Mice carrying the STAT1-F77A mutant are protected from adverse cardiac

In the acute phase after myocardial infarction, immune cells are recruited to ischemic areas of the heart during a first inflammatory phase (reviewed in Bonaventura et al., 2016). This inflammatory reaction begins within the first hours after the insult and is resolved after a few days. It has been noted that the inflammatory environment extends beyond the ischemic tissue and may cause additional damage in the border zone. Shortly after the invasion of the tissue

76

by inflammatory cells, anti-inflammatory signalling starts to facilitate clearing of the inflammation. Following survival of myocardial infarction, patients often suffer from heart failure due to post-infarction left ventricular remodelling (reviewed in Galli and Lombardi, 2016). During this process, fibrotic scar formation of the previously hypoxic tissue is accompanied by an alteration in matrix composition as well as the absence of contracting cardiomyocytes in this area. As a consequence, the left ventricle is often dilated resulting in a permanently reduced ejection fraction.

Figure 22: Increase in left ventricular diameter after myocardial infarction. The diameter of the left ventricular lumen was measured in diastole (A) and systole (B) in wildtype and STAT1-F77A-expressing mice using echocardiographic images obtained in B mode. For all mice analysed, pre-infarction data were acquired. Mice were analysed either during the acute phase (analysis on day 3 after infarction) or kept for monitoring cardiac remodelling (analysis 1 week and 4 weeks after infarction). Shown are only the data acquired from female mice. The numbers of mice included in this experiment is indicated inside the columns. Asterisks indicate statistically significant differences.

Error bars represent standard error of the mean.

In order to evaluate the adaption of the cardiac tissue in the absence of IFNγ signalling, echocardiography was performed on mice, three days, one week and four weeks after ligation of the left anterior descending coronary artery and compared with the measurement obtained before surgery (mice of the day-3 cohort were analysed before and 3 days after surgery, mice of the 4-week cohort were analysed before and 1 week after and 4 weeks after surgery). Based on anatomical measurements of left ventricular dimensions, functional data were computed.

77

Figure 23: Increased left ventricular sectional area due to myocardial infarction. The cross-sectional area of the left ventricular lumen and the epicardial area were measured using echocardiographic images obtained in B mode. (A) Left ventricular cross-sectional area of the lumen in diastole. (B) Measurements obtained in systole. (C) End-systolic epicardial cross-sectional area.

For all mice analysed, pre-infarction data were acquired. Mice were analysed either during the acute phase (analysis on day 3 after infarction) or kept for monitoring cardiac remodelling (analysis 1 week and 4 weeks after infarction). Shown are only the data acquired from female animals. The numbers of mice included are indicated inside the columns. Asterisks indicate statistically significant differences.

Error bars represent standard error of the mean.

Analysis of the entire cohort showed no differences in cardiac remodelling, shape or performance between the wildtype and the STAT1-F77A knockin mouse. Variance of all measurements was high. Separation of the data obtained from male and female mice and separate analysis of these produced a drop in variance. In females carrying the STAT1-F77A knockin allele, the increase in both diastolic and systolic left ventricular diameter was attenuated during short-term follow-up (Figure 22). Similar differences between female wildtype and knockin littermates were detected for the area of luminal and epicardial

cross-78

sections 3 days and 1 week following ligation of the coronary artery (Figure 23). In male mice, no difference was found among the genotypes (data not shown).

Figure 24: Increase in diastolic and systolic volume after myocardial infarction. The end-systolic volume (A) and end-diastolic volume (B) were calculated from the echocardiographic data obtained in B mode. For all mice analysed, pre-infarction data were acquired. Mice were analysed either during the acute phase on day 3 after infarction or kept for monitoring cardiac remodelling at 1 week and 4 weeks following myocardial infarction. Shown are only the data acquired from female mice. Numbers of mice included are indicated inside the columns. Asterisks indicate statistically significant differences. Error bars represent standard error of the mean.

Based on the anatomical measurements of left ventricular dimensions, end-diastolic and end-systolic volumes were computed (Figure 24). Following myocardial infarction, the left ventricle started to dilate massively, almost tripling its original volume by week 4 in the wildtype animals. In line with the above-mentioned data, enlargement of the left ventricle was reduced in the F77A knockin mice during the early phase on day 3 and week 1 and continued as a – however non-significant – trend up to week 4. Therefore, lack in IFNγ-responsiveness significantly ameliorated cardiac remodelling in females.

79

Figure 25: The reduced cardiac function after myocardial infarction is blunted in mice with impaired STAT1 tetramer stabilization. Fractional area shortening (A), fractional shortening (B) and ejection fraction (C) were computed from the echocardiographic data obtained in B mode. For all mice analysed, pre-infarction data were acquired. Mice were analysed either during the acute phase on day 3 after infarction or kept for monitoring cardiac remodelling on week 1 and week 4 after coronary ligation. Shown are only the data acquired from female mice. The numbers of mice included are indicated inside the columns. Asterisks indicate statistically significant differences. Error bars represent standard error of the mean.

In order to test the significance of these findings, fractional area shortening, fractional shortening and the ejection fraction were calculated (Figure 25). In response to the insult, cardiac function was markedly reduced as indicated by a pronounced and immediate drop in all three measurements. In STAT1-F77A female mice, cardiac function was ameliorated in comparison with the wildtype, 1 week after MI surgery. This trend was seen on a non-significant level on day 3 and week 4 after experimental infarction. Again, no non-significant differences were observed from the analysis of male mice. The lack of significant differences among the male cohorts might be attributed to the poor overall survival rates, as described above. Since only a few of the male mice survived, variance was high especially in the

80

wildtype group, suggesting that animals with the highest impact of the infarction were not included in our analysis. Overall, the data revealed that the IFNγ-irresponsive STAT1-F77A mutant mouse line displayed ameliorated cardiac dilation and blunted remodelling resulting in better cardiac performance. This finding suggested that the absence of a functional IFNγ/STAT1-dependent signal response was not only protective due to its direct non-transcriptional effect on apoptosis and autophagy (McCormick et al. 2012) but also due to other transcriptionally related mechanisms.

3.3.4 STAT1 expression is reduced in hearts of STAT1-F77A knockin mice compared