Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Dec;8(6):e00639.
doi: 10.1002/prp2.639.

Poloxamer 188 Protects Isolated Adult Mouse Cardiomyocytes from Reoxygenation Injury

Michele M Salzman  1   2 , Jason A Bartos  3 , Demetris Yannopoulos  3 , Matthias L Riess  1   2   4
Affiliations

Poloxamer 188 Protects Isolated Adult Mouse Cardiomyocytes from Reoxygenation Injury

Michele M Salzman et al. Pharmacol Res Perspect. 2020 Dec.

Abstract

Reperfusion injury is a complex pathological event involving processes that can lead to further disruption of the cell membrane and function following an ischemic event. Return of blood flow allows for the needed reperfusion; however, for a period of time before remaining viable cells stabilize, reperfusion results in additional cellular injury. In cardiomyocytes, loss of membrane integrity allows abnormal influx of extracellular calcium, leading to hyper-contracture and cell death. Methods to improve the membrane integrity of cardiomyocytes overwhelmed by pathological disruptions, such as reperfusion injury, are needed to prevent cell death, because of the myocardium's limited ability to regenerate. Research has shown administration of the copolymer P(oloxamer) 188 before ischemia/reperfusion can protect cardiomyocytes through membrane stabilization. This study sought to determine whether the administration of P188 at the beginning of the clinically more relevant time of reperfusion after ischemia will attenuate any additional damage to cardiomyocytes by stabilizing membrane integrity to allow the cells to maintain function. Using an in-vitro cardiomyocyte model subjected to hypoxia/reoxygenation to simulate ischemia/reperfusion injury, we show that reoxygenation significantly potentiates the injury caused by hypoxia itself. P188, with its unique combination of hydrophobic and hydrophilic chemical properties, and only delivered at the beginning of reoxygenation, dose-dependently protected cardiomyocytes from injury due to reoxygenation by repairing cell membranes, decreasing calcium influx, and maintaining cellular morphology. Our study also shows the hydrophobic portion of P188 is necessary for the stabilization of cell membrane integrity in providing protection to cardiomyocytes against reoxygenation injury.

Keywords: cell membrane stabilizer; heart; ischemia; polyethylene glycol; reperfusion injury; tri-block copolymer.

PubMed Disclaimer

Conflict of interest statement

None of the authors has any potential conflicts of interest.

Figures

Figure 1
Figure 1
A, Diagram showing the general structure of a tri‐block copolymer. P188 (MW 8400 g/mol) has a central portion containing 30 PPO units (represented by y in the diagram) flanked by two side chains consisting of 75 PEO units each (represented by n in the diagram). B, Diagram showing the general structure of a PEG molecule. PEG is a polymer consisting of varying numbers of PEO units (number of units represented by n in the diagram) that make up varying MWs. PEG (MW 8000 g/mol) was used as the control molecule to P188 in our study
Figure 2
Figure 2
A, Representative photo of the general appearance of the isolated adult mouse cardiomyocytes 6 h after plating. At least 50% of the cells show the expected rod‐shaped morphology of cardiomyocytes. B, Representative photo of the typical appearance of the cardiomyocytes ~ 72 h after plating. A confluent layer has formed, with the cells becoming more rounded due to increased cell‐cell contact and limited growth area. Scale bars are 100 µm in length
Figure 3
Figure 3
Potentiation of injury by reoxygenation following hypoxia in the five main indices of cellular function/dysfunction assessed. Reoxygenation following hypoxia significantly decreases (A) cell number/viability as assessed by the CyQUANT Direct Cell Proliferation Assay Kit (data expressed as dot plots of individual experiment data points together with the average number of cells per well ± the standard error of the mean [SEM] bars, number of experiments [N] = 6, 4‐6 replicate wells per treatment per experiment, * vs C/N, Ɨ vs H only), and significantly increases (B) LDH release (data expressed as dot plots of individual data points together with the average absorbance units [AU] per well ± SEM bars, N = 6, 4‐6 replicate wells per treatment per experiment, * vs C/N, Ɨ vs H only), (C) membrane damage as assessed by FM1‐43 incorporation (data expressed as dot plots of individual data points together with the average relative fluorescent units [RFU] per well ± SEM bars, N = 6, 4‐6 replicate wells per treatment per experiment, * vs C/N, Ɨ vs H only), and (D) [Ca2+]i (data expressed as dot plots of individual data points together with the average RFU ± SEM bars, N = 6, 4‐6 replicate wells per treatment per experiment, * vs C/N, Ɨ vs H only. (E) Representative photos of each group (C/N, H only, H/R) show what was consistently observed in the experiments conducted; C/N group shows the normal condition and morphology of the cells at the end of the experiment, the H only group shows irregularly shrunk and rounded cells (small black arrows) and larger areas with no cells present (large black arrows), the H/R group shows more areas with no cells present, irregularly shrunk and rounded cells, as well as disintegrated cells (small white arrows). Scale bars are 100 µm in length
Figure 4
Figure 4
Assessment of cell number and viability. (A) Using the CyQUANT assay, the significant decrease in cell number/viability during H/R compared to cells under C/N conditions was significantly attenuated with P188 concentrations of 30 µmol/L‐1 mmol/L. The dashed line represents the average hypoxia only level of ~72  000 cells/well. No significant effect of P188 was observed under C/N conditions. Data expressed as dot plots of individual data points together with the average number of cells/well ± SEM bars, N = 10, 4‐6 replicate wells per treatment per experiment, * vs C/N, Ɨ vs H/R media only. (B) No significant effect of PEG on cell number/viability as assessed by the CyQUANT assay was observed under C/N or H/R conditions. Data expressed as dot plots of individual data points together with the average number of cells/well ± SEM bars; N = 5, 4‐6 replicate wells per treatment per experiment, * vs C/N. (C) Using the MTS assay as another assessment of cell number/viability in a subset of experiments with P188, the significant decrease in cell number/viability during H/R compared to cells under C/N conditions was significantly attenuated with 100 µmol/L and 300 µmol/L P188. No significant effect of P188 was observed under C/N conditions. Data expressed as dot plots of individual data points together with the average AU/well ± SEM bars, N = 5, 4‐6 replicate wells per treatment per experiment, * vs C/N, Ɨ vs H/R media only. (D) Measurement of caspase‐3 activity was used as an additional assessment of cell viability in another subset of experiments with P188. An increase in caspase‐3 activity during H/R was observed compared to cells in C/N conditions. P188 concentrations of 100 µmol/L and 300 µmol/L showed a trend in decreasing caspase‐3 activity. No effect of P188 was observed under C/N conditions. Data expressed as dot plots of individual data points together with the average AU/well ± SEM bars, N = 3, 4‐6 replicate wells per treatment per experiment, * vs C/N, Ɨ vs H/R media only
Figure 5
Figure 5
Assessment of cell membrane injury and repair. (A) The significant increase in LDH release during H/R compared to cells under C/N conditions was significantly decreased with P188 concentrations of 100 µmol/L‐1 mmol/L. The dashed line represents the average hypoxia only level of ~ 1.7 AU/well. No significant effect of P188 was observed under C/N conditions. Data expressed as dot plots of individual data points together with the average AU/well ± SEM bars, N = 10, 4‐6 replicate wells per treatment per experiment, *vs C/N, Ɨ vs H/R media only. (B) No significant effect of PEG on LDH release was observed under C/N or H/R conditions. Data expressed as dot plots of individual data points together with the average AU/well ± SEM bars, N = 5, 4‐6 replicate wells per treatment per experiment, *vs C/N. (C) The significant increase in FM1‐43 incorporation during H/R compared to cells under C/N conditions was significantly decreased with P188 concentrations of 30 µmol/L‐1 mmol/L. The dashed line represents the average hypoxia only level of ~ 11 900 RFU/well. No significant effect of P188 was observed under C/N conditions. Data expressed as dot plots of individual data points together with the average RFU/well ± SEM bars, N = 10, 4‐6 replicate wells per treatment per experiment, *vs C/N, Ɨ vs H/R media only. (D) No significant effect of PEG on FM1‐43 incorporation was observed under C/N or H/R conditions. Data expressed as dot plots of individual data points together with the average RFU/well ± SEM bars, N = 5, 4‐6 replicate wells per treatment per experiment, *vs C/N
Figure 6
Figure 6
Measurement of [Ca2+]i. (A) The significant increase in [Ca2+]i during H/R compared to cells under C/N conditions was significantly decreased with P188 concentrations of 100 µmol/L and 300 µmol/L. The dashed line represents the average hypoxia only level of ~ 9,200 RFU/well. No significant effect of P188 was observed under C/N conditions. Data expressed as dot plots of individual data points together with the average RFU/well ± SEM bars; N = 10, 4‐6 replicate wells per treatment per experiment, *vs C/N, Ɨ vs H/R media only. (B) No significant effect of PEG on [Ca2+]i was observed under C/N or H/R conditions. Data expressed as dot plots of individual data points together with the average RFU/well ± SEM bars, N = 5, 4‐6 replicate wells per treatment per experiment, *vs C/N
Figure 7
Figure 7
Cellular morphology. Representative photos of each group (C/N ± P188, H/R ± P188) show what was consistently observed in the experiments conducted. C/N group shows the normal condition and morphology of the cells at the end of the experiment, and the addition of P188 during reoxygenation did not significantly change the appearance of cells in C/N conditions. The H/R group shows large areas with no cells present (large black arrows), irregularly shrunk and rounded cells, as well as disintegrated cells (small white arrows); addition of P188 during reoxygenation prevented the majority of cells from shrinking and rounding, and most cells appeared to have nearly normal morphology. Cells that underwent H/R and given PEG during reoxygenation appeared the same as the H/R only group shown in the figure. Scale bars are 100 µm in length
See this image and copyright information in PMC

References

    1. Gottlieb RA. Cell death pathways in acute ischemia/reperfusion injury. J Cardiovasc Pharmacol Ther. 2011;16:233‐238. - PMC - PubMed
    1. Kalogeris T, Baines CP, Krenz M, Korthuis RJ. Cell biology of ischemia/reperfusion injury. Int Rev Cell Mol Biol. 2012;298:229‐317. - PMC - PubMed
    1. Martindale JJ, Metzger JM. Uncoupling of increased cellular oxidative stress and myocardial ischemia reperfusion injury by directed sarcolemma stabilization. J Mol Cell Cardiol. 2014;67:26‐37. - PMC - PubMed
    1. Maneechote C, Palee S, Chattipakorn SC, Chattipakorn N. Roles of mitochondrial dynamics modulators in cardiac ischemia/reperfusion injury. J Cell Mol Med. 2017;21:2643‐2653. - PMC - PubMed
    1. Garcia‐Dorado D, Ruiz‐Meana M, Inserte J, Rodriguez‐Sinovas A, Piper HM. Calcium‐mediated cell death during myocardial reperfusion. Cardiovasc Research. 2012;94:168‐180. - PubMed

Publication types

LinkOut - more resources