Application of OMD (osteomodulatory protein) in ejection fraction reduction type heart failure

Abstract

The invention discloses an application of OMD (osteomodulatory protein) in ejection fraction reduction type heart failure, which reveals that the OMD can effectively relieve cardiac dysfunction, cardiac hypertrophy and myocardial fibrosis caused by aortic arch constriction for the first time through experiments, and has a remarkable treatment effect on the ejection fraction reduction type heart failure. The discovery indicates that the OMD has a huge potential of being developed into a recombinant protein treatment drug for resisting ejection fraction reduction type heart failure. It is similarly worthy of mentioning that it is found that the OMD can serve as a novel biomarker of the ejection fraction reduction type heart failure for the first time, and a new means is provided for diagnosis of related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of osteomodulin OMD in heart failure with reduced ejection fraction. Background Art

[0002] Heart failure is a group of complex clinical syndromes caused by abnormal changes in cardiac structure and / or function due to various reasons, resulting in disorders of ventricular systolic and / or diastolic function. It is a serious manifestation or late stage of various heart diseases, with high mortality and re-hospitalization rates.

[0003] The 2024 edition of the Guidelines for the Diagnosis and Treatment of Heart Failure in China divides heart failure into four categories based on left ventricular ejection fraction (LVEF): heart failure with preserved ejection fraction (HFpEF) (LVEF ≥ 50%), heart failure with mildly reduced ejection fraction (HFmrEF) (LVEF 41%-49%), and heart failure with improved ejection fraction (HFimpEF): previous LVEF ≤ 40%, but after treatment LVEF > 40% and increased by ≥ 10% from baseline and heart failure with reduced ejection fraction (HFrEF) (LVEF ≤ 40%). The 2024 edition of the Guidelines for the Diagnosis and Treatment of Heart Failure in China officially recommends the new quadruple therapy (ARNI / ACEI / ARB, beta-blocker, mineralocorticoid receptor antagonist (MRA) and sodium-glucose co-transporter 2 inhibitor (SGLT2i)) as the basic treatment for HFrEF patients, and points out that quadruple drug therapy can reduce the all-cause mortality of HFrEF patients by 73%. The DAPA-HF study showed that despite receiving quadruple therapy, 14.3% of patients still experienced a primary outcome event during the trial (N Engl J Med. 2019; 381(21): 1995-2008.). New drugs or new treatment options are still needed to further improve patient prognosis and quality of life.

[0004] The diagnosis and evaluation of heart failure rely on medical history, physical examination, laboratory tests, cardiac imaging and functional tests. Clinical blood markers have become an indispensable and important tool in the diagnosis and treatment of heart failure due to their advantages of improving diagnostic accuracy, assisting in efficacy evaluation, and being easy to detect and operate. Plasma natriuretic peptides (B-type natriuretic peptide (BNP) or N-terminal pro-BNP (NT-proBNP)) are widely used for screening, diagnosis, severity and prognosis of heart failure. However, in addition to heart failure, a variety of cardiovascular factors / diseases (acute coronary syndrome, cardiomyopathy, valvular heart disease, pericardial disease, atrial fibrillation, myocarditis, cardiac surgery, cardioversion, myocardial toxicity, etc.) and non-cardiovascular factors / diseases (old age, anemia, renal insufficiency, sleep apnea-hypopnea syndrome, severe pneumonia, pulmonary hypertension, pulmonary embolism, sepsis, severe burns and stroke, etc.) can lead to increased natriuretic peptide levels, which is a major limitation of this indicator. Currently, biomarkers including soluble growth stimulation expressed gene 2 protein (sST2), galectin 3 and growth differentiation factor 15 are constantly emerging, which are helpful for risk stratification and prognosis assessment of patients with heart failure. Therefore, the exploration of new blood markers has important potential.

[0005] The extracellular matrix (ECM) of myocardial cells not only provides support for myocardial cells, but also affects cell replication, growth, death and other life activities, and works with myocardial cells to maintain the integrity of cardiac structure and function. The largest proteomic study of heart failure to date found that in addition to fibrillar collagen, interstitial proteoglycans were also deposited in ischemic heart failure tissues, and members of the small leucine-rich proteoglycan (SLPRs) family were the main components (Circulation 2021; 144: 2021-34.). SLRPs belong to one of the proteoglycan families and are a unique class of proteins located in the extracellular matrix. They are covalently linked to one or more glycosaminoglycans (GAGs) and a core protein. Its main feature is that it has leucine-rich repeat sequences. The family has 17 members, which are divided into 5 subfamilies based on conservation and homology at the protein and genomic levels, cysteine-rich regions, etc. (International journal of molecular sciences 2021; 22.). Among the matrix proteins of the SLRPs family, fibromodulin (Fmod) and biglycan (Bgn) have been directly reported in heart failure (PloS one 2018; 13: e0201422., Connective tissue research 2008; 49: 129-32.). However, there is no clear report on the relationship between osteomodulin (OMD) and heart failure. Osteomodulin OMD is mainly present in the extracellular matrix of mineral tissues including bones and teeth (Bone 2000; 27: 265-70.). An unbiased plasma proteome analysis of a recent cohort study related to cardiovascular disease showed that osteomodulin OMD is a potential circulating biomarker associated with cardiovascular risk factors and type 2 diabetes (Circulation 2016; 134: 270-85., Diabetescare 2020; 43: 843-51.). Recently, researchers have also reported that the levels of osteomodulin OMD in the circulation and tissues are consistent with vascular calcification in the body, and emphasized the potential of osteomodulin OMD as a clinical biomarker (Clinical and translational medicine 2022; 12: e682.). Therefore, the correlation between osteomodulin OMD and the occurrence and development of heart failure with reduced ejection fraction and its potential as a diagnostic and treatment indicator are worth exploring. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention aims to provide an application of osteomodulin OMD in heart failure with reduced ejection fraction.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides a marker for detecting heart failure with reduced ejection fraction, wherein the marker comprises osteopontin OMD.

[0009] The present invention also provides an application of a marker in preparing a kit for detecting heart failure with reduced ejection fraction, wherein the marker comprises osteomodulin OMD.

[0010] The present invention also provides a kit for detecting heart failure with reduced ejection fraction, wherein the kit comprises a reagent for detecting osteomodulin OMD.

[0011] The present invention also provides a medicine for treating heart failure with reduced ejection fraction, wherein the medicine comprises osteomodulin OMD recombinant protein.

[0012] The present invention also provides an application of osteomodulin OMD in preparing a medicine for treating heart failure with reduced ejection fraction.

[0013] The beneficial effect of the present invention is that the present invention reveals for the first time through experiments that osteomodulin OMD can effectively alleviate abnormal cardiac function, cardiac hypertrophy and myocardial fibrosis caused by aortic arch coarctation, and has a significant therapeutic effect on heart failure with reduced ejection fraction. This discovery indicates that osteomodulin OMD has great potential to be developed into a recombinant protein therapeutic drug for heart failure with reduced ejection fraction. It is also worth mentioning that the present invention discovered for the first time that osteomodulin OMD can be used as a new biomarker for heart failure with reduced ejection fraction, providing a new means for the diagnosis of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the results of detecting the expression of OMD in the heart and serum of mice with reduced ejection fraction caused by aortic constriction (TAC) in an embodiment of the present invention;

[0015] Figure 2 This is a schematic diagram of the protective effect of the OMD recombinant protein in the present invention on abnormal cardiac function, cardiac hypertrophy and myocardial fibrosis in a mouse model of heart failure with reduced ejection fraction induced by TAC modeling;

[0016] Figure 3 This is a schematic diagram of the protective effect of the OMD recombinant protein on the hypertrophy of primary cardiomyocytes of neonatal rats induced by ISO stimulation in an example of the present invention;

[0017] Figure 4 This is a schematic diagram of the diagnostic value of serum OMD levels in detecting human heart failure with reduced ejection fraction in an embodiment of the present invention. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with the accompanying drawings. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to this embodiment.

[0019] This example aims to verify the therapeutic and detection effects of osteomodulin OMD on heart failure with reduced ejection fraction through experiments.

[0020] 1. Establishing a mouse model of heart failure with reduced ejection fraction caused by aortic arch constriction (TAC) is a commonly used model for studying heart failure after pressure overload.

[0021] Ten-week-old male C57B6 / L mice were used to construct the model group and the sham operation group (Sham).

[0022] The model group was constructed, and mice were anesthetized with 1.5% isoflurane, fixed on a thermostatic pad, and then connected to a small animal ventilator via an endotracheal tube. The chest cavity was opened in front of the mouse sternum to expose the aortic arch. A 6-0 nylon suture was inserted between the left common carotid artery and the brachiocephalic trunk and passed through the aortic arch. Subsequently, a 27G constriction needle was placed near the blood vessel and ligated with a nylon suture. The constriction needle was immediately removed, the chest was closed and sutured. The incision site was disinfected, and ventilation was stopped after the mouse resumed spontaneous breathing.

[0023] A sham operation group (Sham) was constructed as a control group, in which a 6-0 nylon suture was passed through the aortic arch without ligation, the suture was pulled out, the chest was closed and sutured. The incision site was disinfected, and ventilation was stopped after the mice recovered spontaneous breathing.

[0024] The model group was treated with aortic constriction (TAC) for 4 weeks, which resulted in heart failure with reduced ejection fraction. After 4 weeks, the apical tissues of the model group and the sham group were collected, with 3 mice in each group. Then, transcriptome sequencing analysis was performed on the collected apical tissues, and it was found that compared with the sham group, the top three mRNA levels in the model group that increased most significantly were Fmod, Bgn, and Omd, such as Figure 1 As shown in A. Figure 1 In FIG. 1 , A is a volcano map of genes with significant differences in transcriptome sequencing results of apical tissues of the Sham group and the model group treated with TAC for 4 weeks. This example mainly focuses on OMD.

[0025] Furthermore, RT-qPCR and Western Blot were used to detect the mRNA and protein levels of Omd gene in the heart tissue of another batch of model group treated with TAC for 4 weeks and its corresponding Sham group. It was found that the mRNA and protein levels of Omd gene in the myocardial tissue of mice in the model group were significantly increased, such as Figure 1 As shown in BD. Figure 1 In the figure, B shows the difference in mRNA level of Omd gene in cardiac tissue between Sham group and model group treated with TAC for 4 weeks, C shows the difference in protein level of Omd in cardiac tissue between Sham group and model group treated with TAC for 4 weeks, and D shows the statistical graph of C.

[0026] Furthermore, the mouse osteomodulin detection kit (SEKM-0248) of Beijing Solebow Technology Co., Ltd. was used to detect the content of OMD in the serum of the model group treated with TAC for 4 weeks and its corresponding Sham group. It was found that the serum OMD expression level of the model group was increased, such as Figure 1 As shown in E. Figure 1 In (E), the expression difference of OMD in serum between the Sham group and the model group treated with TAC for 4 weeks.

[0027] 2. The following animal groups were set up: 10-week-old male C57B6 / L mice were randomly divided into Sham group (n=6), TAC model group (n=6), and TAC+OMD model group (n=6), where n is the number of mice in each group. OMD recombinant protein (10825-H08H) was purchased from Beijing Sino Biological Technology Co., Ltd.

[0028] The modeling process of the sham group was as follows: the construction process was the same as that of the sham group in the aforementioned experiment 1, and physiological saline was infused through the tail vein starting from the third week after the construction (200ul / 2day / mouse);

[0029] The modeling process of the TAC modeling group was as follows: the construction process was the same as that of the model group in the aforementioned experiment 1, and physiological saline was infused through the tail vein starting from the third week after the construction (200ul / 2day / mouse);

[0030] The modeling process of the TAC+OMD modeling group was as follows: the construction process was the same as that of the model group in the aforementioned experiment 1, and OMD recombinant protein (3 μg / 2day / mouse) was infused through the tail vein starting from the third week after the construction was completed;

[0031] The conditions of the mice were observed and recorded every 3 days during the modeling period, and the mice were killed after 4 weeks.

[0032] One day before the mice were killed, the heart function of the mice was detected using an ultra-high resolution small animal ultrasound imaging system, and it was found that the exogenous recombinant protein OMD could alleviate TAC-induced heart dysfunction in mice. Figure 2 As shown in A and B. Figure 2In the figure, A shows the echocardiograms of mice in the Sham group, TAC model group and TAC+OMD model group at the end point of modeling, and B shows the left ventricular ejection fractions of mice in the Sham group, TAC model group and TAC+OMD model group at the end point of modeling.

[0033] After the mice were killed, serum and heart tissue were collected, and the heart weight and left lower limb tibia length were measured. It was found that exogenous OMD recombinant protein could downregulate the heart weight / tibia length ratio of mice, such as Figure 2 As shown in C.

[0034] The heart tissue was embedded in paraffin and the size of cardiomyocytes was detected by H&E staining. It was found that exogenous OMD recombinant protein could alleviate the cardiomyocyte hypertrophy caused by TAC modeling. Figure 2 As shown in D and E.

[0035] Masson staining was used to detect cardiac interstitial fibrosis, and it was found that exogenous OMD recombinant protein could significantly alleviate cardiac interstitial fibrosis in mice induced by TAC modeling. Figure 2 As shown in F and G.

[0036] Total protein was extracted from mouse heart tissue homogenate, and protein expression levels of β-MyHC, ANP, and Col lagen I (COL-1) were detected by Western blot. It was found that exogenous OMD recombinant protein could alleviate myocardial hypertrophy and fibrosis caused by TAC modeling. Figure 2 As shown in H.

[0037] RNA was extracted from mouse heart tissue homogenate, and the mRNA levels of Myh7, Nppa, and Co1a1 were detected by RT-qPCR. It was found that exogenous OMD recombinant protein could alleviate myocardial hypertrophy and fibrosis caused by TAC modeling. Figure 2 As shown in I.

[0038] Figure 2In the figure, A is the echocardiogram of mice in Sham group, TAC model group and TAC+OMD model group, B is the ejection fraction of mice in Sham group, TAC model group and TAC+OMD model group, C is the heart weight / tibia length ratio of mice in Sham group, TAC model group and TAC+OMD model group, D is the H&E staining of cardiac paraffin sections of mice in Sham group, TAC model group and TAC+OMD model group, E is the statistical graph of D, F is the Masson staining of cardiac paraffin sections of mice in Sham group, TAC model group and TAC+OMD model group, G is the statistical graph of F, H is the protein expression levels of β-MyHC, ANP and COL-1 in the heart tissue of mice in Sham group, TAC model group and TAC+OMD model group, I is the mRNA levels of Myh7, Nppa and Co1a1 in the heart tissue of mice in Sham group, TAC model group and TAC+OMD model group.

[0039] 3. Set up the following cell groups: Ctrl group, Ang II model group, low-dose OMD+Ang II model group, and high-dose OMD+Ang II model group.

[0040] Isolation and culture of neonatal rat cardiomyocytes: Heart tissue from newborn Sprague-Dawley (SD) rats aged 0 to 2 days was obtained and separated using trypsin. Primary cardiomyocytes were separated from fibroblasts by differential adherence culture. The cell suspension was inoculated in a culture dish containing DMEM high-glucose medium containing 10% fetal bovine serum and allowed to stand for 1 hour. At this time, the cells attached to the wall were mainly fibroblasts. Subsequently, the non-adherent cells, i.e., primary cardiomyocytes, were collected, counted, and inoculated on the culture plate.

[0041] The modeling process of the Ctrl group was as follows: after culturing neonatal rat primary cardiomyocytes for 24 hours, sterile water was added and incubated for another 24 hours;

[0042] The modeling process of the Ang II modeling group was as follows: after culturing primary cardiomyocytes of neonatal rats for 24 hours, they were stimulated with Ang II (1uM) for 24 hours;

[0043] The modeling process of the low-dose OMD+Ang II modeling group was as follows: neonatal rat primary cardiomyocytes were cultured for 24 hours, recombinant protein OMD (50 ng / ml) was added for incubation for 1 hour, and then Ang II (1 uM) was stimulated for 24 hours;

[0044] The modeling process of the high-dose OMD+Ang II modeling group was as follows: neonatal rat primary cardiomyocytes were cultured for 24 hours, recombinant protein OMD (100 ng / ml) was added for incubation for 1 hour, and then Ang II (1uM) was stimulated for 24 hours;

[0045] Total cell protein was collected, and the protein and mRNA expression levels of β-MyHC and ANP were detected by Western blot and RT-qPCR. It was found that exogenous OMD recombinant protein could alleviate the hypertrophic injury of primary cardiomyocytes of neonatal rats induced by Ang II stimulation, such as Figure 3 As shown in A and B.

[0046] Figure 3 A shows the protein expression levels of Myh7 and Nppa in primary cardiomyocytes of neonatal rats in the Ctrl group, Ang II model group, low-dose OMD+Ang II model group, and high-dose OMD+Ang II model group; B shows the mRNA expression levels of Myh7 and Nppa in primary cardiomyocytes of neonatal rats in the Ctrl group, Ang II model group, low-dose OMD+Ang II model group, and high-dose OMD+Ang II model group.

[0047] 4. Preliminary recruitment of 21 patients with heart failure with reduced ejection fraction (HFrEF) and 24 patients without heart failure (NoHF). All enrolled patients were confirmed by symptoms and echocardiogram results by at least two cardiologists. The clinical data of all enrolled patients came from the hospital's electronic medical record system. The level of circulating OMD protein in the blood was measured by enzyme-linked immunosorbent assay kit (E15132h, EIAab, Wuhan, China). The results showed that the serum OMD expression level in HFrEF patients was significantly higher than that in NoHF patients, such as Figure 4 As shown in A. ROC curve analysis showed that serum OMD level can be used to diagnose HFrEF (AUC = 0.770, P < 0.001), with a sensitivity of 66.7% and a specificity of 87.5%. Figure 4 As shown in B. Correlation analysis showed that serum OMD level was significantly correlated with plasma NT-proBNP and left ventricular ejection fraction. The higher the plasma NT-proBNP level, the lower the left ventricular ejection fraction and the higher the serum OMD level. Figure 4 As shown in C and D. The above suggests that OMD has the potential to be a new diagnostic marker for heart failure with reduced ejection fraction.

[0048] Figure 4 In the figure, A is the expression difference of OMD between HFrEF and NoHF patients, B is the area under the curve (AUC) of serum OMD expression level in HFrEF and NoHF patients, C is the relationship between serum OMD level and plasma NT-proBNP, and D is the relationship between serum OMD level and left ventricular ejection fraction (LVEF), an indicator of cardiac echocardiography.

[0049] For those skilled in the art, various corresponding changes and modifications can be made according to the above technical solutions and concepts, and all of these changes and modifications should be included in the protection scope of the claims of the present invention.

Claims

1. A marker for detecting heart failure with reduced ejection fraction, characterized in that: The markers include osteomodulin OMD.

2. Use of a marker in the preparation of a kit for detecting heart failure with reduced ejection fraction, characterized in that: The markers include osteomodulin OMD.

3. A kit for detecting heart failure with reduced ejection fraction, characterized in that: The kit includes reagents for detecting osteomodulin OMD.

4. A drug for treating heart failure with reduced ejection fraction, characterized in that: The drug comprises osteomodulin OMD recombinant protein.

5. Application of osteomodulin OMD in the preparation of drugs for treating heart failure with reduced ejection fraction.

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