Lysosomal reacidification via degradation of PLGA nanoparticles in a lipotoxic cardiomyopathy model

Abstract

Event Abstract Back to Event Lysosomal reacidification via degradation of PLGA nanoparticles in a lipotoxic cardiomyopathy model Frederick M. Zasadny1, 2, James A. Ankrum1, 2 and E Dale Abel2, 3, 4 1 University of Iowa, Biomedical Engineering, United States 2 University of Iowa, Fraternal Order of Eagles Diabetes Research Center, United States 3 University of Iowa Carver College of Medicine, Internal Medicine, United States 4 University of Iowa, Biochemistry, United States Introduction: Lipotoxic cardiomyopathy is one of several cardiovascular complications that cause one third of deaths worldwide[1]. Specifically, obesity related lipid-overload impairment of autophagy causes pathophysiological changes in heart function and structure. There is an increase in fatty acid uptake and utilization in the heart that increases toxic lipid species[2]. These cardiotoxic lipid species have recently been shown to deacidify lysosomes in the heart, the terminus of autophagy[3]. When deacidified, the proteolytic enzymes in the lysosome cannot process autophagic vesicles. Thus, there is an accumulation of autophagic vesicles in heart muscle tissue which impairs cardiac function. Here, a therapeutic strategy utilizing poly(lactic-co-glycolic acid) (PLGA) nanoparticle degradation to reacidify the lysosome and revert the effects of cardiotoxic lipid species restoring autophagic flux in H9c2 cardiomyocytes is presented. Materials and Methods: Nanoparticle (NP) formulation consists of single emulsion solvent extraction evaporation. Namely, PLGA is dissolved in an organic solvent dichloromethane (DCM) (not miscible in water) which is then added to polyvinyl alcohol (PVA) (miscible in water). Upon emulsification of the solution, nanoparticles of PLGA surrounded by DCM are formed suspended in the PVA solution. DCM then evaporates leaving behind hardened nanoparticles in PVA. H9c2 cardiomyocytes (CM’s) were grown to 80% confluence in six well plates and then differentiated for four days with 10nM all-trans retinoic acid. On the third day of differentiation, CM’s were treated with PLGA NP’s for ~15 hours, upon which the cells were treated with 500uM palmitate (Palm) for 4 hours. After treatments, Western Blot, fluorescence microscopy and other assays assessed autophagic flux and PLGA NP activity. For microscopy, PLGA NP’s were labeled with DiD stain to enable colocalization studies with the lysosome (LysoTracker) and autophagic vesicle stains (Cyto-ID). Proteolytic enzyme (Cathepsin L) activity in the lysosome was assessed with Magic Red fluorescence, decreasing with palmitate treatment. Results and Discussion: H9c2 cardiomyocytes treated with palmitate increased Cyto-ID florescence of autophagic vesicles indicating correct modeling of obesity related lipid-overload impairment of autophagy, i.e., a blockade of autophagic flux. Colocalization studies with DiD and LysoTracker stains indicate that the PLGA NP’s are indeed phagocytosed and transported to the lysosome. PLGA NP treatment increased Cathepsin L activity when CM’s are also treated with Palm compared to just Palm treatment, as shown in Figure 1. This indicates that the hydrolytic degradation of PLGA NP’s into lactic and glycolic acid is functionally reacidifying the lysosomes in H9c2 cardiomyocytes and seemingly restoring Cathepsin L activity in the lysosome. Western blot indicates a decrease in the accumulation of autophagic vesicle protein build-up caused by palmitate treatment when co-treated with PLGA NP’s suggesting a restoration of autophagic flux. Conclusions: PLGA NP’s functionally revert obesity related lipid-overload impairment of autophagy in cardiomyocytes with respect to reacidification of impaired lysosomes indicated by restored and increased Cathepsin L activity. Future studies include optimization of PLGA NP design including the conjugation of cardiomyocyte specific moieties that will enable in vivo targeting of heart tissue.