Acute exposure of ceramide enhances cardiac contractile function in isolated ventricular myocytes

Abstract

1. The sphingolipid ceramide, a primary building block for all other sphingolipids, is associated with growth arrest, apoptosis, and lipotoxic dysfunction. Interestingly, ceramide may attenuate high glucose-induced myocyte dysfunction, produce Ca2+ influx, and augment smooth muscle contraction. To determine the role of ceramide on cardiac excitation-contraction (E-C) coupling, electrically paced adult rat ventricular myocytes were acutely exposed to a cell-permeable ceramide analog (10 pm-100 microM) and the following indices were determined: peak shortening (PS), time-to-PS, time-to-90% relengthening, and the maximal velocity of shortening and relengthening (+/-dLdt). Intracellular Ca2+ properties were assessed using fura-2AM fluorescent microscopy. 2. Our results revealed a concentration- and time-dependent increase of PS in ventricular myocytes in response to ceramide associated with an increase in +/-dLdt. The maximal increase in PS was approximately 35% from control value and was maintained throughout the first 20 min of ceramide exposure. However, the ceramide-induced increase in PS was not maintained once the exposure time was beyond 20 min. Acute exposure of ceramide significantly enhanced intracellular Ca2+ release, although at a much lower concentration range. The ceramide-induced augmentation of PS was not significantly affected by inhibition of phosphatidylinositol (PI)-3-kinase, protein kinase C (PKC), ceramide-activated protein phosphatase (CAPP), and nitric oxide (NO) synthase. 3. Our data suggest that ceramide acutely augments the contractile function of cardiac myocytes through an alternative mechanism(s) rather than PI-3-kinase, PKC, CAPP, or NO.

Figure 1

(a) Typical experiment showing the effect of ceramide (10 μM) on myocyte shortening in isolated ventricular myocytes. Myocyte shortening and relengthening were recorded at 25°C before and 5 min after ceramide application. (b) Concentration-dependent response to ceramide (0–100 μM) on myocyte shortening in ventricular myocytes. Mean±s.e.m., n=33–41 cells per group, ^*P<0.05 vs control, as determined by repeated measures ANOVA and subsequent Tukey's post hoc test.

Figure 2

Time-dependent augmentation of PS in cardiac ventricular myocytes treated with 10 μM cell-permeable C₂-ceramide. Mean±s.e.m., n=8–16 cells per group, ^*P<0.05 vs control, as determined by repeated measures ANOVA and subsequent Tukey's post hoc test.

Figure 3

(a) Effect of ceramide on baseline [Ca²⁺]_i, represented by FFI. (b) Concentration-dependent response of ceramide (0–100 μM) on intracellular Ca²⁺ transient changes (ΔFFI) in ventricular myocytes. Mean±s.e.m., n=21–24 cells per group, ^*P<0.05 vs control value, as determined by repeated measure ANOVA and subsequent Tukey's post hoc test.

Figure 4

Effect of the PI-3-kinase inhibitor wortmannin (100 nM), the CAPP inhibitor okadaic acid (100 nM), the PKC inhibitor chelerythrine (1 μM), or the NOS inhibitor L-NAME (100 μM) on per cent change in PS in the absence or presence of ceramide (10 μM) (5 min exposure). Mean±s.e.m., n=11–16 cells per group for inhibitors in the absence of ceramide (left four bars) and n=11–30 cells per group for inhibitors in the presence of 10 μM ceramide (right five bars), ^*P<0.05 vs the respective baseline control value for each inhibitor when assessed using a paired t-test. For the rest of the groups, P>0.05 vs 10 μM ceramide exposure (empty bar), determined by one-way ANOVA.