Free Radical Biology & Medicine, Vol. 6, pp. 23-30, 1989 0891-5849/89 $3.00 + .00 Printed in the USA.All fightsreserved. © 1989Pergamon Pressplc @ Original Contribution EFFECT OF AMBIENT OXYGEN CONCENTRATION ON LIPOFUSCIN ACCUMULATION IN CULTURED RAT HEART MYOCYTES--A NOVEL IN VITRO MODEL OF LIPOFUSCINOGENESIS RAJINDAR S. SOHAL,* MASSOUD R. MARZABADI, DIMITRIOS GALARIS, and ULF T. BRUNK Department of Pathology II, University of LinkSping, S-581 85 LinkOping, Sweden (Received 10 December 1987; Accepted 8 February 1988) Abstract--The objective of this study was to elucidate the factors involved in the accumulation of lipofuscin in post-mitotic cells. The hypothesis that oxidative stress accelerates the rate of lipofuscin accumulation was tested by examining the effects of 5%, 20%, and 40% ambient oxygen concentration on lipofuscin content in cultured rat cardiac myocytes. Lipofuscin was quantified by microspectrofluorometry at 7 and 12 days of in vitro age. Lipofuscin-emitted yellow autofluorescence increased in direct relationship to ambient oxygen concentration with age. Transmission electron microscopic examination of the cells after 3, 8, and 12 days in culture indicated a progressive time and oxygen dependent increase in the frequency and size of lipofuscin organelles. The results are interpreted to suggest that oxidative stress is one of the causal factors in the accumulation of lipofuscin. Keywords--Lipofuscin, Heart, Cultured myocytes, Aging, Hyperoxia, Free radicals, Oxygen, Autofluorescence, In vitro INTRODUCTION Accumulation of lipofuscin in long-lived, post-mitotic cells is the most prominent and consistent age-related morphological alteration. 1-4 Several hypotheses have been proposed concerning the causal factors and mech- anisms responsible for the origin of lipofuscin (for re- view, see Ref. 5). The proposed hypotheses can be divided into two different schools of thought. One view is that lipofuscin accumulates due to either the inability or the decline in the efficiency of cells to degrade substances such as dolichol phosphates and polymer- ized lipoproteins. 6,7 The other view is that lipofuscin is formed primarily as a result of oxygen free radical- induced reactions which cause damage and polymeri- zation of lipids and proteins into undegradable resi- dues. 8'9 The key difference between the two views being that the former regards lipofuscin accumulation to be primarily a manifestation of the limitation of cellular waste disposal or digestive ability while the latter con- *Corresponding author and present address: Departmentof Biolog- ical Sciences, Southern Methodist University, Dallas, TX 75275, USA. siders lipofuscin to be a product of damage inflicted by oxygen free radicals to biological molecules. One of the major limitations in the study of the mechanisms of lipofuscinogenesis has been the lack of adequate in vitro models. For example, lipofuscin ac- cumulation is a feature of aging in non-dividing cells such as cardiac myocytes or neurons, whereas, cells usually cultured in vitro are generally highly prolif- erative. The division of cell cytoplasm together with cell growth, associated with cell cycle, tend to dilute the concentration of lipofuscin within cells, which complicates the interpretation of results. Isolated mammalian cardiac myocytes can be main- tained under cultured conditions for relatively pro- longed periods and have been employed for the purpose of studying a variety of heart cell functions (for ref- erences, see 10, 11). In culture, these cells do not divide and exhibit age-related changes which are sim- ilar to those observed in vivo. For example, they pro- gressively accumulate lipofuscin. To our knowledge, this model system has not as yet been exploited for studying the aging of cardiac myocytes. The present study was conducted in an effort to identify the factors involved in the formation of lipo- 23