Optimization of experimental conditions and analysis tools for the study of phosphodiesterase-5 in a model of cultured adult rat cardiomyocytes

[摘要] ENGLISH ABSTRACT : Part 1Introduction: Phosphodiesterases (PDEs) hydrolyse cyclic nucleotides that regulate ischemia-reperfusion injury (IRI) in the heart. Phosphodiesterases-5 (PDE5) inhibition increases cyclic guanosine monophosphate (cGMP) levels and thereby promotes cardioprotection. Cannabidiol is a cannabinoid that can alter cGMP levels and induced protection in whole hearts. Cannabidiol-mediated cardioprotection might be controlled by specific PDEs, possibly PDE5.This study aimed to:Evaluate the role of PDE5 inhibition in IRI.Determine whether PDE5 plays a role in cannabidiol-mediated protection.Methods: Cultured adult rat cardiomyocytes were subjected to 20 minutes ischemia, 60 minutes reperfusion, which included mitochondrial staining to measure mitochondrial function with JC-1, followed by fluorescence microscopy and image analysis. A cardioprotective dose of cannabidiol and time of intervention was sought by administration of cannabidiol (0.001 μM, 1 μM and 100 μM) during ischemia and reperfusion, only ischemia, and only reperfusion, respectively. 10 μM Sildenafil was administered during ischemia only to inhibit PDE5.Results: Ischemia-reperfusion reduced cell viability according to morphology by 79 % and mitochondrial function by 50 %. None of the treatments induced cardioprotection.Conclusion: The lack of cardioprotection from cannabidiol and sildenafil might have been due to (1) the ischemic conditions being too harsh, (2) the analysis program being faulty, or (3) unreliable data from morphology analysis. These three points of concern became the basis for the new objectives investigated in Part 2 of this thesis.Part 2Introduction: Cell viability and mitochondrial function are parameters normally evaluated in cardiomyocytes, and were also used in this study, but cardioprotection could not be found. This raised concerns about the reliability of the image analysis program (ImageJ), the severity of ischemia, and the reliability of the parameters measured. The method used to determine cell viability was especially questioned, because it relies on the researcher to classify rod cells as viable and round cells as dead, which is thus subjective. Morphometry analysis with length over width (L/W) removes the human aspect, allowing cell viability to be determined by classifying cardiomyocytes with L/W ≥ 1.5 as viable. Length on its own is also a morphometric measurement, but is seldom used. Part 2 of this study aimed to:Compare image analysis of ImageJ with that of CellProfiler.Optimize conditions for ischemia-reperfusion and hypoxia-reperfusion.Compare morphology analysis with morphometry analysis.Methods: The sildenafil experimental images from Part 1 were reanalyzed using CellProfiler and the data compared with that found with ImageJ. Ischemia-reperfusion was induced with less harsh conditions for 1 hour, and compared to hypoxia-reperfusion, using cell viability and mitochondrial function. Cell viability was determined by selecting viable cells by rod shape, compared to L/W ≥ 1.5, and length ≥ 55 μm. The average length for hypercontracted cells in the normoxic population was determined, and found to be consistently below 55 μm. Length ≥ 55 μm was chosen as morphometry selection to identify viable cells.Results: Both ImageJ and CellProfiler provided similar data. Cell viability for L/W ≥ 1.5 and length ≥ 55 μm were similar, but higher than morphology, especially for hypoxia-reperfusion, but not for ischemia-reperfusion. L/W ≥ 1.5 and length ≥ 55 μm found differences between normoxia and hypoxia-reperfusion, unlike morphology. The differences can be explained by morphology selecting fewer cells that are perfectly healthy, while morphometry selects more cells with varying degrees of cell injury. Only for ischemia-reperfusion did all parameters provide similar knockdown. This can be explained by ischemia-reperfusion that induced severe injury and hypoxia-reperfusion that induced less injury.Conclusion: The lack of cardioprotection by PDE5 inhibition and cannabidiol was not due to an image analysis error by the program, but might rather be due to ischemia-reperfusion that was too harsh. Conversely, hypoxia-reperfusion induced injury that was not harsh enough. Morphometry selection is biased and unreliable, and morphometry selection should rather be used to evaluate an injured cardiomyocyte population.

[发布机构] Stellenbosch University