A cell-penetrating PHLPP peptide improves cardiac arrest survival in murine and swine models
Jing Li, … , Henry R. Halperin, Terry L. Vanden Hoek
Jing Li, … , Henry R. Halperin, Terry L. Vanden Hoek
Published May 1, 2023
Citation Information: J Clin Invest. 2023;133(9):e164283. https://doi.org/10.1172/JCI164283.
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Research Article

A cell-penetrating PHLPP peptide improves cardiac arrest survival in murine and swine models

Abstract

Out-of-hospital cardiac arrest is a leading cause of death in the US, with a mortality rate over 90%. Preclinical studies demonstrate that cooling during cardiopulmonary resuscitation (CPR) is highly beneficial, but can be challenging to implement clinically. No medications exist for improving long-term cardiac arrest survival. We have developed a 20–amino acid peptide, TAT-PHLPP9c, that mimics cooling protection by enhancing AKT activation via PH domain leucine-rich repeat phosphatase 1 (PHLPP1) inhibition. Complementary studies were conducted in mouse and swine. C57BL/6 mice were randomized into blinded saline control and peptide-treatment groups. Following a 12-minute asystolic arrest, TAT-PHLPP9c was administered intravenously during CPR and significantly improved the return of spontaneous circulation, mean arterial blood pressure and cerebral blood flow, cardiac and neurological function, and survival (4 hour and 5 day). It inhibited PHLPP-NHERF1 binding, enhanced AKT but not PKC phosphorylation, decreased pyruvate dehydrogenase phosphorylation and sorbitol production, and increased ATP generation in heart and brain. TAT-PHLPP9c treatment also reduced plasma taurine and glutamate concentrations after resuscitation. The protective benefit of TAT-PHLPP9c was validated in a swine cardiac arrest model of ventricular fibrillation. In conclusion, TAT-PHLPP9c may improve neurologically intact cardiac arrest survival without the need for physical cooling.

Authors

Jing Li, Xiangdong Zhu, Matt T. Oberdier, Chunpei Lee, Shaoxia Lin, Sarah J. Fink, Cody N. Justice, Kevin Qin, Andrew W. Begeman, Frederick C. Damen, Hajwa Kim, Jiwang Chen, Kejia Cai, Henry R. Halperin, Terry L. Vanden Hoek

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Figure 1

TAT-PHLPP9c activity and the mechanism of action.

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TAT-PHLPP9c activity and the mechanism of action. (A) TAT-PHLPP9c induce...
(A) TAT-PHLPP9c induced AKT phosphorylation at Ser473 in a dose-dependent manner (0.1, 1, 10 μM) in mouse ventricular cardiomyocytes. Total AKT was used as a loading control. (B) Densitometric analysis of AKT phosphorylation. One-way ANOVA with Dunnett’s test was used. #P < 0.05 between TAT-GFP control and TAT-PHLPP9c (at 1 μM and 10 μM). Data are represented as mean ± SD for 3 experiments. (C) TAT-PHLPP9c (10 μM) increased AKT phosphorylation in a time-dependent manner with a peak at 30 minutes in cardiomyocytes. It had no effect on PKCβ2-S660 phosphorylation. GAPDH was used as a loading control. (D) Densitometric analysis of AKT and PKCβ2-S660 phosphorylation in response to TAT-PHLPP9c treatment. One-way ANOVA with Dunnett’s test was used. #P < 0.05 for time 0 to 15 minutes with TAT-PHLPP9c (10 μM); *P < 0.01 for time 0 to 30 minutes with TAT-PHLPP9c. Data are represented as mean ± SD for 3 experiments. (E) TAT-PHLPP9c blocked endogenous PHLPP1 binding to its membrane adaptor NHERF1. Mouse brain lysates (500 μg) were incubated with TAT-PHLPP9c (1 and 10 μM) or 10 μM of TAT for 30 minutes and were precipitated with NHERF1 antibody; immunoblots were analyzed using antibody against PHLPP1. Equal input into the immunoprecipitation reaction was verified by PHLPP1 antibody. TAT-PHLPP9c at 1 to 10 μM, but not TAT vehicle, inhibited endogenous PHLPP1 binding to NHERF1. (F) Densitometric analysis of immunoprecipitation assay of PHLPP1 and NHERF1 interaction. Paired, 2-tailed t test was used. #P < 0.05 between control and 10 μM of TAT-PHLPP9c; P = 0.51 between control and 10 μM of TAT. Data are represented as mean ± SD for 3 experiments.