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, 9 (1), 5651

Serum of Patients With Acute Myocardial Infarction Prevents Inflammation in iPSC-cardiomyocytes

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Serum of Patients With Acute Myocardial Infarction Prevents Inflammation in iPSC-cardiomyocytes

Katherine Sattler et al. Sci Rep.

Abstract

Acute myocardial infarction (MI) evokes a systemic inflammatory response and locally the degradation of the necrotic tissue, followed by scar formation. The mechanisms for containment of the infarct zone are not studied well. The study aimed to examine the response of healthy cardiomyocytes to serum of patients with myocardial infarction. Human iPSC-cardiomyocytes (iPSC-CM) generated from two healthy donors were incubated with serum of patients with MI with and without ventricular fibrillation (VF) or of healthy controls. Different cell adhesion molecules were studied by flow cytometry and immunostaining. Cellular electrophysiology was studied by patch clamp. The cell adhesion molecules CD54/ICAM-1, CD58/LFA-3 and CD321/JAM-A were expressed on iPSC-CM within the plasma membrane. Incubation with serum of MI patients reduced the levels of expression of CD54/ICAM-1 and CD321/JAM-A by 15-20%. VF serum was less effective than serum of MI patients without VF. MI serum or VF serum did not affect resting potential, action potential duration or maximum depolarization velocity. Myocardial infarction serum exerts anti-inflammatory effects on healthy cardiomyocytes without affecting their electrical activity, thus helping to contain the infarct zone and to protect healthy tissue. Ventricular fibrillation during MI drives healthy cardiomyocytes towards a pro-inflammatory phenotype.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(A). mRNA expression of the pluripotency gene POU5F1 and of the cardiac marker troponin I (TNNT2) during differentiation of hiPSC into cardiomyocytes. The fold change over time relative to the expression of the housekeeping gene GAPDH is presented. Mean of 3 technical replicates of 3–6 biological replicates of 3 different differentiation rounds is shown. Whiskers display standard error. (B). Percentage of marker-expressing iPSC-cardiomyocytes (% of TNNT2-positive cells) at day 25 of differentiation. Mean of n = 5–12 measurements per marker. Whiskers display standard deviation. (C). CD162, CD31, CD62, and CD102 (blue) were not found on iPSC-cardiomyocytes.
Figure 2
Figure 2
(A). Expression levels (median fluorescence intensity) of CD58/LFA-3, CD54/ICAM-1 and CD321/JAM-A on iPSC-cardiomyocytes (TNNT2-positive cells) at different times of differentiation. N = 5–10 measurements per marker. The mean ± standard deviation is shown. (B). Representative plots of flow cytometry for CD58/LFA-3, CD54/ICAM-1 and CD321/JAM-A (blue) showing the marker-positive sub-populations at differentiation day 25. The number of each marker subset gives the percentage of positive cells of all cells. (C). Percentage of iPSC-cardiomyocytes (% of TNNT2-positive cells) expressing CD58/LFA-3, CD54/ICAM-1 and CD321/JAM-A at different times of differentiation. N = 5–10 measurements per marker. The mean ± standard deviation is shown.
Figure 3
Figure 3
Immunofluorescence staining demonstrates the localization of CD58/LFA-3 (A, green), CD54/ICAM-1 (B, green) and CD321/JAM-A (C, yellow) in iPSC-cardiomyocytes (A–C: troponin T, red; nuclei, blue). Each marker is also expressed by cells other than cardiomyocytes, which can best be seen with CD58. Images were taken at 40x magnification. Scale bars represent 50 µm with the exception of CD58 (200 µm for better overview of marker expression by different cells).
Figure 4
Figure 4
Expression levels (median fluorescence intensity relative to cell number) on iPSC-cardiomyocytes after incubation with myocardial infarction serum (MI) or control serum (5% or 20% v/v). (A) CD58/LFA-3, (B) CD54/ICAM-1, (C) CD321/JAM-A. N = 13–26 measurements per marker. The line within box represents the median, additionally the 10th, 25th, 75th, and 90th percentile is shown; whiskers represent error bars.
Figure 5
Figure 5
Expression levels (median fluorescence intensity relative to cell number) on iPSC-cardiomyocytes after incubation with 5% or 20% (v/v) myocardial infarction serum with vs. without ventricular fibrillation (VF). (A) CD58/LFA-3, (B) CD54/ICAM-1, (C) CD321/JAM-A. N = 7–15 measurements per marker. The line within box represents the median, additionally the 10th, 25th, 75th, and 90th percentile is shown; whiskers represent error bars.
Figure 6
Figure 6
Incubation with myocardial infarction serum (MI, black) does not change electrophysiological properties (control serum – Ctr, white). RP – Resting potential, APA – action potential amplitude, Vmax – maximum depolarization velocity, APD – action potential duration. The mean of the indicated number of measurements is shown; whiskers display standard deviation.
Figure 7
Figure 7
Incubation with myocardial infarction serum with ventricular fibrillation (VF, black) does not change electrophysiological properties (myocardial infarction serum without ventricular fibrillation – MI, white). RP – Resting potential, APA – action potential amplitude, Vmax – maximum depolarization velocity, APD – action potential duration. The mean of the indicated number of measurements is shown; whiskers display standard deviation.

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