Fatty Acid (FFA) Transport in Cardiomyocytes Revealed by Imaging Unbound FFA Is Mediated by an FFA Pump Modulated by the CD36 Protein

Abstract

Regulation of FFA 3 transport across the cardiomyocyte membrane is essential for cardiovascular health and dysregulation may result in myocardial lipotoxicity (1). However, the mechanisms governing FFA transport across cell membranes are not well understood (2, 3). Proposed mechanisms include rapid flip-flop through the lipid phase of the plasma membrane (4) or a protein-mediated mechanism (2, 3, 5). Proteins reported to be correlated with FFA transport in cardiac or muscle cells include FAT/CD36, FABPpm, and FATP4 (6 -10).In previous studies of FFA transport we found virtually identical characteristics in adipocytes and preadipocytes, suggesting involvement of an unknown protein because FAT/ CD36, FABPpm, and FATP4 are expressed poorly or not at all in preadipocytes (2, 11-13). Transport in these studies was observed by imaging the concentration of intracellular unbound FFA (FFA i ) (11,12,14). This allowed us to directly monitor the movement of unbound FFA (FFA u ) from the extracellular medium and into the cytosol.Because FFA transport plays a vital role in the heart and because our adipocyte/preadipocyte results raise the possibility that novel FFA transport mechanisms might exist in other tissues, we have investigated FFA transport in cardiomyocytes isolated from wild type (WT) adult C57BL/6 mice and FAT/ CD36 Ϫ/Ϫ mice on the C57BL/6 background. We used several methods to monitor FFA transport in this study, so that key findings were confirmed independently of a specific method. These methods included 1) quantitative imaging of FFA i using a sensor of FFA, ADIFAB (acrylodan-labeled rat intestinal fatty acid-binding protein), 2) using the pH indicator BCECF-AM (2Ј,7Ј-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester) to monitor FFA influx and efflux, and 3) a new method to determine FFA uptake by monitoring the extracellular unbound FFA concentration (FFA o ) using ADIFAB in the extracellular medium.This study provides the first measurements of FFA i in cardiac cells and the first direct measurements of FFA influx and efflux across the cardiac plasma membrane. The results suggest that FFAs are pumped into cardiomyocytes and that FAT/CD36 interacts with the transporter/pump so that it significantly modifies the rate of efflux and, to a lesser extent, the rate of influx. The results significantly alter current views of the mechanisms of FFA transport in cardiac myocytes and may provide insights for understanding the role of FFA in cardiac dysregulation. EXPERIMENTAL PROCEDURES

Multiphasic triacylglycerol dynamics in the intact heart during acute in vivo overexpression of CD36

“…3B ). The responses of the saturable exponential component to increased CD36 content indicate that the fi rst phase of TAG enrichment is defi ned by LCFA transport across the sarcolemma in the intact functioning heart, which is consistent with studies showing a slowed exponential uptake of LCFA into isolated cells that are transporter-defi cient from the CD36 null mice ( 15 ).…”

“…To examine whether LCFA transporter kinetics were discernable in the intact functioning heart via 13 C NMR detection of TAG enrichment kinetics and to better understand how LCFA transport might affect TAG turnover, we have used an in vivo model of exogenous gene delivery to overexpress the rat isoform of CD36 in a cardiac-specifi c manner. Dynamic mode 13 C NMR of the intact functioning heart resolved distinct phases of TAG enrichment that can be elucidated as distinct physiological processes and that are consistent with prior observations of LCFA transport across the sarcolemma of isolated cardiomyocytes ( 15 ) with an eventual linear rate of TAG enrichment at steady state. The fi ndings support our hypothesis that early and late phase kinetics of the isotopic enrichment of TAG from 13 C LCFA can be distinguished and refl ect the separate components of LCFA entry into the cardiomyocyte versus TAG turnover and that these separate rate components are tightly coupled and coordinated by reciprocal control of gene expression.…”

“…The time constant ( ) of the initial exponential enrichment of TAG among sham hearts was = 3.18 ± 0.53 min (n = 5), similar to the exponential transport of LCFA transport into isolated cardiac myocytes ( 15 ). CD36 overexpression dramatically accelerated this exponential phase of the observed TAG enrichment with 13 C palmitate, reducing the time constant by 60% compared with shams ( P < 0.05, Fig.…”

“…Recently, LCFA transport into freshly isolated cardiac myocytes was demonstrated to occur via two distinct processes: a fast exponential component [time constant ( ) of 1-2 min] modulated by CD36 expression, and a slow linear component sensitive to inhibitors of LCFA metabolism ( 15 ). Thus, we hypothesized that such kinetic features of LCFA uptake would be refl ected in the NMR-observable dynamics of TAG enrichment with 13 C-enriched LCFA.…”

The role of triacylglycerol in cardiac energy provision

“…Dynamic mode NMR using 13 C-palmitate shows an initial, saturable exponential incorporation into mTAG in the intact heart, representing sarcolemmal receptor-mediated FA uptake, and a slower A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 26 linear rate, determined by the rate of cellular metabolism and sensitive to inhibitors of LCFA metabolism, reflecting TAG turnover [118,120,[159][160][161]. TAG pool turnover is a function of mTAG pool size and FA availability: TAG turnover increases with high fat diet (in association with increased DGAT1 and ATGL expression [120,130]), in diabetes [160] PPA' PGC overexpressing mouse hearts [120,162], but is decreased in cardiac hypertrophy with diminished PPAR activity [118].…”