Baseline characteristics of the TTS patients
According to the ChiTTS registry data and single medical center data, the baseline characteristics of the TTS patients, heart rate (p<0.05), emotional trigger (p<0.01), female–no. (%) (p<0.01), WBC (p<0.05), IL-6 (p<0.01), PCT (p<0.01), ALT (p<0.01), AST (p<0.01) and TG (p<0.01) were significantly associated with decreasing LVEF according to analysis of variance (ANOVA). (Table 1).
The typical characteristics include left ventricular (LV) segment wall motion abnormalities, including apical or global ballooning and mid- or basal-ventricular akinesis/dyskinesis (Figure 1 A-B); moreover, some of the patients had a decreased LVEF, and some thrombosis formed in the apical part of the left ventricle. Apical ballooning is similar to the following feature observed via echocardiography (Figure 1A) and cardiac MRI (Figure 1D), and likewise, global ballooning of the LV was observed (Figure 1D). Myocardial edema was found in the apical and interventricular septum by T2 mapping imaging (Figure 1B, 1E and 1G) and typical apical edema of the LV is shown in Figure 1 G. G1, apical; G2, middle of the LV; G3, papillary muscle levels of the LV; G4, basal segment of the LV. All the coronary arteries of the TTS patients showed no or mild stenosis (no less than 20%) (Figure 1 C and 2F coronary arteries).
Sac/val decreased inflammatory levels in the TTS patients on day 30
Acute sympathetic stress-induced cardiac injury is accompanied by an inflammatory response, such as inflammatory cell infiltration and inflammatory cytokine/chemokine release. The above mentioned abnormal inflammatory marker (increasing WBC, IL-6, PCT, ALT, AST and TG levels) were also analyzed ( Table 1 ). Associated with decreasing LVEF in the TTS patients, after 30 days, WBC (p<0.05) and IL-6 (p<0.01) were significantly altered between patients who did and did not receive sac/val treatment, while no alteration in PCT, ALT, AST or TG level (p>0.05) (Figure 2 and Table 2).
Logistic regression analysis of the risk factors for TTS patients with a decreasing LVEF ≤ 0.45 in the ChiTTS cohort
Logistic regression analysis of the risk factors for an LVEF ≤ 0.45 from the TTS registry revealed that hyperglycemia (p<0.05), emotional stress (p<0.05) and inflammation (p<0.05) were independent risk factors associated with a decreased LVEF (Table 3).
Sac/val increased the percentage of surviving TTS patients during follow-up
During follow-up, all the TTS patients, treated with sac/val, showed a significantly greater percentage of surviving than without sac/val treatment at days 30 (p<0.01) and 1 year (p<0.01) (Figure 3A-B).
A typical patient after severe limb trauma and emotional stress (both physical and stress triggers) experienced ballooning changes and enlargement of the LV (Figure 3 C-D), myocardial stunning (Figure 3D) and worsening global longitudinal strain (GLS) (LVEF of approximately 0.20 and average GLS of approximately -5) (Figure 3E.), along with LV diastolic dysfunction (Figure 3F) on April 26th, 2020. The patient accepted treatment with rhBNP in the acute phase and then treated with sac/val, a β-blocker, and spironolactone. Approximately three months later, the shape of the LV, LVEF, GLS, and diastolic function improved on July 15th, 2020, and approximately 1 year later, the shape of the LV was normal (Figure 3C-D) and GLS (approximately -20) recovered on May 6th, 2021 (Figure 3E) better than before.
Sac/val improved cardiac dysfunction in TTS-like rats
TTS-like rat models were well established (Figure 4A.- C.). Compared with those in the control group, after one hour with a single i.p. injection of ISO (100 mg/kg), a significant decrease in activity, hair erection, mental discomfort, and increased breathing were observed in the rats (data not shown). And the echocardiography showed that the TTS-like models showed typical symptoms of heart failure (Figure 4). The rat pretreated with sac/val at three different concentration (50 mg/Kg, 75 mg/Kg and 100 mg/Kg) or saline for 1 week, followed with ISO injection. On the 1st, 5th and 8th days after ISO injection, the effects of sac/val on the cardiac function were evaluated. Echocardiography revealed akinesis/dyskinesis of LV wall motion, but the LV wall motion did not recover to baseline levels on day 8 (Figure 4 A - C). Compared with those in the control groups, at days 1, 5, and 8, the LVPWd increased on day 8 (Figure 4 D), the LVESD increased at days 1 and 8 (p<0.01 or p<0.05) (Figure 4 E), and the LVEF and FS in the ISO group were significantly lower (p<0.01 or p<0.05) (Figure 4 H and F). No difference existed in the LVEDD between the control group and the ISO group on day 8 (p>0.05) (Figure 4 G). Compared with those in the control groups, the echocardiography parameters, including LVEF, FS, LVESD, and LVPWd, were not significantly different in the ISO + sac/val 50 mg/kg group (p>0.05) (Figure 4 D-H). However, compared with the ISO groups, significant increasing in ISO + sac/val 75 mg/kg group, LVEF on day 5 ( p<0.01 ), day 8 (p<0.05) (Figure 4 H), FS on day 5 ( p<0.01 ) (Figure 4 F), LVPWd on day 8 ( p<0.05 ) (Figure 4 D), and in ISO + sac/val 100 mg/kg group, LVEF on day 5 ( p<0.01 ), day 8 ( p<0.01 ), all FS on day 1, 5, 8 ( p<0.01 ), LVESD on day 8 ( p<0.01 ), LVPWd on day 8 ( p<0.05 ) (Figure 4 D-F, H).
Sac/val attenuates the cardiac inflammatory response in TTS-like rats by regulating BNP level
No significant cell necrosis or inflammatory cell infiltration was observed in the myocardial tissue, the myocardial fibers were arranged neatly, the structure was clearer, the staining was more uniform, and the nucleus was intact in the control group (Figure 5 A). However, in the TTS-like rat models, inflammatory cell infiltration, myocardial cell swelling and myocardial fiber disorders occurred on day 1, day 5 and day 8, especially on day 5 (Figure 5 A). Moreover, IHC staining revealed that the levels of IL-18 (ISO group vs control, p<0.01) and the macrophage marker Mac-3, which indicate macrophage infiltration (ISO group vs control, p<0.01), were increased in the myocardial tissues of the TTS-like rats on day 1 (Figure 5 B-D). However, IL-18 (ISO group vs sac/val 75 or 100 mg/kg + ISO, p<0.01) and macrophage infiltration (ISO group vs sac/val 75 mg/kg+ISO, p<0.05; vs sac/val 75 or 100 mg/kg+ISO, p<0.01) were inhibited by pretreatment with sac/val for 7 days (Figure 5 B-D). Consistent with this finding, sac/val pretreatment also suppressed the ISO-induced upregulation of macrophage chemokines, including IL-6, MCP-1 and TNF-α on day 1, 5 and 8 (p<0.05-0.001, ISO vs Control or ISO+100mg/mL sac/val vs ISO) (Figure 5 E-G).
Sacubitril is a precursor drug that is metabolized into the active product LBQ657 through esterase after entering the body, inhibiting the activity of enkephalinase and thereby reducing the degradation of BNP. Here, we used LBQ657 to treat the neonatal rat cardiomyocytes, and found that the BNP level was significantly increase in cardiomyocytes (Figure 5H). Circulating levels of BNP activity may increase meaningfully early after the initiation of sac/val [15]. Hence, further in vitro studies should be performed to determine the protective effect of rhBNP on ISO-induced cardiac inflammation. Our result indicated that rhBNP inhibited the elevation expression of inflammatory factors (IL-6, MCP-1 and TNF-α) caused by ISO (Figure 5I-K). Together, this data indicated the sac/val attenuates the cardiac inflammatory response in TTS-like rats by regulating BNP level
Sac/val attenuates cardiac inflammation by inhibiting TLR4/NF-κB signaling pathway activity in TTS-like models
To further investigate the mechanism of the effect of sac/val pretreatment on inflammation, in the TTS model. Western blot analysis revealed that the expression of TLR4 and total and phosphorylated NF-κB were significantly increased (p<0.05; at days 1, 5, and 8, the ISO group vs all the other groups). Significant differences in the expression of TLR4 and total and phosphorylated NF-κB were detected after pretreatment with different doses of ISO (p<0.01-0.05; at days 1, 5, and 8, the ISO group vs all the other groups) (Figure 6 A - E).
Moreover, invitro, rhBNP (5mg/mL) inhibited the expression of the inflammatory proteins TLR4 and NF-κB in Neonatal Rat Cardiomyocyte (p<0.05-0.001, the ISO group vs all the other groups), which demonstrated that rhBNP inhibited inflammation via the TLR4/NF-κB signaling pathway (Figure 6 F - H). Together, this data indicated that sac/val attenuates cardiac inflammation by inhibiting TLR4/NF-κB signaling pathway activity in TTS-like models.
Sac/val attenuates myocardial fibrosis by inhibiting collagen synthesis via the TGF-β1/Smad pathway in TTS-like rats
Proinflammatory cytokines are known to contribute to myocardial damage and long-term pathological remodeling, such as myocardial fibrosis. Sirius red and Masson trichrome staining revealed a significant increase in collagen deposition and large areas of collagen fibers in the ISO group, which were interconnected into a meshwork structure (p<0.01). However, this phenomenon was reversed by pretreatment with sac/val. Differences existed in the different dosages of ISO pretreatment, and the protective effect is most pronounced at a concentration of 100 mg/Kg of Sac/val (p<0.05-0.001, the ISO group vs all the other groups) (Figure 7A-D).
Moreover, we detected the expression of col1a1, col3a1, α-SMA (the fibrosis markers) and TGF-β1 by Western blotting. The results showed that the expression of col1a1, col3a1, α-SMA and TGF-β1 increased significantly in the ISO group than in the control group. After pretreatment with sac/val, the expression of col1a1, col3a1, α-SMA and TGF-β1 significantly decreased in response to different doses of ISO (p<0.01-0.05; the ISO group vs all the other groups) (Figure 7E-J) at day 5 and day 8. Meanwhile, IHC staining revealed that type I and type III collagen deposition was significantly increased in the ISO group (p<0.01) and obviously decreased with sac/val pretreatment (Figure 7K-N) at day 5 and day 8. Taken together, these results indicated that sac/val alleviated ISO - induced cardiac fibrosis.
rhBNP inhibits ISO-induced myocardial fibrosis by inhibiting the phosphorylation of Smad2 in myofibroblasts in vivo.
An in vitro study was also performed to determine the mechanism of the protective effect of rhBNP on ISO - induced myocardial fibrosis. Our results showed that rhBNP (5mg/mL) inhibited the total expression and phosphorylation of Smad2 (p-Smad2), Smad2 and α-Sma in myocardial fibroblasts (p<0.05, the rhBNP group vs the ISO group) (Figure 8 A-C). Immunofluorescence showed that the nuclear location of Smad2 was obviously decreased with rhBNP treatment (Figure 8D-E). Together, rhBNP inhibited ISO-induced myocardial fibrosis in fibroblast lines via the TGFβ/Smad2 signaling pathway.