients with sleep apnea may contribute to ECG changes commonly observed in this population and subsequent increases in cardiovascular morbidity and mortality. Support or Funding Information Funding : DK90541 (MJJ), HL120570 (JKL)

Importance: Phenol kits cleared by the Food and Drug Administration (FDA) are indicated as a topical anesthetic for the tympanic membrane (TM) in adults. However, there is no existing literature that reports outcomes to support the safety and use of phenol on the TM of awake children. Objective: Determine if topical phenol is safe and at low risk for complications and therefore be used effectively in awake children to facilitate office otologic procedures as in adults. Design, Setting, and Participants: Children under 21 years of age whose parents agreed to participate in an awake office setting for tympanostomy tube (TT) placement. All children had TT placement after phenol placement on the TM prior to insertion. Main Outcomes and Measures: TM perforation or other signs of TM complications through a minimum of 6-month clinical follow-up, along with assessment of the tolerability of the procedure by the child. Results: A total of 228 children with an age range of 6 months to 15.9 years and 435 TMs completed TT placement using phenol as a local anesthetic while awake in the office. There were no complications reported in the 204 children at the first follow-up visit post TM placement within 3 to 10 weeks. Of the 93 children followed up at least 6 months, there were no TM complications reported. Conclusions: This is the first study to report the outcomes on the use of phenol in an office setting in children. In this large experience, phenol appears to be tolerable and safe for use in young children in the office and is a potential safe choice of topical anesthesia for surgeons if they choose to perform office procedures such as myringotomies or TT placement on children.

The authors have published on a unique subset of patients whose headaches worsened in the Trendelenburg position and who on time-resolved MR angiography demonstrated left renal vein compression (nutcracker physiology) with retrograde left second lumbar vein (L2LV) flow and regional spinal epidural venous plexus (EVP) congestion. We hypothesized that the spinal EVP congestion subsequently causes a secondary congestion of the cerebral venous system, which then leads to an elevation of CSF pressure above that individuals CSF pressure set point. This results in a daily headache from onset. Thus, eliminating the spinal EVP could conceivably improve or eliminate the manifested headache syndrome. We now present a case series of four patients with long-term follow-up utilizing lumbar vein coil embolization as a new therapeutic approach. In each patient, the MR angiography findings were verified by catheter-based venography. Treatment consisted of endovascular embolization of the second lumbar vein. Four patients have had coil embolization of which three are 1 year or longer from their procedure while one is 10 months posttreatment. All patients were women. Duration of daily headache prior to embolization ranged from 4 to 8 years. Post-embolization: Three patients are either headache free or 90–95% improved with substantial pain free time. There were no procedure-related complications. Our results suggest that embolization of L2LV in a specific patient population with nutcracker physiology may substantially improve head pain issues. This is a minimally invasive outpatient technique with no apparent side effects.