Macrophage-Dependent Interleukin-6-Production and Inhibition of IK Contributes to Acquired QT Prolongation in Lipotoxic Guinea Pig Heart

Abstract

In the heart, the delayed rectifier K current, I_K, composed of the rapid (I_Kr) and slow (I_Ks) components contributes prominently to normal cardiac repolarization. In lipotoxicity, chronic elevation of pro-inflammatory cytokines may remodel I_K, elevating the risk for ventricular arrythmias and sudden cardiac death. We investigated whether and how the pro-inflammatory interleukin-6 altered I_K in the heart, using electrophysiology to evaluate changes in I_K in adult guinea pig ventricular myocytes. We found that palmitic acid (a potent inducer of lipotoxicity), induced a rapid (~24 h) and significant increase in IL-6 in RAW264.7 cells. PA-diet fed guinea pigs displayed a severely prolonged QT interval when compared to low-fat diet fed controls. Exposure to isoproterenol induced torsade de pointes, and ventricular fibrillation in lipotoxic guinea pigs. Pre-exposure to IL-6 with the soluble IL-6 receptor produced a profound depression of I_Kr and I_Ks densities, prolonged action potential duration, and impaired mitochondrial ATP production. Only with the inhibition of I_Kr did a proarrhythmic phenotype of I_Ks depression emerge, manifested as a further prolongation of action potential duration and QT interval. Our data offer unique mechanistic insights with implications for pathological QT interval in patients and vulnerability to fatal arrhythmias.

Keywords: QT prolongation; cytokines; guinea pig; inflammation; interleukin-6; lipotoxicity; ventricular myocytes.

Figure 1

Lipotoxicity induces cytokine secretion in RAW264.7 macrophage cells. (A), Schematics of experimental protocol: RAW264.7 macrophage cells were exposed to liptoxicity and different cytokines were assayed and analyzed 24 h after exposure. (B), Column graph represents enzyme-linked immunosorbent assay quantification (in pg/mL), of IL-6, IL-1β, IL-1α and INF-β in unstimulated and stimulated cells. Data points are mean ± S.E.M, n = 5–10 experiments. * Statistical significance at P < 0.05.

Figure 2

Lipotoxicity and IL-6 + IL-6R promotes arrhythmogenesis in guinea pig ventricular myocytes. (A), Cartoon representation of experimental protocol. Guinea pig ventricular myocytes were incubated with PA (1 mM) or IL-6 (20 ng/mL) + IL-6R (25 ng/mL) for 2 h. and ventricular electrical activity was assessed. (B), Exemplar superimposed traces (≥ 20 sequential APs) of ventricular action potentials recorded from isolated control or untreated myocytes. (C), Representative superimposed AP traces before (Black trace), and after incubation with PA (Red trace). (D), Sample APs depicting APD prolongation and beat-to-beat variability in IL-6+IL-6R-treated myocytes compared to untreated myocytes. (E), Averaged APD₉₀ measured in untreated myocytes, PA- and IL-6+IL-6R-treated cells. IL-6+IL-6R promotes arrhythmogenesis manifested as triggered EADs (F), and spontaneous beats (G). Data are presented as mean ± SEMs. (Scale bar: 40 mV × 200 ms). * Statistical significance at P < 0.05. Data were generated from cardiomyocytes from three different guinea pigs.

Figure 3

Lipotoxicity induces QT_c prolongation in guinea pig. (A), Experimental protocol used to assess the effect of lipotoxicity induced by BSA conjugated palmitic acid, injected in guinea pig cranial vena cava (CrVC). QT_c was measured at day 0 and progressively after 3, 7, 10 and 14 days. (B), Representative traces of surface ECG recorded at baseline and in lipotoxic (PA-BSA), and non-lipotoxic (BSA-alone) guinea pigs at day 7. (C), Plot showing progressive changes in QT_c relative to time computed from lipotoxic (●) and non-lipotoxic (◯), guinea pigs. Red horizontal lines indicate zero baseline. Compared to non-lipotoxic controls, lipotoxic guinea pigs displayed significant QT_c prolongation which peaked after seven days and remained sustained thereafter up until day 14. Data are presented as mean ± SEMs. (Scale bars: 0.5 mV × 200 ms). * Statistical significance at P < 0.05.

Figure 4

Lipotoxic adult guinea pigs show vulnerability to ventricular arrhythmogenesis. (A), Cartoon depiction of experimental protocol. Telemetered guinea pigs were exposed to lipotoxicity and altered cardiac rhythms were monitored for 24 h (B), Representative images of cardiac rhythms from adult guinea pigs exposed to ISO demonstrating premature ventricular contractions (PVCs). (C), Quantification of PVCs measured in non-lipotoxic, lipotoxic and lipotoxic +ISO adult guinea pigs. Lipotoxic guinea pigs in the absence and presence of ISO displayed significantly more PVCs than age-matched non-lipotoxic controls. TdP (D) and VF (E) in a free-moving lipotoxic guinea pig exposed to ISO. Data are presented as mean ± SEMs. Scale bar: 0.5 mV × 100 ms (B), 0.5 mV × 10 s (D,E). * Statistical significance at P < 0.05.

Figure 5

Short-term palmitic acid diet feeding prolongs QT_c interval in adult guinea pigs. (A), Experimental protocol. Adult guinea pigs were subjected to either PA-diet or low-fat feeding and changes in QT_c were assessed at baseline and after 10 days by conducting ECG measurements. Progressive body weight (B) and fasting blood glucose (C) changes relative to time measured in guinea pigs fed with either PA-diet (●) or LFD (◯). (D), Representative ECG traces measured in a PA-diet fed guinea pig at the beginning and after 10 days, highlighting QT_c compared to LFD-fed controls (E). Red horizontal lines indicate zero baseline. (F), Computed QT_c values measured in guinea pigs fed a PA-diet or LFD. Data are presented as mean ± SEMs. Scale bar: 0.5 mV × 400 ms. * Statistical significance at P < 0.05.

Figure 6

IL-6 inhibits native I_Kr and I_Ks currents in guinea pig ventricular myocytes. (A), Experimental protocol: Macroscopic I_Kr and I_Ks currents were measured in freshly isolated ventricular myocytes from adult guinea-pig heart under control (or untreated) conditions and after pre-exposure to IL-6 or IL-6 + IL-6R for 2 h. Exemplar I_Kr tail current traces measured in the absence (B), and presence (C), of IL-6 (20 ng/mL) + IL-6R (25 ng/mL). (D), Population I-V curves for I_Kr tail currents. Representative I_Ks current traces measured in the absence (E), and presence of IL-6 alone (F), or IL-6 + IL-6R (G). (H), Population current density-voltage curves for I_Ks measured in basal, IL-6- and IL-6 + IL-6R-treated adult guinea-pig ventricular cardiomyocytes. Insets: voltage protocols used for evoking I_Kr and I_Ks currents. Data are presented as mean ± SEMs. (Scale bar: 0.5pA/pF × 500 ms and 10pA/pF × 1 s for I_Kr and I_Ks currents respectively). * Statistical significance at P < 0.05.

Figure 7

Functional outcome of IL-6-dependent inhibition of I_Kr and I_Ks in simulated electrical activity of human ventricular myocytes. (A), Cartoon illustration summarizing the profound inhibition by IL-6 or IL-6 + IL-6R of I_Kr and I_Ks densities, and predicted implications for QT_c prolongation and arrhythmogenesis in patients using in silico computer modeling. (B), Comparison of AP waveforms in the strand and pseudo-ECG (C), calculation in basal conditions, and in the presence of IL-6 and IL-6 + IL-6R. The stimulation begins on the left side of the strand (1st cell in the endocardium) and the pseudo-ECG is calculated at a virtual electrode located at 2 cm from the last cell in the epicardium. The strand is depicted in yellow, while the blue squares over the strand denote cells selected to for AP waveforms.

Figure 8

IL-6 and IL-6R leads to impaired mitochondrial bioenergetics in human-induced pluripotent stem cell derived cardiomyocytes. (A), Human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) are exposed to IL-6 alone or IL-6 + IL-6R and mitochondrial stress parameters were analyzed after 2 h (B), IL-6- or IL-R + IL-6R-treated hiPSC-CMs displayed severely depressed oxygen consumption rate (or OCR), compared to untreated controls, indicating impaired metabolism. (C), hiPSC-CMs pre-exposed to IL-6 and IL-6 + IL-6R displayed statistically significant depression in ATP production, basal respiration, and maximal respiration when compared to untreated controls. Nonmitochondrial oxygen consumption respiration and spare respiratory capacity were also severely blunted. Proton leak remained essentially unchanged. Data are presented as mean ± SEMs. * Results with P < 0.05 were considered statistically significant.