Cardioprotection by poloxamer 188 is mediated through increased endothelial nitric oxide production

Abstract

Ischemia/reperfusion (I/R) injury significantly contributes to the morbidity and mortality associated with cardiac events. Poloxamer 188 (P188), a non-ionic triblock copolymer, has been proposed to mitigate I/R injury by stabilizing cell membranes. However, the underlying mechanisms remain incompletely understood, particularly concerning endothelial cell (EC) function and nitric oxide (NO) production. We employed human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) and ECs to elucidate the effects of P188 on cellular survival, function, and NO secretion under simulated I/R conditions. iPSC-CMs contractility and iPSC-ECs' NO production were assessed following exposure to P188. Further, an isolated heart model using Brown Norway rats subjected to I/R injury was utilized to evaluate the ex-vivo cardioprotective effects of P188, examining cardiac function and NO production, with and without the administration of a NO inhibitor. In iPSC-derived models, P188 significantly preserved CM contractile function and enhanced cell viability after hypoxia/reoxygenation. Remarkably, P188 treatment led to a pronounced increase in NO secretion in iPSC-ECs, a novel finding demonstrating endothelial protective effects beyond membrane stabilization. In the rat isolated heart model, administration of P188 during reperfusion notably improved cardiac function and reduced I/R injury markers. This cardioprotective effect was abrogated by NO inhibition, underscoring the pivotal role of NO. Additionally, a dose-dependent increase in NO production was observed in non-ischemic rat hearts treated with P188, further establishing the critical function of NO in P188 induced cardioprotection. In conclusion, our comprehensive study unveils a novel role of NO in mediating the protective effects of P188 against I/R injury. This mechanism is evident in both cellular models and intact rat hearts, highlighting the potential of P188 as a therapeutic agent against I/R injury. Our findings pave the way for further investigation into P188's therapeutic mechanisms and its potential application in clinical settings to mitigate I/R-related cardiac dysfunction.

Keywords: Cardiac arrest; Copolymer; Ischemia/reperfusion injury; Myocardial infarction; NO; P188.

Fig. 1

P188-mediated protection of iPSC-CMs under simulated H/R conditions. (A) Schematic diagram illustrating the differentiation process of iPSCs to iPSC-CMs, followed by hypoxic challenge and subsequent reoxygenation phase supplemented with various P188 concentrations. (B) Quantitative assessment of iPSC-CM viability across different P188 concentrations under simulated H/R conditions. The data emphasizes a marked increase in cell survival with increasing P188 concentrations. Cell viability values are normalized to normoxic control cells. (C–E) Quantitative representation showcasing the augmented functionalities of iPSC-CMs, including contraction rate (C), relaxation rate (D), and acceleration (E), upon P188 exposure under simulated H/R conditions. (F) Representative images depicting the distribution of troponin T in iPSC-CMs treated with varying P188 concentrations. Staining was shown as nuclei in blue and troponin in green; scale bar, 60 μm. Data are shown as mean ± SEM. Significance levels in comparison to the non-treated control group were determined using one-way ANOVA with Dunnett’s test: *P < 0.05, ****P < 0.0001.

Fig. 2

Cell death analysis and apoptosis detection in iPSC-CM. (A) Live/dead staining with Calcein-AM (green) and Propidium Iodide (PI, red) of iPSC-CMs after treatment with a concentration series of P188 ranging from 10^− 13 M to 10^− 6 M. Non-treated samples serve as controls. Scale bars, 170 μm. (B) Quantification of the live/dead cell ratio in iPSC-CMs following reoxygenation, with or without P188 treatment. (C) Flow cytometry analysis of Annexin V and PI staining for detection of apoptosis in iPSC-CMs. The stacked peaks represent the population of Annexin V-positive/PI-positive cells, indicating an increasing ratio of late apoptotic/necrotic cells in P188-treated iPSC-CMs. Data are shown as mean ± SEM. Significance levels in comparison to the non-treated control group were determined using one-way ANOVA with Dunnett’s test: *P < 0.05, ****P < 0.0001.

Fig. 3

Effects of P188 on iPSC-ECs viability and intracellular NO production under simulated I/R conditions. (A, B) Schematic representation of the experimental timeline, detailing the differentiation protocol of iPSC-ECs (A) followed by hypoxic exposure and subsequent reoxygenation (B). (C) Representative images of intracellular NO staining, indicated by the DAF-FM dye. Positive intracellular NO production is depicted in green, nuclei are stained in blue, and the endothelial cell marker, CD31, is shown in red. The top row represents cells subjected to hypoxic treatment, while the bottom row shows corresponding cells under normal oxygen conditions, both treated with respective P188 concentrations; scale bars, 100 μm. (D) Quantification of intracellular NO production determined via DAF-FM staining; values normalized to normoxic control cells. (E) Quantification of cell viability post-reperfusion in iPSC-ECs treated with or without P188; values normalized to normoxic control cells. Data are shown as mean ± SEM. Significance levels in comparison to the non-treated control group were determined using one-way ANOVA with Dunnett’s test: *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 4

Effect of P188 on cardiac function and viability in rat isolated heart experiments. (A) Systolic (LVSP), (B) diastolic (LVEDP) and (C) developed left ventricular pressure (LVDP), (D) heart rate (HR), (E) rate pressure product (RPP), (F) coronary flow (CF), (G) dP/dt_min and (H) dP/dt_max, as indices of relaxation and contractility, respectively, and (I) infarct size are shown at 120 min of reperfusion. P188 significantly improved cardiac function and viability when compared to other groups undergoing ischemia/reperfusion. Notably, inhibition of nitric oxide synthase by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) abolished all effects of P188. Since not all data were normally distributed and had equal variance, we display them as box plots with median and interquartile range. Tests were considered statistically significant at P < 0.05 (one symbol; P < 0.01 two symbols; P < 0.001 three symbols; P < 0.0001 four symbols): *vs CON/control, ^†vs. ISC, ^‡vs. P188, ^§vs. P188 + L-NAME.

Fig. 5

Nitric oxide measurement in rat isolated hearts at different P188 concentrations. (A) Fluorescence of the nitric oxide (NO)-sensitive dye 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate serving as an indicator of NO production revealed an increase after administration of P188 which was abolished by the nonspecific NO synthase inhibitor Nω-Nitro-l-arginine methyl ester hydrochloride (L-NAME). (A insert) In the absence of L-NAME, P188 increased the total amount of NO release, measured as the area under the curve (AUC) of the fluorescence signal; an effect which was abolished by L-NAME. (B) There was no increase in NO production in untreated control hearts. *P < 0.05 vs. P-188-NAME.