5-HTT Deficiency in Male Mice Affects Healing and Behavior after Myocardial Infarction

Figure 1. Experimental timeline of the long-term study. MI, myocardial infarction; Echo, echocardiography; SPT, sucrose preference test; EPM, elevated plus maze; OFT, open field test; DLB, dark-light box; SIT, social interaction test.

Figure 2. Age-dependent development cardiac dysfunction in 5-HTT deficient (−/−) mice. (A,B) Echocardiographic assessment of left ventricular dimensions and function revealed a significant increase in end-systolic area (A) and decline in fractional area change (B) in 4–6-month-old 5-HTT−/− mice relative to age-matched controls and 2–3-month-old 5-HTT−/− mice (n = 8–18 per age group and genotype). (C–E) Hemodynamic measurements from 7-month-old mice demonstrated slightly diminished cardiac output (C), increased pressure at the peak rate of left ventricular filling (D) and decreased peak systolic ejection rate (E) in 5-HTT−/− mice as compared to 5-HTT+/+ littermates (n = 5–6/genotype). (F) Myocardial collagen content did not differ between 5-Htt genotypes (n = 6/genotype). Data are shown as mean ± standard error of the mean (SEM). # p < 0.1, * p < 0.05, ** p < 0.01 and *** p < 0.001; two-way analysis of variance (ANOVA) followed by Sidak multiple comparisons test (A,B) or one-way ANOVA with Tukey post hoc test (C–F).

Figure 3. Survival of 5-HTT−/− mice is severely impaired after MI. (A,B) Kaplan–Meier survival curves of (A) all mice subjected to MI surgery show no statistically significant difference between 5-Htt genotypes, but (B) among those animals with large infarcts survival is severely impaired in 5-HTT−/− mice. (C) Histological quantification of the infarct size (fraction of the infarcted left ventricle) 8 weeks after surgery showed that none of the surviving 5-HTT−/− mice had a large (>30%) MI. Dashed line indicates the cut-off value of 30% for stratification into groups with small (<30%) and large (>30%) MI size. Values are shown as mean ± SEM. (A,B) log-rank test, (C) Welch’s ANOVA with Games–Howell post hoc test. * p < 0.05, ** p < 0.01.

Figure 4. Development of left ventricular failure and hypertrophy after MI in mice. (A–C) Assessment of left ventricular dimensions and function by serial echocardiography on postoperative days 1, 21 and 56. Irrespective of 5-Htt genotype, MI mice exhibited a time- and infarct size-dependent increase in end-systolic area (A) and end-diastolic area (B) and a persistent reduction in fractional area change (C) as compared to sham controls, indicating progressive LV dilatation and dysfunction due to heart failure. (D) Analysis of left ventricle-to-body weight (LV/BW) ratio as an index of cardiac hypertrophy revealed significant enlargement in MI > 30% mice relative to MI < 30% and sham animals at 8 weeks post-op. Data are shown as mean ± SEM. * p < 0.05 and *** p < 0.001 for comparisons between groups; + p < 0.05 and +++ p < 0.001 compared to day 1; (A–C) three-way mixed ANOVA and (D) ordinary two-way ANOVA followed by Sidak or Tukey multiple comparisons test.

Figure 5. Body weight, liquid intake and sucrose preference before and after surgery. Presurgical body weights were comparable among genotypes (A). Postoperatively, sham-operated 5-HTT−/− mice gained more weight than respective controls and mice with large infarcts (MI > 30%) exhibited greater weight loss than sham and MI < 30% mice in the first week post-op (B). 5-HTT null mutants drank less than the other two genotypes both before (C) and after surgery (D), and liquid intake was significantly diminished in MI > 30% mice at week 1 post-op (D). Sucrose preference was comparable among genotypes at baseline (E) and did not significantly change postoperatively, neither across genotypes nor between sham vs. MI mice (F). Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. (A,C,E) one-way ANOVA followed Tukey post hoc test; (B,D,F) three-way mixed ANOVA followed by Sidak multiple comparisons test.

Figure 6. Distinct behavioral phenotype of 5-HTT deficient mice in multiple approach-avoidance conflict tests. (A–C) During the 30-min open field test (OFT), 5-HTT−/− traveled significantly shorter distances than the other two 5-Htt genotypes in the entire arena (A) and in the center (B). Furthermore, MI < 30% were more active than sham animals during the first 2 min of the OFT (A) and covered cumulatively longer distances in the center of the arena as compared to sham controls (B). Interestingly, MI > 30% deposited less fecal pellets in the OF than MI < 30% and sham animals (C). (D,E) In the dark-light box (DLB), half of the 5-HTT−/− mice refrained from exploring the bright section (D). Furthermore, 5-HTT−/− mice covered shorter distances (E) and spent cumulatively less time in the light chamber (F) as compared to 5-HTT+/− and 5-HTT+/+ mice. Additionally, 5-HTT+/− mice with small infarcts (MI < 30%) spent significantly more time in the light compartment of the DLB than 5-HTT+/− mice of the other two groups as well as 5-HTT+/+ and 5-HTT−/− mice with MI < 30% (F). (G–I) In the social interaction test (SIT), only 3 out of 8 5-HTT−/− mice explored the unfamiliar stimulus mouse at least once…

Figure 7. Infarct size quantification, electrocardiographic recordings, FACS and gene expression analysis in infarcted mice at day 3 post-op. (A) Evans blue/TTC staining revealed a significantly increased infarct-to-area at risk (INF/AAR) ratio in 5-HTT+/− compared to 5-HTT+/+ and 5-HTT−/− mice. The fraction of AAR and INF relative to left ventricle (LV) was comparable among 5-Htt genotypes. (B) Postoperative electrocardiography (ECG) recordings showed significantly smaller R-, S- and T-wave amplitudes in 5-HTT−/− mice as compared to 5-HTT+/+ littermates. (C) Evaluation of immune cell subsets in the infarct area by FACS analysis revealed no differences between 5-Htt genotypes. (D) Gene expression analysis in the infarcted myocardium revealed significant upregulation of transforming growth factor β (TGF- β), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and matrix metalloproteinase 2 (MMP2) in 5-HTT−/− relative to 5-HTT+/+ mice. Furthermore, matrix metalloproteinase 3 (MMP-3) was downregulated and matrix metalloproteinase 13 (MMP-13) upregulated in 5-HTT+/− mice as compared to 5-HTT+/+ and 5-HTT−/− mice, respectively. Data are shown as mean ± SEM. # p < 0.1, * p < 0.05 and ** p <…