Consistent with the depressed ΔSS, cardiomyocytes isolated from male WT mice after CLP also showed depressed sarcomere velocity (p<0.001) and sarcomere return velocity (p<0.001, Supplemental Figure 2A-B)

1 Supplemental Results

1. Echocardiography examination (Supplemental Figure 1)

We assessed cardiac contractility in vivo, by echocardiography (Supplemental Figure 1A). In male WT mice, left ventricular (LV) ejection fraction was unchanged from baseline at 24 and 48h after CLP (Supplemental Figure 1B). LV internal diameter (Supplemental Figure 1C) was also unchanged from baseline after CLP, indicating that our empirical resuscitation protocol was effective in preventing hypovolemia. The total wall thickness of the LV (Supplemental Figure 1D) was also unchanged after CLP, indicating the absence of myocardial edema. CLP induced a decrease in heart rate, which was evident at the time of the echocardiography assessment (Supplemental Figure 1E).

2. Cardiomyocyte studies in male mice (Supplemental Figure 2).

To identify the cellular mechanisms responsible for the development of cardiomyopathy after CLP, we studied cardiomyocytes isolated from male WT mice at baseline, 24h after sham operations and 24h after CLP (Figure 2A). As shown in the main text (Figure 2), in male WT mice, CLP induced a depression in cardiomyocytes ΔSS, ΔCai and τCa prolongation. Consistent with the depressed ΔSS, cardiomyocytes isolated from male WT mice after CLP also showed depressed sarcomere velocity (p<0.001) and sarcomere return velocity (p<0.001, Supplemental Figure 2A-B). The time to peak of sarcomere shortening and diastolic sarcomere length were unchanged from baseline (Supplemental Figure 2C-D). ΔCai/dt (p<0.001) was also depressed after CLP, whereas Cai transients time to peak and diastolic Ca²⁺ levels were unchanged (Supplemental Figure 2E-G).

Sham operations induced no change in any of the cardiomyocyte parameters measured (Figure 2 and Supplemental Figure 2).

2

To investigate the role of redox stress, we compared male WT and CAT mice. At baseline, cardiomyocyte contractile parameters were similar in male CAT and WT mice (Figure 2 and Supplemental Figure 2), including similar ΔSS, ΔCai and τCa (Figure 2), similar sarcomere velocity, sarcomere return velocity, sarcomere shortening time to peak and diastolic sarcomere length, ΔCai/dt, Cai transient time to peak and diastolic Ca²⁺ levels (Supplemental Figure 2 A- G).

Importantly, cardiomyocytes isolated from CAT male mice showed less severe contractile dysfunction after CLP, as compared to male WT mice. As shown in the main text (Figure 2), the depression of ΔSS and ΔCai after CLP was 55 - 50% less severe in male CAT mice, as compared to male WT mice. Consistently, sarcomere velocity and sarcomere return velocity showed only a non-significant trend towards decrease after CLP in CAT mice

(Supplemental Figure 2A-B). The time to peak of sarcomere shortening and diastolic sarcomere length were unchanged after CLP in CAT mice, similar to the findings in WT male mice

(Supplemental Figure 2C-D). ΔCaⁱ/dt, Cai transients time to peak and diastolic Ca²⁺ levels were also unchanged after CLP in male CAT mice (Supplemental Figure 2E-G).

A direct comparison of male CAT vs. WT mice after CLP revealed that CAT mice showed larger ΔSS (p<0.001) and ΔCaⁱ (p<0.001, Figure 2A-B), larger sarcomere velocity (p<0.001), return velocity (p<0.001) and ΔCaⁱ/dt (p<0.001, Supplemental Figure 2A, B, E). This indicated that male CAT mice were partially protected against the dysregulation of

cardiomyocyte Ca²⁺ handling after CLP, suggesting that this dysregulation is partially the result of increased redox signaling.

4. Cardiomyocyte studies in female mice.

To assess the possible sex-dependence of the mechanisms revealed, we next studied cardiomyocytes isolated from female WT mice, as compared to male WT mice (Figures 3-4).

3

As detailed in the main text, in female WT mice, CLP did not induce a change in ΔSS, ΔCai or τCa (Figure 2). Consistently, sarcomere velocity, sarcomere return velocity, the time to peak of sarcomere shortening, diastolic sarcomere length, ΔCai/dt, the time to peak of the Cai

transients and diastolic Ca²⁺ levels were also unchanged after CLP in WT female mice, as compared to baseline (Supplemental Figure 2A-G).

Interestingly, at baseline, cardiomyocytes isolated from female FVB mice had smaller ΔSS, ΔCai and prolonged τCa, as compared to male WT mice (Figure 2). Consistently,

sarcomere velocity (p<0.001), sarcomere return velocity (p<0.001) and ΔCaⁱ/dt (p<0.001) were also less in female vs. male WT mice (Supplemental Figure 2A, B, E). Sarcomere time to peak was prolonged in female vs. male mice (p<0.001, Supplemental Figure 2C), whereas the diastolic sarcomere length, Cai transients time to peak, and diastolic Ca²⁺ levels were similar in female and male WT mice (Supplemental Figure 2D, F, G).

At baseline, compared to female WT mice, female CAT mice showed similar ΔSS but higher ΔCai and shorter τCa (Figure 2). Consistently, sarcomere velocity and return velocity (Supplemental Figure A-B) were also similar in female WT and CAT mice. ΔCaⁱ/dt showed a trend towards higher values in female CAT mice vs. female WT (p=0.054, Supplemental Figure 2E), whereas sarcomere time to peak, diastolic sarcomere length, Cai transients time to peak and diastolic Ca²⁺ levels were similar in female CAT vs. female WT mice (Supplemental Figure 2C-D, F-G).

4 Supplemental Figure Legends

Supplemental Figure 1. Echocardiography examination.

The echocardiography study was performed in a cohort of 6 mice, studied at baseline and at 24 and 48h after CLP (25 ga needle). A. Representative echocardiography stills illustrating the main parameters measured. LV anterior wall is shown on top, and the posterior wall on the bottom. The anterior wall thickness (AW), posterior wall thickness (PW) and LV internal diameter (ID) were measured in systole (AWS, PWS, and IDS) and diastole (AWD, PWD, and IDD), respectively. Panels show LV EF (B), LV internal diameter in diastole (IDD, C), total wall thickness (TWT, D) and heart rate (E).

Supplemental Figure 2. Cardiomyocyte contractile parameters

Average sarcomere velocity (A), sarcomere return velocity (B), sarcomere shortening time to peak (C), diastolic sarcomere length (D), ΔCai/dt (E), Cai transients time to peak (F) and diastolic Ca²⁺ levels (G) in the 8 groups, as shown. N= 68 cells from 5 mice (68/5) for male WT mice at baseline, 25/3 in male WT sham and 47/3 for male WT mice after CLP. N = 47/4 for male CAT mice at baseline and 49/4 after CLP. N = 34/4 for female WT BL, 40/4 for female WT after CLP, and 27/4 female CAT mice baseline. Data is shown as mean ± SEM. *: p<0.05 for CLP vs. baseline. #: p<0.05 for CAT vs. WT, &: p>0.05 for male vs. female.

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