Studying The Effects Of Altered Protein Kinase a Pathway on the Interaction of Serca2a and Phospholamban (Plb) Observed through a Modified Green Fluorescent Protein (SGP)
Heart failure with a reduced ejection fraction (HFrEF) is characterized by left ventricular enlargement and an ejection fraction <40%. HFrEF is associated with a decreased cardiac pump function and diminished cardiac contractility. Cardiac contractility is regulated by phospholamban (PLB) inhibition of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), a pump-protein that translocates calcium ions into the sarcoplasmic reticulum. The interaction of SERCA2a and PLB protein can be studied with an interposed circularly permutated green fluorescent protein (S-cpGFP-P = SGP). PLB mediated disinhibition of SERCA2a may be directly mediated by higher calcium concentrations in the cytoplasm. HEK 293 cells are transfected with SGP-cDNA to confirm SGP integrity, cultured, and observed using an inverted tissue culture fluorescence microscope (Olympus IX73). Sprague-Dawley rat adult cardiomyocytes are isolated and transfected with the SGP construct and fluorescence will be assessed. Cardiomyocytes are cultured. The intracellular calcium concentration is increased in HEK 293 cells, H9 cells, and adult rat cardiac myocytes using several alternative approaches i.e. electrical stimulation, ionomycin and extracellular calcium modifications. With a rise of intracellular calcium the fluorescent yield of SGP increases to directly reveal a dissociation of PLB from SERCA2a which translates into increased contractility. This mechanism is independent of PLB phosphorylation. This is a first documentation in living cells that the interaction of SERCA2a and PLB is directly regulated by cytoplasmatic calcium levels suggesting an autoregulatory mechanism that likely plays a substantial role in physiology as well as heart failure as it increases sarcoplasmatic calcium loads and contractility.