Angiotensin converting enzyme inhibitors, among them captopril, improve survival following myocardial infarction (MI). The mechanisms of captopril action remain inadequately understood due to its diverse effects on multiple signalling pathways at different time periods following MI. Here we aimed to establish the role of captopril in late-stage post-MI remodelling. Left anterior descending artery (LAD) ligation or sham surgery was carried out in male C57BL/6J mice. Seven days post-surgery LAD ligated mice were allocated to daily vehicle or captopril treatment continued over four weeks. To provide comprehensive characterization of the changes in mouse heart following MI a 3D light sheet imaging method was established together with automated image analysis workflow. The combination of echocardiography and light sheet imaging enabled to assess cardiac function and the underlying morphological changes. We show that delayed captopril treatment does not affect infarct size but prevents left ventricle dilation and hypertrophy, resulting in improved ejection fraction. Quantification of lectin perfused blood vessels showed improved vascular density in the infarct border zone in captopril treated mice in comparison to vehicle dosed control mice. These results validate the applicability of combined echocardiographic and light sheet assessment of drug mode of action in preclinical cardiovascular research. Although timely primary coronary percutaneous intervention has substantially improved patient survival post myocardial infarction (MI), the often-concomitant cardiac dysfunction and heart failure affect a significant number of patients. Infarction leads to rapid replacement of cardiomyocytes by fibrotic scar tissue and remodelling of both the infarcted area and the viable myocardium are critical determinants of long-term survival. Despite extensive research, pharmacological treatment options to limit adverse cardiac remodelling and restore myocardial function are insufficient and evaluation of new cardioprotective strategies remains an important objective.4. The beneficial effects arise from attenuation of LV dilation, reduction of preload and afterload, improved oxygen supply, systemic neurohormonal changes and altered cytokine environment6. In early phase following MI ACEi inhibit fibrosis and can thereby have a limiting effect on infarct size7. Newer ACEi  with improved pharmacokinetics and comparable beneficial effects on patient survival have been developed8. Better understanding of the dynamic phases in post-MI remodelling and the role the existing drugs play in these can lead to the development of new therapeutics.Angiotensin converting enzyme inhibitors (ACEi), including captopril, became the first-line therapeutics for acute MI in 1970s and have been consistently shown to improve patient survival and left ventricular (LV) ejection fraction (EF) after acute MI9. Consistent with clinical findings, chronic captopril treatment attenuated left ventricular dilation and improved long-term survival in rat LAD-ligation model11. Similarly, captopril has been shown to exert beneficial effects on aortic flow parameters and myocardial strain following LAD ligation in mice, increasing overall survival13. Since young mice have been shown to survive extensive MI with limited concomitant mortality12 it opens up possibilities to characterize the effect of pharmaceuticals in late-stage post-MI remodelling.Left anterior descending artery (LAD) ligation in mice and rats remains the most common technique in preclinical pharmacological research to assess the efficacy of treatment options for MI16. Angiogenesis in the infarcted heart involves the growth of new capillaries from the endocardium and through the border zone, lying at the interface of healthy and infarcted tissue18. To date, both vascular density and border zone topology have remained challenging to analyse on tissue sections, due to the highly irregular shape and extent of the infarct19. Optical clearing of mouse and even human organs and tissues combined with light sheet fluorescence microscopy (LSFM) has opened new possibilities for visualizing cells and activated signalling pathways in 3D24, but shortage of automated quantitative analysis platforms is still a bottleneck and no studies have so far demonstrated the sensitivity of the method in preclinical pharmacological cardiovascular research. The current study was aimed at developing a light sheet imaging platform for myocardial infarction analysis and to use it for demonstrating the mode of action of delayed captopril treatment.Microvascular rarefaction in the infarcted heart limits blood supply to cardiomyocytes and has emerged as a promising therapeutic area of intervention7 days after surgery LAD ligated mice (n = 24) were randomized into treatment groups by both LV internal diameter in diastole (LVIDd) as well as ejection fraction (EF), measured by use of transthoracic echocardiography Fig. a. EnrolmMale C57BL/6 J mice  were single-housed in a controlled environment  with a 12 h light/dark cycle. The animals were acclimatized for at least one week before surgery. All animals had ad libitum access to chow diet  and tap water throughout the study period. Body weight and 24-h water intake were measured daily. The studies were approved by The Danish Animal Experiments Inspectorate , conformed to the European Parliament Directive on the Protection of Animals Used for Scientific Purposes (2010/63/EU) and comply ARRIVE guidelines.25. In brief, a small cutaneous incision was made on the left side of the chest wall and the pectoral muscles were bluntly dissected to visualize the fourth intercostal space. A small hole was pierced through the intercostal muscles with a haemostat and the ribs were retracted. With a gentle grip the heart was pushed through the opening and the LAD was identified and ligated using Perma-hand silk suture 7-0 (Ethicon). Subsequently, the heart was quickly returned to the thoracic cavity and the pneumothorax was manually evacuated. Finally, the skin was sutured, and the mouse was allowed to recover. Buprenorphine analgesia was administered for two days after surgery. Sham surgery was performed by exposing the heart and inserting the suture, but without tightening the ligature around the LAD artery.Before surgery, mice received acetaminophen and buprenorphine analgesia. Surgery was performed under 2% isoflurane as described by Gao et al.Seven days after LAD ligation or sham surgery, the resultant infarction was assessed by echocardiography using a Vevo 3100 system  equipped with a MX400 20–46 MHz linear array transducer . The mice were anesthetized (Isoflurane 2%) and were placed in a supine position on a heating pad and chest hair was removed. Body temperature measured by a rectal probe and heart rate was monitored continuously during the examination. Two-dimensional (2D) parasternal long- and short-axis views were obtained to visualize the location and extent of the MI. Subsequently, 2D-guided M-mode in the parasternal short axis view was used to calculate EF and LV internal diameters in systole and diastole  using Vevolab software . Only LAD-ligated mice with presence of an anterior wall myocardial infarction extending from the mid-papillary level into the apex were included in the study. EF and LV internal diameters in systole and diastole (LVIDs and LVIDd) were measured by 2D-guided M-mode from the parasternal short axis view and EF was used for randomization into study groups.After four weeks of treatment EF, LVIDs and LVIDd were re-assessed using the same procedures as described for the enrolment echocardiograph, including placement of the M-mode plane for the evaluation of EF to mimic the plane used during enrolment. Furthermore, LV end-diastolic volume was estimated by tracing the inner perimeter of the LV in parasternal long axis view in end-diastole.LVID and EF percent change from week 0 was calculated as: (LVID or EF in week 4—LVID or EF in week 0)/ LVID or EF in week 0 × 100%. All echocardiographic examinations and post-hoc analyses were performed by the same examiner blinded to the identity of the mouse.Captopril  was dissolved in tap water. Cherry flavoured Kool-Aid  was added to the drinking water to increase palatability in captopril as well as vehicle treated mice.26. Mice were placed in isoflurane chamber, maintaining 3.5% isoflurane concentration. After 7 min anaesthesia was confirmed by lack of active paw reflex. While maintaining mice under terminal isoflurane anaesthesia rib cage was opened to expose the heart. Room temperature freshly prepared cardioplegic solution  was injected into the right atrium with a 27G needle to induce diastole and stop the heart. Euthanasia was performed by cutting through the aorta and vena cava and removing the heart. Once removed, the heart was perfused through the aorta using the above described cardioplegic solution to remove fluorescent lectin from cardiac chambers. Hearts were subsequently perfused with freshly prepared cold glyoxal solution for rapid fixation27 and immersion-fixed overnight at 4 °C. For tissue clearing, the samples were dehydrated in methanol/water series: 20%, 40%, 60%, 80%, 100%; 1 h each at room temperature. The samples were next transferred to fresh 100% methanol and incubated for two days at room temperature on a horizontal shaker. Next, the samples were incubated by shaking in 66% dicloromethane (DCM)/33% methanol overnight. They were washed twice for 1 h by shaking in 100% DCM at room temperature and taken to dibenzyl ether (DBE). Hearts were weighed after transferring to DBE.Mice received a 100 µl tail vein injection of 1 mg/ml Lycopersicon Esculentum (Tomato) lectin conjugated to DyLight 649  that binds to glycoproteins and reliably labels perfused blood vesselsIn initial experiments comparing the different fixation conditions the hearts were excised as above, washed in room temperature phosphate buffered saline for 1–2 min  and then immersed in either 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF) or glyoxal-based fixative and fixed overnight. The samples were cleared as indicated above.Hearts were imaged using a Lavision ultramicroscope II  with Zyla 4.2PCL10 sCMOS camera , SuperK EXTREME supercontinuum white-light laser EXR-15  and MV PLAPO 2XC  objective. Samples were mounted in the same orientation and fixed by the posterior wall with soft neutral silicone gel to a custom-made transparent silicone sample holder. Hearts were imaged in a DBE filled chamber. ImSpector microscope controller software (v7) was used . Whole-heart imaging was carried out at 1.2 × magnification, using 560 ± 20 nm excitation (84% software-controlled laser power cut-off at 100% NKT laser output) and 650 ± 25 nm emission wavelengths at 217 ms exposure time in a z-stack at 15 μm intervals. 9 horizontal focusing steps were merged in contrast-based blending. For whole heart imaging, Olympus dipping cap  was used.Vasculature imaging was carried out at 4 × magnification, using 630 ± 15 nm excitation wavelength and 680 ± 15 nm emission wavelength . Autofluorescence was captured using 560 ± 20 nm excitation (65% software-controlled laser power cut-off at 100% NKT laser output) and 650 ± 25 nm emission wavelengths. 209 ms exposure time and horizontal focusing in 7 steps was used and z-stacks were captured at 7 µm interval. Optically corrected Lavision dipping cap (LV OM DCC20) with 5.7 mm working distance was used for high resolution imaging.28.Bitplane Imaris software  was used for 3D tissue rendering and for manual segmentation of the LV chamber to generate training dataset for a deep-learning model. For enhanced accuracy, manual segmentation was performed sequentially on two imaging planes (YZ and XY) on a total of 28 hearts, which were augmented in 3D in random orientation to generate 112 volumes for deep learning model development. 100 volumes were used as training set and 12 for validation purposes. U-net was used for deep learning30, which measures the ratio of the total area in the apical part of the LV chamber to the fitted cone and hence more precisely detects local dilation that can remain undetected in sphericity and volume analyses. If the heart is fully conical the index would equal to one. Apical geometry was analysed in long axis 2-chamber views of 2D-projections acquired from 3D image stacks. The 2D-projections were performed along the third principal axis of the LV segmentation obtained by the deep-learning model. Diastole was assessed by visual observation of the imaged heart and by setting volume (above 10 mm3) and dimension thresholds (long axis length above 4 mm). Hearts that were below these volume and size thresholds were assessed as not fully diastolic and were excluded from statistical analysis. Image analysis was therefore performed on 8 sham—vehicle, 10 LAD—vehicle and 10 LAD—captopril treated mice. Cardiac curvature maps were derived by converting the LV chamber volume to surface mesh and visualizing it in Meshlab31. Lower half of the mesh  was isolated from each mesh to quantify the distribution of curvature across the surface.LV chamber morphology, volume analyses, vasculature analysis and wall thickness characterization were carried out in Python. Infarction induced changes in the LV apical morphology was characterised using the apical conicity index32, that was tuned to enhance bright vessels with an approximate lumen diameter below 10 µm. Subsequent thresholding was applied to the vessel-enhanced image to create the final vasculature segmentation. Ventricular wall thickness was estimated locally using a maximal sphere fitting approach33. Correlative analysis of vascular density and wall thickness was applied only on the LV wall and excluded the septum and right ventricle.For vasculature and wall thickness analysis, pre-segmentation was applied in Imaris to exclude scar tissue and suture material outside the heart. Vessel segmentation was carried out by applying vessel enhancement filter in PythonAll statistical analyses were performed in GraphPad Prism (version 8.4.2). Tukey–Kramer test was used for statistical comparison of multiple groups after ANOVA. Pairwise comparison was done using two-tailed t-test. All data are presented as mean values and standard errors of the mean (s.e.m).First, we wished to establish whether delayed captopril treatment has a beneficial effect on cardiac function. To model MI the LAD was ligated in 6-week-old mice and pharmacological intervention was started 1 week after the surgery, thereby preventing early ACEi activity on cardiac fibroblasts and allowing fibrotic scar formation.Seven days after sham surgery or LAD ligation, the survival rate was 100% and 63%, respectively. No mortality was observed during the 4 weeks of drug treatment and daily water intake was similar between the groups , whereas Sham—vehicle and LAD—vehicle had similar terminal body weight Table . Heart wWe next used echocardiography to characterize MI-induced changes in the LV wall. In accordance with diastolic dysfunction LV anterior wall (LVAW) thickness was significantly reduced in LAD-ligated groups Fig. h–i. TherWe next established a protocol for rapid fixation and clearing of the heart for light sheet microscopy, with the aim of maintaining high quality cardiac morphology, avoiding tissue degradation, and keeping all analysed hearts in comparable diastolic phase. The choice of the fixative is of key significance in order to maintain correct cardiac morphology . Since intracardiac perfusion fixation damages the heart and fails to keep the hearts in diastolic phase we analysed cardiac morphology in hearts immersion fixed in PFA, NBF and glyoxal, followed by clearing in DBE. This analysis showed that hearts immersion fixed in PFA and NBF suffered from significant irregularities in myocardial morphology as well as from high autofluorescence of blood . In contrast, hearts immersion fixed in glyoxal maintained correct cardiac morphology and had low autofluorescence of blood . We further optimized the protocol by implementing retrograde perfusion of the excised hearts with cardioplegic solution before glyoxal fixation, thereby enabling to maintain the hearts in diastole for comparable image analysis. The resulting cleared hearts had low uniform autofluorescence, making it feasible to image entire sample at high resolution in approximately 30 min. Light sheet imaging of LAD-ligated hearts provided initial visual overview of the infarct zone due to its thinner ventricular walls and resulting greater optical transparency .3 (± 0.5) in the Sham—vehicle group to 24.6 mm3 (± 3.2) in LAD—vehicle and 22.5 mm3 (± 1.8) in the LAD—Captopril group. The increase in LAD—vehicle group was significant in comparison to Sham—vehicle (p < 0.01)  in the Sham—vehicle group to 14.58 mm3 (± 1.97) and 12.71 mm3 (± 1.22) in the LAD—vehicle and LAD—Captopril group, respectively .To enable reproducible analysis of cardiac morphology for drug efficacy studies, a deep learning-based 3D image analysis platform was developed to automatically segment the LV chamber and provide quantitative endpoints on its morphology. Deep learning computational model provided accurate segmentation of chamber volume . Representative images illustrate cross-morphological differences between study groups , border zone  and neighbouring myocardial tissue with healthy cardiomyocytes . A 3D local thickness mask was generated for all samples  system in the infarct border zone67, which has known roles in promoting microvasculature remodelling and angiogenesis68. These results highlight localized effect of captopril in the infarct border zone.Our data from 3D imaging shows that delayed captopril treatment leads to higher vascular density in the infarcted heart border zone but has neither positive nor negative effect on vascular density in the infarct zone and in the posterior cardiac wall. Still little is known about the dynamic response of microvasculature to MI, although angiogenesis has emerged as a lucrative target in pharmacological researchUsing correlative functional and light sheet imaging, this study provides detailed characterization of the effect of captopril on postinfarction remodelling in a mouse chronic heart failure model. Captopril helps to preserve cardiac morphology, limits LV wall dilation, improves border zone vascularisation and results in improved EF following MI.Supplementary Information.Supplementary Video 1.Supplementary Video 2.


The changes in right ventricular (RV) contractility of Kawasaki disease (KD) still remain unclear.We aimed to determine whether RV systolic dysfunction can be detected by cardiac magnetic resonance (CMR) feature tracking and to find its association with coronary artery lesions .Peak systolic myocardial longitudinal, radial and circumferential strain and the strain rate  in the global RV and three levels  were measured in 66 patients with convalescent KD. A total of 20 controls were included. Comparisons were made with controls and among KD subgroups divided with coronary artery lesions.RVSC (−10.575% vs. −10.760%), RVSL (−18.150% vs. −18.712%) and RVSRC (−0.815/s vs. −0.924/s) were slightly lower in KD group without significant difference. All the strain and strain rate presented lowest in the basal level. In subgroup comparison, lower RVSL and RVSRL were observed in the giant coronary artery aneurysm (CAA) group; RVSR (15.844% vs. 16.897%), RVSRR (1.245/s vs. 1.322/s) and RVSRC (−0.715/s vs. −0.895/s) were lower in thrombosed group; RVSRL (−1.27/s vs. −1.503/s) were lower in stenosis group. All the comparison in subgroups did not reach significant difference. From the analysis of receiver operating characteristic curve, RVSRL had a better ability to identify KD with giant CAA and stenosis. For the identification of thrombosis, RVSRC had a better ability.Lower strain and strain rates of RV were detected in convalescent KD. More pronounced in those with persisting coronary artery lesions. It was reported that coronary artery lesions (CALs) is the dominant common complication, mainly including coronary artery aneurysm (CAA), thrombosis and stenosis. The incidence of CAA is about 5% and huge CAA can usually form thrombosis and ischemic cardiac disease. In the chronic stage, the reconstruction of coronary artery continues and induces stenosis and micro‐flow impairment.Myocarditis, the most common non‐CAL, accounts for 50%–70% of patients in the acute phase, followed by myocardial fibrosis. For KD, the routine cardiac function often displays normal with routine measurements,Compared with echocardiography, cardiac MR feature tracking (CMR‐FT) allows for the quantitative assessment of regional and global myocardial mechanic deformation that is related to myocarditis and myocardial fibrosis. Myocarditis has been found in the entire heart, and the fibrosis continues to the convalescent stage. Previous studies have summarized the left ventricular (LV) myocardial functional impairments in the acute and chronic phase in detail. However, the abnormal mechanics in right ventricle (RV) of KD patients still remains undefined. This is the first study to apply CMR‐FT in KD to examine mechanic deformation in RV and to discover its association with CALs. Our goal was to define the variation of RV myocardial deformation within KD cohorts and to assess if variation in myocardial deformation may help identify subgroups with higher risk.22.1The study was conducted in Children's Hospital of Fudan University in Shanghai, China. For patients with KD, when they were admitted into the hospital, the first exam was echocardiography. After finding CALs on echocardiography, CMR was performed for myocardial and coronary artery evaluation and digital subtraction angiography (DSA) was conducted as a golden standard for CALs. From May 2013 and May 2020, totally 78 KD patients with CMR were collected in our center. We reviewed all the images and chose 66 patients in our cohort to evaluate the RV contractility impairment. We excluded five patients without good CMR imaging quality to evaluate the CALs, three patients without adequate CMR sequence for strain analysis, and two cases without DSA to confirm the CALs. A total of 20 cases with matched age and sex were enrolled in the control group. The study was approved by the Ethics Committee of our hospital and all patients signed written informed consents. The phases were defined as follow: acute phase, day 1–14, subacute phase, day 15–42, and convalescent/chronic phase, after day 43.2.2All patients and controls experienced CMR at 1.5‐T MR . Imaging was conducted under oral sedation for the patients under 8‐year‐old or who could not cooperate to complete the CMR.2, repetition time (TR) 48–84 ms, temporal resolution 50 ms, flip angle 62°, voxel size 1.2 × 1.2 × 6 mm3. The images were acquired during free breathing with navigator triggering for respiratory compensation.A combination of 16‐elements phased array surface coil and spine coil were used. Initially, a survey examination in three orthogoal planes was performed to localization of the heart, and then an interactive sequence was performed to define patient‐specific relevant orientations . Electrocardiography (ECG)‐gated two‐dimensional steady‐state free precession (SSFP) segmented cine images in short axial and longitude plans with full coverage of the LV and RV were acquired for functional assessment and CMR‐FT assessment. The imaging parameters were listed below: field‐of‐view (FOV) = 240 × 240 mm2) with T2 preparation to suppress the signal from the myocardium and fat suppression to suppress the high signal from epicardial fat. The whole heart and the CALs in the main coronary arteries were viewed in this sequence, including the right coronary artery (RCA), the left coronary artery (LCA), the left anterior descending artery (LAD), the left circumflex artery (CCX).A 3D volume of axial images covering the heart and aortic arch were acquired using SSFP sequence , as previously described.The quantification of the LV and RV volume indices were normalized to the body surface area. The 3D SSFP images were applied to evaluate the presence of aneurysms, stenosis and thrombi of RCA. According to 2017 AHA standardAll the CMR studies were analyzed by 1 radiologist who was blinded to the patient clinical data. In addition, random samples of 20 cases were re‐evaluated by the same radiologist to assess intra‐observer and by another investigator for inter‐observer agreement. There exist no consensus normal values for myocardial deformation in children, so we compared the KD group with the control group.2.4t‐test and one‐way ANOVA were adopted to compare the strain and strain rate between subgroups. Diagnostic accuracy of CMR parameters was evaluated through receiver operating characteristic (ROC) curve analysis. For area, under the curve (AUC), a value of 0.9–1.0 was considered excellent, 0.75–0.9 marked good, 0.6–0.75 stood for moderate and 0.5–0.6 was poor. Best cut‐off values, sensitivity and specificity were derived from ROC curves. Intra‐ and inter‐observer variability was assessed using Cronbach's α. Statistical analysis was performed with SPSS software . A p‐value of ≤.05 was regarded statistically significant.All data were reported as mean ± SD. Unpaired Student's 33.1In Table 3.2Totally, 66 CMR exams were carried out and routine CMR indices of RV cardiac function were normal, without any wall motion abnormality Table . A total3.3Compared with controls, RVSC, RVSL and RVSRC were slightly lower in KD group Table . The abo3.4Comparisons were made between KD subgroups subdivided by z score of CAAs in the RCA. Among 66 patients, 10 did not experience CAA in the RCA, 12 with small CAA, 29 with median size of CAAs, and 15 with giant CAAs. When KD patients were subdivided into four groups according to z score of CAA, lower RVSL, RVSRC and RVSRL were observed in the giant CAA group. However, no statistically significant difference was illustrated among the subgroups (Table When we compared patients suffering from thrombi (n = 13) with those without thrombi (n = 53), RVSR, RVSRR and RVSRC were lower in thrombosed group, but did not reach statistical difference.When patients catching with stenosis (n = 4) were compared with those without stenosis (n = 62), RVSRL were lower in stenosis group, but did not reach statistical significant difference.3.5ROC curve revealed that RVSRL had a better ability to identify KD from giant CAA and stenosis. The cut‐off value for RVSRL was −1.405  and −1.15  respectively. For the identification of thrombosis, RVSRC had a better ability with a cut‐off of −0.565 . The results were listed in Table 3.6Twenty subjects were randomly selected from KD group to assess inter‐ and intra‐observer agreement of CMR‐FT analysis. We evaluated the intra‐observer and inter‐observer agreement by Cronbach's α. If the value is higher than 0.8, the reliability is high. If the value is between 0.7 and 0.8, the reliability is good. If the value is between 0.6 and 0.7, the reliability is acceptable. If the value is less than 0.6, the reliability is poor. According to CMR‐based strain parameters, we identified the good reliability in the inter‐ and intra‐observer agreement, except for the inter‐observer agreement of RVSRR (Cronbach's α = 0.651).4As a systemic vasculitis of medium sized vessels, perhaps the pericardium, myocardium, valves and the coronary arteries were inflamed in KD.It has been proved that the strain analysis was superior to routine cardiac function for evaluation of subclinical LV systolic dysfunction in many diseases, including KD.In our study, a considerable proportion of patients suffer from normal EF and abnormal RV strain indices, indicating that myocardial mechanic deformation impairment starts before RV cardiac dysfunction. KD patients were revealed to have lower RVSC, RVSL and RVSRC compared with age‐matched normal controls. The results did not reach statistic difference, which may be caused by the preserved EF in this group. Meanwhile, we also found that the proximal segments presented more obvious strain abnormality, which was related with the CAAs that mostly existed in the proximal segments of RCA. The paper had unraveled the non‐uniform mechanics of RV, characterized by increased strain values at the apex.In subgroup analysis, a more pronounced decrease was shown in those with persisting CALs versus those without persisting CALs.For echocardiography, currently, the qualitative evaluation of RV cardiac function is a diagnostic challenge due to the unusual shape and uneven contractility pattern.Although the number of patients in this study was large enough, it still existed several limitations. (1) We did not include KD children without CALs, so we missed the data of this subgroup. (2) Because of the difficulty of assessment to the thin‐walled RV, the evaluation of RV myocardial perfusion and scarring was not conducted. (3) The accuracy of CMR and echocardiography of strain analysis was not compared. Therefore, future studies should be conducted to solve these problems.5In KD, a reduction in strain of RV was observed in the convalescent stage, detecting subclinical functional abnormalities. A more pronounced decrease was displayed in those with persisting CALs. These results provide new insight into the effects of strain analysis on ventricular mechanics and may prompt modifications in management.The authors declare that they have no competing interests.Informed consent forms were signed by the parents and ethics approval was approved by the institutional review boards of the Children's Hospital of Fudan University.Table S1. AUC of RV strain and strain rate parameters for detection of huge CAA, thrombosis and stenosis. AUC = aera under curve; CAA = coronary artery aneurysms; RVSR = right ventricular strain radial; RVSC = right ventricular strain circumferential; RVSL = right ventricular strain longitudinal; RVSRR = right ventricular strain rate radial; RVSRC = right ventricular strain rate circumferential; RVSRL = right ventricular strain rate longitudinal. *presenting the item with highest AUC.Click here for additional data file.


Right ventricular dysfunction after CABG is associated with poor peri- and postoperative outcomes. We aimed to identify clinical and experimental predictors for preoperative inapparent right ventricular dysfunction and therefore hypothesized that reduced myofilament force development as well as altered levels of biomarkers might predict inapparent right ventricular dysfunction.From 08/2016 to 02/2018, 218 patients scheduled for CABG were divided into two groups . Baseline serum samples for biomarkers , clinical laboratory and transthoracic echocardiographic parameters were evaluated. To examine the myocardial apparatus of the right ventricle intraoperative right auricular tissue was harvested for stepwise skinned fiber force measurements.2, p = 0.013), larger LA area  and reduced LVEF . Furthermore, higher serum ADMA  and higher serum Pentraxin-3 levels  were observed in these patients. Skinned fiber force measurements showed significant lower values at almost every step of calcium concentration . Multivariable analysis revealed DM , preoperative AF , GFR , albumin , Pentraxin-3 , depressed LVEF , lower force values:  as predictors for clinical inapparent right heart dysfunction.Patients with TAPSE < 20 mm had a higher incidence of DM , preoperative AFib , reduced GFR (67 ± 18 vs. 77 ± 24 ml/min/1.73 mThese preliminary data showed that inapparent right heart dysfunction in CAD is already associated with reduced force development of the contractile apparatus. Right ventricular dysfunction in patients undergoing coronary artery bypass surgery (CABG) is associated with poor perioperative and postoperative outcomes –3. IdentThe relative paucity of translational studies on the relationship between TAPSE, intrinsic myocardial function and biomarkers at the level of the myocardial apparatus in symptomatic CAD patients prior to CABG, contribute to this uncertainty. We hypothesized that patients with impaired evident (TAPSE < 16 mm) and inapparent (TAPSE > 16 mm and < 20 mm) right heart dysfunction may have impaired induced myocardial force values and could be identified with altered levels of biomarkers underlying myocardial remodeling processes. For assessment of the myocardial contractility, we chose the skinned human fiber method, which is “a canonical functional probe of the intrinsic myocardial regulatory complex” and offers the possibility to examine the contractile properties of the myocardial apparatus (actin and myosin) directly by withdrawal of membrane-dependent processes –11.This is important since membrane-dependent processes are likely to influence contractile kinetics by enzymatic modifications or membrane receptor mediated effects. This can be excluded in the skinned fiber mode, as direct observations of the myofilaments offer insights into the process of (impaired) contraction. Considering that atrial tissue has comparable contractile properties to ventricular tissue, results derived from skinned atrial tissue may also have relevance for the heart as a whole and is a well-established method in literature , 13.The aim of the study was to evaluate if clinical inapparent or “low range normal” right ventricular function (TAPSE between 16 and 20 mm) is already associated with altered serum concentrations of selected biomarkers, altered right auricular force values (as signs of impaired function of the contractile apparatus) and clinical parameters of the patients.Two-hundred eighteen patients undergoing elective on pump CABG between August 2016 and February 2018 were enrolled in this study. All patients were informed and gave their written consent to participate. The study was approved by the University Hospital ethics committee . Patients with valve pathologies, reoperations, emergency indications or pulmonary hypertension of any origin were excluded.All patients received a routine preoperative echocardiographic examination . Measurements and analysis were performed by trained echocardiography technician, blinded to patients. We measured left and right atrial end-systolic areas, systolic pulmonary artery pressure (sPAP), TAPSE, left ventricular ejection fraction (LVEF), according to echocardiographic guidelines for assessment of right heart function . We diviPreoperative clinical chemistry included creatinine, albumin, glomerular filtration rate (GFR) and N-terminal pro brain natriuretric peptid (NT-proBNP). We performed enzyme-linked immunosorbant assay (ELISA) for the following markers of fibrosis and inflammation, taken from serum blood samples of the patients. All ELISAs were conducted with the ELISA reader Tecan Infinity Pro : Galectin, Transforming Growth Factor ß (TGFß), N Acyl-Symmetric Dimethylarginine (N-Acyl-SDMA), Arginine, Asymmetric Dimethylarginine (ADMA)  and Pentraxin .We measured the calcium-induced force of right atrial skinned human fibers as previously described –10. We rThe trabeculae were then cut small bundles for the experimental cycle. The experimental set-up has been previously described –10. BrieDemographic variables, comorbidities, blood tests, ventricular function, operative data, 30-day, and mid-term outcomes in patients undergoing elective CABG were compared between patients with TAPSE ≤ 20 versus ≥ TAPSE. Continuous and categorical variables were compared using t-test and Fisher’s exact test, respectively. Multivariable linear and logistic regression models were constructed to identify factors associated with reduced right heart function (TAPSE ≤ 20 mm), postoperative atrial fibrillation (POAF) and length of stay. Candidate variables were selected a priori following a review of existing literature as well as the clinical and research experiences of the investigators , 8–10. MDeath notification was confirmed by medical record (evidence of life or death).218 patients who underwent elective CABG were evaluated. The age (mean ± SD) of the patient cohort was 68 ± 9 years and 187 (86%) were male. The clinical characteristics of patients are summarized in Table p < 0.001), higher incidence of DM , greater prevalence of AF , larger LA size , decreased LVEF , reduced preoperative glomerular filtration rates , lower preoperative Albumin serum concentrations  and higher serum concentrations of Pentraxin-3  and increased ADMA levels .Patients with reduced TAPSE (Group 2) presented with a significant higher Euro Score II . Furthermore patients developed significant more often POAF  and prolonged LOS . As of July 2019, the mean (± SD) follow-up was 14 ± 6 months in group 2 and 13 ± 6 months in group 1. Of the 218 patients, two died (0.9%) within 30 days of surgery while the overall follow-up rate for mid-term outcomes (> 30 days) presented in Table Operative and postoperative data are depicted in Table p = 0.069). At all other steps of calcium concentration were significant lower in the group with reduced TAPSE.Table Higher incidence of DM odds ratio (OR) 2.53; 95% CI, 1.12–5.73), higher EuroScore II  greater prevalence of preoperative AF , decreased GFR  and lower albumin level , higher level of Pentraxin-3 , poorer LVEF  and lower calcium-induced force values: pCa 5.4 .Predictors of known reduced right heart function, such as higher prevalence of significant preoperative AF , advanced age , higher EuroScore II  appeared to be predictors for POAF. Moreover, larger LA diameter , abnormal TAPSE , higher level of ADMA  and more arterial and venous grafts, respectively  appeared to be significant risk for POAF (≤ 30 d). Abnormal TAPSE  and larger RA  appear to influence the odds of prolonged LOS  showed significant impairment of the contractile apparatus utilizing our experimental model of skinned human right auricular fibers. A significant reduction in calcium-induced force values was observed at every force step generated by increasing calcium concentration. For TAPSE, cut-off point of < 16 mm according to guidelines, predicted impaired RV function with high specificity but low sensitivity . TherefoOur data show that experimental force development significantly decreases in patients with moderate to severe RV dysfunction. These results confirm that even in the absence of clinical symptoms, right heart dysfunction appears to be present in elective CABG patients. Thus, this subpopulation of “asymptomatic” surgical candidates may have a priori a greater incidence of developing RV failure. Available evidence does suggest that chronic ischemia of the inferior, lateral LV walls and ventricular septum increase the likelihood of RV dysfunction –19. ThisAnother finding is that DM, preoperative Afib, higher EuroScore II, lower albumin level, decreased GFR, and reduced LVEF were associated with reduced right heart function, which is in concert with literature –24. Our Gorter and coworkers demonstrated that Afib is associated with right heart dysfunction as result of decreased RA emptying fraction and reduced RA reservoir strain . SimilarRecent studies have described the association of impaired renal function and decreased serum albumin levels in right-sided HF, which may be the result of subclinical remodeling process of the right atrium and ventricle , 24. In To date, right-sided HF pathophysiology in symptomatic CHD for CABG remains unknown and there is no ability to determine who is at risk of progression or who will recover after CABG. To overcome these pitfalls, we analyzed a series of candidate markers for fibrosis and inflammation in baseline serum blood samples and correlated these factors with reduced right heart function. Significantly higher normalized concentrations of Pentraxin-3 in patients with reduced TAPSE vs. with normal TAPSE was significantly positively associated with reduced right heart function. Pentraxin-3 is a protein mediator of innate immunity produced by VSMCs and ECs  and obseIn this study, reduced right heart function had no impact on clinical outcome parameters aside from significant longer hospital stay, which is inevitable in patients with reduced right heart function . ReducedSeveral limitations must be noted. First, the sample size is still too small to draw conclusions concerning predictors for postoperative right heart impairment and follow-up in patients with CAD undergoing CABG procedure. Second, the higher incidence of diabetes and Afib in the group with reduced TAPSE might have also influenced the contractile capacity of the skinned fibers. Third, operator-specific treatment of the tissue samples with possible damage to the trabeculae cannot be excluded, although harvesting protocols were designed with a high degree of standardization. Furthermore in the scientific statement from the American Heart Association on evaluation and management of right-sided heart failure  cardiac However, TAPSE has been established as an easy and fast obtainable screening method for right heart function with good correlation with more sophisticated techniques estimating RV systolic function (radionuclide-derived RV EF or 2D RF EF) . Our patThese preliminary data showed that a TAPSE of < 20 mm is associated with signs of right heart dysfunction at the level of myofilament apparatus. Increased Pentraxin-3 levels were associated with a TAPSE < 20 mm assuming that myofilament function is already decreased in a low normal range of TAPSE, which is echocardiographically supposed to be “normal” and could help identify patients at risk for right dysfunction immediately after CABG. However, larger studies and longer follow-up are mandatory to confirm our findings and delineate their clinical impact.


Heart failure (HF) is a major public health problem in the United States as well as worldwide. Chronic heart failure is a syndrome of reduced cardiac output resulting from impaired ventricular function, impaired filling, or a combination of both. Associated symptoms include dyspnea, fatigue, and decreased exercise tolerance. HF has a marked effect on morbidity and mortality, given limited therapeutic choices. The first line of therapeutic agents indicated in heart failure are beta-blockers. Other drugs and therapeutic modalities employed in HF treatment include angiotensin-receptor blockers (ARBs), sacubitril (neprilysin inhibitor) combination with the ARB, valsartan, small doses of aldosterone receptor antagonists (ARAs) in the setting of angiotensin-converting enzyme (ACE) inhibitors, and beta-blockers. Additionally, the sodium-glucose transporter-2 inhibitor, dapagliflozin in the setting of ACE inhibitors, ARBs, or sacubitril-valsartan plus beta-blocker have been employed. Other therapeutic modalities have included loop diuretics, digoxin, the hydralazine-isosorbide dinitrate combination, ivabradine, the inotropes, dobutamine, milrinone, and dopamine. Decreased cardiac contractility is central to the systolic HF. Therapeutic agents employed to increase cardiac contractility in HF are limited because of their mechanistic-related adverse effect profiles. Omecamtiv mecarbil (OM) is a first of its class cardiac myosin activator that increases the cardiac contractility by specifically binding to the catalytic S1 domain of cardiac myosin, to be employed in heart failure treatment. This agent has demonstrated benefit in reducing heart rate, peripheral vascular resistance, mean left arterial pressure, and left ventricular end-diastolic pressure in the animal models. Additionally, OM is known to improve systolic wall thickening, stroke volume (SV), and cardiac output (CO). OM increases systolic ejection time (SET), cardiac myocyte fractional shortening without significant increase of LV dP/dtmax, myocardial oxygen consumption, and myocyte intracellular calcium. The benefits of OM have been demonstrated through key trials, as (i) The Acute Treatment with Omecamtiv mecarbil to Increase Contractility in Acute Heart Failure (ATOMIC-AHF), and (ii) The Chronic Oral Study of Myosin Activation to Increase Contractility in Heart Failure (COSMIC-HF). The Global Approach to Lowering Adverse Cardiac Outcomes Through Improving Contractility in Heart Failure (GALACTIC-HF) trial is ongoing and can help provide further clinical data. OM provides a novel mechanism and therapeutic approach to managing patients with HF. Preclinical and clinical data suggest that OM capability can improve cardiac function, decrease ventricular wall stress, reverse ventricular remodeling, and promote sympathetic withdrawal. Heart failure (HF) is a significant public health challenge, with a prevalence of more than 5.8 million individuals in the United States (US) and greater than 23 million patients worldwide . Acute dOverview of chronic heart failureIn chronic heart failure (CHF), also known as congestive heart failure, there is decreased capacity of the heart to circulate and propel adequate blood and oxygenation throughout the body . CHF is Based on ejection fraction, heart failure can be categorized into three broad categories. These include: (1) Heart failure with reduced ejection fraction (HFrEF), EF > 40%; (2) heart failure with preserved ejection fraction (HFpEF), EF >50%; and (3) heart failure with mid-range EF (HFmrEF), also referred to as HFpEF-borderline and HFpEF-improved with EF in HFrEF improved > 40%; EF of 40-49% and 41-49% based on US and European guidelines, respectively , 8. The Cardiomyopathy frequently results in heart failure . There aDrug therapy in heart failureCurrent heart failure therapy concepts rely on two mechanisms. The most successful pathway is blockage of neurohormonal activation with ACE inhibitors, beta-blockers, and/or aldosterone receptor blockers . The secDirect cardiac sarcomere contractility activation has been demonstrated to improve cardiac performance without adverse effects. Sarcomere consists of thin and thick interdigitating filaments. Myosin is the core of the thick filament and uses chemical energy derived from ATP hydrolysis for contraction. Myosin motors act on the thin filaments, which consist of actin and the regulatory complex, troponin-tropomyosin. Free calcium levels in resting muscles low thus, regulatory proteins prevent myosin from interacting with actin. Calcium is routinely and transiently released from the sarcoplasmic reticulum into the cytoplasm with every heartbeat. Calcium then binds to troponin, allowing myosin to interact with actin filaments for the contraction to occur. As calcium is removed from the cytoplasm, the muscle relaxes. Direct activation of cardiac sarcomere can be conceptually achieved either via sensitizing the regulatory proteins to calcium or activating cardiac myosin directly . This coVarious markers such as heart rate (HR), blood pressure (BP) are essential for monitoring therapies and disease progression to prevent morbidity in heart failure patients. Other markers used to monitor drug therapy in heart failure include cardiac output (CO) and systemic vascular resistance (SVR). For example, the employment of vasodilators in heart failure increases CO via decreasing SVR, with inotropes deployed for blood pressure support. Mean arterial pressure (MAP) could also be readily monitored in this population, given that MAP = CO SVR. Cardiac index (CI) and brain natriuretic peptide (BNP) have also been used as markers to monitor heart failure therapy. OM is known to improve systolic wall thickening, stroke volume (SV), and CO ,20.Beta-blockersThe first line of drugs indicated in heart failure are beta-blockers, which are recommended for use, regardless of NYHA class. Beta-blockers block the SNS activation by blocking epinephrine and norepinephrine and provide benefits in controlling heart rate and reducing the risk of arrhythmias . IndividAngiotensin-converting enzyme inhibitorsAngiotensin-converting enzyme (ACE) inhibitors are recommended for use in all individuals with left ventricular systolic dysfunction or HfrEF, irrespective of functional class, except in those cases of intolerance or a contraindication, such as angioedema exists. Mortality reduction in heart failure observed with these agents has hovered around 30% .Angiotensin-receptor blockersAngiotensin-receptor blockers (ARBs) are recommended in individuals with intolerable adverse effects from ACE inhibitors (predominantly cough). Decreased mortality in various randomized controlled trials has ranged from 30%-45% -27. ACE Neprilysin inhibitors and angiotensin-receptor blockers (ARBs)The employment of sacubitril (neprilysin inhibitor) and use with the ARB, valsartan in HF patients with mild to severe symptoms, NYHA class II-IV has further reduced the risk for cardiovascular death or heart failure associated hospitalization by 20% .Beta-blockers, hydralazine and nitratesIn individuals who cannot tolerate ACE inhibitors or ARBs, the combination of hydralazine and isosorbide dinitrate should be considered; however, the decrease in mortality is not as profound as with ACE inhibitors and ARBs . This beAldosterone antagonistsThe addition of small doses of aldosterone receptor antagonists (ARAs) in the setting of ACE inhibitors and beta-blockers may provide added diuresis, improved symptoms, and ejection fraction. These agents also reduce morbidity and mortality for patients in NYHA Class II-IV -34.Sodium-glucose-transporter-2 inhibitorThe employment of the sodium-glucose transporter-2 inhibitor, dapagliflozin in the setting of ACE inhibitors, ARBs, or sacubitril-valsartan plus a beta-blocker has also been shown to reduce all-cause mortality and worsening HF in adults with NYHA Class II-IV with or without diabetes mellitus .Loop diureticsLoop diuretics reduce blood volume, which decreases edema and congestion in patients with HF. Most patients will need a loop diuretic for symptom relief. These are the only agents that have demonstrated short-term symptomatic benefits. Additionally, diuretics decrease pulmonary capillary wedge pressure as well as increase exercise capacity .DigoxinDigoxin has been used in the treatment of HF for decades. It can cause a small increase in cardiac output, improves the patient symptoms, and helps in decreasing HF-related hospitalizations. It is best reserved for individuals with symptomatic NYHA class II to IV heart failure; however, it provides no survival advantage in comparison to placebo . DigoxinIvabradineIvabradine has been demonstrated to decrease hospitalization risk in patients with mild to moderate symptoms, within NYHA classes II-III who are in normal sinus rhythm with a resting heart rate greater than or equal to 70 beats per minute (BPM) . Its abiInotropic agentsThe use of intravenous inotropes can be helpful in afterload reduction for hospitalized patients unresponsive to oral agents, with decompensated heart failure with low cardiac output and evidence of end-organ hypoperfusion or hypotension. However, these agents' routine use for inpatient management of acute decompensated HF is limited due to a lack of evidence for a survival benefit and concerns regarding the risk of increased mortality and adverse side effects . AdditioSeveral agents described above have been used in the management of heart failure, with some successes. Unfortunately, progress in the development of therapeutic agents for heart failure management has been plodding. Omecamtiv mecarbil is a novel agent, recently granted Fast Track designation by the Food and Drug Administration for the potential treatment of chronic heart failure in HFrEF.Omecamtiv mecarbil (OM)Myocardial contractility is at the core of HF progression, compromising cardiac output (CO), initially upon effort, and at rest in later stages of the disease . OmecamtOM has demonstrated benefit in reducing heart rate, peripheral vascular resistance, mean left arterial pressure, and left ventricular end-diastolic pressure in previous experimental canine studies. OM has also been shown to improve the CO, Stroke Volume, (SV) and systolic wall thickening. Besides OM was also shown to increase systolic ejection time (SET) as well as cardiac myocyte fractional shortening without a significant increase in LV dP/dtmax, myocardial oxygen consumption, and myocyte intracellular calcium .The population that may benefit from OM was described in the GALACTIC-HF trial as those with EF of less than or equivalent to 28% . AdditioOM provides a novel mechanism and therapeutic approach to managing patients with HF. Preclinical and clinical data demonstrated capability in improved cardiac function, decreased ventricular wall stress, the reversal of ventricular remodeling, and promotion of sympathetic withdrawal. Prior experimentation attempts at increasing cardiac contractility with oral agents in HF have resulted in an increased risk of mortality at pharmacologically effective doses. Via a novel mechanism of action, OM can increase cardiac contractility without predisposing patients to adverse effects seen with other inotropic agents. Additionally, OM has not demonstrated limitations such as adverse effects on blood pressure, heart rate, potassium homeostasis, or renal function that have typically limited the employment of the current HF therapies. Ongoing and future clinical trials will continue to evaluate OM’s value in HF therapy and its benefit in preventing HF events and CV death.


Many novel therapies to treat myocardial infarction (MI), yielding promising results in animal models, nowadays failed in clinical trials for several reasons. The most used animal MI model is based on permanent ligation of the left anterior descending (LAD) coronary artery in healthy mice resulting in transmural MI, while in clinical practice reperfusion is usually accomplished by primary percutaneous coronary interventions (PCI) limiting myocardial damage and inducing myocardial ischemia–reperfusion (MI-R) injury. To evaluate a more similar murine MI model we compared MI-R injury to unreperfused MI in hypercholesterolemic apolipoprotein (APO)E*3-Leiden mice regarding effects on cardiac function, left ventricular (LV) remodeling and inflammation. Both MI-R and MI resulted in significant LV dilation and impaired cardiac function after 3 weeks. Although LV dilation, displayed by end-diastolic (EDV) and end-systolic volumes (ESV), and infarct size (IS) were restricted following MI-R compared to MI , cardiac function was not preserved. LV-wall thinning was limited with non-transmural LV fibrosis in the MI-R group (66.7%). Two days after inducing myocardial ischemia, local leucocyte infiltration in the infarct area was decreased following MI-R compared to MI (36.6%), whereas systemic circulating monocytes were increased in both groups compared to sham (130.0% following MI-R and 120.0% after MI). Both MI-R and MI models against the background of a hypercholesterolemic phenotype appear validated experimental models, however reduced infarct size, restricted LV remodeling as well as a different distributed inflammatory response following MI-R resemble the contemporary clinical outcome regarding primary PCI more accurately which potentially provides better predictive value of experimental therapies in successive clinical trials. Patients usually suffer from comorbidities like hypertension, hypercholesterolemia, and type 2 diabetes mellitus, amongst other risk factors of MI2. Current guidelines aim for timely reperfusion by primary percutaneous coronary interventions (PCI)3 to limit myocardial damage and reduce infarct size resulting in a better clinical outcome4. Paradoxically, restoration of myocardial blood flow initiates myocardial reperfusion injury by a series of events which apparently affects post-ischemic infarct healing, LV remodeling and effects of applied treatment opportunities6.Cardiovascular disease remains the main cause of death worldwide. Etiologically, atherosclerosis, a chronic inflammatory process, leads to the formation of lipid-rich lesions in the vascular wall, the atherosclerotic plaque. The vast majority of myocardial infarctions (MI) are a consequence of the rupture of such an inflamed atherosclerotic plaque resulting in sudden occlusion of a coronary artery9 which has additionally been underscored by a position paper of the European Society of Cardiology (ESC)10 and united in several practical guidelines with the aim of increasing rigor and reproducibility in preclinical research12. Yet until recently, most data regarding MI have been generated in animals lacking human comorbidity, spared of atherosclerosis with its associated heightened inflammatory phenotype13. However, a close relation has been clarified, since leukocytosis predicts cardiovascular events and precipitates plaque rupture14. In addition, atherosclerosis is associated with chronic monocytosis associated with increased levels of lymphocyte antigen (Ly)-6Chi monocytes in response to hypercholesterolemia15. Following MI, an inflammatory phenotype with Ly-6Chi monocytes and M1-type macrophages initially dominates which passes into a reparative phenotype with Ly-6Clo monocytes and M2-type macrophages16, making these cells main contributors in the murine post-ischemic acute inflammatory response affecting myocardial wound healing17. Even though these cells are necessary in post-ischemic wound repair, excessive Ly-6Chi monocytosis resulted in impaired infarct healing and accelerated deterioration of ejection fraction (EF) in unreperfused MI, underscoring the need for a balanced and coordinated response19. In addition, most animal models used for studying novel therapeutic strategies are typically characterized by permanent LAD coronary artery occlusion, whereas in contemporary clinical practice patients with acute MI receive rapid reperfusion therapy defined in the guideline as within 90 min from first medical contact with the emergency medical system till reperfusion3.To investigate the etiology of MI and treatment opportunities, animal models are indispensable. The selection of an appropriate animal model to ensure optimal translation of novel cardioprotective therapies from bench to bedside is of the utmost importance20. As a result, promising experimental results derived from animal studies may be viewed with some caution. This is also supported by years of experience in which numerous therapeutic strategies, for several reasons, have failed their translation into successful clinical trials, deviating from promising results in preclinical animal studies22. When using preclinical animal experiments, it is of exceptional importance to guarantee reproducibility, pay attention to cardiovascular risk factors, comorbidities, and comedications, as well as addressing long-term effects of adjunct cardioprotection after completing the entire process of scar maturation23. Ideally, animal models resemble the clinical setting exhibiting a human-like atherosclerotic phenotype as a major comorbidity24 and with respect to physiological cardiovascular parameters, in which they are exposed to temporarily myocardial ischemia followed by reperfusion experiencing human-like infarct distribution. Therefore, large animal studies remain most appropriate and are preferred over rodent models26, however, these are expensive and practically more difficult to implement. This study aims to investigate the effects of MI-R compared to unreperfused MI on cardiac function, LV remodeling and the post-ischemic inflammatory response against the background of a hypercholesterolemic phenotype in APOE*3-Leiden mice which are known to develop advanced aortic atherosclerotic lesions resembling their human counterparts when exposed to a hypercholesterolemic phenotype as a result of a cholesterol-enriched Western-type diet27, and to test its suitability as an experimental murine MI-R model with respect to cost effectiveness and practical ease of use.This scientific knowledge contributes to discussions about the relevancy of animal-derived data and whether these studies resemble the clinical setting with their human counterparts accurately which has been explicitly addressed beforep = 0.94) and triglyceride  levels did not differ between the MI-R and MI group 3 weeks after surgery , pointing to more subtle myocardial damage and limited LV remodeling and compensatory cardiac hypertrophy. Body weight (BW) was not affected between the MI and MI-R group  (Table Cardiac hypertrophy was assessed by determination of the heart to body weight (HW/BW) ratio. The HW/BW-ratio was significantly less increased in the MI-R group compared to the MI group . This effect likely resulted from augmented LV remodeling in the 3 weeks following surgery, since initial infarct size after 2 days was similar in the MI and MI-R groups  and end-systolic volume  were significantly smaller in the MI-R group compared to the MI group, consistent with limited cardiac remodeling. Again, initial LV dimensions at day 2 showed no differences between both groups  which implicates impaired or at least a difference in LV remodeling during the 3 weeks following MI , which was preceded by a similar EF (p = 0.49) after 2 days at baseline  after 3 weeks showed a significantly enlarged infarct size as assessed by contrast-enhanced MRI in the MI group when compared to MI-R . This observation explains the non-significant difference in relative LV fibrous content since transmural infarction in the MI group caused substantial LV wall thinning , indicating compensatory concentric hypertrophy  and unreperfused MI  compared to sham . On the other hand, the percentage circulating reparative Ly-6Clo monocytes . Although not significantly, Ly-6Chi monocytes  seems to be overrepresented 2 days after MI-R compared to MI (2.4 ± 0.5% vs. 1.7 ± 0.7%) and Ly-6Clo monocytes  2 days after MI compared to MI-R (2.1 ± 0.5% vs. 1.6 ± 0.5%). Circulating eosinophils were markedly decreased in the MI-R  and MI  group compared to sham (18.4 ± 2.1%) animals  analysis showed significantly increased circulating monocytes  2 days after MI-R . In addition, leukocyte infiltration compared to sham (4.9 ± 0.4 per 0.25 mm2) was increased in both groups . Furthermore, the distribution pattern in the chronic inflammatory phase after 3 weeks differed markedly regarding a resolution of infiltrated leukocytes in general as compared to 2 days following MI. Although, a significantly increased number of local leukocyte infiltration in the border zones and infarct area was demonstrated following MI-R  and MI  as compared to sham . In vivo studies were performed in hypercholesterolemic female APOE*3-Leiden mice41 on a semisynthetic Western-type diet supplemented with 0.4% cholesterol  after completing a dietary run-in period of 4 weeks at the induction of myocardial ischemia. Female rather than male APOE*3-Leiden mice were used because of their higher and stable plasma cholesterol and triglyceride levels, confined to the VLDL/LDL-sized lipoprotein fraction43. Plasma levels of TC and TG were determined after a 4-h fasting period using commercially available enzymatic kits according to the manufacturer’s protocols .All animal experiments were approved by the Institutional Committee for Animal Welfare of the Leiden University Medical Center (LUMC) and conformed to the 44. Briefly, after endotracheal intubation and ventilation mice were kept anesthetized with 1.5–2% isoflurane. Subsequently, a left thoracotomy was performed and the LAD coronary artery was ligated permanently in the MI group or during 45 min followed by permanent reperfusion in the MI-R group. Analgesia was obtained with buprenorfine s.c. pre- and post-operative. Sham operated animals were operated similarly but without ligation of the LAD (sham).Myocardial infarction was induced with either MI-R injury or unreperfused MI model by ligation of the LAD coronary artery at day 0 in 12–14 weeks old female APOE*3-Leiden mice as described previouslyFor short-term experiments, mice were euthanized 2 days after surgery to study the effects on the acute post-ischemic inflammatory response, resulting in the following groups: MI (n = 6), MI-R (n = 5), and sham (n = 8). For long-term experiments, mice were followed for 3 weeks following surgery to assess the effects on cardiac function and post-ischemic inflammation, resulting in the following groups: MI (n = 16), MI-R (n = 15), and sham (n = 13).LV dimensions and function were serially assessed 2 days and 3 weeks after surgery with cardiac MRI by using a 7-T MRI  equipped with a combined gradient and shim coil to obtain contrast-enhanced and cine MRI images. Initial infarct size was determined at day 2 to distinguish for any possible existing effect. Effects related to both infarction models regarding infarct size were determined by repetition of contrast-enhanced MRI after 3 weeks. LV dimensions were measured after 2 days and 3 weeks and cardiac function was determined. Image reconstruction was performed using Bruker ParaVision 5.1 software.LV endo- and epicardial borders were delineated manually with the MR Analytical Software System (MASS) for mice . End-diastolic and end-systolic phases and the contrast enhanced areas were identified automatically, and the percentage of infarcted myocardium, LV-EDV, LV-ESV, LV-EF, and LV stroke volume (SV) were computed.To study the systemic effects whole blood was analyzed for circulating inflammatory cells at day 2. Hematological values were obtained using a semi-automatic hematology analyzer F-820 . For FACS analysis, whole blood was incubated on ice with directly conjugated antibodies directed against Ly-6C-FITC , Ly-6G-PE , CD11b-APC , CD115-PerCP , and CD45R-APC-Cy7 .After 3 weeks mice were euthanized and blood samples were collected for analysis. Subsequently, the heart and lungs were quickly excised. Hearts were weighted as an indication of congestive heart failure and immersion-fixated and embedded in paraffin.Sirius Red staining was used to determine LV fibrous content, as a measure of infarct size, and LV wall thickness. All measurements were performed using the ImageJ2 × 2.1.4.5 O software program . For analysis of the cardiac inflammatory response sections were stained using antibodies against leukocytes .t tests. A value of p < 0.05 was considered to represent a significant difference. Statistical procedures were performed using IBM SPSS 26.0  and GraphPad Prism 8.0  also used for the representation of figures.Values were expressed as mean ± SEM. Comparisons of parameters between the MI, MI-R, and sham groups were made using 1-way analysis of variance (ANOVA) with Tukey’s correction or 2-way ANOVA with repeated measures and Tukey’s post-test in case of multiple time points. Comparisons between MI and MI-R were made using unpaired Supplementary Information 1.


In an analysis stratified by setting, the highest mortality risk benefit was observed in case of acute myocardial infarction: RR 0.75; 95%CI 0.68–0.83; p < 0.0001; I2 = 0%. There were no apparent differences between two techniques in terms of complications. In heterogeneous populations of critically ill adults in CS and supported with VA-ECMO, the adjunct of LV unloading is associated with lower early mortality and higher rate of weaning.During veno-arterial extracorporeal membrane oxygenation (VA-ECMO), the increase of left ventricular (LV) afterload can potentially increase the LV stress, exacerbate myocardial ischemia and delay recovery from cardiogenic shock (CS). Several strategies of LV unloading have been proposed. Systematic review and meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement included adult patients from studies published between January 2000 and March 2019. The search was conducted through numerous databases. Overall, from 62 papers, 7581 patients were included, among whom 3337 (44.0%) received LV unloading concomitant to VA-ECMO. Overall, in-hospital mortality was 58.9% (4466/7581). A concomitant strategy of LV unloading as compared to ECMO alone was associated with 12% lower mortality risk (RR 0.88; 95% CI 0.82–0.93;  Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) provides life support for patients with refractory cardiogenic shock and significantly improves their survival working as a bridge to either recovery or other long-term treatments ,6,7,8,9.A well recognized limitation of the retrograde aortic flow while on VA-ECMO is the increase of left ventricular (LV) afterload , which cSeveral LV unloading strategies have been described and proposed in order to minimize the risk of these complications , howeverThe aim of this study was to comprehensively assess the impact on early outcomes of different strategies of LV unloading in patients undergoing VA-ECMO and sustaining advanced cardiogenic shock by various etiologies.This systematic review and meta-analysis was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . The PRIHuman studies were included if they assessed survival after VA-ECMO or weaning from VA-ECMO support instituted for refractory cardiogenic shock. Research centers were checked to avoid potential overlapping patients and those reporting on smaller samples of patients were excluded. Reviews and case reports were not considered. Two independent reviewers (M.K. and K.Z.) selected the studies for inclusion, extracted studies, as well as patient characteristics of interest and relevant outcomes. Two authors (M.K. and K.Z.) independently assessed the trials’ eligibility and risk of bias. Risk of bias at the individual study level was assessed using the Risk of Bias in Not-randomized Studies-of Interventions (ROBINS-I) tool . Any divThe primary endpoints were in-hospital/30-day survival and weaning from VA-ECMO. Secondary endpoints were in-hospital cerebrovascular events (CVE), brain death, limb complications, reoperation for bleeding, sepsis and acute kidney failure w/wo continuous veno-venous hemofiltration (CVVH). Outcome definitions were the ones adopted by the investigators of the included studies.I2 test. To control for the anticipated heterogeneity among observational studies, absolute values and means were pooled using inverse variance random effects models. The primary endpoints were assessed in relation to the specific setting according to etiology of cardiogenic shock which included: (1) postcardiotomy shock (PCS), (2) acute myocardial infarction (AMI), (3) myocarditis and (4) mixed cohort of different etiologies including both postcardiotomy shock and AMI and other etiologies. Number needed to treat (NNT) was calculated for these subgroups. Secondary analysis focused on specific left ventricular unloading strategy: (1) intra-aortic balloon pump (IABP), (2) LV venting with cannula in left atrium or ventricle or (3) percutaneous ventricular assist device . We performed separate analysis of studies with propensity score matching or presenting propensity score adjusted odds ratio (OR) of primary endpoints. We investigated if use of different unloading strategies had influence on complication rates, ECMO duration and weaning rates by means of meta-regression analyses [Statistical analyses were performed in Comprehensive Meta-Analysis, v. 2.0  and Review Manager 5.3 . The results are expressed as pooled untransformed proportion risk ratios (RR) with their 95% confidence intervals (CI). Heterogeneity across studies was evaluated using the analyses . Similaranalyses .The study selection process and reasons for exclusion of some studies are described in Patients undergoing VA-ECMO had a mean age of 57.8 years and 71.0% were male. Follow-up across the studies varied between 30-day and in-hospital survival. Populations included patients on VA-ECMO support for cardiogenic shock secondary to mixed etiologies , PCS  and AMI ; VA-ECMO was employed for myocarditis in 4 series enrolling 103 patients.p < 0.0001; I2 = 40%; All 63 included series (7581) contributed to the analysis of overall mortality; constructed funnel plot did not reveal any signs of publication bias or big study effect : overallp < 0.00001; I2 = 0%; NNT = 15. A mortality risk benefit of 11% was demonstrated in studies including mixed indication for LV unloading + ECMO: RR (95%CIs): 0.90 (0.81–1.00); p = 0.04; I2 = 48%; NNT = 11; In patients with postcardiotomy cardiogenic shock, LV unloading on top of ECMO was associated with 7% non-significantly lower mortality risk; RR (95%CIs): 0.93 (0.85–1.01); p = 0.09; I2 = 29%; NNT = 125. No differences were seen between LV-unloading + ECMO as compared to ECMO alone in patients with myocarditis; NNt = 9. Significant statistical differences as of extent of benefit were demonstrated between subgroups (pinteraction = 0.01). No impact on early mortality was found according to the type of cannulation, peripheral and central, in a meta-regression analysis, The highest mortality risk benefit (25%) was observed in the subgroup of patients undergoing LV unloading + ECMO for AMI: RR (95%CIs): 0.75 (0.67–0.83); p < 0.00001; I2 = 38%: weaning was possible in 60.4% (1789/2964) of included patients with corresponding rates of 75.3% (821/1090) and 51.7% (968/1874) for LV unloading + ECMO and ECMO alone; p = 0.05; I2 = 0%. Differences between subgroups were not statistically significant.Seventeen studies with nearly 3000 patients reported on weaning rates in subsets receiving LV unloading + ECMO as compared to ECMO therapy alone. In the overall analysis, LV unloading was associated with 35% higher probability of weaning from ECMO: RR (95%CIs): 1.35 (1.21–1.51); p = 0.92; I2 = 0%); p = 0.66; I2 = 7%; p = 0.50; I2 = 0% : 1.27 (0.14–1.42); p < 0.0001; I2 = 32%); p = 0.20; I2 = 41%. Additionally, Impella device was independently associated with higher chance of weaning from ECMO: RR (95%CIs): 1.65 (1.05–2.59); p = 0.03; I2 = 74%  reported on LV unloading by direct LV venting catheters: a statistical trend of 32% reduced mortality risk was demonstrated for ECMO + LV venting as compared to ECMO alone: RR (95%CIs): 0.68 (0.45–1.03); I2 = 74% .p = 0.004; I2 = 42%; Analyses were repeated as sensitivity for primary endpoints mortality and weaning from ECMO this time included only studies that reported effect estimates for propensity matched cohorts only: 5 studies  providedp < 0.001; I2 = 0%; Weaning rates for comparison LV unloading + ECMO and ECMO alone adjusted for propensity were reported in 4 studies ; again, Sensitivity analyses performed by deleting each study, one at a time, and repeating the calculations did not change the direction nor magnitude of the treatment effect, suggesting absence of big-study effect.VA-ECMO is an established treatment able to provide a mechanical circulatory support for patients in cardiogenic shock, aiming a bridge to decision or to myocardial recovery ,6,7,8,9.ECLS institution increases the left ventricle afterload with a rise in LV end-systolic volume and reduction in LV stroke volume. If peripheral resistance and LV contractility are fixed, increase in LV end-diastolic volume is the only way to overcome the afterload via the Frank–Starling mechanism. In this case, higher levels of VA-ECMO flow cause a progressive rise in LV end-diastolic pressure, LA pressure, pulmonary capillary wedge pressure, that are associated with a further reduced LV stroke volume ,27,28. HIf the overload is extreme and LV contractile impairment significant, the LV is unable to provide a sufficient flow against the increased afterload and the aortic valve may remain closed even during systole, causing blood stasis in the left ventricle, left atrium and aorta, and accounting for intracardiac thrombosis which has been reported in up to 6% of the cases ,31. The Definition of LV distension during VA-ECMO is lacking in the literature. Truby et al.  attempt These differences in definitions and assessments may account for the high variability of LV distension rate in the literature. Camboni et al.  reportedDrugs administration is the first line treatment of left ventricle distension. Inotropes can be administered to increase LV contractility while vasodilators may reduce the peripheral resistances and decrease left ventricle afterload. A careful fluid balance (diuretics/fluid restriction) avoiding fluid overload can reduce the risk of pulmonary edema. Ventilatory optimization, including higher PEEP, prolonged expiration time and lower tidal volume, may further improve the venous drainage.When medical treatment is not successful, the non-pharmacological management of LV distension, acting with a “direct” or indirect” mechanism, can be obtained through a surgical or percutaneous strategy .IABP has been the most used technique to unload the left ventricle during ECMO support . The IABThe use of Impella in combination with VA-ECMO  has been shown to provide improved weaning and survival rates compared to ECMO alone strategy and to established risks scores ,48,49,50®). Direct left ventricle unloading can be also achieved or by a surgical cannulation of the ventricle apex [Other unloading strategies have been reported in the literature and address the endpoints of this meta-analysis . Brieflycle apex ,57 and tcle apex ,58 or pecle apex ,57, incrHemodynamic responses to ECMO are different among patients and are affected by clinical presentation, associated comorbidities and the cardiovascular system coupling. This high variability may explain the difficulties in driving robust conclusions in terms of efficacy and safety of LV unloading during VA-ECMO.p = 0.10). IABP was not associated with improved survival in AMI patients , PCS  when placed prior to ECMO initiation , or when routinely inserted . Recently, Russo et al. [p < 0.00001). Bleeding, limb ischemia, renal replacement therapy, multiorgan failure and stroke or transient ischemic attack were not demonstrably different in patients treated with VA-ECMO with versus without left ventricular unloading. Hemolysis was the only secondary outcome higher in patients who underwent VA-ECMO with left ventricular unloading .Up to date and to the best of our knowledge other two meta-analysis have been published on LV unloading strategy ,37. In 2As analysis of only non-randomized studies, our analysis shared similar limitations with these reports which included experiences with small populations and lacked some critical information about the timing of ECMO institution, the timing of LV unloading adjunct, or the weaning protocols. Most importantly, none of the studies report exact criteria for therapy escalation e.g., addition of IABP or Impella device to ECMO. In addition, observational nature of these studies promotes selection bias. However, compared to previous meta-analyses, that present certain methodological flaws , the current study, including 62 studies and more than 7500 patients, represents the first comprehensive approach addressing LV unloading strategies during ECMO support.We found that, regardless the strategy  and the etiology , and suggests, within the limitations of this analysis, a more pronounced positive impact of LV unloading in the peripheral VA-ECMO setting.The analysis of weaning, additionally included as a sensitivity analysis, might give presumptive underlying evidence of true reasons for improved survival after VA-ECMO support. The possibility of providing an adequate oxygen delivery associated with the reduction of myocardial injury and the relief of pulmonary congestion, thus enhancing arterial oxygenation and reducing pulmonary complications, may explain the higher rate of survival in patients who received an adjunct treatment able to prevent or solve left ventricular distension during VA-ECMO support.During veno-arterial extracorporeal membrane oxygenation, the increase of left ventricular afterload can negatively impact the recovery from cardiogenic shock. In this meta-analysis including 7581 patients on VA-ECMO support, the adjunct of left ventricular unloading was associated with 35% higher probability of weaning and 12% lower risk of mortality.


Coronary heart disease is a serious cardiovascular disease. There is coronary atherosclerosis, resulting in lumen stenosis, blockage, and then the symptoms of insufficient blood supply and hypoxia in the myocardium. Chronic heart failure is a kind of syndrome with abnormal ventricular filling and ejection function, which is the final stage of the development of coronary heart disease. At present, the treatment plan of Western medicine can significantly reduce the hospitalization rate, but it is still not satisfactory for the prognosis and mortality of patients. Shenfu injection has advantages in the treatment of heart failure in patients with coronary heart disease, but there is a lack of standard clinical studies to verify it, so the purpose of this randomized controlled study is to evaluate the efficacy and safety of Shenfu injection combined with sodium nitroprusside in the treatment of chronic heart failure in patients with coronary heart disease.This is a prospective randomized controlled trial to study the efficacy and safety of Shenfu injection combined with sodium nitroprusside in the treatment of chronic heart failure in patients with coronary heart disease. The patients will be randomly divided into a treatment group and the control group according to 1:1, in which the treatment group is treated with Shenfu injection combined with sodium nitroprusside, and the control group is treated with sodium nitroprusside alone. Both groups will be treated with standard treatment for 7 days and followed up for 30 days to pay attention to their efficacy and safety indexes. The observation indexes include TCM syndrome score, N-terminal pro-brain natriuretic peptide, left ventricular ejection fraction, brain natriuretic peptide, left ventricular end-systolic diameter, left ventricular end-diastolic diameter, stroke volume, adverse reactions and so on. We will use SPSS 25.0 software for data analysis.This study will evaluate the efficacy and safety of Shenfu injection combined with sodium nitroprusside in the treatment of chronic heart failure in patients with coronary heart disease. The results of this experiment will provide a clinical basis for Shenfu injection combined with sodium nitroprusside in the treatment of chronic heart failure in coronary heart disease.DOI 10.17605/OSF.IO/4KNG3 The European Society of Cardiology found that there are more than 15 million patients with heart failure for every 1 billion people, with a total prevalence rate of 2% to 3% in the general population and 10% to 20% in people aged 70 to 80.Chronic heart failure is characterized by abnormal cardiac structure and function, which lead to the decrease of ventricular ejection fraction and ventricular filling, which cannot meet the metabolic needs of body tissue. It is a critical cardiac disease and the main cause of death in heart patients. Sodium nitroprusside is a vasoactive drug, which can effectively dilate small arteries and veins, selectively relax vascular smooth muscle and reduce peripheral vascular resistance, temporarily change hemodynamic variables, reduce blood pressure, relieve cardiac load, and increase cardiac output. However, long-term use of nitric oxide donor sodium nitroprusside can lead to outflow tissue damage through protein nitrification, resulting in an increase in intraocular pressure, and drug withdrawal cannot recover.Sodium nitroprusside has a certain value in improving the prognosis of patients with chronic heart failure.,8 According to the theory of traditional Chinese medicine, the main pathogenesis of chronic heart failure is yang-deficiency of heart and kidney. Shenfu injection is made from ginseng and aconite extract. Modern medical research has found that Shenfu injection can reduce peripheral vascular resistance, improve microcirculation, protect cardiomyocytes, and protect vascular endothelium. At the same time, Shenfu injection has small clinical adverse reactions and a safe wide range of dose. Therefore, we intend to use this randomized controlled trial to evaluate the efficacy and safety of Shenfu injection combined with sodium nitroprusside in the treatment of chronic heart failure in patients with coronary heart disease.Traditional Chinese medicine in the treatment of chronic heart failure in patients with coronary heart disease can effectively alleviate the symptoms of heart failure and improve the quality of life of patients.22.1This is a prospective randomized controlled trial to study the efficacy and safety of Shenfu injection combined with sodium nitroprusside in the treatment of coronary heart disease with chronic heart failure. This trail will follow the comprehensive test reporting standards, and the flow chart is shown in Fig. 2.2This research scheme is in line with the Helsinki Declaration and approved by the Clinical Research Ethics Committee of our hospital. This research scheme has been registered on OSF (registration number: DOI 10.17605/OSF.IO/4KNG3). Before being randomly divided into groups, all patients need to sign a written informed consent form, and they are free to choose whether to continue the trial at any time.2.3 Doppler ultrasound indicates that the left ventricular ejection fraction is less than 50%, and the condition was in stable stage; age 40 to 79 years old; all patients signed consent.Inclusion criteria: meet the diagnostic criteria of coronary heart disease, refer to the 2017 Chinese guidelines for Prevention and treatment of Cardiovascular Diseases; meet the diagnostic criteria of chronic heart failure,Exclusion criteria: patients with other serious cardiovascular diseases; patients with other serious diseases such as malignant tumors; patients with severe mental illness; patients who are allergic to or have contraindications to medication in this study; unable to understand the study plan or unwilling to participate after explanation.2.4α = 1.96, 1−β = 0.9, μβ = 1.28. δ = π1−π2 sample size estimation formula for comparison of 2 sample rates:This study was a pilot clinical trial. According to the analysis and calculation of the related literature published in the past 10 years, the therapeutic efficiency of Western medicine is 72.3%, and the effective rate of traditional Chinese medicine combined with Western medicine is 92.1%. Using the sample size estimation formula compared with multiple sample rates, set the significance test level α = 0.05, then μThe sample size of each group is calculated to be 73. Considering shedding and other factors, the expansion is about 20%, and it is calculated that 182 patients need to be included. According to the simple randomized method, the patients included in the study are divided into control group (n = 91) and the observation group (n = 91).2.5In this study, through prehospital recruitment, in-hospital inpatients to screen patients who meet the criteria, patients and their families approve the study program and sign the informed consent form. The patients will be randomly assigned to the treatment group and the control group. Among them, the treatment group will receive Shenfu injection  combined with intravenous drip of sodium nitroprusside (sodium nitroprusside 3 μ g / kg/min), while the control group will only receive intravenous drip of sodium nitroprusside. Patients in both groups will receive the same routine care and drug treatment, such as angiotensin converting enzyme inhibitor/angiotensin II receptor blocker, beta blocker, diuretic, etc. If necessary, the attending physician can adjust the program according to the patient's condition, and all interventions will be recorded in detail for final result analysis. We will set up a special drug manager, and the nurse will be responsible for preparing the medicine and preparing the solution. Therefore, the study is not blind to nurses, but to researchers, patients, and statisticians. All patients will receive an intravenous injection once a week. The health status of each patient is evaluated before and after treatment, including efficacy and safety indicators, and all patients are followed up by telephone for 30 days. Follow-up included cardiovascular events and rehospitalization.2.61.), N-terminal pro-brain natriuretic peptide (NT-pro BNP), left ventricular ejection fraction, brain natriuretic peptide (BNP).Main outcome indicators: TCM syndrome score , left ventricular end-systolic diameter, left ventricular end-diastolic diameter.3.Adverse reactions: including abnormal liver function and uncomfortable symptoms  during treatment.2.7The data are collected according to the evaluation criteria before the beginning of treatment, 72 hours after the treatment and at the end of treatment. Thirty days after the end of treatment, each patient is followed up by outpatient or telephone. The reasons for the loss of follow-up cannot be collected. All data will be collected jointly by 1 or 2 assistants. Personal information about potential participants and registered participants will be collected, shared, and stored in a separate storeroom to protect before, during, and after confidentiality. The access to the database is limited to the researchers of this research group.2.8t test is used between groups, paired t test is used within groups, rank sum test is used for those who do not conform to normal distribution, and χ2 test is used for counting data. When P < 0.05, it is statistically significant.In this study, SPSS25.0 statistical analysis software is used for data analysis, and the measurement data are expressed by (3 In the increasingly severe trend of population aging in China, the incidence rate is increasing. The proportion of patients with chronic heart failure caused by coronary heart disease is about 46.8%, with high morbidity and mortality. Its main manifestation is the decrease of ventricular filling and pumping function. At present, Western medicine is mainly treated with cardiotonic drugs, diuretics, and vasodilators, which can reduce the hospitalization rate and risk factors to a certain extent. But it still cannot achieve the ideal effect. In recent years, traditional Chinese medicine has accumulated rich experience in the treatment of chronic heart failure, and the effect is remarkable, which can significantly improve the symptoms of patients and delay the development of the disease. Most patients with heart disease have sleep disorders. A study shows that massage and aromatic massage can improve the sleep quality of these patients. The combination of traditional Chinese and Western medicine in the treatment of chronic heart failure of coronary heart disease has become a trend of clinical treatment.Chronic heart failure is a common manifestation of coronary heart disease and an end-stage manifestation of all heart diseases. Ginsenosides, the main active ingredient in ginseng, account for 4% of the total ginseng, which can improve the immunity of the body; the active ingredients of aconite have cardiotonic and antiarrhythmic effects; and the combination of the 2 drugs can protect blood vessels and heart. Myocardial ischemia-reperfusion in patients with coronary heart disease can cause aseptic inflammation. The participation of inflammatory mediators will lead to persistent injury of cardiomyocytes, and toll-like receptors are the receptors of inflammatory pattern recognition.–29 Shenfu injection can significantly reduce the recognition of Toll-like receptor inflammation and the expression of inflammatory factors, reduce cardiomyocyte injury, improve cardiac function, and less adverse clinical reactions.Ginseng and aconite, the main components of Shenfu injection, have the effect of restoring yang and saving adversity, replenishing qi, and relieving detoxification, which has been significantly effective in the treatment of heart failure of coronary heart disease for a long time, and its protective effect on heart has been proved.The purpose of this randomized controlled trial is to verify that Shenfu injection can significantly improve the clinical symptoms and cardiac function in patients with chronic heart failure in patients with coronary heart disease. Since there is no standard clinical study to evaluate the efficacy of Shenfu injection combined with sodium nitroprusside in patients with heart failure of coronary heart disease, we intend to evaluate its efficacy and safety through prospective randomized controlled trials.This study also has some limitations: due to the short planned follow-up time, we are unable to understand the impact of long-term results, so we may extend the follow-up time if necessary. And due to the influence of treatment, this study cannot achieve strict double-blindness, which may affect the results to a certain extent.Data collection: Binbin Guo and Tian YangFunding acquisition: Wenpeng Xu.Funding support: Wenpeng XuInvestigation: Tian Yang and Jinniang NanResources: Jinniang Nan and Qianghui HuangSoftware operating: Qianghui HuangSoftware: Qianghui Huang.Supervision: Chenxiu Wang and Wenpeng XuWriting – original draft: Binbin Guo and Tian YangWriting – review & editing: Binbin Guo and Wenpeng Xu