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Recombinant Activated Protein C (rhAPC) Affects Lipopolysaccharide-Induced Mechanical Compliance Changes and Beat
Aysegül Temiz Artmann1, Eylem Kurulgan Demirci1,2, Ipek Seda Fırat1
1Institute for Bioengineering, University of Applied Sciences Aachen/Campus Juelich, Juelich, Germany.
Background:
Septic cardiomyopathy increases mortality by 70% to 90% and results in mechanical dysfunction of cells.
Methods:
Here, we created a LPS-induced in-vitro sepsis model with mouse embryonic stem cell-derived cardiomyocytes (mESC-CM) using the CellDrum technology which simultaneously measures mechanical compliance and beat frequency of mESCs. Visualization of reactive oxygen species (ROS), actin stress fibers, and mRNA quantification of endothelial protein C receptor (EPCR) and protease-activated receptor 1 (PAR1) before/after LPS incubation were used for method validation. Since activated protein C (APC) has cardioprotective effects, samples were treated with human recombinant APC (rhAPC) with/-out LPS predamage to demonstrate the application in therapeutic studies.
Results:
Twelve hours LPS treatment (5 μg/mL) increased ROS and decreased actin stress fiber density and significantly downregulated EPCR and PAR1 compared to control samples (0.26, 0.39-fold respectively). rhAPC application (5 μg/mL, 12 h) decreased ROS and recovered actin density, EPCR, and PAR1 levels were significantly upregulated compared to LPS predamaged samples (4.79, 3.49-fold respectively). The beat frequencies were significantly decreased after 6- (86%) and 12 h (73%) of LPS application. Mechanical compliance of monolayers significantly increased in a time-dependent manner, up to eight times upon 12-h LPS incubation compared to controls. rhAPC incubation increased the beat frequency by 127% (6h-LPS) and 123% (12h-LPS) and decreased mechanical compliance by 68% (12h-LPS) compared to LPS predamaged samples.
Conclusion:
LPS-induced contraction dysfunction and the reversal effects of rhAPC were successfully assessed by the mechanical properties of mESC-CMs. The CellDrum technology proved a decent tool to simulate sepsis in-vitro.
Insights
This study developed an in-vitro sepsis model using mouse embryonic stem cell-derived cardiomyocytes to assess sepsis-induced heart dysfunction. Activated protein C (APC) treatment reversed these detrimental effects, highlighting its therapeutic potential.
Area of Science:
- Cardiovascular Research
- Stem Cell Biology
- Sepsis Pathophysiology
Background:
- Septic cardiomyopathy significantly elevates mortality rates (70-90%) due to cellular mechanical dysfunction.
- Understanding sepsis-induced cardiac impairment is crucial for developing effective treatments.
Purpose of the Study:
- To establish an in-vitro sepsis model using mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs).
- To evaluate the therapeutic potential of activated protein C (APC) in mitigating sepsis-induced cardiac dysfunction.
- To validate the CellDrum technology for assessing cardiomyocyte mechanical properties.
Main Methods:
- Developed an LPS-induced in-vitro sepsis model with mESC-CMs using CellDrum technology.
- Measured cardiomyocyte mechanical compliance and beat frequency.
- Assessed reactive oxygen species (ROS), actin stress fibers, and specific mRNA levels (EPCR, PAR1).
- Treated cells with human recombinant APC (rhAPC) before or after LPS exposure.
Main Results:
- LPS treatment increased ROS, decreased actin density, and downregulated EPCR/PAR1.
- LPS significantly reduced cardiomyocyte beat frequency and increased mechanical compliance.
- rhAPC treatment reduced ROS, restored actin density, and upregulated EPCR/PAR1.
- rhAPC reversed LPS-induced decreases in beat frequency and increases in mechanical compliance.
Conclusions:
- CellDrum technology effectively simulates in-vitro sepsis and assesses LPS-induced cardiomyocyte dysfunction.
- rhAPC demonstrates significant cardioprotective effects, reversing sepsis-induced mechanical dysfunction in mESC-CMs.
