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.

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.

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.

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.

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.

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.

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.

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.

High-dose erythropoietin during cardiac resuscitation lessens postresuscitation myocardial stunning in swine.

We investigated the metabolic and functional myocardial effects of erythropoietin (EPO) administered during resuscitation from cardiac arrest using an open-chest pig model of ventricular fibrillation and resuscitation by extracorporeal circulation, after having reported in rats a reversal of postresuscitation myocardial dysfunction associated with activation of mitochondrial protective pathways. Ventricular fibrillation was induced in 16 male domestic pigs and left untreated for 8 minutes, after which extracorporeal circulation was started and maintained for 10 additional minutes, adjusting the extracorporeal flow to provide a coronary perfusion pressure of 10 mmHg. Defibrillation was accomplished and the extracorporeal flow was adjusted to secure a mean aortic pressure of 40 mmHg or greater during spontaneous circulation for up to 120 minutes. Pigs were randomized 1:1 to receive EPO (1200 U/kg) or 0.9% NaCl before starting extracorporeal circulation. Severe postresuscitation myocardial dysfunction developed in both groups. However, recovery of myocardial function-comparing baseline with 120 minutes postresuscitation-was better in pigs treated with EPO than NaCl, as shown for left ventricular ejection fraction (from 45 ± 8% to 36 ± 9% in EPO, not significant; and from 46 ± 8% to 26 ± 8% in NaCl, P < 0.001) and for peak systolic pressure/end-systolic volume (from 2.7 ± 0.8 mmHg/mL to 2.4 ± 0.7 mmHg/mL in EPO, not significant; and from 3.0 ± 1.1 mmHg/mL to 1.8 ± 0.6 mmHg/mL, P < 0.001 in NaCl). The EPO effect was associated with significantly higher myocardial O2 consumption (12 ± 6 mL/min/unit of tissue vs 6 ± 2 mL/min/unit of tissue, P < 0.017) without effects on myocardial lactate consumption. Thus, EPO administered during resuscitation from ventricular fibrillation lessened postresuscitation myocardial stunning-an effect that could be useful clinically to help promote postresuscitation hemodynamic stability.