Free Fatty Acid Effects on the Atrial Myocardium: Membrane Ionic Currents Are Remodeled by the Disruption of T-Tubular Architecture

Abstract

Background: Epicardial adiposity and plasma levels of free fatty acids (FFAs) are elevated in atrial fibrillation, heart failure and obesity, with potentially detrimental effects on myocardial function. As major components of epicardial fat, FFAs may be abnormally regulated, with a potential to detrimentally modulate electro-mechanical function. The cellular mechanisms underlying such effects of FFAs are unknown.

Objective: To determine the mechanisms underlying electrophysiological effects of palmitic (PA), stearic (SA) and oleic (OA) FFAs on sheep atrial myocytes.

Methods: We used electrophysiological techniques, numerical simulations, biochemistry and optical imaging to examine the effects of acutely (≤ 15 min), short-term (4-6 hour) or 24-hour application of individual FFAs (10 μM) on isolated ovine left atrial myocytes (LAMs).

Results: Acute and short-term incubation in FFAs resulted in no differences in passive or active properties of isolated left atrial myocytes (LAMs). 24-hour application had differential effects depending on the FFA. PA did not affect cellular passive properties but shortened (p<0.05) action potential duration at 30% repolarization (APD30). APD50 and APD80 were unchanged. SA had no effect on resting membrane potential but reduced membrane capacitance by 15% (p<0.05), and abbreviated APD at all values measured (p≤0.001). OA did not significantly affect passive or active properties of LAMs. Measurement of the major voltage-gated ion channels in SA treated LAMs showed a ~60% reduction (p<0.01) of the L-type calcium current (ICa-L) and ~30% reduction (p<0.05) in the transient outward potassium current (ITO). A human atrial cell model recapitulated SA effects on APD. Optical imaging showed that SA incubated for 24 hours altered t-tubular structure in isolated cells (p<0.0001).

Conclusions: SA disrupts t-tubular architecture and remodels properties of membrane ionic currents in sheep atrial myocytes, with potential implications in arrhythmogenesis.

Fig 1. Saturated free fatty acids, but not a mono-unsaturated fatty acid, shorten action potential duration in atrial cells.

Panels A, C, E; Atrial myocyte action potential recordings in control solution (CTL, black) and following incubation in solutions containing 10 μm palmitic acid (PA, blue), stearic acid (SA, red) or oleic acid (OA, green). Average APD30, 50, & 80 measurements in CTL and following incubation in PA (Panel B, n = 9, 12; *p<0.05), SA (Panel D, n = 22, 24; ***p<0.001) and OA (Panel F, n = 6, 5). CTL solution in this and subsequent Figs, represent vehicle control, see Methods section. Scale bars: 100 msec and 20 mV.

Fig 2. Voltage-gated ionic currents are differentially affected by stearic acid.

Panel A: Representative traces of sodium currents (I_Na) in control (CTL, black) and in SA (10 μm; red) treated cells. Inset: voltage-clamp protocol for current activation. Plots of current density (Panel B), and of voltage-dependence of current activation and inactivation (Panel C), n = 5, 7). Panel D: Representative traces (selected) of calcium current (I_{Ca, L}) in CTL and in SA (10 μm; red) treated cells. Inset: voltage-clamp protocol for current activation. Plots of current density (Panel E), and of voltage-dependence of current activation and inactivation (Panel F). Incubation of myocytes in SA caused a significant reduction of I_{Ca, L} density (n = 24, 22; *p<0.05). Panel G: Transient outward potassium currents (I_TO) in CTL and in SA treated cells. Inset: voltage-clamp protocol. Panel H: Current-density measurements showing effects of SA on peak I_TO, but not on the steady state current (I_SS) (Panel I) (n = 8, 8; *p<0.05).

Fig 3. Human atrial action potential model simulation of stearic acid effects on ionic currents in isolated atrial cells.

Panel A: Model generated action potential under control conditions (left) and following SA-induced changes in ionic currents (see text for details) (right). Panel B: Measurements of APD30, 50 & 80 from ten consecutive action potential simulations under control (CTL, black) and with SA treatment (white).

Fig 4. Cav1.2 Localization and whole cell protein content is not affected by stearic acid.

Transmitted light image of an atrial myocyte (top), Cav1.2 protein staining (green), α-actinin staining (red) and a merged image of Cav1.2, α-actinin and DAPI staining (bottom) under control (CTL; Panel A) and following incubation in stearic acid (SA; Panel B). Inset: 20 μm section of the merged image and corresponding intensity profile of Cav1.2 and α-actinin. Scale bars 20 μm. Panel C: Quantification of the intensity profiles shows SA did not alter the mean distance between intensity peaks for Cav1.2 (left) or α-actinin (right; n = 12, 12). Panel D: (left) Western blot for Cav1.2, with GAPDH as a control. Pane D: (right) normalized densitometry plot of Cav1.2 protein levels in CTL and SA treated cell lysates (n = 3).

Fig 5. Stearic Acid disrupts t-tubules in atrial myocytes.

Panel A (top): Coordinate axes for reference and schematic diagrams illustrating the fields of view. Panel A: subpanels (a, c, e & g) are XY planar views and (b, d, f & h) are 40 μm ZY cross-sectional views of the same cell. (a & b) Di-8-ANEPPS staining of t-tubules in freshly dissociated (t = 0) atrial myocytes (n = 19). After 24 hrs in culture, Control (CTL) cells shown in (c) and (d) retain t-tubule structures (n = 20). (e) and (f) T-tubular structure after chronic incubation of myocytes in PA (n = 19). (g) and (h) reduction in t-tubules after 24 hr incubation of myocytes in SA (n = 25). Arrows indicate the lateral membrane and arrowheads highlight an individual t-tubule in both views. (*) identifies the nuclear region. Scale bars: 20 μm (XY) and 5 μm (ZY). Panel B: Quantification of t-tubules in using the ratio of the t-tubule region and total cell fluorescence. SA reduced the presence of t-tubule structures in LA myocytes (CTL vs. SA; ***p<0.0001, n = 25), CTL vs. PA; p = ns (n = 19)). Panel C: SA reduced the capacitance of atrial myocytes following incubation in SA (Panel C, n = 83 *p<0.05), but did not alter 2D surface area of myocytes (n = 68).