Improved exercise capacity in cyclophilin-D knockout mice associated with enhanced oxygen utilization efficiency and augmented glucose uptake via AMPK-TBC1D1 signaling nexus.

We previously reported in HEK 293T cells that silencing the mitochondrial peptidyl prolyl isomerase cyclophilin-D (Cyp-D) reduces Vo2. We now report that in vivo Cyp-D ablation using constitutive Cyp-D knockout (KO) mice also reduces Vo2 both at rest (∼15%) and during treadmill exercise (∼12%). Yet, despite Vo2 reduction, these Cyp-D KO mice ran longer (1071 ± 77 vs. 785 ± 79 m; P = 0.002), for longer time (43 ± 3 vs. 34 ± 3 min; P = 0.004), and at higher speed (34 ± 1 vs. 29 ± 1 m/s; P ≤ 0.001), resulting in increased work (87 ± 6 vs. 58 ± 6 J; P ≤ 0.001). There were parallel reductions in carbon dioxide production, but of lesser magnitude, yielding a 2.3% increase in the respiratory exchange ratio consistent with increased glucose utilization as respiratory substrate. In addition, primary skeletal muscle cells of Cyp-D KO mice subjected to electrical stimulation exhibited higher glucose uptake (4.4 ± 0.55 vs. 2.6 ± 0.04 pmol/mg/min; P ≤ 0.001) with enhanced AMPK activation (0.58 ± 0.06 vs. 0.38 ± 0.03 pAMPK/ß-tubulin ratio; P ≤ 0.01) and TBC1 (Tre-2/USP6, BUB2, Cdc16) domain family, member 1 (TBC1D1) inactivation. Likewise, pharmacological activation of AMPK also increased glucose uptake (3.2 ± 0.3 vs. 2.3 ± 0.2 pmol/mg/min; P ≤ 0.001). Moreover, lactate and ATP levels were increased in these cells. Taken together, Cyp-D ablation triggered an adaptive response resulting in increased exercise capacity despite less oxygen utilization associated with increased glucose uptake and utilization involving AMPK-TBC1D1 signaling nexus.-Radhakrishnan, J., Baetiong, A., Kaufman, H., Huynh, M., Leschinsky, A., Fresquez, A., White, C., DiMario, J. X., Gazmuri, R. J. Improved exercise capacity in cyclophilin-D knockout mice associated with enhanced oxygen utilization efficiency and augmented glucose uptake via AMPK-TBC1D1 signaling nexus.

Sodium-Hydrogen Exchanger Isoform-1 Inhibition: A Promising Pharmacological Intervention for Resuscitation from Cardiac Arrest.

Out-of-hospital sudden cardiac arrest is a major public health problem with an overall survival of less than 5%. Upon cardiac arrest, cessation of coronary blood flow rapidly leads to intense myocardial ischemia and activation of the sarcolemmal Na+-H+ exchanger isoform-1 (NHE-1). NHE-1 activation drives Na+ into cardiomyocytes in exchange for H+ with its exchange rate intensified upon reperfusion during the resuscitation effort. Na+ accumulates in the cytosol driving Ca2+ entry through the Na+-Ca2+ exchanger, eventually causing cytosolic and mitochondrial Ca2+ overload and worsening myocardial injury by compromising mitochondrial bioenergetic function. We have reported clinically relevant myocardial effects elicited by NHE-1 inhibitors given during resuscitation in animal models of ventricular fibrillation (VF). These effects include: (a) preservation of left ventricular distensibility enabling hemodynamically more effective chest compressions, (b) return of cardiac activity with greater electrical stability reducing post-resuscitation episodes of VF, (c) less post-resuscitation myocardial dysfunction, and (d) attenuation of adverse myocardial effects of epinephrine; all contributing to improved survival in animal models. Mechanistically, NHE-1 inhibition reduces adverse effects stemming from Na+-driven cytosolic and mitochondrial Ca2+ overload. We believe the preclinical work herein discussed provides a persuasive rationale for examining the potential role of NHE-1 inhibitors for cardiac resuscitation in humans.

Cyclophilin-D: a resident regulator of mitochondrial gene expression.

Cyclophilin-D (Cyp-D) is a mitochondrial matrix peptidyl-prolyl isomerase. Because cyclophilins can regulate nuclear gene expression, we examined whether Cyp-D could regulate mitochondrial gene expression. We demonstrated in HEK 293T cells that transfected Cyp-D interacts with mitochondrial transcription factors B1 and B2 (TFB2M) but not with mitochondrial transcription factor A. We also demonstrated that Cyp-D interacts in vivo with TFB2M. Genetic silencing of Cyp-D and pharmacologic inhibition of Cyp-D markedly reduced mitochondrial transcription to 18 ± 5% (P < 0.05) and 24 ± 3% (P < 0.05) of respective controls. The level of interaction between Cyp-D and TFB2M correlated with the level of nascent mitochondrial RNA intensity (r = 0.896; P = 0.0156). Cyp-D silencing down-regulated mitochondrial transcripts initiated from the heavy strand promoter 2 [i.e., NADH dehydrogenase 1 (ND1) by 11-fold, P < 0.005; cytochrome oxidase 1 (COX1) by 4-fold, P < 0.001; and ATP synthase subunit 6 (ATP6) by 6.5-fold, P < 0.005); but not NADH dehydrogenase 6 (ND6)], which is initiated from the light strand promoter. Cyp-D silencing reduced mitochondrial membrane potential and cellular oxygen consumption (from 59 ± 5 to 34 ± 1 µmol oxygen/min/10(6) cells, P < 0.001); the latter without a statistically significant reversal after uncoupling electron transport from ATP synthesis, consistent with down-regulation of electron transport complexes. Accordingly, these studies provide novel evidence that Cyp-D could play a key role in regulating mitochondrial gene expression.

Ubiquinol (reduced Coenzyme Q10) in patients with severe sepsis or septic shock: a randomized, double-blind, placebo-controlled, pilot trial.

INTRODUCTION: We previously found decreased levels of Coenzyme Q10 (CoQ10) in patients with septic shock. The objective of the current study was to assess whether the provision of exogenous ubiquinol (the reduced form of CoQ10) could increase plasma CoQ10 levels and improve mitochondrial function. METHODS: We performed a randomized, double-blind, pilot trial at a single, tertiary care hospital. Adults (age ≥18 years) with severe sepsis or septic shock between November 2012 and January 2014 were included. Patients received 200 mg enteral ubiquinol or placebo twice a day for up to seven days. Blood draws were obtained at baseline (0 h), 12, 24, 48, and 72 h. The primary outcome of the study was change in plasma CoQ10 parameters (total CoQ10 levels, CoQ10 levels relative to cholesterol levels, and levels of oxidized and reduced CoQ10). Secondary outcomes included assessment of: 1) vascular endothelial biomarkers, 2) inflammatory biomarkers, 3) biomarkers related to mitochondrial injury including cytochrome c levels, and 4) clinical outcomes. CoQ10 levels and biomarkers were compared between groups using repeated measures models. RESULTS: We enrolled 38 patients: 19 in the CoQ10 group and 19 in the placebo group. The mean patient age was 62 ± 16 years and 47% were female. Baseline characteristics and CoQ10 levels were similar for both groups. There was a significant increase in total CoQ10 levels, CoQ10 levels relative to cholesterol levels, and levels of oxidized and reduced CoQ10 in the ubiquinol group compared to the placebo group. We found no difference between the two groups in any of the secondary outcomes. CONCLUSIONS: In this pilot trial we showed that plasma CoQ10 levels could be increased in patients with severe sepsis or septic shock, with the administration of oral ubiquinol. Further research is needed to address whether ubiquinol administration can result in improved clinical outcomes in this patient population. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT01948063. Registered on 18 February 2013.

Vitamin C compromises cardiac resuscitability in a rat model of ventricular fibrillation.

Resuscitation from cardiac arrest is partly limited by progressive reduction in left ventricular distensibility, leading to decreased hemodynamic efficacy of cardiopulmonary resuscitation (CPR). Reduction in left ventricular distensibility has been linked to loss of mitochondrial bioenergetic function that can result from oxidative injury. Attenuation of oxidative injury by administration of vitamin C during CPR may help maintain left ventricular distensibility and favor resuscitability and survival. Ventricular fibrillation was electrically induced in 2 series of 16 rats each and left untreated for 10 minutes. Resuscitation was attempted by 8 minutes of CPR and delivery of electrical shocks. Dehydroascorbate (DHA)-an oxidized form of vitamin C that enters the cell via glucose transporters-was used in series 1 and ascorbic acid (AA)-the reduced form of vitamin C that enters the cell via specialized AA transporters-in series 2. In each series, rats were randomized 1:1 to receive a 250 mg/kg right atrial bolus of DHA or AA or vehicle immediately before chest compression. Left ventricular distensibility-measured as the ratio between coronary perfusion pressure and compression depth-was numerically lower (not significant) in rats that received DHA (1.6 ± 0.2 vs. 1.9 ± 0.7 mm Hg/mm) and AA (1.8 ± 0.6 vs. 1.9 ± 0.3 mm Hg/mm). In addition, resuscitability was compromised by DHA (2/8 vs. 7/8; P = 0.041) and by AA (0/8 vs. 5/8; P = 0.026). AA levels in mitochondria were no different than control. Vitamin C failed to preserve left ventricular distensibility during CPR and had detrimental effects on resuscitability, suggesting possible disruption of protective signaling mechanisms during oxidative stress by vitamin C.

Enhanced Oxygen Utilization Efficiency With Concomitant Activation of AMPK-TBC1D1 Signaling Nexus in Cyclophilin-D Conditional Knockout Mice.

We have previously reported in HEK 293 T cells and in constitutive cyclophilin-D (Cyp-D) knockout (KO) mice that Cyp-D ablation downregulates oxygen consumption (VO2) and triggers an adaptive response that manifest in higher exercise endurance with less VO2. This adaptive response involves a metabolic switch toward preferential utilization of glucose via AMPK-TBC1D1 signaling nexus. We now investigated whether a similar response could be triggered in mice after acute ablation of Cyp-D using tamoxifen-induced ROSA26-Cre-mediated (i.e., conditional KO, CKO) by subjecting them to treadmill exercise involving five running sessions. At their first treadmill running session, CKO mice and controls had comparable VO2 (208.4 ± 17.9 vs. 209.1 ± 16.8 ml/kg min-1), VCO2 (183.6 ± 17.2 vs. 184.8 ± 16.9 ml/kg min-1), and RER (0.88 ± 0.043 vs. 0.88 ± 0.042). With subsequent sessions, CKO mice displayed more prominent reduction in VO2 (genotype & session interaction p = 0.000) with less prominent reduction in VCO2 resulting in significantly increased RER (genotype and session interaction p = 0.013). The increase in RER was consistent with preferential utilization of glucose as respiratory substrate (4.6 ± 0.8 vs. 4.0 ± 0.9 mg/min, p = 0.003). CKO mice also performed a significantly higher treadmill work for given VO2 expressed as a power/VO2 ratio (7.4 ± 0.2 × 10-3 vs. 6.7 ± 0.2 10-3 ratio, p = 0.025). Analysis of CKO skeletal muscle tissue after completion of five treadmill running sessions showed enhanced AMPK activation (0.669 ± 0.06 vs. 0.409 ± 0.11 pAMPK/ß-tubulin ratio, p = 0.005) and TBC1D1 inactivation (0.877 ± 0.16 vs. 0.565 ± 0.09 pTBC1D1/ß-tubulin ratio, p < 0.05) accompanied by increased glucose transporter-4 levels consistent with activation of the AMPK-TBC1D1 signaling nexus enabling increased glucose utilization. Taken together, our study demonstrates that like constitutive Cyp-D ablation, acute Cyp-D ablation also induces a state of increased O2 utilization efficiency, paving the way for exploring the use of pharmacological approach to elicit the same response, which could be beneficial under O2 limiting conditions.

Targeted Delivery of Electrical Shocks and Epinephrine, Guided by Ventricular Fibrillation Amplitude Spectral Area, Reduces Electrical and Adrenergic Myocardial Burden, Improving Survival in Swine.

Background We previously reported that resuscitation delivering electrical shocks guided by real-time ventricular fibrillation amplitude spectral area (AMSA) enabled return of spontaneous circulation (ROSC) with fewer shocks, resulting in less myocardial dysfunction. We now hypothesized that AMSA could also guide delivery of epinephrine, expecting further outcome improvement consequent to less electrical and adrenergic burdens. Methods and Results A swine model of ventricular fibrillation was used to compare after 10 minutes of untreated ventricular fibrillation a guidelines-driven (n=8) resuscitation protocol, delivering shocks every 2 minutes and epinephrine every 4 minutes, with an AMSA-driven shocks (n=8) protocol, delivering epinephrine every 4 minutes, and with an AMSA-driven shocks and epinephrine (ADSE; n=8) protocol. For guidelines-driven, AMSA-driven shocks, and ADSE protocols, the time to ROSC (mean±SD) was 569±164, 410±111, and 400±80 seconds (P=0.045); the number of shocks (mean±SD) was 5±2, 3±1, and 3±2 (P=0.024) with ADSE fewer than guidelines-driven (P=0.03); and the doses of epinephrine (median [interquartile range]) were 2.0 (1.3-3.0), 1.0 (1.0-2.8), and 1.0 (0.3-3.0) (P=0.419). The ROSC rate was similar, yet survival after ROSC favored AMSA-driven protocols (guidelines-driven, 3/6; AMSA-driven shocks, 6/6; and ADSE, 7/7; P=0.019 by log-rank test). Left ventricular function and survival after ROSC correlated inversely with electrical burden (ie, cumulative unsuccessful shocks, J/kg; P=0.020 and P=0.046) and adrenergic burden (ie, total epinephrine doses, mg/kg; P=0.042 and P=0.002). Conclusions Despite similar ROSC rates achieved with all 3 protocols, AMSA-driven shocks and ADSE resulted in less postresuscitation myocardial dysfunction and better survival, attributed to attaining ROSC with less electrical and adrenergic myocardial burdens.

Constitutive cyclophilin-D ablation in mice increases exercise and cognitive-behavioral performance under normoxic and hypoxic conditions.

We recently reported that constitutive ablation of cyclophilin-D (Cyp-D) in mice reduces oxygen consumption (VO2) while paradoxically increasing exercise endurance, thereby demonstrating increased O2 utilization efficiency. This response was associated with augmented glucose uptake and glucose utilization, in part mediated through adenosine monophosphate-activated kinase signaling. We now hypothesized that Cyp-D knock-out (KO) mice might also exhibit improved cognitive-behavioral performance and that these favorable adaptive responses may persist under hypoxic conditions. We therefore assessed under normoxic (20.9% O2, simulating ground O2 levels) and hypoxic (8% O2, simulating 7600 m altitude O2 levels) conditions exercise capacity and cognitive-behavioral performance. We used a treadmill test to assess exercise capacity, a pole-test to assess agility, an elevated-plus-maze test to assess anti-anxiety, and a passive avoidance test to assess learning and memory retention. Compared to wild type, Cyp-D KO mice showed comparable treadmill work under normoxia (48 ± 12 vs 47 ± 9 Joules) but increased treadmill work (12 ± 1 vs 8 ± 1 Joules; p = 0.02) under hypoxia. Cyp-D KO mice displayed increased pole-descending time (17 ± 3 vs 8 ± 2 s; p ≤ 0.05) under normoxia but shorter pole-descending time (21 ± 3 vs 37 ± 4 s; p ≤ 0.01) under hypoxia. In addition, the Cyp-D KO mice demonstrated increased elevated plus-maze open arm time (91 ± 31 vs 23 ± 12 s; p ≤ 0.05) under hypoxia and increased latency to enter dark chamber (261 ± 23 vs 185 ± 42 s; p ≤ 0.05) under normoxia. Thus, our experiments showed that under normoxia Cyp-D KO mice displayed anti-anxiety behavior and improved learning and memory retention. Under hypoxia, Cyp-D KO mice displayed increased exercise capacity, increased agility, and increased anti-anxiety consistent with our previously reported findings of increased O2 utilization efficiency. Identifying interventions to elicit these effects could be beneficial in a myriad of physiological and clinical conditions in which increasing O2 utilization efficiency would be advantageous.

Targeting mitochondria for resuscitation from cardiac arrest.

Reversal of cardiac arrest requires reestablishment of aerobic metabolism by reperfusion with oxygenated blood of tissues that have been ischemic for variable periods of time. However, reperfusion concomitantly activates a myriad of pathogenic mechanisms causing what is known as reperfusion injury. At the center of reperfusion injury are mitochondria, playing a critical role as effectors and targets of injury. Studies in animal models of ventricular fibrillation have shown that limiting myocardial cytosolic Na+ overload attenuates mitochondrial Ca2+ overload and maintains oxidative phosphorylation, which is the main bioenergetic function of mitochondria. This effect is associated with functional myocardial benefits such as preservation of myocardial compliance during chest compression and attenuation of myocardial dysfunction after return of spontaneous circulation. Additional studies in similar animal models of ventricular fibrillation have shown that mitochondrial injury leads to activation of the mitochondrial apoptotic pathway, characterized by the release of cytochrome c to the cytosol, reduction of caspase-9 levels, and activation of caspase-3 coincident with marked reduction in left ventricular function. Cytochrome c also "leaks" into the bloodstream attaining levels that are inversely proportional to survival. These data indicate that mitochondria play a key role during cardiac resuscitation by modulating energy metabolism and signaling apoptotic cascades and that targeting mitochondria could represent a promising strategy for cardiac resuscitation.

Limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation prevents excess mitochondrial Ca2+ accumulation and attenuates myocardial injury.

BACKGROUND: intracellular Na+ accumulation during ischemia and reperfusion leads to cytosolic Ca2+ overload through reverse-mode operation of the sarcolemmal Na+ -Ca2+ exchanger. Cytosolic Ca2+ accumulation promotes mitochondrial Ca2+ (Ca2+ m) overload, leading to mitochondrial injury. We investigated whether limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation (VF) attenuates Ca2+ m overload and lessens myocardial dysfunction in a rat model of VF and closed-chest resuscitation. METHODS: hearts were harvested from 10 groups of 6 rats each representing baseline, 15 min of untreated VF, 15 min of VF with chest compression given for the last 5 min (VF/CC), and 60 min postresuscitation (PR). VF/CC and PR included four groups each randomized to receive before starting chest compression the new NHE-1 inhibitor AVE4454B (1.0 mg/kg), the Na+ channel blocker lidocaine (5.0 mg/kg), their combination, or vehicle control. The left ventricle was processed for intracellular Na+ and Ca2+ m measurements. RESULTS: limiting sarcolemmal Na+ entry attenuated cytosolic Na+ increase during VF/CC and the PR phase and prevented Ca2+ m overload yielding levels that corresponded to 77% and 71% of control hearts at VF/CC and PR, without differences among specific Na+ -limiting interventions. Limiting sarcolemmal Na+ entry attenuated reductions in left ventricular compliance during VF and prompted higher mean aortic pressure (110 +/- 7 vs. 95 +/- 11 mmHg, P < 0.001) and higher cardiac work index (159 +/- 34 vs. 126 +/- 29 g x m x min(-1) x kg(-1), P < 0.05) with lesser increases in circulating cardiac troponin I at 60 min PR. CONCLUSIONS: Na+ -limiting interventions prevented excess Ca2+ m accumulation induced by ischemia and reperfusion and ameliorated myocardial injury and dysfunction.

In vivo opening of the mitochondrial permeability transition pore in a rat model of ventricular fibrillation and closed-chest resuscitation.

Opening of the mitochondrial permeability transition pore (mPTP) is considered central to reperfusion injury. Yet, most of our knowledge comes from observations in isolated mitochondria, cells, and organs. We used a rat model of ventricular fibrillation (VF) and closed-chest resuscitation to examine whether the mPTP opens in vivo and whether cyclosporine A (CsA) attenuates the associated myocardial injury. Two series of 26 and 18 rats each underwent 10 minutes of untreated VF before attempting resuscitation. In series-1, rats received 50 µCi of tritium-labeled 2-deoxyglucose ([3H]DOG) harvesting their hearts at baseline (n=5), during VF (n=5), during resuscitation (n=6), and at post-resuscitation 60 minutes (n=5) and 240 minutes (n=5). mPTP opening was estimated measuring the ratio of mitochondria to left ventricular intracellular [3H]. In series-2, rats received 10 mg/kg of CsA or vehicle before resuscitation, measuring mitochondrial NAD+ content to indirectly assess mPTP opening. In Series-1, the mPTP opening ratio vs baseline (10.4 ± 1.9) increased during VF (16.8 ± 2.4, NS), closed-chest resuscitation (20.8 ± 6.3, P<0.05), and at post-resuscitation 60 minutes (20.9 ± 4.7, P<0.05) and 240 minutes (25.7 ± 11.0, P<0.01). In series 2, CsA failed to attenuate reductions in mitochondrial NAD+ and did not affect plasma cytochrome c, plasma cardiac troponin I, myocardial function, and survival. We report for the first time in an intact rat model of VF that mPTP opens during closed-chest resuscitation consistent with previous observations in mitochondria, cells, and organs of mPTP opening upon reperfusion. CsA, at the dose of 10 mg/kg neither prevented mPTP opening nor attenuated post-resuscitation myocardial injury.

Early and sustained vasopressin infusion augments the hemodynamic efficacy of restrictive fluid resuscitation and improves survival in a liver laceration model of hemorrhagic shock.

BACKGROUND: Current management of hemorrhagic shock favors restrictive fluid resuscitation before control of the bleeding source. We investigated the additional effects of early and sustained vasopressin infusion in a swine model of hemorrhagic shock produced by liver laceration. METHODS: Forty male domestic pigs (32-40 kg) had a liver laceration inflicted with an X-shaped blade clamp, 32 received a second laceration at minute 7.5, and 24 received two additional lacerations at minute 15. Using a two-by-two factorial design, animals were randomized 1:1 to receive vasopressin infusion (0.04 U/kg per minute) or vehicle intraosseously from minute 7 until minute 240 and 1:1 to receive isotonic sodium chloride solution (12 mL/kg) intravenously at minute 30 or no fluids. RESULTS: Kaplan-Meier curves showed greater survival after vasopressin with isotonic sodium chloride solution (8/10) compared to vasopressin without isotonic sodium chloride solution (4/10), vehicle with isotonic sodium chloride solution (3/10), or vehicle without isotonic sodium chloride solution (3/10), but the differences were not statistically significant (p = 0.095 by log-rank test). However, logistic regression showed vasopressin to elicit a statistically significant benefit on survival (p = 0.042). Vasopressin augmented mean aortic pressure between 10 and 20 mm Hg without intensifying the rate of bleeding from liver laceration, which was virtually identical to that of vehicle-treated animals (33.9 ± 5.1 and 33.8 ± 4.8 mL/kg). Vasopressin increased systemic vascular resistance and reduced transcapillary fluid extravasation, augmenting the volume of isotonic sodium chloride solution retained (6.5 ± 2.7 vs 2.4 ± 2.0 mL/kg by minute 60). The cardiac output and blood flow to the myocardium, liver, spleen, kidney, small bowel, and skeletal muscle at minute 120 and minute 180 were comparable or higher in the vasopressin group. CONCLUSIONS: Early and sustained vasopressin infusion provided critical hemodynamic stability during hemorrhagic shock induced by liver laceration and increased the hemodynamic efficacy of restrictive fluid resuscitation without intensifying bleeding or compromising organ blood flow resulting in improved 240-minute survival.

Real-Time Ventricular Fibrillation Amplitude-Spectral Area Analysis to Guide Timing of Shock Delivery Improves Defibrillation Efficacy During Cardiopulmonary Resuscitation in Swine.

BACKGROUND: The ventricular fibrillation amplitude spectral area (AMSA) predicts whether an electrical shock could terminate ventricular fibrillation and prompt return of spontaneous circulation. We hypothesized that AMSA can guide more precise timing for effective shock delivery during cardiopulmonary resuscitation. METHODS AND RESULTS: Three shock delivery protocols were compared in 12 pigs each after electrically induced ventricular fibrillation, with the duration of untreated ventricular fibrillation evenly stratified into 6, 9, and 12 minutes: AMSA-Driven (AD), guided by an AMSA algorithm; Guidelines-Driven (GD), according to cardiopulmonary resuscitation guidelines; and Guidelines-Driven/AMSA-Enabled (GDAE), as per GD but allowing earlier shocks upon exceeding an AMSA threshold. Shocks delivered using the AD, GD, and GDAE protocols were 21, 40, and 62, with GDAE delivering only 2 AMSA-enabled shocks. The corresponding 240-minute survival was 8/12, 6/12, and 2/12 (log-rank test, P=0.035) with AD exceeding GDAE (P=0.026). The time to first shock (seconds) was (median [Q1-Q3]) 272 (161-356), 124 (124-125), and 125 (124-125) (P<0.001) with AD exceeding GD and GDAE (P<0.05); the average coronary perfusion pressure before first shock (mm Hg) was 16 (9-30), 10 (6-12), and 3 (-1 to 9) (P=0.002) with AD exceeding GDAE (P<0.05); and AMSA preceding the first shock (mV·Hz, mean±SD) was 13.3±2.2, 9.0±1.6, and 8.6±2.0 (P<0.001) with AD exceeding GD and GDAE (P<0.001). The AD protocol delivered fewer unsuccessful shocks (ie, less shock burden) yielding less postresuscitation myocardial dysfunction and higher 240-minute survival. CONCLUSIONS: The AD protocol improved the time precision for shock delivery, resulting in less shock burden and less postresuscitation myocardial dysfunction, potentially improving survival compared with time-fixed, guidelines-driven, shock delivery protocols.

Plasma Cytochrome c Detection Using a Highly Sensitive Electrochemiluminescence Enzyme-Linked Immunosorbent Assay.

BACKGROUND: Cytochrome c is an intermembrane mitochondrial protein that is released to the bloodstream following mitochondrial injury. METHODS AND RESULTS: We developed an electrochemiluminescence immunoassay to measure cytochrome c in human and rat plasma, which showed high sensitivity with broad dynamic range (2-1200 ng/mL in humans and 5-500 ng/mL in rat) and high assay reproducibility (inter-assay coefficient <6% in humans and <10% in rat). In patients after blunt trauma, plasma cytochrome c directly correlated with injury severity. In rats after cardiac resuscitation, plasma cytochrome c inversely correlated with survival and responsiveness to mitochondrial protective interventions. CONCLUSIONS: The cytochrome c assays herein presented have high sensitivity, wide dynamic range, and high reproducibility well suited for biomarker of mitochondrial injury.

Ventricular Fibrillation Waveform Changes during Controlled Coronary Perfusion Using Extracorporeal Circulation in a Swine Model.

BACKGROUND: Several characteristics of the ventricular fibrillation (VF) waveform have been found predictive of successful defibrillation and hypothesized to reflect the myocardial energy state. In an open-chest swine model of VF, we modeled "average CPR" using extracorporeal circulation (ECC) and assessed the time course of coronary blood flow, myocardial metabolism, and myocardial structure in relation to the amplitude spectral area (AMSA) of the VF waveform without artifacts related to chest compression. METHODS: VF was induced and left untreated for 8 minutes in 16 swine. ECC was then started adjusting its flow to maintain a coronary perfusion pressure of 10 mmHg for 10 minutes. AMSA was calculated in the frequency domain and analyzed continuously with a 2.1 s timeframe and a Tukey window that moved ahead every 0.5 s. RESULTS: AMSA progressively declined during untreated VF. With ECC, AMSA increased from 7.0 ± 1.9 mV·Hz (at minute 8) to 12.8 ± 3.3 mV·Hz (at minute 14) (p < 0.05) without subsequent increase and showing a modest correlation with coronary blood flow of borderline statistical significance (r = 0.489, p = 0.0547). Myocardial energy measurements showed marked reduction in phosphocreatine and moderate reduction in ATP with increases in ADP, AMP, and adenosine along with myocardial lactate, all indicative of ischemia. Yet, ischemia did not resolve during ECC despite a coronary blood flow of ~ 30% of baseline. CONCLUSION: AMSA increased upon return of coronary blood flow during ECC. However, the maximal level was reached after ~ 6 minutes without further change. The significance of the findings for determining the optimal timing for delivering an electrical shock during resuscitation from VF remains to be further explored.

Adverse postresuscitation myocardial effects elicited by buffer-induced alkalemia ameliorated by NHE-1 inhibition in a rat model of ventricular fibrillation.

Major myocardial abnormalities occur during cardiac arrest and resuscitation including intracellular acidosis-partly caused by CO2 accumulation-and activation of the Na+-H+ exchanger isoform-1 (NHE-1). We hypothesized that a favorable interaction may result from NHE-1 inhibition during cardiac resuscitation followed by administration of a CO2-consuming buffer upon return of spontaneous circulation (ROSC). Ventricular fibrillation was electrically induced in 24 male rats and left untreated for 8 min followed by defibrillation after 8 min of cardiopulmonary resuscitation (CPR). Rats were randomized 1:1:1 to the NHE-1 inhibitor zoniporide or vehicle during CPR and disodium carbonate/sodium bicarbonate buffer or normal saline (30 ml/kg) after ROSC. Survival at 240 min declined from 100% with Zoniporide/Saline to 50% with Zoniporide/Buffer and 25% with Vehicle/Buffer (P = 0.004), explained by worsening postresuscitation myocardial dysfunction. Marked alkalemia occurred after buffer administration along with lactatemia that was maximal after Vehicle/Buffer, attenuated by Zoniporide/Buffer, and minimal with Zoniporide/Saline [13.3 ± 4.8 (SD), 9.2 ± 4.6, and 2.7 ± 1.0 mmol/l; P ≤ 0.001]. We attributed the intense postresuscitation lactatemia to enhanced glycolysis consequent to severe buffer-induced alkalemia transmitted intracellularly by an active NHE-1. We attributed the worsened postresuscitation myocardial dysfunction also to severe alkalemia intensifying Na+ entry via NHE-1 with consequent Ca2+ overload injuring mitochondria, evidenced by increased plasma cytochrome c Both buffer-induced effects were ameliorated by zoniporide. Accordingly, buffer-induced alkalemia after ROSC worsened myocardial function and survival, likely through enhancing NHE-1 activity. Zoniporide attenuated these effects and uncovered a complex postresuscitation acid-base physiology whereby blood pH drives NHE-1 activity and compromises mitochondrial function and integrity along with myocardial function and survival.

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique.

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes - but is not limited to - hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

Vasopressin Infusion with Small-Volume Fluid Resuscitation during Hemorrhagic Shock Promotes Hemodynamic Stability and Survival in Swine.

INTRODUCTION: Current management of hemorrhagic shock (HS) in the battlefield and civilian settings favors small-volume fluid resuscitation before controlling the source of bleeding. We investigated in a swine model of HS the effects of vasopressin infusion along with small-volume fluid resuscitation; with erythropoietin (EPO) and HS severity as additional factors. METHODS: HS was induced in 24 male domestic pigs (36 to 41 kg) by blood withdrawal (BW) through a right atrial cannula modeling spontaneous bleeding by a mono-exponential decay function. The initial 12 pigs received no fluids; the last 12 pigs received normal saline (NS) half the BW volume. Pigs were randomized 2:1 to receive intraosseously vasopressin (0.04 U/kg·min-1) or vehicle control from minute 7 to minute 210. Pigs assigned to vasopressin were further randomized 1:1 to receive EPO (1,200 U/kg) or vehicle control and 1:1 to have 65% or 75% BW of their blood volume. Shed blood was reinfused at 210 minutes and the pigs recovered from anesthesia. RESULTS: Survival at 72 hours was influenced by vasopressin and NS but not by EPO or % BW. Vasopressin with NS promoted the highest survival (8/8) followed by vasopressin without NS (3/8), NS without vasopressin (1/4), and neither treatment (0/4) with overall statistical significance (log-rank test, p = 0.009) and each subset different from vasopressin with NS by Holm-Sidak test. Vasopressin increased systemic vascular resistance whereas NS increased cardiac output. CONCLUSION: Vasopressin infusion with small-volume fluid resuscitation during severe HS was highly effective enabling critical hemodynamic stabilization and improved 72 hour survival.

Estrogen fails to facilitate resuscitation from ventricular fibrillation in male rats.

Administration of 17ß-estradiol has been shown to exert myocardial protective effects in hemorrhagic shock. We hypothesized that similar protective effects could help improve resuscitation from cardiac arrest. Three series of 18, 40, and 12 rats each, underwent ventricular fibrillation for 8 minutes followed by 8 minutes of chest compression and delivery of electrical shocks. In series-1, rats were randomized 1:1 to receive a bolus dose of 17ß-estradiol (1 mg/kg) or 0.9% NaCl before chest compression; in series-2, rats were randomized 1:1:1:1 to receive a continuous infusion of 0.9% NaCl or a 17ß-estradiol solution designed to attain a plasma level of 10(0), 10(2), or 10(4) nM during chest compression; and in series-3, rats were randomized 1:1 to receive a continuous infusion of 17ß-estradiol to attain a plasma level of 10(2) nM or 0.9% NaCl during chest compression, providing inotropic support during the post-resuscitation interval using dobutamine infusion. 17ß-estradiol failed to facilitate resuscitation in each of the 3 series. In series-1 and series-2, resuscitability and short-term survival was reduced in 17ß-estradiol groups attaining statistical significance in series-2 when the three 17ß-estradiol groups were combined (p = 0.035). In series-3, all rats were resuscitated and survived for 180 minutes aided by dobutamine which partially reversed post-resuscitation myocardial dysfunction but without additional benefits on myocardial function in the 17ß-estradiol group. The present study failed to support a beneficial effect of 17ß-estradiol for resuscitation from cardiac arrest and raised the possibility of detrimental cardiac effects compromising initial resuscitability and subsequent survival in a male rat model of ventricular fibrillation and closed chest resuscitation.

Effects of intraosseous erythropoietin during hemorrhagic shock in swine.

OBJECTIVE: To determine whether erythropoietin given during hemorrhagic shock (HS) ameliorates organ injury while improving resuscitation and survival. METHODS: Three series of 24 pigs each were studied. In an initial series, 50% of the blood volume (BV) was removed in 30 minutes and normal saline (threefold the blood removed) started at minute 90 infusing each third in 30, 60, and 150 minutes with shed blood reinfused at minute 330 (HS-50BV). In a second series, the same HS-50BV protocol was used but removing an additional 15% of BV from minute 30 to 60 (HS-65BV). In a final series, blood was removed as in HS-65BV and intraosseous vasopressin given from minute 30 (0.04 U/kg min(-1)) until start of shed blood reinfusion at minute 150 (HS-65BV+VP). Normal saline was reduced to half the blood removed and given from minute 90 to 120 in half of the animals. In each series, animals were randomized 1:1 to receive erythropoietin (1,200 U/kg) or control solution intraosseously after removing 10% of the BV. RESULTS: In HS-50BV, O2 consumption remained near baseline yielding minimal lactate increases, 88% resuscitability, and 60% survival at 72 hours. In HS-65BV, O2 consumption was reduced and lactate increased yielding 25% resuscitability. In HS-65BV+VP, vasopressin promoted hemodynamic stability yielding 92% resuscitability and 83% survival at 72 hours. Erythropoietin did not affect resuscitability or subsequent survival in any of the series but increased interleukin-10, attenuated lactate increases, and ameliorated organ injury based on lesser troponin I, AST, and ALT increases and lesser neurological deficits in the HS-65BV+VP series. CONCLUSIONS: Erythropoietin given during HS in swine failed to alter resuscitability and 72 hour survival regardless of HS severity and concomitant treatment with fluids and vasopressin but attenuated acute organ injury. The studies also showed the efficacy of vasopressin and restrictive fluid resuscitation for hemodynamic stabilization and survival.