Survival and Neurologic Outcomes From Pharmacologic Peptide Administration During Cardiopulmonary Resuscitation of Pulseless Electrical Activity.
Jun 27, 2024DOI: 10.1161/jaha.123.033371Journal ArticleAHA
Written by: Matt T OberdierJing LiDaniel I AmbinderMasahito SuzukiEthan TumarkinSarah FinkLuca NeriXiangdong ZhuCody N JusticeTerry L Vanden HoekHenry R Halperin
The data associated with this study are available upon reasonable request from the corresponding author. Twenty‐four adult female Yorkshire pigs (30–35 kg; Archer Farms; Darlington, MD) were tranquilized with 22 mg/kg ketamine and mechanically ventilated with 100% O2 and 1% to 2.5% isoflurane in the supine position. Access to both femoral veins and arteries was acquired via 8 French sheaths. Pigtail catheters were placed in the descending thoracic aorta and right atrium for continuous blood pressure measurements. External defibrillator electrodes were placed on both sides of the chest.Five minutes after cessation of isoflurane and immediately after discontinuation of mechanical ventilation, PEA was induced by inflating balloon catheters at proximal positions in the right coronary and left anterior descending arteries to full occlusion as verified by fluoroscopy (Figure 1). PEA was determined via real‐time waveform inspection and defined as no pulsatility and a mean pressure <30 mm Hg with normal sinus rhythm. PEA was successfully induced in all study animals, with none being removed for failed induction. PEA was allowed to persist, on average, for between 7 and 8 minutes. To resolve differences among pressure depression period and extent of depression, an index of ischemic severity was defined as the quotient of mean pressure to duration where lower values are less favorable for survival.Controls received two 0.5‐mg doses of epinephrine at 1 and 3 minutes after the start of ACLS, whereas peptide‐treated animals also received two 7.5‐mg/kg doses of TAT‐PHLPP9c. Between doses and corresponding to 2 minutes after the start of ACLS, the occluding balloons were deflated and removed. ACLS indicates advanced cardiovascular life support; KCl, potassium chloride; PEA, pulseless electrical activity; and ROSC, return of spontaneous circulation.After the allotted PEA period, advanced cardiovascular life support (ACLS) was initiated with mechanical ventilation and vest CPR36 at an inflation pressure of 200 mm Hg, a rate of 60 per minute, and a 40% duty cycle. Vest CPR is the standard technique in our laboratory because it is consistent over time and more reproducible than manual CPR, and thus it limits bias. In our experience, vest CPR is also less likely to damage the sternum and ribs than a mechanical piston device. The settings used for vest CPR are standard in our laboratory and were the same throughout all studies.Twelve control animals were administered boluses of 0.5 mg epinephrine via the venous line after 1 and 3 minutes of support. Twelve peptide‐treated animals were also given boluses of 7.5 mg/kg TAT‐PHLPP9c at 1 and 3 minutes of CPR while the control swine were given an equal volume of only saline. The balloons were deflated and removed after 2 minutes of support. When necessary, shocks were administered to reestablish normal electrical rhythms. ACLS continued until ROSC was achieved or for 5 minutes without vascular tone or for 9 minutes with vascular tone, which was defined as diastolic pressures >20 mm Hg after administration of the second bolus of epinephrine.
Animals were recovered following anesthesia and classified as neurologically intact or not intact according to a porcine adaptation of the modified Rankin Scale for neurologic disability,37 which is similar to a scale used by Berg et al that considers behavior, motor function, and cerebral performance.38 Based on behavior norms for healthy swine, scores from 0 to 6 indicate increasing disability from normal to death (0=no symptoms; 1=mild to moderate lethargy, ambulating, and eating; 2=severe lethargy, ambulating, and eating; 3=awake, ambulating, and not eating; 4=awake, not ambulating or eating; 5=coma; 6=dead). Animals with ROSC that were neurologically intact were kept alive overnight then euthanized via potassium chloride infusion while sedated. The study complied with national standards for animal care and use and was approved by the Johns Hopkins Medicine Animal Care and Use Committee (Protocol No. SW19M406).
In 10 animals (5 controls and 5 peptide treatments), the myocardial concentration of nicotinamide adenine dinucleotide (NAD+) was determined with a NAD+/NADH quantification kit (Cayman Chemical; Ann Arbor, MI), following the manufacturer's instructions. To measure NAD+ in myocardial tissue, tissue lysates were extracted with 100 μL of 0.5 M perchloric acid for 30 minutes and then neutralized with 100 μL of 0.55 M K2CO3. The neutralized extracts were centrifuged at 10 000 g for 1 minute and supernatants were used for NAD+ assay. To detect NAD+ content, the extracted supernatant (100 μL) was transferred into a 96‐well plate, incubated with 100 μL of reaction solution for 90 minutes, and measured at 450 nanometers with a microplate reader (Thermo Scientific; Waltham, MA). NAD+ concentrations were calculated according to an NAD+ standard curve and expressed as pmol/mg protein for tissue lysates.
Summary statistics are presented as means and SDs. For continuous data, normality was determined via the Jarque–Bera test. To determine differences for normally distributed data, 2‐tailed unpaired t tests were performed, whereas Mann–Whitney tests were used to determine differences for nonparametric data. Fisher's exact test was used to determine differences between control and peptide groups for ROSC and neurologic outcomes. P values <0.05 were considered significant.
Results
During the prearrest and PEA periods, mean aortic, right atrial, and coronary perfusion pressures were the same among the control and peptide groups (Table). During CPR, mean right atrial and vest pressures were also the same among the control and peptide groups; however, mean aortic (26.4 versus 40.8 mm Hg; P=0.001), mean aortic systolic (48.9 versus 68.2 mm Hg; P=0.008), mean aortic diastolic (16.3 versus 28.9 mm Hg; P=0.001), and coronary perfusion (27.9 versus 13.3 mm Hg; P=0.002) pressures were significantly higher in the peptide group. Ten minutes after ROSC, mean right atrial pressure was significantly lower in the peptide group (7.6 versus 4.2 mm Hg; P=0.036). Thirty minutes after ROSC, mean right atrial (10.8 versus 5.5 mm Hg; P=0.0171), mean right atrial systolic (12.7 versus 7.9 mm Hg; P=0.048), and mean right atrial diastolic (8.6 versus 3.2 mm Hg; P=0.024) pressures were significantly lower in the peptide group.
Summary of Mean, Systolic, and Diastolic Aortic, Right Atrial, and Vest Pressures During Various Periods Throughout the Experiment for the Control and Peptide Groups
For the PEA period, duration is reported and used in conjunction with mean aortic pressure to define an ischemic index. All units are mm Hg unless otherwise noted. Data are reported as controls vs peptide.
CPR indicates cardiopulmonary resuscitation; PEA, pulseless electrical activity; R‐10, 10 min after ROSC; R‐30, 30 min after ROSC; and ROSC, return of spontaneous circulation.
Indicates P value determined by Mann–Whitney test.
The duration (P=0.70) and severity of PEA, determined by aortic mean arterial pressure (P=0.93) and the ischemic index (P=0.85), were comparable among the control and peptide groups. However, on average, PEA duration (7.5 versus 7.8 minutes) and the ischemic index (3.2 versus 3.1) were biased against the peptide group. Additionally, the duration of CPR was longer for the control than the peptide group (6.1 versus 4.8 minutes; P=0.047).
ROSC was more common in the peptide group, but this difference was not significant (8 control versus 12 peptide; P=0.093). In the post‐ROSC period, defibrillation shocks were more commonly administered to controls than peptide‐treated animals (35 versus 16, respectively) but in an equal number of animals per group (7 each). Also in the post‐ROSC period, a total of 5 additional doses of epinephrine were administered to 3 control animals to assist in achieving a life‐sustaining mean blood pressure, compared with none in the control group. Fully intact 24‐hour, neurologic survival was significantly more frequent in the peptide group (0 control versus 11 peptide; P<0.00001) (Figure 2). At the time of euthanasia, NAD+ levels in heart tissues from controls and peptide‐treated animals were compared, and a significantly higher NAD+ concentration was observed in the controls than in the peptide‐treated animals (1001.6 versus 481.9 pmol/mg, respectively; P=0.009) (Figure 3).
Degrees of neurologic disability were graded on the modified Rankin Scale adapted for swine where 0=no symptoms; 1=mild to moderate lethargy, ambulating, and eating; 2=severe lethargy, ambulating, and eating; 3=awake, ambulating, and not eating; 4=awake, not ambulating or eating; 5=coma; and 6=dead.
Data are presented as means and SDs. N=5 in both control and the treatment groups. #P=0.009 as determined by the Mann–Whitney test. NAD indicates nicotinamide adenine dinucleotide; and SCA, sudden cardiac arrest.
Discussion
To our knowledge, this is the first preclinical study using a pharmacologic intervention beyond vasopressors and adrenergics to treat PEA‐SCA that demonstrates improved neurologic outcomes. It is significant that this study was performed in a PEA model because the destabilizing event leading to arrest was ischemia, while in the prior study by Li et al the peptide was evaluated in a ventricular fibrillation model in which arrest was electrically induced.23 In general, PEA‐SCA is associated with ischemia39, 40 while ischemia is only 1 of several possible causes of ventricular fibrillation.41 Because PEA and ventricular fibrillation have independent causes, their ROSC and recovery mechanisms are also potentially distinct. Therefore, it was essential that peptide effectiveness be tested in the context of multiple pathophysiologic conditions.
In the PEA study here, the outcomes for the controls are worse than those observed for out‐of‐hospital PEA (0% versus 6% survival),2 respectively, and those of the peptide group surpass the current best‐case in‐hospital cardiac arrest survival rates that occur for patients on extracorporeal membrane oxygenation (92% versus 30%,42 respectively). However, the objective of testing the preclinical efficacy of the peptide does not depend on replicating clinical outcomes, but, rather, the goal of the study was to test the peptide during conditions in which PEA was the initiating cause. Therefore, it can be viewed that the peptide resulted in exceedingly positive neurologic outcomes despite the control conditions being demanding enough to result in less survival than what is clinically observed.
Independent of outcomes relative to those that are observed in human populations, our findings suggest that the peptide had a stronger neurologic than a cardiac effect as evidenced by the statistically significant improvement in neurologic outcomes as compared with the better but not statistically significant increase in survivors. The increase in NAD+ in cardiac tissue strongly suggests a cardiac effect, and more peptide‐treated survivors were demonstrated in a murine study by Li et al.23 Therefore, the peptide may have also demonstrated a statistically significant improvement in survival if there were more animals per group or the average PEA period were longer, likely resulting in a decrease in the number of control survivors.
Despite dramatic differences in neurologic outcomes, this study was biased in favor of the control group due to its shorter average PEA duration and higher average ischemic index, but these differences were not statistically significant. Also, for controls, more effort was expended to resuscitate those animals as evidenced by the significantly longer CPR duration with comparable vest pressures and more defibrillation attempts in the same number of animals per group. However, rather than suggesting a treatment bias, the significantly higher coronary perfusion pressures for the peptide treated animals during CPR is a potential short‐term effect of the peptide because the independent variables of this segment of the experiment (vest pressure and epinephrine dosage) were the same for the control and peptide animals, while coronary perfusion pressure was a dependent measure. Therefore, average or statistically significant differences in survival, neurologic outcomes, and NAD+ concentrations are attributable to the peptide and not differences in coronary perfusion pressures.
During the recovery phase, the higher right atrial pressures in the control group may indicate reduced cardiac function as a result of cardiac stunning. Such a condition may have been reduced or eliminated in the peptide‐treated group. However, echocardiography or cardiac output data are not available to directly support this theory.
In addition to previously established benefits (improved cerebral blood flow and cardiac function, better glucose usage, and decreased plasma taurine and glutamate),23 increased metabolic resiliency was demonstrated via higher myocardial NAD+ concentration in the peptide‐treated group. This finding demonstrates that TAT‐PHLPP9c administration favorably influences multiple metabolic factors, and the observation in myocardial tissue in particular might explain a means by which the treatment group may have had less cardiac stunning as evidenced by higher coronary perfusion pressures and lower right atrial pressures.
An unexpected finding of this study was that mean aortic, mean aortic systolic, and mean aortic diastolic pressures during chest compressions were higher in the peptide group even though the vest pressures were the same. Such data suggest that the peptide may have a systemic effect minutes after being administered.
TAT‐PHLPP9c has several major advantages over conventional intra‐arrest cooling. A peptide could be administered easily during CPR in an out‐of‐hospital setting by first responders. A peptide also avoids the hours it takes to achieve steady state cooling6 and to induce the corresponding molecular cascade in humans. Further, unlike physical cooling, molecular induction does not depend on identifying an optimal target temperature, for which there remains ongoing uncertainty.4, 5 However, it is of potential future interest to perform a follow‐up study in which outcomes of arrest are compared in TAT‐PHLPP9c versus intra‐arrest physical cooling groups.
There are 4 limitations of this study. First, the findings are based on short‐term recovery, which is significant because outcomes the day after resuscitation are not always consistent with survival in the months and years later, as demonstrated by patients given epinephrine for out‐of‐hospital cardiac arrest who had an increased rate of resuscitation but also had diminished long‐term survival.43, 44 Second, treatment versus placebo was not blinded or randomized, but there are sufficient data to show that there was no bias favoring the treatment group. Third, the animals in this study were all female because in the experience of our laboratory, males are not survivable and are therefore poor models for survival and outcome studies. As such, animal welfare was respected by not including males. This lack of diversity limits a generalization that includes males; however, the Akt pathway does not involve interactions with sex‐specific effectors, so it is reasonable to think that the peptide acts similarly in males. Fourth, this study does not directly measure previously studied biomarkers, and thus the underlying physiologic effects remain speculative; however, Akt‐activated metabolic recovery was definitively shown to be improved in mice,23 and similar physiology is expected in swine. Fifth, the neurologic outcomes and molecular findings with NAD+ do not directly support one another because timely collection of brain tissue was not possible and cardiac tissue was instead procured. Nonetheless, the findings of increased myocardial tissue NAD+ concentrations with peptide treatment are consistent with an effect of increased Akt signaling and support translational relevance.
Future studies will investigate other molecular targets that may be used to activate the Akt pathway during cardiac arrest. Also, clinical trials are now warranted because, together with the data from Li et al,23 TAT‐PHLPP9c has been shown to improve survival in multiple preclinical models of the major subtypes of cardiac arrest (asystole, ventricular fibrillation, and PEA).
Conclusions
When administered intravenously during standard ACLS in a swine model of PEA, TAT‐PHLPP9c resulted in improved survival with full neurologic function, higher aortic pressures, and higher NAD+ concentration in myocardial tissue. The peptide's effectiveness has been demonstrated in multiple preclinical models of the major subtypes of cardiac arrest, justifying clinical trials.
Source of Funding
This project was sponsored by the National Heart, Lung, and Blood Institute (HL‐R01‐147031) and the Chicago Biomedical Consortium in conjunction with the Searle Funds at the Chicago Community Trust (Grant A‐008). Support was also provided to C.N.J. via training awards from the National Heart, Lung, and Blood Institute (T32HL139439 and F30HL165836).
Disclosures
None.
DA
Dr. Daniel Ambinder
Cardiology · Interventional Cardiology & StructuralUniversity of Maryland School of Medicinedaniel
