A method for constructing a mouse model of heart failure with preserved ejection fraction

Abstract

The application discloses a kind of construction methods of the ejection fraction reserve type heart failure mouse model, in view of the outstanding technical problems that existing HFpEF animal model is difficult to simulate the multiple risk factor comorbidity characteristics of human, lack unified evaluation standard, and difficult to accurately locate pathological stage, the application first establishes the multi-dimensional evaluation system of HFpEF animal model covering four dimensions of complication load, pathological biochemical index, diastolic function and myocardial structure reconstruction;Multiple attack strategy of high-fat diet combined with L-NAME and STZ is used, C57BL / 6 wild type mice and SAM fast aging mice are used as objects, and a series of HFpEF models covering two types of clinical backgrounds of non-aging metabolic syndrome and senile comorbidity are constructed respectively.The model series provided by the application has the characteristics of low cost, simple operation, stable phenotype and covering different disease stages, which provides a reliable experimental platform for HFpEF pathogenesis research and treatment drug screening.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically a method for constructing a mouse model of heart failure with preserved ejection fraction. Background Technology

[0002] Heart failure with preserved ejection fraction (HFpEF) is one of the most heterogeneous and challenging subtypes of heart failure, accounting for approximately 50% of all heart failure cases. Its development is not driven by a single factor, but rather by the long-term combined effects of multiple risk factors, including aging, hypertension, obesity, dyslipidemia, and chronic low-grade inflammation. Currently, there are no specific drugs for treating HFpEF. Most drugs effective against heart failure with reduced ejection fraction (HFrEF) have failed to reduce the morbidity and mortality of HFpEF, highlighting the urgent need to develop targeted treatment strategies.

[0003] Reliable animal models are a crucial bridge for translating basic research findings into clinical practice. However, existing animal models of heart failure with HFpEF have significant limitations: pressure overload models (such as transverse aortic arch coarctation) primarily simulate myocardial hypertrophy, but often deteriorate into ventricular dilation and reduced ejection fraction as the disease progresses, making it difficult to maintain the core phenotype of "preserved ejection fraction"; single metabolic intervention models (such as a simple high-fat diet) can induce obesity and insulin resistance, but the impairment of cardiac function is slow and mild, making it difficult to fully represent the typical diastolic dysfunction of HFpEF; gene editing models have high technical barriers and are expensive, and single-gene interventions cannot fully reproduce the complex pathological features of HFpEF driven by multiple factors. These model limitations are a major reason why many interventions that are effective in animal experiments fail in clinical trials for HFpEF patients—for example, sildenafil can significantly improve diastolic function in transverse aortic arch coarctation models, but failed to meet the primary endpoint in clinical trials for HFpEF patients.

[0004] In recent years, animal model research has gradually evolved towards a "multiple-hit" strategy, which involves superimposing multiple risk factors to simulate the complex clinical background of HFpEF. However, existing protocols still have considerable room for optimization in terms of the combination of hit factors, timing, and model stability.

[0005] Furthermore, another significant bottleneck in the field of HFpEF animal models is the lack of unified, standardized, and quantifiable evaluation criteria. The diagnosis of HFpEF relies on a comprehensive assessment of echocardiographic parameters, natriuretic peptide levels, and diastolic function indicators. However, existing studies use varying evaluation indicators and inconsistent thresholds across different laboratories, resulting in inconsistent model quality and severely limiting cross-study comparisons and clinical translation efficiency.

[0006] In summary, existing technologies have at least the following shortcomings: a lack of stable animal models that can comprehensively simulate the characteristics of multiple risk factors and multiple comorbidities driving HFpEF; a lack of unified, quantifiable, and animal-appropriate model evaluation criteria; and a lack of systematic screening methods for accurately locating the disease stage of different models' pathological processes. Summary of the Invention

[0007] To address the aforementioned shortcomings, this invention first establishes a multidimensional evaluation standard for HFpEF suitable for animal-level studies; second, it employs a multi-hit strategy to construct a series of HFpEF models covering non-aging and aging backgrounds in C57BL / 6 mice and SAM rapidly aging mice, respectively; finally, it uses transcriptome sequencing and protein validation to precisely locate the pathological processes of different models, providing a stable and reliable model tool for the study of HFpEF pathogenesis and the screening of treatment strategies.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] A method for constructing a mouse model of heart failure with preserved ejection fraction includes the following steps: treating mice with a multi-hit strategy, which includes metabolic intervention and intervention with at least one cardiovascular and metabolic stressor; performing multi-dimensional phenotypic evaluation on the treated mice, which includes four dimensions: comorbidity burden evaluation, pathological and biochemical index evaluation, diastolic function evaluation, and myocardial structural remodeling evaluation; and determining whether the model has been successfully constructed based on the results of the multi-dimensional phenotypic evaluation.

[0010] Furthermore, the metabolic intervention is a high-fat diet.

[0011] Furthermore, the cardiovascular and metabolic stressors are selected from one or more of L-NAME, streptozotocin, and high-salt drinking water.

[0012] Furthermore, the mice are C57BL / 6 wild-type mice or SAM rapidly aging mice.

[0013] Furthermore, when the mouse is a C57BL / 6 wild-type mouse, the multi-hit strategy is a dual-hit strategy of high-fat diet combined with L-NAME drinking water.

[0014] Furthermore, when the mice are C57BL / 6 wild-type mice, the multiple-attack strategy is a triple-attack strategy consisting of a high-fat diet combined with L-NAME drinking water and intraperitoneal injection of streptozotocin starting in the 9th week of feeding.

[0015] Furthermore, when the mouse is a rapidly aging SAM mouse, the multiple attack strategy is a triple attack strategy of high-fat diet combined with high-salt water intake.

[0016] Furthermore, when the mice are SAM rapidly aging mice, the multiple-attack strategy is a quadruple-attack strategy consisting of a high-fat diet combined with L-NAME drinking water and intraperitoneal injection of streptozotocin starting in the 9th week of feeding.

[0017] Furthermore, the indicators for assessing the comorbidity burden include: systolic blood pressure ≥130 mmHg; total cholesterol ≥80% higher than the normal control group, or triglycerides ≥50% higher than the normal control group; fasting blood glucose ≥11.1 mmol / L, or peak blood glucose ≥16.7 mmol / L 30 / 60 minutes after glucose loading in a glucose tolerance test; serum creatinine ≥50% higher than the normal control group, or blood urea nitrogen ≥60% higher than the normal control group.

[0018] The pathological and biochemical indicators evaluated included: myocardial interstitial collagen volume fraction ≥10%; myocardial tissue IL-6 level ≥50% higher than normal control group; any one of serum ANP, BNP, or NT-proBNP ≥100% higher than normal control group; lung tissue wet / dry weight ≥15% higher than normal control group; and exercise tolerance ≥25% lower than normal control group.

[0019] In the evaluation of diastolic function, a left ventricular ejection fraction ≥50% is a prerequisite, and the indicators include: a left heart catheterization measurement of LVEDP ≥50% higher than the normal control group; at least two of the ultrasound indicators showing changes: an increase of ≥20% in E / e', a prolongation of ≥20% in IVRT, a decrease of ≥15% in E / A, and a decrease of ≥20% in the absolute value of dp / dt max compared to the normal control group; and at least two of the speckle tracking indicators showing changes: a decrease of ≥15% in GLS, a decrease of ≥25% in left atrial strain, and a decrease of ≥10% in left ventricular circumferential strain compared to the normal control group.

[0020] The indicators for evaluating myocardial structural remodeling include: changes in at least two of the following ultrasound indicators: IVSd increased by ≥20%, LVPWd increased by ≥20%, RWTd increased by ≥30%, and left atrial diameter increased by ≥30%; heart weight / tibia length ratio was ≥30% higher than that of the normal control group, or myocardial cell HE staining cross-sectional area was ≥20% higher than that of the normal control group.

[0021] Application of a mouse model of heart failure with preserved ejection fraction in screening drugs for the treatment of heart failure with preserved ejection fraction.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention establishes a multidimensional HFpEF phenotypic evaluation system applicable to the animal level, covering four major modules: comorbidity burden, pathochemical indicators, diastolic function parameters, and myocardial structural remodeling, which can provide a unified quantitative basis for the systematic identification of model phenotypes.

[0024] Based on the above evaluation system, this invention employs a multi-pronged attack strategy combining a high-fat diet with L-NAME and STZ. Using C57BL / 6 mice and SAM aging mice as subjects, it can construct a series of HFpEF models covering both non-aging metabolic syndrome and comorbid HFpEF clinical backgrounds, providing differentiated model selection for drug screening with different pathogenesis mechanisms. This method requires no gene editing, is low-cost, easy to promote, and the model phenotypes more closely match the pathological characteristics of multiple metabolic disorders comorbid in clinical HFpEF.

[0025] This invention, through transcriptome sequencing and protein validation, for the first time precisely locates the pathological process of each model at the molecular level, clarifying the early, progressive, and terminal / severe stages of HFpEF corresponding to each model. This provides scientific guidance for researchers to accurately select the most suitable model according to experimental objectives, effectively reducing the risk of clinical translation failure due to model mismatch with disease stage. Attached Figure Description

[0026] Figure 1 Effects of multiple blows on blood pressure in C57BL / 6 mice (A) Systolic blood pressure (B) Diastolic blood pressure

[0027] Figure 2 Effects of multiple hits on blood glucose in C57BL / 6 mice.

[0028] Figure 3 Effects of multiple hits on lipid metabolism in C57BL / 6 mice (A) TC level (B) TG level.

[0029] Figure 4 Typical echocardiograms of C57BL / 6 mice subjected to multiple blows: (A) M-mode echocardiogram of male mice at the horizontal level; (B) Tissue Doppler imaging of male mice; (C) M-mode echocardiogram of female mice at the horizontal level; (D) Tissue Doppler imaging of female mice.

[0030] Figure 5 Effects of multiple blows on cardiac function in C57BL / 6 mice (A) Left ventricular ejection fraction (B) E / e' (C) Cardiac output (D) Left ventricular end-diastolic interventricular septal thickness (E) Left ventricular end-diastolic posterior wall thickness (F) Relative wall thickness.

[0031] Figure 6 Effects of multiple blows on cardiac function in C57BL / 6 mice (A) End-diastolic left ventricular diameter (B) End-systolic left ventricular diameter (C) Short axis shortening rate.

[0032] Figure 7 Effects of multiple blows on heart failure biomarkers and inflammatory factors in C57BL / 6 mice (A) Serum ANP level (B) BNP level in cardiac homogenate (C) Serum NT-proBNP level (D) IL-6 level in myocardial tissue

[0033] Figure 8 Effects of multiple blows on cardiac tissue morphology in C57BL / 6 mice (A) HE staining (B)

[0034] Masson staining (C) collagen volume fraction.

[0035] Figure 9 Effects of multiple blows on blood pressure in SAM mice (A) Systolic blood pressure (B) Diastolic blood pressure

[0036] Figure 10 The effect of multiple hits on blood glucose in SAM mice.

[0037] Figure 11 Effects of multiple hits on lipid metabolism in SAM mice (A) TC (B) TG levels.

[0038] Figure 12 Typical echocardiogram of multiple-hit SAM mice (A) Horizontal M-mode echocardiogram (B) Tissue Doppler imaging.

[0039] Figure 13 Effects of multiple blows on cardiac function in SAM mice: (A) Left ventricular ejection fraction (B) E / e' level (C) Cardiac output level (D) Relative wall thickness (E) Left ventricular end-diastolic interventricular septal thickness (F) Left ventricular end-diastolic posterior wall thickness (G) Left ventricular end-diastolic diameter (H) Left ventricular end-systolic diameter (I) Short axis shortening rate.

[0040] Figure 14 Effects of multiple blows on heart failure biomarkers and inflammatory factors in SAM mice (A) Serum ANP level (B) BNP level in cardiac homogenate (C) Serum NT-proBNP level (D) IL-6 level in myocardial tissue

[0041] Figure 15 Effects of multiple blows on cardiac tissue morphology in SAM mice (A) HE staining (B) Masson staining (C) Quantitative analysis of collagen volume fraction.

[0042] Figure 16 Scoring results of each model

[0043] Figure 17 GO enrichment analysis of DEGs between the male double-hit group and the control group.

[0044] Figure 18Bubble plot of KEGG enrichment analysis of DEGs between the male double-hit group and the control group.

[0045] Figure 19 GO enrichment analysis of DEGs between the male triple-hit group and the control group.

[0046] Figure 20 Bubble chart of KEGG enrichment analysis of DEGs between the male triple-hit group and the control group.

[0047] Figure 21 Effects of multiple hits on the expression of inflammation initiation markers in cardiac tissue of C57BL / 6 mice (A) TLR4 expression (immunohistochemical staining) (B) MyD88, Pho-NF-κB, and VCAM-1 expression (Western blot).

[0048] Figure 22 Effects of multiple hits on the expression of key proteins in the stress signaling pathway in the cardiac tissue of C57BL / 6 mice (Western blot).

[0049] Figure 23 Effects of multiple-hit C57BL / 6 mouse cardiac tissue fibrosis markers TGF-β, α-SMA, and Collagen III expression (immunohistochemical staining).

[0050] Figure 24 GO enrichment analysis of DEGs between the SAM triple-strike group and the control group.

[0051] Figure 25 Bubble chart of KEGG enrichment analysis of DEGs between the SAM triple-hit group and the control group.

[0052] Figure 26 GO enrichment analysis plot of DEGs between the SAM quadruple hit group and the control.

[0053] Figure 27 Bubble chart of KEGG enrichment analysis of DEGs between the SAM quadruple hit group and the control group.

[0054] Figure 28 Effects of multiple hits on the expression of inflammatory initiation markers in the cardiac tissue of SAM mice (A) TLR4 expression (immunohistochemical staining) (B) MyD88, Pho-NF-κB, and VCAM-1 expression (Western blot).

[0055] Figure 29 Effects of multiple blows on the expression of key proteins in stress amplification in the cardiac tissue of SAM mice (Western blot).

[0056] Figure 30 Effects of multiple hits on the expression of TGF-β, α-SMA and Collagen Ⅲ, markers of cardiac fibrosis in SAM mice (immunohistochemical staining). Detailed Implementation

[0057] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0058] Example 1: Evaluation Method of HFpEF Animal Model

[0059] This embodiment provides a multi-dimensional systematic evaluation method for the HFpEF animal model.

[0060] Prerequisites and exclusion criteria: LVEF ≥ 50%. This is a prerequisite for all models and is not included in the scoring. If the model animal's LVEF < 50%, it will be excluded.

[0061] The evaluation method includes the following four evaluation dimensions:

[0062] (1) Assessment of comorbidity burden:

[0063] The systolic / diastolic blood pressure (hypertension) of the model animals was measured, with a systolic blood pressure ≥130 mmHg.

[0064] Total cholesterol and triglycerides (hyperlipidemia): Total cholesterol is ≥80% higher than the normal control group, or triglycerides are ≥50% higher than the normal control group.

[0065] Fasting blood glucose and glucose tolerance: Fasting blood glucose ≥11.1 mmol / L; Glucose tolerance: Peak blood glucose ≥16.7 mmol / L 30 / 60 minutes after glucose load.

[0066] Serum creatinine and blood urea nitrogen (kidney injury): serum creatinine was ≥50% higher than that of the normal control group, and blood urea nitrogen was ≥60% higher than that of the normal control group.

[0067] The evaluation of the comorbidities of the model animals is based on a comprehensive assessment. If the conditions are met, the animals are considered to have comorbidities. Three comorbidities are awarded as 3 points, two comorbidities as 2 points, one comorbidity as 1 point, and no comorbidities as 0 points.

[0068] (2) Evaluation of pathological and biochemical indicators:

[0069] Masson staining was used to assess the myocardial interstitial collagen volume fraction (myocardial fibrosis). A myocardial interstitial collagen volume fraction ≥ 10% was scored as 2 points, and 0 points as otherwise scored.

[0070] IL-6 (inflammation) in myocardial tissue was detected by ELISA. If the IL-6 level in the model group was ≥50% higher than that in the normal control group, 1 point was awarded if yes and 0 points if no.

[0071] Serum ANP, BNP, and NT-proBNP levels were detected by ELISA. If any one of these levels in the model group was ≥100% higher than that in the normal control group, 1 point was awarded; otherwise, 0 points were awarded.

[0072] The degree of lung congestion was assessed by the wet-to-dry weight ratio of lung tissue. If the model group showed an increase of ≥15% compared to the normal control group, 1 point was awarded if yes and 0 points if no.

[0073] Exercise endurance was assessed using a treadmill forced exercise experiment. If the exercise endurance of the model group decreased by ≥25% compared to the normal control group, 1 point was awarded if yes and 0 points if no.

[0074] (3) Evaluation of diastolic function:

[0075] If LVEDP is measured by left ventricular catheterization and is ≥50% higher than that of the normal control group, 1 point is awarded if yes and 0 points if no.

[0076] Echocardiography was used to measure E / A, E / e', isovolumetric relaxation time (IVRT), and dp / dt max. In the model group, E / e' was ≥20% higher than in the normal control group; IVRT was ≥20% longer than in the normal control group; E / A was ≥15% lower than in the normal control group; and the absolute value of dp / dt max was ≥20% lower than in the normal control group. One point was awarded for changes in two or more of these indicators.

[0077] GLS, left atrial strain, and left ventricular circumferential strain were measured using speckle tracking technology. The model group showed a decrease in GLS of ≥15%, a decrease in left atrial strain of ≥25%, and a decrease in left ventricular circumferential strain of ≥10% compared to the normal control group. One point was awarded for changes in two or more of the above indicators.

[0078] (4) Evaluation of myocardial structural remodeling:

[0079] IVSd, LVAWd, RWTd, and left atrial diameter were measured by echocardiography. Compared with the normal control group, IVSd increased by ≥20%, LVPWd increased by ≥20%, RWTd increased by ≥30%, and left atrial diameter increased by ≥30%. Two or more of these conditions were recorded as 2 points.

[0080] By calculating the heart weight / tibia length ratio and measuring the cross-sectional area of ​​myocardial cells by HE staining, the model group had a heart weight / tibia length ratio that was ≥30% higher than the normal control group, and the cross-sectional area of ​​myocardial cells measured by HE staining was ≥20% higher than the normal control group. One point was awarded for having one or more of these criteria.

[0081] The evaluation results of the above four dimensions together constitute the HFpEF phenotypic feature map of the model animal. A score of ≥9 points is considered a successful model.

[0082] Example 2: Construction and Phenotypic Evaluation of the C57BL / 6 Mouse Multiple Hit HFpEF Model

[0083] This embodiment uses C57BL / 6 wild-type mice as subjects. The HFpEF animal model was constructed by using a "double-hit" strategy of high-fat diet (HFD) combined with L-NAME drinking water and a "triple-hit" strategy of adding streptozotocin (STZ) intraperitoneal injection to the "double-hit" strategy. The phenotype of the mice was systematically evaluated.

[0084] Animal grouping and modeling

[0085] After acclimatization, C57BL / 6 mice were randomly divided into groups of 10 mice each according to their body weight. The grouping and treatment methods are as follows:

[0086] (1) Male normal control group (M-NC): C57BL / 6 male mice were fed growth maintenance diet and ordinary drinking water throughout the entire process.

[0087] (2) Male double hit group (M-2 hit): C57BL / 6 male mice were fed a high-fat diet with 60% fat as the energy source throughout the entire process, combined with 0.5 g / L L-NAME drinking water.

[0088] (3) Male triple hit group (M-3 hit): C57BL / 6 male mice were injected intraperitoneally with STZ at a dose of 50 mg / kg starting from week 9 of feeding, for 5 consecutive days. Ten days after the last injection of STZ, the fasting blood glucose of the mice was measured. The mice were considered to have successfully established a diabetic model if the blood glucose was ≥11.1 mmol / L (mice with fasting blood glucose <11.1 mmol / L were excluded). The mice were fed until the end of week 15 for subsequent experiments.

[0089] (4) Female normal control group (F-NC): C57BL / 6 female mice were fed growth maintenance diet and ordinary drinking water throughout the entire process.

[0090] (5) Female double hit group (F-2 hit): C57BL / 6 female mice were fed a high-fat diet with 60% fat as the energy source throughout the entire process, combined with 0.5 g / L L-NAME drinking water.

[0091] (6) Female triple hit group (F-3 hit): C57BL / 6 female mice, treated in the same way as the M-3 hit group.

[0092] Model Evaluation

[0093] Fifteen weeks after modeling, phenotypic identification was performed on mice in each group. Evaluation indicators included blood pressure, fasting blood glucose, blood lipids, echocardiography, heart failure biomarkers, myocardial inflammatory factors, and histopathological changes.

[0094] See Figure 1 After 15 weeks of modeling, the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the mice were significantly higher than those of the normal control group.

[0095] During the triple-strike treatment, a high-fat diet combined with intraperitoneal injection of STZ induced diabetic symptoms in mice. See [link to article / document]. Figure 2 The fasting blood glucose levels in the triple-hit group mice were significantly higher than those in the normal control group and the double-hit group, and the fasting blood glucose levels in the male triple-hit group were significantly higher than those in the female triple-hit group.

[0096] In establishing a high-fat HFpEF model characterized by hyperlipidemia, a high-fat diet was used as the sole diet for the model group mice. After 15 weeks of modeling, the levels of total cholesterol (TC) and triglycerides (TG) in the liver homogenate of the mice significantly increased, with a more pronounced increase in the triple-hit group (see [reference needed]). Figure 3 .

[0097] The above experimental results show that the animals in the double-hit model group have symptoms of hyperlipidemia and hypertension; the animals in the triple-hit model group have symptoms of hyperlipidemia, hypertension, and hyperglycemia.

[0098] HFpEF is a subtype of heart failure characterized primarily by left ventricular diastolic dysfunction, see [link to relevant documentation]. Figure 4 Echocardiography of mice in all model groups showed changes in the structure of the left ventricle to a certain extent.

[0099] Echocardiographic statistical results showed that... Figure 5Compared with the control group, the left ventricular ejection fraction (LVEF) of mice in each model group did not change significantly and remained within the preservation range (LVEF > 50%). Further assessment of left ventricular diastolic function revealed that the E / e' ratio was significantly higher in male double-hit, triple-hit, and female triple-hit model groups than in the same sex control group, indicating that the left ventricular diastolic capacity of these three model groups was impaired and accompanied by increased filling pressure. Cardiac output (CO) was measured under resting conditions, and the results showed no statistically significant difference between the model groups and the control group, suggesting that the overall pumping capacity of the model mice could still be maintained under resting conditions. The left ventricular end-diastolic interventricular septal thickness (IVSd) in male model group mice and the left ventricular end-diastolic posterior wall thickness (LVPWd) in female double-hit mice were significantly increased compared with the normal control group; the relative wall thickness (RWT) was significantly increased in male triple-hit and female double-hit groups. The results suggest that male double-strike, male triple-strike, and female double-strike mice may have exhibited left ventricular concentric remodeling or hypertrophy, accompanied by impaired diastolic function. This structural change, characterized primarily by increased wall thickness rather than ventricular dilation, is consistent with the classic features of HFpEF. These results suggest that sex and strike intensity may influence the degree and pattern of HFpEF structural remodeling.

[0100] Left ventricular circumference parameters are important ultrasound indicators for evaluating ventricular structural remodeling, see... Figure 6 There were no significant differences in left ventricular diameter at end-diastole (LVIDd), left ventricular diameter at end-systole (LVIDs), and fractional shortening (FS) between the model groups and the same-sex control group, indicating that the model established in this study conforms to the pathological characteristics of non-expansive remodeling of HFpEF.

[0101] See Figure 7 Serum ANP and NT-proBNP levels were significantly elevated in all model groups of mice. Simultaneously, BNP levels in cardiac homogenates were significantly increased compared to the control group. This indicates that mice in both the double-hit and triple-hit groups exhibited pathological changes such as increased cardiac load and increased ventricular wall tension. IL-6 levels in cardiac homogenates were significantly elevated in all model groups, with a more significant increase in IL-6 levels observed in male model groups compared to female model groups.

[0102] See Figure 8HE staining results showed that the myocardial tissue structure of C57BL / 6 male control mice was normal. In the male double-hit group, a small number of areas of myocardial cells were disordered, with a small number of inflammatory cells and fibrous tissue proliferation visible in the interstitial spaces, and some myocardial cells were edematous. In the male triple-hit group, some areas of myocardial tissue showed fibrous tissue proliferation and inflammatory cells, with mild hypertrophy of the surrounding myocardial cells. The myocardial tissue structure of C57BL / 6 female control mice was normal. In the female double-hit model group, the myocardial tissue structure was very slightly abnormal, with relatively orderly arrangement of myocardial cells, occasional myocardial cell edema, and a small number of inflammatory cell infiltrations in the interstitium; no obvious fibrosis was observed. In the female triple-hit model, the heart structure was mildly damaged, with disordered arrangement of myocardial cells in some areas, accompanied by fibrous tissue proliferation and inflammatory cell infiltration, hypertrophy of the surrounding myocardial cells, increased diameter, apoptosis of some myocardial cells, and cell condensation.

[0103] See Figure 8 Masson staining results showed that, compared with the normal control group, the double-hit model group had significantly increased blue-stained collagen fibers in the myocardial interstitium and some perivascular areas, and widened myocardial fiber gaps, indicating that myocardial interstitial fibrosis had begun to form. The triple-hit model group showed further aggravation of collagen fiber deposition compared with the double-hit model group, manifested by an expansion of the blue-stained area in the myocardial interstitium, with obvious collagen fiber proliferation and tissue remodeling in some areas, indicating that the degree of myocardial fibrosis was further aggravated. Multiple hits can effectively induce myocardial fibrosis and myocardial remodeling.

[0104] The above results indicate that the combination of multiple factors better simulates the clinical characteristics of HFpEF driven by multiple risk factors and multiple comorbidities than a single factor. In the C57BL / 6 mouse model, the male triple-hit group (hyperlipidemia, hypertension, hyperglycemia) is the preferred model that is more stable and better reflects the clinical comorbid characteristics of HFpEF.

[0105] Example 3: Construction and Phenotypic Evaluation of a Multiple-Hit HFpEF Mouse Model under Aging Background

[0106] This embodiment uses SAM mice as the subject, constructs an HFpEF animal model under aging background using a multi-hit strategy, and conducts a systematic evaluation of its phenotype.

[0107] Animal grouping and modeling

[0108] After acclimatization, SAM-R1 and SAM-P8 mice were randomly divided into 3 groups of 8 mice each, according to their body weight:

[0109] (1) Normal control group (NC): SAM-R1 mice were fed growth maintenance diet and ordinary drinking water throughout the entire process.

[0110] (2) Triple-hit model group: SAM-P8 mice were fed a high-fat diet with 60% fat as the energy source throughout the entire process, and were given 1% NaCl solution in their drinking water.

[0111] (3) Quadruple-hit model group (4-hit): SAM-P8 mice were fed a high-fat diet with 60% fat as the energy source throughout the entire process, and were given 0.5 g / L L-NAME solution in drinking water. Starting from the 9th week of feeding, STZ was injected intraperitoneally at a dose of 50 mg / kg for 5 consecutive days. Ten days after the last injection of STZ, fasting blood glucose was measured in the mice. If the blood glucose was ≥11.1 mmol / L, the diabetes model was considered to be successfully established. The mice were fed until the end of the 15th week for subsequent experiments.

[0112] Model Evaluation

[0113] Fifteen weeks after modeling, phenotypic identification was performed on mice in each group. Evaluation indicators included blood pressure, fasting blood glucose, blood lipids, echocardiography, heart failure biomarkers, myocardial inflammatory factors, and histopathological changes.

[0114] See Figure 9 In the model mice, both systolic and diastolic blood pressure were significantly elevated compared to the normal control group. Fasting blood glucose levels were significantly elevated in the SAM quadruple-hit group mice. Figure 10 The levels of TC and TG in the liver homogenate of model mice were significantly increased compared with those in the normal control group, and the increase was more pronounced in the SAM quadruple hit group. Figure 11 The above results indicate that the animals in the SAM triple-strike model group exhibited signs of aging, hyperlipidemia, and hypertension; the animals in the SAM quadruple-strike model group exhibited signs of aging, hyperlipidemia, hypertension, and hyperglycemia.

[0115] See Figure 12 Echocardiography of the model group mice showed that the structure of the left ventricle had changed to a certain extent, characterized by thickening of the left ventricular wall and no obvious enlargement of the ventricle.

[0116] See Figure 13Compared with the control group, no significant changes were observed in LVEF in any of the model groups, while E / e' was significantly increased. There was no statistically significant difference in CO between the model groups and the control group, indicating that while the overall left ventricular systolic function was preserved in the SAM triple-hit and SAM quadruple-hit groups, varying degrees of diastolic dysfunction were present. IVSd was significantly increased in the SAM triple-hit model group compared to the normal control group; LVPWd and RWT were not significantly increased in the model group compared to the normal control group, suggesting that the SAM triple-hit group mice exhibited a tendency for left ventricular concentric remodeling accompanied by diastolic dysfunction. LVIDd and LVIDs were not significantly changed compared to the same-sex control group in any of the model groups, remaining within the normal range. However, FS was significantly decreased in the quadruple-hit model group compared to the normal control group. These results suggest that the SAM triple-hit model group mice preserved overall left ventricular systolic function at rest without significant pathological changes in systolic function, while the SAM quadruple-hit model group mice showed a decrease in left ventricular systolic function, exhibiting pathological changes of impaired systolic function.

[0117] See Figure 14 The levels of natriuretic peptides in the circulation and heart tissue of mice were detected. Compared with the control group, the serum levels of ANP and NT-proBNP in each model group were significantly increased. Simultaneously, the BNP level in the heart homogenate was significantly increased. These experimental results indicate that mice in the SAM triple-hit and SAM quadruple-hit groups may have experienced pathological changes such as cardiac overload and increased ventricular wall tension. The IL-6 level in the heart homogenate of the model group mice was significantly higher than that of the normal control group, and the increase in IL-6 level in the heart homogenate of the SAM quadruple-hit model group was even more significant.

[0118] See Figure 15 In the control group, the myocardial tissue structure of mice was normal, with clear and orderly outlines of cardiomyocytes and no abnormalities observed. In the SAM triple-hit group, the myocardial tissue structure of mice showed mild abnormalities, with disordered arrangement of cardiomyocytes in some areas, a small number of hypertrophied cells with increased diameter, and minimal fibrous tissue proliferation, inflammatory cell infiltration, and fat vacuoles. In the SAM quadruple-hit group, the overall myocardial tissue structure of mice was moderately abnormal, with diffuse infiltration of numerous inflammatory cells in the interstitium, disordered arrangement of cardiomyocytes, and mild hypertrophy; fibrous tissue proliferation was also observed in the interstitium. Masson staining results showed that the myocardial fibers in the control group were neatly arranged, and the muscle bundle structure was intact. In the SAM triple-hit group, the cardiomyocytes were disordered, the interstitial spaces were widened, and there was increased collagen fiber deposition in the myocardial interstitium and perivascular space; collagen fiber deposition was further aggravated in the SAM quadruple-hit group, manifested as more extensive interstitial fibrosis and focal collagen aggregation, with more significant disordered myocardial tissue structure. These results suggest that with the increase of multiple-hit factors, cardiac remodeling in mice gradually worsens, with the most significant myocardial fibrosis observed in the SAM quadruple-hit group.

[0119] The SAM triple-hit model mainly manifests as aging, hypertension, hyperlipidemia, preserved ejection fraction, diastolic dysfunction, myocardial fibrosis, and vascular injury, which is closer to the typical HFpEF-like phenotype in the context of aging. The SAM quadruple-hit model further adds abnormal glucose metabolism and L-NAME-related endothelial damage, showing more severe metabolic disorders, inflammatory response, myocardial injury and fibrotic changes, and decreased FS, suggesting that it may be in a state of severe HFpEF or a transition from HFpEF to early systolic dysfunction.

[0120] Example 4

[0121] This embodiment utilizes the comprehensive evaluation method established in Example 1 to conduct a systematic evaluation and comparison of the C57BL / 6 mouse model groups constructed in Example 2 and the SAM mouse model groups constructed in Example 3.

[0122] See Figure 16 The results showed that the male C57BL / 6 triple-strike model had the best overall performance, and compared with SAM mice, this model was less expensive and easier to generalize. Among the models, the female C57BL / 6 dual-strike model showed no significant changes in any evaluation index and had the weakest overall performance, making it unsuitable for preclinical studies.

[0123] Example 5: Evaluation and Precise Screening of Pathological Progression Using a Transcriptomics-Based HFpEF Model

[0124] Transcriptome sequencing analysis was performed on myocardial tissue from C57BL / 6 male double-hit and triple-hit groups. Compared with the control group, 151 differentially expressed genes were detected in the double-hit group, and 569 differentially expressed genes were detected in the triple-hit group. These results suggest that multiple hits can induce changes in myocardial tissue transcription levels, but the triple-hit strategy can induce more widespread and significant gene expression disturbances, indicating a stronger perturbation effect on the myocardial transcriptional regulatory network.

[0125] See Figure 17 GO enrichment results in the male double-hit group showed that DEGs were enriched in biological processes related to signal transduction, endogenous stimulus response, and tissue remodeling. Molecular alterations in the male double-hit group were mainly associated with metabolism, neurohumoral regulation, and early pathological cardiac remodeling. See [link to relevant documentation]. Figure 18 KEGG analysis showed that DEGs were mainly enriched in the neuroendocrine-metabolic regulatory network. Therefore, the male double-hit group corresponds at the molecular level to the early, mild pathological stage of HFpEF, characterized by hypermetabolic stress, abnormal hormonal response, and neurohumoral imbalance.

[0126] See Figure 19Unlike the male double-hit group, the GO enrichment results of DEGs in the male triple-hit group showed strong immune inflammatory activation. The enriched entries can be summarized into three functional modules: ① Immune initiation module, including "response to external stimulus," indicating activation of the innate immune sensing system. ② Immune cell activation module, including "leukocyte activation," "lymphocyte activation," and "cell activation," indicating recruitment and activation of leukocytes and lymphocytes in the myocardium, further amplifying local pathological effects. ③ Immune regulation module, such as "regulation of immune system process" and "positive regulation of immune response," suggesting continuous activation of the immune regulatory network in this model, forming a persistent inflammatory microenvironment. The myocardial transcriptional disorder induced by male triple-hit is no longer limited to the early stages of metabolic stress but has shifted to a pathological state centered on excessive immune inflammatory activation, indicating further aggravation of HFpEF pathological damage. See Figure 20 KEGG enrichment further confirmed that the male triple-hit model more closely resembles the pathological features of HFpEF in terms of low-grade chronic inflammation, endothelial activation, enhanced immune-related pathways, and tissue remodeling. Therefore, the male triple-hit group corresponds to the continuous evolution of systemic chronic inflammation driven by comorbidities, followed by coronary microvascular endothelial activation, myocardial fibrosis, and diastolic dysfunction. Thus, compared to the male double-hit model, the male triple-hit model is closer to the core pathological essence of HFpEF and is more suitable as the preferred model for subsequent mechanistic studies, consistent with the pathological stage of HFpEF progression.

[0127] Based on transcriptome functional enrichment results and myocardial tissue pathological changes, this study further analyzed at the protein level the key signaling pathways that may be involved in the process of multiple hits inducing HFpEF-like pathological changes.

[0128] See Figure 21As shown, multiple hits can induce upregulation of TLR4, a receptor related to innate immune recognition in myocardial tissue, with a more pronounced activation of inflammatory recognition in the male triple hit group. MyD88 levels were elevated in both the female and male triple hit groups, while there was no significant difference in the double hit group, suggesting that MyD88-mediated innate immune signaling is more significantly activated under triple hit conditions. Multiple hits can significantly activate the NF-κB inflammatory transcriptional program, with triple hits inducing a more intense inflammatory response. VCAM-1 expression was significantly elevated in both model groups, indicating that multiple hits, especially triple hits, can significantly enhance the expression of adhesion molecules in myocardial tissue, reflecting a more pronounced tendency for endothelial activation and inflammatory cell adhesion recruitment.

[0129] See Figure 22 In both the male double-hit and female / male triple-hit groups, the Pho-JNK / JNK, Pho-Erk1 / 2 / Erk1 / 2, and Pho-AKT / AKT ratios in myocardial tissue were significantly increased. This indicates that the JNK-mediated stress kinase signaling pathway and the Erk1 / 2-mediated myocardial remodeling signaling pathway were significantly activated, while the AKT pathway was compensatorily activated, participating in the regulation of myocardial cell survival and metabolic adaptive changes. The activation of these pathways showed the most significant upward trend in the male triple-hit group. The expression level of GSDMD-N protein in both the female and male triple-hit groups was significantly increased compared with the control group, suggesting that triple-hit can upregulate the expression level of pyroptosis effector proteins in myocardial tissue and induce myocardial pyroptosis.

[0130] Notably, the Pho-JNK / JNK and Pho-Erk1 / 2 / Erk1 / 2 ratios did not show significant changes in the female double-hit group, suggesting that while a high-fat diet combined with L-NAME intervention may initiate myocardial inflammation and fibrosis-related changes in female mice to some extent, it is insufficient to activate the MAPK stress signaling pathway and induce significant myocardial stress damage. However, under the combined intervention of a high-fat diet, L-NAME, and STZ triple-hit, the superposition of multiple pathological factors—lipid metabolism disorder, suppressed endogenous NO synthesis, and abnormal glucose metabolism—synergistically enhances the myocardial tissue's response to stress stimuli, ultimately mediating significant activation and signal amplification of the myocardial MAPK signaling pathway, thus exacerbating myocardial pathological damage.

[0131] See Figure 23 In the model group mice, the positive areas of Collagen III, TGF-β, and α-SMA in the heart tissue all increased to varying degrees. The results suggest that multiple hits can induce innate immune activation, enhanced pro-fibrotic signals, myofibroblast activation, and increased collagen deposition in myocardial tissue, thereby promoting myocardial fibrotic remodeling, and the changes were more pronounced in the male triple hit group.

[0132] The protein level analysis results indicated that the male triple-hit group exhibited a HFpEF cascade pathological process of "inflammatory initiation-stress amplification-fibrotic remodeling", which is consistent with the findings of transcriptomics.

[0133] Compared with the control group, a total of 925 differentially expressed genes were detected in the SAM triple-hit group, and a total of 1483 differentially expressed genes were detected in the SAM quadruple-hit group. Both multiple hits can induce changes in the transcriptional level of cardiac tissue, and the quadruple-hit strategy can induce more extensive and significant gene expression disorders, with a stronger perturbation effect on the myocardial transcriptional regulatory network.

[0134] See Figure 24 GO enrichment results showed that the molecular alterations in the SAM triple-hit group were mainly related to extracellular matrix remodeling, tissue structure changes, enhanced stress response, and abnormal ion transport. This group is no longer limited to simple metabolic or neurohumoral stress, but tends to form a complex pathological state with myocardial fibrosis remodeling, abnormal cell adhesion, and functional dysregulation.

[0135] See Figure 25 KEGG enrichment results generally revealed significant metabolic abnormalities, extracellular matrix remodeling, activated immune inflammation, and altered neurohumoral signaling. This suggests that mouse heart tissue already possesses a clear structural remodeling basis dominated by abnormal collagen deposition, accompanied by enhanced innate immune inflammatory responses and complex neurohumoral molecular network imbalances. These results support the view that the SAM triple-hit group is characterized by a complex pathological state involving metabolic reprogramming, fibrotic remodeling, enhanced inflammatory responses, and multi-pathway network imbalances. Therefore, at the molecular level, the SAM triple-hit group corresponds to the pathological stage of HFpEF progression characterized by hypermetabolic stress, abnormal hormone responses, and fibrotic remodeling.

[0136] See Figure 26 Unlike the SAM triple attack group, the SAM quadruple attack group showed explosive immune activation, a strong defensive stress response, and active cytokine signaling in GO enrichment results. This suggests that the myocardial tissue in this group had a greatly enhanced defensive response to strong external interference and biological stimuli, and that the tissue had undergone comprehensive immune activation and high stress under multiple pathological attacks. This indicates that the SAM quadruple attack group has more prominent features of immune inflammatory activation, enhanced cytokine signaling, and upregulated defensive response, confirming that the pathological process has progressed from a simple stress stage in the early and middle stages to a complex immune inflammatory stage, and that the HFpEF pathological stage has further progressed.

[0137] See Figure 27KEGG results showed significant immune inflammatory responses, intercellular communication, and activation of pro-inflammatory signaling pathways, indicating significant release of inflammatory factors and leukocyte chemotaxis in the myocardial tissue of the SAM quadruple attack group. This reflects that under multiple attack conditions, the myocardial microenvironment is accompanied by significant immune cell infiltration and amplification of inflammatory signal cascades. The enrichment of pathways related to infection or autoimmune diseases suggests the activation of antigen-presenting cells such as macrophages in myocardial tissue, the involvement of the innate and adaptive immune systems, and the potential immune homeostasis imbalance induced by metabolic stress. This may be the pathological basis for promoting ventricular remodeling, impaired diastolic function, and microvascular dysfunction in HFpEF.

[0138] Based on transcriptome functional enrichment results and myocardial tissue pathological changes, this study further validated at the protein level the key signaling pathways that may be involved in the process of multiple hits inducing HFpEF-like pathological changes.

[0139] See Figure 28 Multiple-hit therapy induced upregulation of TLR4, a receptor related to innate immune recognition in myocardial tissue, with a more pronounced activation of inflammatory recognition in the SAM quadruple-hit group. The expression of MyD88, Pho-NF-κB-p65, and VCAM-1 proteins was increased in the cardiac tissue of both the SAM triple and quadruple-hit groups, suggesting that innate immune recognition, inflammatory transcriptional activation, and vascular inflammatory effects jointly contribute to the formation of the myocardial inflammatory microenvironment in the model group. The overall inflammatory activation was more pronounced in the SAM quadruple-hit group than in the SAM triple-hit group, consistent with the aforementioned transcriptomic findings of enhanced immune-inflammatory characteristics in the SAM quadruple-hit group, suggesting that the SAM quadruple-hit model may possess stronger characteristics for initiating myocardial inflammation and outputting inflammatory effects.

[0140] At the level of amplified inflammatory stress, see Figure 29 Both triple and quadruple SAM attacks significantly increased the Pho-JNK / JNK, Pho-Erk1 / 2 / Erk1 / 2, and Pho-AKT / AKT ratios and GSDMD-N expression levels in myocardial tissue, with the quadruple attack group showing the most significant increase. This suggests that, in the context of aging, both triple and quadruple attack strategies can amplify myocardial inflammatory stress signals, upregulate the expression levels of pyroptosis effector proteins in myocardial tissue, and exacerbate myocardial damage.

[0141] See Figure 30 Immunohistochemical results showed that both the SAM triple-hit and SAM quadruple-hit groups exhibited enhanced expression of fibrosis-related proteins, with the SAM quadruple-hit group showing a greater increase. These results suggest that multiple hits can induce enhanced pro-fibrotic signals in myocardial tissue, activation of myofibroblasts, and increased collagen deposition, thereby promoting myocardial fibrotic remodeling.

[0142] Both the triple and quadruple-strike strategies combined with aging can induce myocardial inflammation initiation, stress amplification, and fibrotic remodeling, but there are differences in the activation intensity between the two groups. This suggests that in the context of aging, multiple stimuli such as high-fat diet and hypertension are sufficient to induce myocardial tissue into a HFpEF-like pathological state characterized by stress response, low-grade inflammation, and structural remodeling. The SAM quadruple-strike group can further amplify the processes of local myocardial inflammation recognition, inflammatory transcription, endothelial adhesion, pyroptosis execution, and extracellular matrix deposition on the basis of aging, lipid metabolism disorders, and hypertension. In other words, the quadruple-strike group has stronger inflammatory driving and fibrotic progression characteristics than the triple-strike group.

[0143] In summary, the pathological process of the C57BL / 6 male double-hit model is metabolic / neurological stress-inflammatory initiation-mild fibrotic remodeling, which is consistent with the early clinical pathological stage of HFpEF. The male triple-hit group formed a more complete cascade pathological process of inflammation initiation-stress amplification-fibrotic remodeling, which is closer to the pathogenesis of clinically comorbid HFpEF and is consistent with the clinically advanced pathological stage of HFpEF.

[0144] Both the SAM triple and quadruple strikes conform to the pathological process of inflammation initiation-stress amplification-fibrotic remodeling. The SAM triple strike is more consistent with the pathological stage of HFpEF clinical progression, while the SAM quadruple strike, with its worsening symptoms and decreased FS, corresponds to the pathological stage of HFpEF clinical terminal stage.

[0145] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A method for constructing a mouse model of heart failure with preserved ejection fraction, characterized in that, The study includes the following: mice are treated using a multi-pronged approach, which includes metabolic intervention and intervention with at least one cardiovascular and metabolic stressor; the treated mice are then subjected to a multi-dimensional phenotypic evaluation, which includes four dimensions: comorbidity burden evaluation, pathological and biochemical indicator evaluation, diastolic function evaluation, and myocardial structural remodeling evaluation; based on the results of the multi-dimensional phenotypic evaluation, the model is determined to have been successfully constructed.

2. The method according to claim 1, characterized in that, The metabolic intervention involved feeding the animal a high-fat diet.

3. The method according to claim 1, characterized in that, The cardiovascular and metabolic stressors are selected from one or more of L-NAME, streptozotocin, and high-salt drinking water.

4. The method according to claim 1, characterized in that, The mice were either C57BL / 6 wild-type mice or SAM rapidly aging mice.

5. The method according to claim 4, characterized in that, When the mice are C57BL / 6 wild-type mice, the multi-hit strategy is a double-hit strategy of high-fat diet combined with L-NAME drinking water.

6. The method according to claim 4, characterized in that, When the mice are C57BL / 6 wild-type mice, the multi-hit strategy is a triple-hit strategy consisting of a high-fat diet combined with L-NAME drinking water and intraperitoneal injection of streptozotocin starting in the 9th week of feeding.

7. The method according to claim 4, characterized in that, When the mice are SAM rapidly aging mice, the multi-hit strategy is a triple-hit strategy of high-fat diet combined with high-salt water intake.

8. The method according to claim 4, characterized in that, When the mice are SAM rapidly aging mice, the multi-hit strategy is a quadruple-hit strategy consisting of a high-fat diet combined with L-NAME drinking water and intraperitoneal injection of streptozotocin starting in the 9th week of feeding.

9. The method according to claim 1, characterized in that, The indicators for assessing the comorbidity burden include: systolic blood pressure ≥130 mmHg; total cholesterol ≥80% higher than the normal control group, or triglycerides ≥50% higher than the normal control group; fasting blood glucose ≥11.1 mmol / L, or peak blood glucose ≥16.7 mmol / L 30 / 60 minutes after glucose loading in a glucose tolerance test; serum creatinine ≥50% higher than the normal control group, or blood urea nitrogen ≥60% higher than the normal control group. The pathological and biochemical indicators evaluated included: myocardial interstitial collagen volume fraction ≥10%; myocardial tissue IL-6 level ≥50% higher than normal control group; any one of serum ANP, BNP, or NT-proBNP ≥100% higher than normal control group; lung tissue wet / dry weight ≥15% higher than normal control group; and exercise tolerance ≥25% lower than normal control group. In the evaluation of diastolic function, a left ventricular ejection fraction ≥50% is a prerequisite, and the indicators include: a left heart catheterization measurement of LVEDP ≥50% higher than the normal control group; at least two of the ultrasound indicators showing changes: an increase of ≥20% in E / e', a prolongation of ≥20% in IVRT, a decrease of ≥15% in E / A, and a decrease of ≥20% in the absolute value of dp / dt max compared to the normal control group; and at least two of the speckle tracking indicators showing changes: a decrease of ≥15% in GLS, a decrease of ≥25% in left atrial strain, and a decrease of ≥10% in left ventricular circumferential strain compared to the normal control group. The indicators for evaluating myocardial structural remodeling include: changes in at least two of the following ultrasound indicators: IVSd increased by ≥20%, LVPWd increased by ≥20%, RWTd increased by ≥30%, and left atrial diameter increased by ≥30%; heart weight / tibia length ratio was ≥30% higher than that of the normal control group, or myocardial cell HE staining cross-sectional area was ≥20% higher than that of the normal control group.

10. The use of the mouse model of heart failure with preserved ejection fraction constructed by the method according to any one of claims 1-9 in screening drugs for the treatment of heart failure with preserved ejection fraction.

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