TNF-alpha and IL-1beta increase Ca2+ leak from the sarcoplasmic reticulum and susceptibility to arrhythmia in rat ventricular myocytes

Abstract

Sepsis is associated with ventricular dysfunction and increased incidence of atrial and ventricular arrhythmia however the underlying pro-arrhythmic mechanisms are unknown. Serum levels of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) are elevated during sepsis and affect Ca2+ regulation. We investigated whether pro-inflammatory cytokines disrupt cellular Ca2+ cycling leading to reduced contractility, but also increase the probability of pro-arrhythmic spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). Isolated rat ventricular myocytes were exposed to TNF-alpha (0.05 ng ml(-1)) and IL-1beta (2 ng ml(-1)) for 3 hr and then loaded with fura-2 or fluo-3 to record the intracellular Ca2+ concentration ([Ca2+](i)). Cytokine treatment decreased the amplitude of the spatially averaged Ca2+ transient and the associated contraction, induced asynchronous Ca2+ release during electrical stimulation, increased the frequency of localized Ca2+ release events, decreased the SR Ca2+ content and increased the frequency of spontaneous Ca2+ waves at any given cytoplasmic Ca2+. These data suggest that TNF-alpha and IL-1beta increase the SR Ca2+ leak from the SR, which contributes to the depressed Ca2+ transient and contractility. Increased susceptibility to spontaneous SR Ca2+ release may contribute to arrhythmias in sepsis as the resulting Ca2+ extrusion via NCX is electrogenic, leading to cell depolarisation.

2010 Elsevier Ltd. All rights reserved.

Figures

Fig. 1

(A) Fast time base records of cell length and Ca2+ transients from cells following 180 min incubation in either normal Tyrode's solution (NT) or NT supplemented with 0.05 ng ml−1 TNF-α and 2 ng ml−1 IL-1β. Mean data for control (n = 80) and treated, cells (n = 82) on contraction amplitude (B) and magnitude of the cytosolic Ca2+ transient (C). (D) The effects of 180 min exposure to TNF-α (0.05 ng ml−1, n = 13) and IL-1β (2 ng ml−1, n = 14) alone on contraction and the Ca2+ transient amplitude. *P < 0.05.

Fig. 2

Mean data describing contraction amplitude (A), Ca2+ transient amplitude (B), SR Ca2+ content (C) and diastolic fluorescence ratio (D) of control (n = 27) and treated (n = 24) cells during 1 and 3 Hz stimulation. At 1 Hz, cytokine treatment significantly decreased cell contraction, the Ca2+ transient and SR Ca2+ content (*P < 0.05 vs 1 Hz control, t-test). At 3 Hz all parameters were significantly increased (+P < 0.001, paired t-test vs 1 Hz) in both control and treated cells however cell contraction, the Ca2+ transient and SR Ca2+ content remained significantly depressed compared to control (#P < 0.05 vs 3 Hz control, t-test). Rate-dependent decreases in the time for half decay of contraction (E) and the Ca2+ transient (F) were observed at 3 Hz in both control and treated cells (*P < 0.05, paired t-test vs 1 Hz) but this was unaffected by cytokine treatment.

Fig. 3

(A) Typical line-scan images of Ca2+ sparks from myocytes under control conditions (left) or following incubation with TNF-α and IL-1β (right). Surface plots are from the regions of each line-scan indicated. Selected line profiles are also shown below each image (1,2). These were produced by averaging 3 pixels centred on the peak of the Ca2+ sparks. (B) Mean data describing spark frequency (s−1) per 100 μm, amplitude (F/F0), duration (FDHM) and width (FWHD) in control and treated cells (n = 19 per group). ***P < 0.001 vs control, **P < 0.05 vs control, t-test.

Fig. 4

(A) Typical line-scan images obtained during field stimulation from control (left) and treated (right) cells displaying synchronous or asynchronous Ca2+ release, respectively. (B) Line profiles positioned transversely across the image (i and iv), at the point of stimulation. (C) Superimposed longitudinal line profiles indicating the time course and amplitude of the Ca2+ transients at the points indicated (ii and iii for control (left) and v and vi for treated cells (right)). All line profiles were obtained by averaging over 3 pixels.

Fig. 5

(A) Representative data showing field stimulated Ca2+ transients interspersed with local Ca2+ release (red bars) and propagated waves (black bars) in a cytokine-treated cell. (B) Line-scan image and surface plot from a control cell following field stimulation (upper panel). Control cells typically exhibit a quiescent period immediately after each stimulation, when Ca2+ sparks were not apparent or occurred at very low frequency. Also shown is a line-scan image and corresponding surface plot from a cell treated with TNF-α and IL-1β under similar conditions (lower panel). Treated cells typically exhibited Ca2+ sparks during and immediately after each stimulated response. (C) Cumulative data showing changes in spontaneous Ca2+ spark frequency (s−1) per 100 μm, from control (left) and treated (right) cells stimulated at 6 s intervals. In control cells (n = 20) spark frequency progressively increased from a very low level immediately after the initial stimulation. In treated cells (n = 28) there was no clear temporal relationship between the time and spark frequency. ***Value significantly different from that at 1 s (P < 0.001). Vertical bar indicates 25 μm. ‘S’ indicates field stimulation.

Fig. 6

(A) Representative record of spontaneous Ca2+ waves recorded in a control unstimulated cell at extracellular [Ca2+] of 1, 2 and 3 mM. (B) Mean data describing the frequency of spontaneous Ca2+ waves in control (n = 7–9) and treated (n = 8) cells. Frequency was increased significantly (**P < 0.01) in treated cells compared to control cells at any given extracellular Ca2+ and in both control and treated cells between 1 and 3 mM extracellular Ca2+ (+P < 0.01).