We used two different approaches to analyze cardiomyocyte data. The first approach employed a two-way ANOVA test followed by a one-sided, unpaired Student t test, with a Bonferroni correction for multiple comparisons (GraphPad Prism 6.00 for Windows). For this analysis, we considered each cell to be an independent observation (n = number of cells). Because this assumption was not unequivocally confirmed by an Interclass Correlation Coefficient test in each group studied, we used a second statistical method that applied a random intercepts model with post hoc tests using Tukey multiple comparisons (R Studio statistical software). The main conclusions of the study were the same with either approach. The statistical values shown in the figures were obtained using the Tukey test.
Echocardiography experiments were performed before and after LPS challenge in the same mice, using a paired approach. Six to ten echocardiography stills were recorded and measurements averaged for each heart. Groups were compared using two-way ANOVA, followed by a paired, one-sided t test, with a Bonferoni correction (GraphPad Prism 6.00 for Windows).
Survival analysis in Figure 5 was performed using a Mantel–Cox test (GraphPad Prism 6.00 for Windows).
The main goal of this study was to identify and compare the cellular mechanisms responsible for LPS-induced cardiomyopathy in male versus female C57Bl/6J mice. For this, we isolated LV cardiomyocytes from male and female mice, at baseline and 14 ± 2 h after LPS challenge. We measured sarcomere shortening and ΔCai in cardiomyocytes externally paced at 5 Hz and 37°C. Figure 1A shows typical experimental recordings from cardiomyocytes isolated from male and female mice, at baseline and after LPS challenge.
Sarcomere shortening is depressed after LPS challenge in both male and female WT mice
At baseline, cardiomyocytes isolated from female mice showed sarcomere shortening (Fig. 1B), Departure Velocity (Fig. 1C), and Return Velocity (Fig. 1D) that were not significantly different than in male mice. Sarcomere shortening Time-To-Peak was also similar in male and female mice (Fig. 1E) at baseline, whereas diastolic sarcomere length was slightly larger in female versus male mice (Fig. 1F).
Consistent with our previous findings (5), in male mice, LPS challenge induced a profound depression of cardiomyocyte sarcomere shortening (Fig. 1B), Departure Velocity (Fig. 1C), and Return Velocity (Fig. 1D). The Time-To-Peak of sarcomere shortening was not changed (Fig. 1E), and neither was diastolic sarcomere length (Fig. 1F).
In female WT mice, LPS challenge decreased sarcomere shortening (Fig. 1B) and Departure velocity (Fig. 1C), to a level not significantly different than in male mice. LPS also decreased Return Velocity, with borderline significance (P = 0.10, Fig. 1D). There was no change in sarcomere shortening Time-To-Peak (Fig. 1E) or diastolic sarcomere length after LPS challenge in female mice (Fig. 1F).
LPS depresses ΔCai and SERCA in WT male, but not in female WT mice
At baseline, ΔCai (Fig. 2A), ΔCai/dt (Fig. 2B), τCa (Fig. 2C), Ca2+ transient Time-To-Peak (Fig. 2D), and diastolic Ca2+ level (Fig. 2E) were similar in male and female mice. In male mice, LPS administration induced a depression of ΔCai (Fig. 2A), and ΔCai/dt (Fig. 2B). τCa was prolonged after LPS (Fig. 2C), indicating that SERCA function is inhibited, consistent with our previous findings (5). Ca2+ transient Time-To-Peak was prolonged after LPS (Fig. 6D), while diastolic Ca2+ level was unchanged (Fig. 2E).
Importantly, in female mice, LPS challenge did not induce a significant decrease in ΔCai and ΔCai/dt, as it did in male mice (Fig. 2A). As a result, ΔCai and ΔCai/dt were significantly greater in WT female versus WT male mice after LPS challenge (Fig. 2A). τCa was not changed, indicating that SERCA function was intact in female WT mice, as opposed to male mice (Fig. 2C). Ca2+ transient Time-To-Peak and diastolic Ca2+ level were unchanged after LPS (Fig. 2, D and E).
In female mice, sGCα1 deficiency protects against the depression of cardiomyocytes sarcomere shortening after LPS
Figure 3A shows typical sarcomere shortening and Ca2+ transients of cardiomyocytes isolated from WT and sGCα1−/− female mice, at baseline and after LPS challenge. At baseline, sarcomere shortening (Fig. 3B), Departure Velocity (Fig. 3C), Return Velocity (Fig. 3D) and sarcomere shortening Time-To-Peak (Fig. 3E), and diastolic sarcomere length (Fig. 3F) were similar in female sGCα1−/− and WT mice.
After LPS, sarcomere shortening (Fig. 3B). Departure Velocity (Fig. 3C), and Return Velocity (Fig. 3D) were not significantly decreased in female sGCα1−/− mice (although a nonsignificant trend toward a decrease was evident). Sarcomere shortening Time-To-Peak and diastolic sarcomere length were unchanged after LPS in sGCα1−/− female mice (Fig. 3, E and F).
In female sGCα1−/− mice, LPS challenge does not change ΔCai
At baseline, ΔCai was not significantly different in sGCα1−/− and WT female mice at both baseline and after LPS (although a trend toward larger values in sGCα1−/− mice was evident, with borderline significance, P = 0.06, Fig. 4, A and B). τCa was similar, indicating that SERCA function is not changed (Fig. 4C). Diastolic Ca2+ levels and Ca2+ transient Time-To-Peak were also unchanged (Fig. 4, D and E).
LPS challenge did not induce any change in ΔCai, ΔCai/dt, τCa, diastolic Ca2+ levels or Ca2+ transient Time-To-Peak in sGCα1−/− female mice (Fig. 4, A–E). This was identical to the findings in WT female mice. There was a borderline trend (P = 0.09) toward an increased ΔCai in LPS-challenged sGCα1−/− versus WT female mice (Fig. 4A).
LPS depresses LV EF to a similar degree in WT male and female mice
Next, we studied cardiac performance by echocardiography. We compared mice at baseline and 14 ± 2 h after challenge with 25 μg/g LPS (same protocol as for cardiomyocyte studies). To preserve euvolemia, all LPS-challenged mice received a bolus of warm Normal Saline (0.5 mL, ip) 20 min before the beginning of the echocardiography study. Figure 5 illustrates typical echocardiography stills recorded in male and female, WT and sGCα1−/− mice, at baseline and after LPS challenge.
LV EF (Fig. 6A) was similar at baseline in male and female mice of both genotypes. After LPS administration, LVEF was decreased in both sexes and genotypes, signifying the development of cardiomyopathy. LVEF decrease after LPS was similar in WT male, WT female, and sGCα1−/− male mice, but was less pronounced in sGCα1−/− female mice. The partial protection of sGCα1−/− female mice to LPS was consistent with the less pronounced decrease in cardiomyocyte sarcomere shortening (Fig. 3).
LV TWT (Fig. 6B) was similar at baseline in male and female mice, either WT or sGCα1−/−. LPS challenge did not change TWT in either groups. LV ID (Fig. 6C) was similar at baseline in the four groups. LPS induced a slight decrease in LV ID in male WT mice (and a similar trend in female WT mice), signifying the persistence of a slight degree of hypovolemia after LPS challenge, despite ip resuscitation. LV ID was not changed after LPS challenge in sGCα1−/− mice, either male or female.
At baseline, heart rate (Fig. 6D) was similar in the four groups. LPS challenge was associated with a slight decrease in heart rate, in both male and female WT mice and in sGCα1−/− female mice (with a similar trend in sGCα1−/− male mice).
In female mice, sGCα1 deficiency confers protection against LPS-induced mortality
Next, we aimed to determine whether the differences in cardiomyocyte function and cardiac EF identified above play a role in determining mouse mortality after LPS challenge. For this, we used a range of LPS doses between 4 μg/g and 100 μg/g body weight.
In WT male mice (Fig. 7A), mortality increased with increasing doses of LPS, from zero mortality after a dose of 4 μg/g LPS to 100% mortality after a dose of 20 μg/g LPS.
In WT female mice (Fig. 7B), mortality was zero after challenge with 7 or 10 μg/g LPS, which was significantly less than in WT males. Challenge with 20 μg/g LPS, however, induced 100% mortality, similar to WT males. Female protection against LPS-induced mortality was, therefore, manifested only at lower doses (that induce a mild disease state), but not at higher doses.
In male sGCα1−/− mice (Fig. 7C), LPS-induced mortality was not significantly different than in WT males, although a trend toward a worsening was seen for 4 μg/g LPS (P = 0.070).
Female sGCα1−/− mice (Fig. 7D) showed an improved survival after LPS challenge, as compared with WT females (most clearly seen for a dose of 20 μg/g) and sGCα1−/− males (for doses between 7 and 20 μg/g). The improved survival of female sGCα1−/− mice, compared with female WT or male sGCα1−/− mice, was consistent with the less severe depression in LV EF (Figs. 5 and 6) and cardiomyocyte sarcomere shortening (Fig. 3) found above.
This is the first head-to-head comparison of female versus male C57Bl/6J mice from the point of view of cardiomyocyte Ca2+ handling, at baseline and after challenge with LPS. Extending our previous studies in male mice (5, 9), we found that LPS depresses cardiomyocyte sarcomere shortening in female mice also, to a level comparable with that observed in male mice. Importantly, cardiomyocyte dysfunction was the result of different cellular mechanisms in male and female mice. In male mice, LPS acts by inhibiting SERCA function, which contributes to a decrease in ΔCai(5, 9). In contrast, in female mice, ΔCai and SERCA were not changed after LPS, and the depression of cardiomyocyte sarcomere shortening was due to mechanisms downstream of the Ca2+ transient, possibly represented by an impairment of the myofilaments function. Upstream of Ca2+ handling, sGCα1-synthesized cGMP plays opposite roles in male and female mice. We have previously found that, in male mice, cGMP plays protective roles (5, 9), by opposing SERCA redox-dependent inhibition (5). On the contrary, in female mice, cGMP plays a pathological role, by mediating the development of contractile dysfunction.
LPS depressed cardiomyocytes sarcomere shortening in male and female WT mice
At baseline, cardiomyocytes isolated from male and female mice had similar sarcomere shortening, ΔCai and τCa. LPS challenge depressed sarcomere shortening in both male and female mice, to comparable levels. However, the underlying mechanisms underlying sarcomere shortening depression were different in male and female mice (see below).
Distinct Ca2+ handling abnormalities underlie the development of LPS-induced cardiomyopathy in male and female mice
In male mice, sarcomere shortening depression induced by LPS was associated with a profound decrease in ΔCai and SERCA inhibition. These results were consistent with our previous findings (5) that, in male C57Bl/6 mice, LPS induced SERCA dysregulation, as a results oxidative modifications (sulphonylation) of one reactive Cys residue (Cys674-SO3H). Downstream of the Ca2+ transient, we have previously shown that there were no changes in myofilament function after LPS, and the decrease in sarcomere shortening occurred proportionally to the decrease in ΔCai(5).
In contrast, in female mice, the depression of cardiomyocytes sarcomere shortening after LPS occurred in the absence of changes in ΔCai and SERCA. This indicated the presence of deficits in mechanisms downstream of the Cai transients, whose exact nature remains to be determined. The most likely hypothesis is a decrease in myofilament sensitivity for Ca2+, following troponin I hyperphosphoration, as previously demonstrated in male mice after challenge with lower doses (6 μg/g) of LPS (4). Alternative mechanisms include a decrease in myofilament maximal tension (13) or an increase in the passive resistance of the cell, such as internal cell viscosity (mainly determined by the density of microtubules) (14) or the myofilament (titin) elastic resistance (15).
Differential roles for cGMP in male and female hearts
A second important observation was that sGCα1 deficiency modulated cardiomyocyte response to LPS differently in male and female mice.
In male mice, we have previously reported that sGCα1 deficiency is associated with increased SERCA Cys674-SO3H formation and SERCA inhibition, and a more profound decrease in ΔCai and sarcomere shortening after LPS challenge (5, 9). This indicated that, in male mice, cGMP generated by sGC plays a protective, antioxidant role (5) after LPS challenge.
In the current study, in female mice, sGCα1 deficiency was associated with improved survival rates, less severe LVEF depression, and less severe depression of cardiomyocyte sarcomere shortening, as compared with WT female mice. This indicated, therefore, that in female mice, the depression of cardiomyocyte sarcomere shortening after LPS is partially mediated by the release of cGMP from sGCα1. Mechanistically, one possible pathway involved could be, for example, the inhibition of the enzyme phosphodiesterase 3 (PDE3) by cGMP (16), which would lead to an increase in cAMP levels and subsequent phosphokinase A-dependent phosphorylation of TnI. TnI hyperphosphorylation has been suggested as the causative factor leading to the decrease in myofilament sensitivity for Ca2+ in some models of sepsis-induced cardiomyopathy (4). Whether this is indeed the causative pathway in LPS-challenged female mice remains to be determined.
Female sGCα1−/− mice have increased survival rates, and less severe depression of LVEF than WT female or male WT and sGCα1−/− mice
In WT mice, female mice had preserved survival rates after challenge with low doses of LPS (7–10 μg/g). This is in accord with a previously published observation that challenge with 4 μg/g LPS induces a less severe depression of LVEF in female versus male mice (17) and with the general belief that female sex confers a relative protection against sepsis (18). However, challenge with higher doses of LPS (15–20 μg/g) induced similar mortality rates in male and female mice, associated with a similar decrease in LVEF. It seems likely, therefore, that the relative protection conferred by female sex is limited to less severe forms of disease.
Importantly, sGCα1−/− female mice showed better survival and a less pronounced decrease in LV EF than WT female and sGCα1−/− male mice. This was consistent with the less pronounced decrease in cardiomyocyte sarcomere shortening observed, and strongly suggested that in female mice, sGC-released cGMP plays a significant pathophysiological role in LPS-induced cardiomyopathy and mortality. In contrast, after challenge with high doses of LPS, male sGCα1−/− mice had similar mortality rates and degree of LV EF depression as male WT mice (see Limitations).
A unitary hypothesis (Fig. 8) can explain how redox- and cGMP mechanisms may mediate LPS-induced cardiomyopathy separately in male and female mice. We can assume that both mechanisms are initially activated by nitric oxide (NO) released by the inducible NO synthase (NOS2) in both male and female myocardium. In male mice, oxidative stress dominates (19, 20), and cause SERCA oxidation (5, 21) and ΔCai depression, while it prevents cGMP formation by inhibiting sGC (22–24). Female mice, however, are known to have more potent myocardial antioxidant defenses, as the result of higher levels of antioxidant enzymes (Mn-superoxide dismutase and glutathione peroxidase), and decreased mitochondrial ROS production (25). Therefore, in the female myocardium, it is possible that LPS does not increase oxidative stress sufficiently to depress SERCA and ΔCai. In the same time, redox-dependent sGC inhibition (22) is also absent, and the levels of cGMP increase, triggering myofilaments dysfunction downstream. Clearly, future experiments are needed to explore this hypothesis, which, if confirmed, may have significant therapeutic implications.
Significant attention has been devoted to the concept of sepsis “endotypes” (26); subclasses of patients that fall under the diagnosis of septic shock but differ significantly (as the result of different genetic background, sepsis etiology, or comorbidities) to the result that they require different therapeutic strategies. Here, we suggest, for the first time, that distinct etiological endotypes may exist within the diagnosis of sepsis-induced cardiomyopathy. If the sex differences found here also apply to humans, it is possible that male and female patients may require different therapeutic strategies. Women with sepsis-induced cardiomyopathy may benefit from therapeutic strategies that increase myofilament sensitivity (27) and/or inhibit sGC activity (28). In contrast, male patients may benefit from drugs that potentiate antioxidant defenses (29, 30) as well as any approaches that stimulate SERCA and/or L-type Ca2+ channels and accelerate Ca2+ handling.
It is important to realize that attempting to treat sepsis-induced cardiomyopathy without knowing the exact cause of cardiomyocyte dysfunction may do more harm than good. For example, attempting to compensate a decrease in ΔCai by increasing myofilament sensitivity to Ca2+(27) may improve the contractility, but at the expense of worsening cardiac diastolic compliance. On the reverse, increasing ΔCai (usually with positive intropic drugs such as dobutamine or digoxin) in patients in whom the deficit lies at the level of myofilaments may induce life-threatening ventricular arrhythmias.
Limitations and future directions
The current experiments were designed to reproduce those used in our previous study in male mice (5). We used the same WT mice (from same vendor), and sGCα1−/− mice (from our own colony) as before (5). We used the same LPS type (from E. coli, 0111:84, Sigma), dose (25 μg/g) and incubation time (14 ± 2 h). In these conditions, we replicated or main previous findings (5), such as the observation that, in male mice, LPS induces a profound decrease in cardiomyocyte ΔCai, SERCA function and sarcomere shortening, associated with a decrease in LVEF and significant mortality. However, we acknowledge a number of minor differences between our current and previous results. For example, mortality rate was generally less in our previous study (9) (50% after a dose of 25 μg/g LPS) than here (100% after a dose of 20 μg/g LPS), for WT male mice. Also, the decrease in LVEF after LPS was more pronounced in sGCα1−/− versus WT male mice in our previous (9), but was similar in sGCα1−/− and WT male mice in this study. These inconsistencies could be attributed to differences in animal care and housing, or batch of LPS, and do not influence our main conclusions.
The female mice used in this study were not selected on the basis of the estrus cycle. Given the short duration of the estrus cycle in mice (4–5 days), we did not expect any phenotypical changes at the level of the cardiomyocytes to occur cyclically so rapidly. However this may be possible, and it remains for the future to determine whether the sex differences identified here apply across the female estrus cycle or are cycle phase dependent.
We also acknowledge that the current results will have to be replicated in other sepsis-induced cardiomyopathy models (31) and species, before its main conclusions could be translated in clinical research.
Summary and conclusions
The present study is the first investigation of myocardial Ca2+ handling in endotoxemic female mice. Consistent with a wealth of previous data (18), we show that female sex is associated with improved survival rates after challenge with lower doses of LPS. However, higher doses of LPS were associated with similar survival rates and a similar decrease in LVEF in male and female WT mice. LPS induces the depression of cardiomyocyte sarcomere shortening in both female and male mice. Importantly, cardiomyocyte dysfunction occurs through different mechanisms in the two sexes. While in male mice, LPS induces SERCA inhibition and depressed ΔCai, in female mice Ca2+ handling is preserved, and the deficits appear primarily confined downstream of the Cai transient, possibly at the level of the myofilaments. Upstream of Ca2+ handling, cGMP generated by sGC also plays opposite roles in the two sexes, being protective in males and pathological in females. These sex differences suggest that different therapeutic strategies may be required for cardiac protection in septic men and women.
Statistical design and analysis was performed with the assistance of Timothy Houle, PhD and Sara Burns, MS (Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital).
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Keywords:© 2016 by the Shock Society
Calcium handling; excitation contraction coupling; myofilaments; sepsis; sepsis-induced cardiomyopathy; septic shock; SERCA