Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter.

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP3Rs) and the mitochondrial Ca2+ uniporter (MCU) are key proteins that regulate intracellular Ca2+ concentration. Mitochondrial Ca2+ accumulation activates Ca2+-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP3R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca2+ signaling. Our results show that TKO cells (exhibiting total loss of Ca2+ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca2+ signaling is dispensable for the bioenergetic needs of both IP3R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.

Redox regulation of type-I inositol trisphosphate receptors in intact mammalian cells.

A sensitization of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release is associated with oxidative stress in multiple cell types. These effects are thought to be mediated by alterations in the redox state of critical thiols in the IP3R, but this has not been directly demonstrated in intact cells. Here, we utilized a combination of gel-shift assays with MPEG-maleimides and LC-MS/MS to monitor the redox state of recombinant IP3R1 expressed in HEK293 cells. We found that under basal conditions, ∼5 of the 60 cysteines are oxidized in IP3R1. Cell treatment with 50 µm thimerosal altered gel shifts, indicating oxidation of ∼20 cysteines. By contrast, the shifts induced by 0.5 mm H2O2 or other oxidants were much smaller. Monitoring of biotin-maleimide attachment to IP3R1 by LC-MS/MS with 71% coverage of the receptor sequence revealed modification of two cytosolic (Cys-292 and Cys-1415) and two intraluminal cysteines (Cys-2496 and Cys-2533) under basal conditions. The thimerosal treatment modified an additional eleven cysteines, but only three (Cys-206, Cys-767, and Cys-1459) were consistently oxidized in multiple experiments. H2O2 also oxidized Cys-206 and additionally oxidized two residues not modified by thimerosal (Cys-214 and Cys-1397). Potentiation of IP3R channel function by oxidants was measured with cysteine variants transfected into a HEK293 IP3R triple-knockout cell line, indicating that the functionally relevant redox-sensitive cysteines are predominantly clustered within the N-terminal suppressor domain of IP3R. To our knowledge, this study is the first that has used proteomic methods to assess the redox state of individual thiols in IP3R in intact cells.

Standardized Rehabilitation and Hospital Length of Stay Among Patients With Acute Respiratory Failure: A Randomized Clinical Trial.

IMPORTANCE: Physical rehabilitation in the intensive care unit (ICU) may improve the outcomes of patients with acute respiratory failure. OBJECTIVE: To compare standardized rehabilitation therapy (SRT) to usual ICU care in acute respiratory failure. DESIGN, SETTING, AND PARTICIPANTS: Single-center, randomized clinical trial at Wake Forest Baptist Medical Center, North Carolina. Adult patients (mean age, 58 years; women, 55%) admitted to the ICU with acute respiratory failure requiring mechanical ventilation were randomized to SRT (n=150) or usual care (n=150) from October 2009 through May 2014 with 6-month follow-up. INTERVENTIONS: Patients in the SRT group received daily therapy until hospital discharge, consisting of passive range of motion, physical therapy, and progressive resistance exercise. The usual care group received weekday physical therapy when ordered by the clinical team. For the SRT group, the median (interquartile range [IQR]) days of delivery of therapy were 8.0 (5.0-14.0) for passive range of motion, 5.0 (3.0-8.0) for physical therapy, and 3.0 (1.0-5.0) for progressive resistance exercise. The median days of delivery of physical therapy for the usual care group was 1.0 (IQR, 0.0-8.0). MAIN OUTCOMES AND MEASURES: Both groups underwent assessor-blinded testing at ICU and hospital discharge and at 2, 4, and 6 months. The primary outcome was hospital length of stay (LOS). Secondary outcomes were ventilator days, ICU days, Short Physical Performance Battery (SPPB) score, 36-item Short-Form Health Surveys (SF-36) for physical and mental health and physical function scale score, Functional Performance Inventory (FPI) score, Mini-Mental State Examination (MMSE) score, and handgrip and handheld dynamometer strength. RESULTS: Among 300 randomized patients, the median hospital LOS was 10 days (IQR, 6 to 17) for the SRT group and 10 days (IQR, 7 to 16) for the usual care group (median difference, 0 [95% CI, -1.5 to 3], P = .41). There was no difference in duration of ventilation or ICU care. There was no effect at 6 months for handgrip (difference, 2.0 kg [95% CI, -1.3 to 5.4], P = .23) and handheld dynamometer strength (difference, 0.4 lb [95% CI, -2.9 to 3.7], P = .82), SF-36 physical health score (difference, 3.4 [95% CI, -0.02 to 7.0], P = .05), SF-36 mental health score (difference, 2.4 [95% CI, -1.2 to 6.0], P = .19), or MMSE score (difference, 0.6 [95% CI, -0.2 to 1.4], P = .17). There were higher scores at 6 months in the SRT group for the SPPB score (difference, 1.1 [95% CI, 0.04 to 2.1, P = .04), SF-36 physical function scale score (difference, 12.2 [95% CI, 3.8 to 20.7], P = .001), and the FPI score (difference, 0.2 [95% CI, 0.04 to 0.4], P = .02). CONCLUSIONS AND RELEVANCE: Among patients hospitalized with acute respiratory failure, SRT compared with usual care did not decrease hospital LOS. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00976833.

Computer clinical decision support that automates personalized clinical care: a challenging but needed healthcare delivery strategy.

How to deliver best care in various clinical settings remains a vexing problem. All pertinent healthcare-related questions have not, cannot, and will not be addressable with costly time- and resource-consuming controlled clinical trials. At present, evidence-based guidelines can address only a small fraction of the types of care that clinicians deliver. Furthermore, underserved areas rarely can access state-of-the-art evidence-based guidelines in real-time, and often lack the wherewithal to implement advanced guidelines. Care providers in such settings frequently do not have sufficient training to undertake advanced guideline implementation. Nevertheless, in advanced modern healthcare delivery environments, use of eActions (validated clinical decision support systems) could help overcome the cognitive limitations of overburdened clinicians. Widespread use of eActions will require surmounting current healthcare technical and cultural barriers and installing clinical evidence/data curation systems. The authors expect that increased numbers of evidence-based guidelines will result from future comparative effectiveness clinical research carried out during routine healthcare delivery within learning healthcare systems.

Enabling a learning healthcare system with automated computer protocols that produce replicable and personalized clinician actions.

Clinical decision-making is based on knowledge, expertise, and authority, with clinicians approving almost every intervention-the starting point for delivery of "All the right care, but only the right care," an unachieved healthcare quality improvement goal. Unaided clinicians suffer from human cognitive limitations and biases when decisions are based only on their training, expertise, and experience. Electronic health records (EHRs) could improve healthcare with robust decision-support tools that reduce unwarranted variation of clinician decisions and actions. Current EHRs, focused on results review, documentation, and accounting, are awkward, time-consuming, and contribute to clinician stress and burnout. Decision-support tools could reduce clinician burden and enable replicable clinician decisions and actions that personalize patient care. Most current clinical decision-support tools or aids lack detail and neither reduce burden nor enable replicable actions. Clinicians must provide subjective interpretation and missing logic, thus introducing personal biases and mindless, unwarranted, variation from evidence-based practice. Replicability occurs when different clinicians, with the same patient information and context, come to the same decision and action. We propose a feasible subset of therapeutic decision-support tools based on credible clinical outcome evidence: computer protocols leading to replicable clinician actions (eActions). eActions enable different clinicians to make consistent decisions and actions when faced with the same patient input data. eActions embrace good everyday decision-making informed by evidence, experience, EHR data, and individual patient status. eActions can reduce unwarranted variation, increase quality of clinical care and research, reduce EHR noise, and could enable a learning healthcare system.

Variation in the rapid shallow breathing index associated with common measurement techniques and conditions.

BACKGROUND: The rapid-shallow-breathing index (RSBI) is widely used to evaluate mechanically ventilated patients for weaning and extubation, but it is determined in different clinical centers in a variety of ways, under conditions that are not always comparable. We hypothesized that the value of RSBI may be significantly influenced by common variations in measurement conditions and technique. METHODS: Sixty patients eligible for a weaning evaluation after >or=72 hours of mechanical ventilation were studied over 15 months in a medical intensive care unit. RSBI was measured while the patients were on 2 different levels of ventilator support: 5 cm H2O continuous positive airway pressure (CPAP) versus T-piece. RSBI was also calculated in 2 different ways: using the values of minute ventilation and respiratory rate provided by the digital output of the ventilator, versus values obtained manually with a Wright spirometer. Finally, RSBI was measured at 2 different times of the day. RESULTS: RSBI was significantly less when measured on 5 cm H2O CPAP, compared to T-piece: the medians and interquartile ranges were 71 (52-88) breaths/min/L versus 90 (59-137) breaths/min/L, respectively (P<.001). There were no significant differences in the value of RSBI obtained using ventilator-derived versus manual measures of the breathing pattern. RSBI was also not significantly different in the morning versus evening measurements. CONCLUSIONS: RSBI can be significantly affected by the level of ventilator support, but is relatively unaffected by both the technique used to determine the breathing pattern and the time of day at which it is measured.

Redox regulation of ER and mitochondrial Ca2+ signaling in cell survival and death.

Physiological signaling by reactive oxygen species (ROS) and their pathophysiological role in cell death are well recognized. This review focuses on two ROS targets that are key to local Ca2+ signaling at the ER/mitochondrial interface - notably, inositol trisphosphate receptors (IP3Rs) and the mitochondrial calcium uniporter (MCU). Both transport systems are central to molecular mechanisms in cell survival and death. Methods for the measurement of the redox state of these proteins and for the detection of ROS nanodomains are described. Recent results on the redox regulation of these proteins are reviewed.

Influence of Prehospital Function and Strength on Outcomes of Critically Ill Older Adults.

OBJECTIVES: To understand the influence of prehospital physical function and strength on clinical outcomes of critically ill older adults. DESIGN: Secondary analysis of prospective cohort study. SETTING: Health, Aging and Body Composition (Health ABC) Study. PARTICIPANTS: Of 3,075 older adult Health ABC participants, we identified 575 (60% white, 61% male, mean age 79) with prehospital function or grip strength measurements within 2 years of an intensive care unit stay. MEASUREMENTS: The primary analysis evaluated the association between prehospital walk speed and mortality, and secondary analyses focused on associations between function or grip strength and mortality or hospital length of stay. Function and grip strength were analyzed as continuous and categorical predictors. RESULTS: Slower prehospital walk speed was associated with greater risk of 30-day mortality (for each 0.1 m/s slower, odds ratio = 1.13, 95% confidence interval (CI) = 1.04-1.23, P = .004). Grip strength, chair stands, and balance had weaker, non-statistically significant associations with 30-day mortality. Participants with slower prehospital walk speed (hazard ratio (HR) = 0.94, 95% CI = 0.90-0.98, P = .005) and weak grip strength (HR = 0.85, 95% CI = 0.73-0.99, P = .03) were less likely to be discharged from the hospital alive. All function and strength measures were significantly associated with 1-year mortality. CONCLUSION: Slow prehospital walk speed was strongly associated with greater 30-day mortality and longer hospital stay in critically ill older adults, and measures of function and strength were associated with 1-year mortality. These data add to the accumulating evidence on the relationship between physical function and critical care outcomes.

Peak pressures during manual ventilation.

INTRODUCTION: Manual (bag) ventilation sometimes achieves better oxygenation than does a mechanical ventilator. We speculated that clinicians might generate very high airway pressure during manual ventilation (much higher than the pressure delivered by a mechanical ventilator), and that the high airway pressure causes alveolar recruitment and thus improves oxygenation. Such high pressure might injure alveoli in some patients. METHODS: We tested the hypothesis that manual ventilation may involve substantially higher pressure than is delivered by a mechanical ventilator. We asked experienced respiratory therapists to manually ventilate a lung model that was set to represent several typical clinical scenarios. RESULTS: We found that the peak airway pressure generated by the therapists was sometimes in excess of 100 cm H(2)O. CONCLUSIONS: The high airway pressure during manual ventilation would be considered extreme in the context of conventional mechanical ventilation, which raises questions about whether manual ventilation causes barotrauma.

Cardiopulmonary resuscitation among mechanically ventilated patients.

PURPOSE: To evaluate the outcomes, including long-term survival, after cardiopulmonary resuscitation (CPR) in mechanically ventilated patients. METHODS: We analyzed Medicare data from 1994 to 2005 to identify beneficiaries who underwent in-hospital CPR. We then identified a subgroup receiving CPR one or more days after mechanical ventilation was initiated [defined by ICD-9 procedure code for intubation (96.04) or mechanical ventilation (96.7x) one or more days prior to procedure code for CPR (99.60 or 99.63)]. RESULTS: We identified 471,962 patients who received in-hospital CPR with an overall survival to hospital discharge of 18.4 % [95 % confidence interval (CI) 18.3-18.5 %]. Of those, 42,163 received CPR one or more days after mechanical ventilation initiation. Survival to hospital discharge after CPR in ventilated patients was 10.1 % (95 % CI 9.8-10.4 %), compared to 19.2 % (95 % CI 19.1-19.3 %) in non-ventilated patients (p < 0.001). Among this group, older age, race other than white, higher burden of chronic illness, and admission from a nursing facility were associated with decreased survival in multivariable analyses. Among all CPR recipients, those who were ventilated had 52 % lower odds of survival (OR 0.48, 95 % CI 0.46-0.49, p < 0.001). Median long-term survival in ventilated patients receiving CPR who survived to hospital discharge was 6.0 months (95 % CI 5.3-6.8 months), compared to 19.0 months (95 % CI 18.6-19.5 months) among the non-ventilated survivors (p < 0.001 by logrank test). Of all patients receiving CPR while ventilated, only 4.1 % were alive at 1 year. CONCLUSIONS: Survival after in-hospital CPR is decreased among ventilated patients compared to those who are not ventilated. This information is important for clinicians, patients, and family members when discussing CPR in critically ill patients.

Inpatient transfers to the intensive care unit: delays are associated with increased mortality and morbidity.

OBJECTIVE: To examine if delayed transfer to the intensive care unit (ICU) after physiologic deterioration is associated with increased morbidity and mortality. DESIGN: Inception cohort. SETTING: Community hospital in Ogden, Utah. PATIENTS: Ninety-one consecutive inpatients with noncardiac diagnoses at the time of emergent transfer to the ICU. We determined the time when each patient first met any of 11 pre-specified physiologic criteria. We classified patients as "slow transfer" when patients met a physiologic criterion 4 or more hours before transfer to the ICU. Patients were followed until discharge. INTERVENTIONS: None. MEASUREMENTS: In-hospital mortality, functional status at hospital discharge, hospital resources. MAIN RESULTS: At the time when the first physiologic criterion was met on the ward, slow- and rapid-transfer patients were similar in terms of age, gender, diagnosis, number of days in hospital prior to ICU transfer, prehospital functional status, and APACHE II scores. By the time slow-transfer patients were admitted to the ICU, they had significantly higher APACHE II scores (21.7 vs 16.2; P =.002) and were more likely to die in-hospital (41% vs 11%; relative risk [RR], 3.5; 95% confidence interval [95% CI], 1.4 to 9.5). Slow-transfer patients were less likely to have had their physician notified of deterioration within 2 hours of meeting physiologic criteria (59% vs 31%; P =.001) and less likely to have had a bedside physician evaluation within the first 3 hours after meeting criteria (23% vs 83%; P =.001). CONCLUSIONS: Slow transfer to the ICU of physiologically defined high-risk hospitalized patients was associated with increased risk of death. Slow response to physiologic deterioration may explain these findings.

Ventilation of patients with acute lung injury and acute respiratory distress syndrome: has new evidence changed clinical practice?

OBJECTIVES: A recent randomized trial of mechanical ventilation in acute lung injury (ALI)/adult respiratory distress syndrome (ARDS) demonstrated a 22% relative reduction in mortality rate using 6 mL/kg predicted body weight tidal volume vs. 12 mL/kg predicted body weight tidal volume. We determined whether publication of these findings changed clinical practice. DESIGN: Retrospective cohort, 12 months before (Pre) and 12 months after publication (Post) of a randomized trial supporting the use of a 6 mL/kg predicted body weight tidal volume strategy. SETTING: Three tertiary care hospitals in northern New England. PATIENTS: From a sample of 943 patients receiving prolonged mechanical ventilation between 1998 and 1999 (Pre) and between 2000 and 2001 (Post), 300 patients meeting the American-European Consensus Conference definition of ALI or ARDS were selected for analysis. INTERVENTIONS: The tidal volume, tidal volume/kg predicted body weight, and proportion receiving tidal volume/kg > or =6 mL/kg and < or =12 mL/kg predicted body weight were recorded at noon the first day after the diagnosis of ALI or ARDS was established. MEASUREMENTS AND MAIN RESULTS: Pre and Post mean tidal volume (+/- sd) size and tidal volume size/kg predicted body weight were 759 +/- 158 mL (median 750 mL) vs. 639 +/- 138 mL (median 600 mL, p <.001) and 12.3 +/- 2.7 mL/kg (median 11.7 mL/kg) vs. 10.6 +/- 2.4 mL/kg (median 10.7 mL/kg, p <.001) respectively. Pre and Post plateau pressures and peak airway pressures were similar. CONCLUSION: Publication of a trial demonstrating large mortality reductions using small tidal volume was associated with significant reductions in tidal volume delivered to patients with ALI/ARDS. However, wide variation in practice persists, and the proportion of patients receiving tidal volumes within recommended limits (< or =8 mL/kg) remains modest.