Loss of chondroitin sulfate proteoglycan sulfation allows delayed sympathetic reinnervation after cardiac ischemia–reperfusion

Figures

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  FIGURE 1. Sympathetic reinnervation of the cardiac scar 40 days after MI. Immunohistochemistry for tyrosine hydroxylase (cyan) identified sympathetic neurons in sections from hearts obtained 40 or 10 days after MI. (a) Example images from Veh‐treated hearts 40 days after MI, ISP‐treated hearts 40 days after MI, and Veh‐treated hearts 10 days after MI. (b) Quantification of TH+ innervation density expressed as % area, n = 6 40d + veh (3F, 3 M), n = 5 40d + ISP (3F, 2 M), and n = 3 10d (1F, 2 M); open symbols = female, closed symbols = male. Two‐way ANOVA, Tukey's post‐test to compare all groups, select comparisons shown, ns, not significant, ****p‐value<0.0001. Scale bar 100 μm. Inset scale bar 50 μm.

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  FIGURE 2. Early reinnervation does not alter cardiac function or infarct size 40 days after MI. Functional parameters were similar in vehicle (VEH)‐ and ISP‐treated mice 40 days after MI. (a) Cardiac output (C.O.), (b) stroke volume, (c) ejection fraction, and (d) fractional shortening. Data are Mean ± SD, n = 5 animals/group (VEH 2F, 3 M; ISP 3F, 2 M); open symbols = female, closed symbols = male; unpaired t‐test. (e) Infarct size is similar 40 days after MI in VEH‐ or ISP‐treated hearts. Mean ± SD, VEH n = 6 (3F, 3 M), ISP n = 5 (3F, 2 M); open symbols = female, closed symbols = male; unpaired t‐test. Sample image of LV with infarct outlined in a white line. Scale bar = 500 μm.

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  FIGURE 3. Arrhythmia susceptibility is similar 40 days after MI with vehicle or ISP treatment (a) Example PVC after administration of Isoproterenol + caffeine in a vehicle‐treated animal 40 days after MI. (b) Number of PVCs occurring after injection of Iso/caffeine. Data are mean ± SD, n = 5–6 (VEH 2F, 3 M; ISP 3F, 3 M); open symbols = female, closed symbols = male. Unpaired t‐test with Welch's correction; ns‐ not significant; p = 0.8. (c) Arrhythmia scores after injection of Iso/caffeine. Arrhythmia scores are based on the most severe arrhythmia observed in each heart (0 = no PVCs, 1 = single PVCs) See methods for additional details. n = 5–6 (VEH 2F, 3 M; ISP 3F, 3 M); open symbols = female, closed symbols = male; Mann–Whitney test; ns‐ not significant; p = 0.9.

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  FIGURE 4. Chondroitin 4,6‐sulfation is reduced to control levels 40 days after MI. Western blot quantification of (a) 4‐sulfation (4S CS‐GAGs), (b) 6‐sulfation (6S CS‐GAGs), and (c) NG2 core protein and in the cardiac scar 40 days after MI compared to unoperated LV tissue. For visual comparison, two samples in the blots are from D7 post‐MI when sulfation levels are high in the infarct; these were quantified but not used for statistical comparison due to sample size. n = 8 animals for D40 post‐MI vehicle treated (4F, 4 M) and n = 4 animals for unoperated left‐ventricle group (2F, 2 M); unpaired t‐test (Welch's correction), ns, not significant, *p < 0.05. (d) Western blot images of a–c.