Colitis induced ventricular alternans increases the risk for ventricular arrhythmia

Figures

• 

  Fig. 1.. Mouse model of DSS induced active colitis. A. Change in disease activity index (DAI) plotted over time of DSS exposure (3.5 % days 1–7) and remission (days 8–28) for mice treated with DSS (n = 72) and mice treated with DSS + ACE inhibitor (I(ACE): 3 mg/kg/day; days 5–10 ip; n = 15). B. Change in body weight of CTL mice (n = 6) and mice treated with DSS (n = 15) or DSS + I(ACE) (n = 15) until day of sacrifice. C. Colon length two days after the end of DSS treatment from CTL (n = 9), DSS (n = 15) and DSS + I(ACE) (n = 15). Data are presented as mean ± SD. Statistical analysis was conducted with one-way ANOVA on ranks and Dunn’s multiple comparisons test.

• 

  Fig. 2.. Active colitis induces ventricular electrophysiological remodeling. A. Representative ECG recording from CTL (black) and DSSA (red) mice 2 days after the end of DSS treatment. Scatter plots of B. HR, C. QT interval, and D. corrected QT interval (QTc) from CTL ( = 23 mice) and DSSA ( = 42 mice) ECGs show prolonged QT and QTc intervals during active colitis. Data are presented as mean ± SD and statistical analysis was performed with unpaired Student’s t-test.

• 

  Fig. 3.. Active colitis promotes the dispersion of ventricular repolarisation. A. Representative electrograms from a CTL (black) and DSSA (red) heart, recorded on a single MEA electrode. FPdur, a measure of APD is indicated in the upper trace. B. Representative contour plots of excitation spread in CTL (top) and DSSA (bottom) hearts. C. Spontaneous heart rate (HR) and ventricular D. FPdur, E. FPdisp, and F. conduction velocity (θ) quantified from Langendorff perfused hearts isolated from CTL (, n = 7) and DSSA (, n = 7) mice. Data are presented as mean ± SD and statistical analysis was performed with unpaired Student’s t-test.

• 

  Fig. 4.. Dispersion of ventricular repolarization does not depend on APD prolongation. A. Representative APs recorded at 1 Hz stimulation from CTL (top) and DSSA (bottom) isolated VMs. Scatter plots of B. AP amplitude, C. AP maximum upstroke velocity (Vmax), and D. AP duration at 50 % (APD50) and E. 70 % (APD70) repolarization from CTL ( = 20/8 cells/mice) and DSSA VMs ( = 20/9 cells/mice). Data are presented as mean ± SD and statistical analysis was performed with Mann-Whitney U test (D,E) or Student’s t-test (B,C).

• 

  Fig. 5.. Colitis increases the risk for ventricular alternans. A. Representative MEA recording from DSSA hearts with the red line indicating areas alternating out of phase. B. Magnification of electrogram recorded from the MEA electrode specified. C. Average FPdur of alternating (alt) and non-alternating (non-alt) electrodes recorded from DSSA ventricles (n = 4 hearts). D. Alternans ratio (AR) and E. discordance index of CTL (, n = 4 mice) and DSSAventricles (, n = 5 mice) plotted in dependence on pacing frequency. Data are presented as mean ± SEM and were analyzed using paired Student’s t-test (C) or mixed-effect ANOVA. A significant interaction between groups (CTL, DSSA) and pacing frequency was determined. (D: F(4,24) = 23.01, p < 0.0001; E. F(4,24) = 6.028, p = 0.0017).

• 

  Fig. 6.. Colitis increases the risk for CaT alternans. A. Percentage of CTL (black, 8 of 38 cells from 3 mice) and DSSA (red, 15 of 31 cells from 3 mice) myocytes with (, ) and without (,) pacing induced alternans. B. Representative (ΔF/F0) plots from CTL (black) and DSSA (red) myocytes exhibiting CaT alternans. C. Alternans ratio for CTL (: n = 38/3, cells/mice) and DSSA ( : n = 31/3, cells/mice) myocytes plotted against pacing frequency. Data are presented as mean ± SEM (C) and analyzed using either a two-proportion z-test (A) or mixed-effect ANOVA (C) where a significant interaction between groups (CTL, DSSA) and pacing frequency was determined, F(8, 412) = 5.150, p ≤0.001.

• 

  Fig. 7.. Colitis prolongs the CaT duration. Representative (ΔF/F0) plots from CTL (black) and DSSA (red) myocytes during A. field stimulation (1 Hz). Quantification of B. CaT amplitude and C. CaT decay constant (τCaT) from CTL (: 38/4 cells/mice) and DSSA ( : 43/6 cells/mice) VMs. D. Representative (ΔF/F0) plots from caffeine (10 mM) induced CaTs induced in CTL (black) and DSSA (red) VMs. Quantification of E. caffeine transient amplitude (CaTcaff) and F. caffeine transient decay constant (τcaff). Data presented as mean ± SD. Significance was determined by unpaired Mann-Whitney U test (B,E) or Student’s t-test (C,F).

• 

  Fig. 8.. SERCA activity is attenuated during active colitis. A. Representative Western blots of ventricular protein lysate from CTL (n = 4) and DSSA (n = 4) mice stained for PLB, pPLBSer16 and pPLBThr17 (top). Poinceau S stain of the same blot (bottom) was used for normalization to total protein levels. Quantification of B. PLB normalized to total protein and the ratio of C. pPLBSer16/PLB and D. pPLBThr17/PLB. E. ΔF/F0 plots showing CaT alternans in DSSA myocyte before and after Istaroxime (ISTAX) perfusion. F. ISTAX induced change in AR of DSSA VMs ( : n = 6/2 cells/mice). G. Quantification of AngII in serum of CTL (: n = 10) and DSSA ( : n = 5) mice. Data presented as mean ± SD. Statistical significance was determined by paired (F) or unpaired (G) Student’s t-test.

• 

  Fig. 9.. ACE inhibition normalizes colitis-induced risk for alternans. A. CaT amplitude and B. τCaT, from CTLI(ACE) (: 22/2 cells/mice) and DSSI(ACE) ( : 33/4 cells/mice) as well as C. fractional release of VMs from CTL (13/2 cells/mice), DSSA (12/2 cells/mice) and DSSI(ACE) (9/2 cells/mice). D. Percentage of CTLI(ACE) and DSSI(ACE) VMs with (closed) and without (open) pacing induced alternans. Quantification of MEA recordings for E. alternans ratio (AR) and F. discordance index of CTL (n = 4), DSSA (n = 5), and DSSI(ACE) (n = 4) ventricles plotted in dependence on pacing frequency. G. Representative Western blots of ventricular protein lysate from CTL (n = 4) and DSSI(ACE) (n = 4) hearts stained for PLB, p-PLBSer16 and p-PLBThr17 (top). Poinceau S stain of the same blot (bottom) was used for normalization to total protein levels. Quantification of H. PLB normalized to total protein and the ratio of I. p-PLBSer16/PLB and J. p-PLBThr17/PLB. Data presented as mean ± SD (A-C, H–I) or mean ± SEM (E,F). Significance was determined by Student’s t-test (A,B, H-J), one-way ANOVA and Tukey’s multiple comparisons test (C); mixed-effect ANOVA (E,F) where a significant interaction between groups…