Method Article

Modeling and Evaluation of Murine Diabetic Cardiomyopathy Model

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DOI:

10.3791/67189

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November 29th, 2024

 ,  ,  ,  ,  , 
1Department of Cardiology, West China Hospital, Sichuan University, 2Laboratory of Cardiac Structure and Function, Institute of Cardiovascular Diseases, West China Hospital, Sichuan University, 3Cardiac Structure and Function Research Key Laboratory of Sichuan Province, West China Hospital, Sichuan University

* These authors contributed equally

In This Article

Summary

This protocol outlines a method for inducing diabetic cardiomyopathy through a combination of high-fat diet feeding and streptozotocin injection. This approach aims to provide a reliable framework for scientific investigation into diabetic cardiomyopathy and to explore potential avenues for clinical treatment applications.

Abstract

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus (T2DM), remain poorly understood. The myocardial toxicity associated with T2DM is attributed to factors such as lipotoxicity, glucotoxicity, oxidative stress, reduced cardiac efficiency, and lipoapoptosis. Compared to rats, mice offer greater accessibility, cost-effectiveness, and broader applicability for animal experiments. Insulin resistance and impaired insulin secretion are crucial factors in the pathophysiology of T2DM. We introduce a novel nongenetic murine model that replicates the progression of human DbCM induced by a combination of high-fat diet (HFD) feeding and streptozotocin (STZ) injection. In this study, we used wild-type C57BL/6J mice, administering an HFD regimen for 12 weeks, followed by intraperitoneal injections of STZ for an additional 12 weeks to induce characteristic manifestations of T2DM. We conducted oral glucose tolerance tests and measured serum insulin concentrations to confirm the development of insulin resistance and insufficient insulin secretion. Cardiac structure and function were rigorously assessed through noninvasive transthoracic echocardiography. Pathological characteristics were evaluated through Masson's trichrome staining and wheat germ agglutinin (WGA) staining, revealing pathological features related to DbCM. Therefore, we provide a robust and versatile method for establishing a nongenetic murine model of DbCM.

Introduction

Type 2 diabetes mellitus (T2DM) is a progressively escalating global health concern, standing as a leading cause of morbidity and mortality among affected individuals. The prevalence of T2DM is closely related to the rising epidemic of obesity1,2. More than one-third of patients with T2DM exhibit a distinct cardiovascular phenotype termed diabetic cardiomyopathy (DbCM), characterized by myocardial dysfunction that occurs independently of coronary artery disease, hypertension, and valvular heart disease3. Emerging evidence suggests that approximately 20% of diabetic patients are predisposed to developing heart failure3, a condition closely associated with their prognosis4. Several pathophysiological mechanisms of DbCM have been proposed, including inflammation5, cardiac remodeling and dysfunction6, mitochondrial dysfunction7, oxidative stress8, and metabolic disturbances9. Despite extensive research, the complete spectrum of mechanisms and their individual contributions to DbCM remain incompletely understood10. Therefore, well-established preclinical animal models are essential for advancing the study of this condition.

T2DM is characterized by insulin resistance and progressive insulin insufficiency11, with most being overweight or obese12. In this study, we establish a stable and modified murine model of DbCM based on previous studies, combining high-fat diet (HFD) feeding and streptozotocin (STZ) injection. In T2DM, insulin resistance and pancreatic β-cell destruction contribute to the disease's pathophysiology12. This model leverages two prominent risk factors of T2DM. Specifically, mice fed with HFD develop insulin resistance, and subsequent STZ injections further impair pancreatic islet β-cell function, significantly leading to the progression of T2DM-like pathophysiology. STZ, isolated from Streptomyces achromogenes, was first described in 1963 for its selective destruction of pancreatic islet β-cells and its diabetogenic properties13. There are two STZ treatment models: administering a relatively high dose of STZ results in a short-term cardiomyopathy model, while repeated low-dose STZ injections induce a T2DM model that naturally progresses to DbCM. As a result of the latter method, animals develop hyperglycemia, polydipsia, and polyuria, all of which are characteristics of human T2DM and develop DbCM naturally.

This study found that wild-type C57BL/6J mice fed with HFD followed by intraperitoneal STZ injections (30 mg/kg) exhibited cardiac function, morphology, and histology consistent with the characteristics of DbCM by the end of the experiment. This approach provides an effective method for establishing a murine DbCM model.

Protocol

All procedures followed institutional guidelines for animal research in accordance with the Guide for the Care and Use of Laboratory Animals outlined by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1996). All animal experiments were approved by the Ethics Committee of Animal Care and the Ethics Committee of Sichuan University. Male C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co. (China). Throughout the experimental duration, mice were housed under controlled conditions of 24 °C and a regulated 12 h light/dark cycle, with ad libitum access to food and water.

Mouse study timeline diagram, ND vs. HFD/STZ, OGTT, insulin, citrate, STZ, experimental setup.
Figure 1: Schematic experiment timeline. The mice used in HFD/STZ and ND groups were both at approximately 7-8 weeks of age. The body weight and random blood glucose level of each mouse were recorded on a weekly basis. At the 12-week and 24-week feeding intervals, the metabolic phenotype of mice was evaluated by an oral glucose tolerance test (OGTT) and serum insulin level measurement. Additionally, echocardiography was assessed at baseline (week 0), 12 weeks, and 24 weeks. Following the 24-week feeding period, the mice were euthanized. Please click here to view a larger version of this figure.

1. HFD feeding

  1. Feed male C57BL/6J mice (7-8 weeks old) either with a normal diet (ND) or HFD (60% kcal fat) for 24 weeks to induce obesity.
  2. Record the body weight of each mouse every 4 weeks. Ensure the weight curve exhibits a similar upward pattern before 12 weeks, with a steeper slope observed in the HFD group.

2. STZ solution preparation and injection

NOTE: Store STZ at -20 °C to prevent degradation due to its high instability at room temperature (RT). Blend STZ with citrate buffer immediately within 5 min before injection.

  1. Prepare citrate buffer. Weigh 2.1 g of citric acid (relative molecular mass: 210.14) and dissolve it thoroughly in 100 mL of distilled water to prepare solution A; weigh 2.94 g of sodium citrate (relative molecular mass: 294.10) and dissolve it thoroughly in 100 mL of distilled water to prepare solution B. Mix solution A and B in a 1: 1.32 ratio to obtain the citric acid working solution. Filter out impurities using a 0.45 µm filter membrane.
  2. Weigh 1 g of STZ and dissolve it thoroughly in 100 mL of the prepared mixed citric acid working solution. Store the resulting STZ solution at 4 °C. Ensure the injection of the STZ solution occurs within 30 min whenever feasible.
  3. Using a 1 mL syringe, administer intraperitoneal injections of a solution containing 30 mg/kg of STZ daily for 7 consecutive days to mice in the HFD/STZ group while providing an equal volume of citrate buffer (0.1 mmol/L, pH = 4.5) to mice in the ND group.
  4. Assay blood glucose levels 1 week after the final injection. Classify mice exhibiting random blood glucose concentrations exceeding 16.7 mmol/L as T2DM mice.
    1. Carefully position the mouse onto a restraining device, ensuring its tail extends fully outside.
    2. Prior to blood collection, sanitize the tail's blood collection site thoroughly with a 70% alcohol swab, maintaining sterility and cleanliness.
    3. With a sharp-tipped needle, insert it into the tail vein, advancing from the tip towards the base at a depth of 3-4 mm. Once the needle is securely in place, gently squeeze the top of the tail to encourage blood flow.
    4. Collect approximately 10 µL of blood from a mouse and carefully apply the collected blood onto the dedicated test strip. Once the blood is absorbed, the glucose meter will promptly display the blood glucose reading.

3. Oral glucose tolerance test (OGTT)14

  1. Before OGTT, fast each mouse overnight for 14 h, while it has unrestricted access to water.
  2. Prepare approximately 10 mL of a 20% glucose solution. Administer a dose of 1 g/kg glucose via oral gavage to each mouse during the OGTT.
  3. Monitor blood glucose levels at several time points: immediately (0 min), 15 min, 30 min, 60 min, and 120 min post-administration.

4. Echocardiography assessment of cardiac function

  1. Conduct echocardiography on each mouse at baseline (0 weeks), 12 weeks, and 24 weeks after feeding with either HFD or ND.
  2. Prior to echocardiography, anesthetize the mouse via inhalation of 3% isoflurane which is administered with 100% oxygen using an anesthetic machine. Ensure the mouse shows no reaction to skin pinching using a toothed tweezer or stimulation of its toes and tail. Apply veterinary ointment to the eyes to prevent dryness during anesthesia.
  3. Position the mouse on a heating pad to maintain body temperature, and secure its claws to an electrode to ensure a stable supine position. Adjust the isoflurane concentration between 1%-2% for maintenance anesthesia to maintain a target heart rate of approximately 450 beats per minute.
  4. Remove the mouse's fur using depilatory cream and apply ultrasonic gel to its chest. Remove the depilatory cream and ultrasonic gel with sterile saline following echocardiography.
  5. Evaluate the cardiac function and structural parameters with a 50 MHz probe.
    1. To obtain an optimal left ventricle (LV) long-axis view, position the probe on the left side of the animal's chest.
    2. Depending on the individual anatomy, rotate the probe counterclockwise between 15° and 45° relative to the left parasternal line, with the notch directed toward its right shoulder. Adjust the x-axis and y-axis under the B-mode15.
      NOTE: An appropriate LV long-axis view should include (i) the aortic valve and aortic root; (ii) the LV chamber is positioned in the center of the view; (iii) the base-to-apex axis should be parallel to the transducer16.
    3. Press M-mode 2x to display the measuring line. Position this line at the level of the papillary muscle. Subsequently, take measurements for at least three consecutive heartbeats to ensure accuracy. Tap Save Clip to save the cine loop in the series.
    4. Identify the end-systolic dimension as the phase that coincided with the ECG T wave, and determine the end-diastolic dimension as the phase corresponding to the ECG R wave.
    5. Tilt the operating pad, ensuring the upper left corner is lowest, and the lower right corner is highest. Adjust the probe to penetrate along the direction of the mouse's cardiac apex, parallel to the long axis of the heart, to get a transapical four-chamber view.
    6. Successively select C (Color Doppler) and PW (PW Doppler) modes, place the sample volume at the highest velocity point, adjust the sampling direction to match the direction of blood flow, and record mitral valve hemodynamic information (E and A wave)16. Tap Save Clip to save the cine loop in the series.
  6. Halt isoflurane inhalation to allow the mice to regain consciousness. Return the animals to their cages, housed in a controlled environment with a 12-h light/dark cycle at both the 0 and 12 week marks. Following the 24-week echocardiographic assessment, humanely euthanize the mice through cervical dislocation following isoflurane inhalation.
  7. Perform the analysis of the ejection fraction (EF), fraction shortening (FS), left ventricular posterior wall (LVPW), left ventricular internal diameter (LVID), interventricular septum (IVS), and E/A ratio using the workstation dongle.

5. Histological staining

  1. After completing the echocardiographic analysis at the 24-week mark, euthanize the mouse by spinal cord dislocation following isoflurane inhalation.
  2. Dissect the mouse using ophthalmic scissors and tweezers. Sever the ribs carefully to ensure complete exposure of the heart.
  3. Perfuse the heart through the apex with saline until the liver becomes pallor, indicating successful perfusion. Remove the heart and rinse it thoroughly in saline to ensure complete blood removal.
  4. Perform paraffin embedding17.
    1. Fix the heart with 4% formalin at RT for at least 24 h.
    2. Place the dehydration box containing the heart in a dehydrator to undergo a gradual dehydration process involving gradient alcohol and wax leaching at 65 °C.
  5. Prepare tissue sections17.
    1. Place the trimmed wax block containing papillary muscle into a microtome for slicing, with a thickness of 4 µm, and store at RT for pathological staining.
  6. Perform stain using Masson's trichrome18 and wheat germ agglutinin (WGA)19 staining.

Results

This study involved random allocation of mice into two groups: the ND group and the HFD/STZ group, with 6 mice in each group. Subsequent to the final echocardiography and OGTT tests 24 weeks after feeding, the mice were euthanatized to harvest their heart tissues for a histological assessment.

HFD/STZ caused an obvious body weight gain, reaching its peak in 12 weeks, and was significantly higher compared with the ND group. Following the administration of STZ, a notable decrease in body weight was observed among the mice in the HFD/STZ group. In contrast, the mice in the ND group maintained a steady increase in their body weight over the subsequent 12-week period (Figure 2A). The random blood glucose levels of the HFD/STZ group were always significantly higher than the ND group during the whole feeding time (Figure 2B). The OGTT curve of the HFD/STZ group was significantly higher than that of the ND group. It was apparent that the blood glucose peak in both groups reached in 15 min and then declined (Figure 2C,D). During the course of the study, serum insulin levels in the ND group were initially higher than in the HFD group in the first 12 weeks. However, after giving STZ, a reversed trend was observed with levels of serum insulin (Figure 2E,F).

Hearts exposed to HFD/STZ displayed both diastolic and systolic dysfunction compared with ND group, presenting as weak amplitude ofinterventricular septum (IVS) and left ventricular posterior wall (LVPW) pulsation (Figure 3), decreased ejection fraction (EF) andfractional shortening (FS) (Figure 4A,B), increased end-diastolic left ventricular internal dimension (LVID), incrassated IVS and LVPW (Figure 4C,D,E), and decreased E/A ratio (Figure 4F) in HFD/STZ group in 24 weeks.

Cardiac remodeling stemming from metabolic disorders is shown in Figure 5A,B. A histological examination uncovered increases in cardiac fibrosis and the proportion of cardiomyocyte-occupied area within the HFD/STZ group, compared with the ND group (Figure 5C).

Graphs of body weight, glucose levels, and insulin comparison in ND and HFD/STZ mice over time.
Figure 2: Body weight, random blood glucose, OGTT, and serum insulin level change during feeding time. (A) Body weight shift of mice during feeding time. The weight curve exhibited a similar upward pattern before 12 weeks, with a steeper slope observed in the group. Following the 12-week period, a notable reduction in weight was observed in the HFD/STZ group, whereas the ND group maintained its upward trajectory in weight gain. (B) Random blood glucose levels changed within feeding time. The blood glucose level remained stable in the ND group, while it continued to rise in the HFD/STZ group, with a significant increase after 12 weeks. (C) and (D) Blood glucose levels during OGTT at 12-week and 24-week feeding. (E,F) Serum insulin level at 12 weeks and 24 weeks for ND and HFD/STZ group. After 12 weeks of experimentation, the insulin level in the HFD/STZ group exhibited a notable elevation compared to the ND group. However, by the 24-week mark, this trend was reversed, with the insulin level in the HFD/STZ group being significantly lower than that of the ND group. Data are presented as mean ± SD. P values were calculated by unpaired t-test. **: P < 0.01, ***: P < 0.001, ****: P < 0.0001; ND (n = 6) vs HFD/STZ (n = 6). Please click here to view a larger version of this figure.

Ultrasound analysis; cardiac function comparison; ND vs. HFD/STZ; weeks 0, 12, 24; heart study chart.
Figure 3: Representative M-mode echocardiography and E/A wave of the mitral valve at 0, 12, and 24 weeks. There were differences in the heights of E and A peaks between the two groups, with E peak > A peak at 0 week and E peak almost equal to A peak at 24 weeks in the HFD/STZ group. The M-mode color Doppler ultrasound showed that the amplitude of IVS and LVPW pulsation was weaker in the HFD/STZ group at 12 and 24 weeks. Please click here to view a larger version of this figure.

Cardiovascular measurements graph; ND vs HFD/STZ over time, EF, FS, LVID, IVS, LVPW, E/A ratio.
Figure 4: Quantitative results of cardiac structure and function. (A) Ejection fractions (EF); (B) Fraction shorting (FS); (C) Left ventricular end-diastolic internal diameter (LVID, d); (D) Interventricular end-diastolic septum (IVS, d); (E) Left ventricular end-diastolic posterior wall (LVPW, d); (F) E/A ratio. At 12 weeks, the HFD/STZ group exhibited diastolic dysfunction, manifesting as increased LVID and E/A ratio and normal EF. By 24 weeks, both systolic and diastolic functions declined, showing thickening of LVPW and IVS, significant decreases in EF and FS, further increases in LVID, and further decreases in the E/A ratio. Data are presented as mean ± SD. P values were calculated by unpaired t-test. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001; ND (n = 6) vs HFD/STZ (n = 6). Please click here to view a larger version of this figure.

Histology comparison, ND vs HFD/STZ, muscle fibers; DAPI/WGA, Masson staining, bar graph results.
Figure 5: Mice in the HFD/STZ group lead to cardiac remodeling after 24 weeks. (A) Representative WGA staining of heart tissues in each group. (B) Representative Masson staining of heart tissues in each group. (C,D) Quantified cross-sectional area and fibrotic area of the LV. Significantly increased fibrosis and cardiac hypertrophy were observed in the HFD/STZ group compared to the ND group. Data are presented as mean ± SD. P values were calculated by unpaired t-test. ***: P < 0.001, ****: P < 0.0001; ND (n=6) vs HFD/STZ (n=6). Please click here to view a larger version of this figure.

Discussion

Given the widespread prevalence of diabetes mellitus and its associated cardiovascular complications globally, there is an urgent need to uncover the underlying molecular mechanisms and develop preventative and therapeutic strategies for this condition20. The pathogenesis of DbCM, one of the cardiovascular complications for patients with T2DM, remains unclear, with no effective approaches to prevent and treat21. The absence of reliable preclinical models that accurately mimic the pathophysiological processes of the human heart highlights the significance of developing a stable and effective model specifically designed to investigate DbCM mechanisms.

The protocol we present outlines a well-established and stable method for generating a DbCM mouse model, which serves as a robust tool for studying T2DM-associated myocardial dysfunction. HFD feeding to induce insulin resistance is generally regarded as an effective approach to model many complications associated with human diabetes22. In this method, HFD is combined with low-dose STZ injections, an agent that induces pancreatic islet damage. This method can better simulate the process of insulin resistance and relative insulin insufficiency.

Generally, preclinical models of DbCM fall into two categories: genetic and diet-pharmacologically induced models. The ob/ob mouse23,24, the db/db mouse25,26, and the Zucker diabetic fatty rats27 are the three most widely used genetic models28,29. These three rodent models develop increased body weight, hyperglycemia, impaired glucose handling, and insulin resistance due to the intervention of leptin signaling or its receptor. However, leptin has cardioprotective properties30, which can potentially complicate the interpretation of results from these models since alterations in leptin signaling may directly affect cardiac function. Additionally, mutations in the leptin receptor are rarely the cause of T2DM in humans31. Moreover, genetic models are also costly and present important differences from the human condition32. Compared with rats, mice are more readily available, cost-effective, and widely used in animal experiments. Some studies use pure diet-indued methods to establish the T2DM model, such as HFD and/or sugar (high sucrose or high fructose), which may cause obesity, insulin resistance, and diabetes33,34,35,36. However, the severity of hyperglycemia and insulin resistance is less pronounced compared to genetic models like the ob/ob or db/db mice37.

Combining diet-induced with diabetogenic agent STZ is considered more suitable for medical research than diet-based models alone. Although the general procedures of the HFD/STZ DbCM model have been described in several studies, we have made several improvements. Although T2DM predominates among diabetes cases in humans, creating an animal model for it presents greater challenges compared to T1DM. The pivotal factor lies in the dosage and frequency of STZ injections, which significantly impact model establishment. Excessive STZ doses result in a model closely resembling T1DM and lead to higher mortality rates.

Previous studies indicated that administering multiple low-dose injections of STZ at a dosage of 50 mg/kg better models T2DM31,38,39. However, these studies often have relatively short HFD feeding durations, ranging from 3 weeks with varying fat content. A study by Ahlke Heydemann40 showed that prolonged HFD feeding results in excess lipid storage, leading to lipid toxicity, obesity, decreased metabolic flexibility, oxidative stress, and chronic inflammation-conditions similar to those observed in DbCM heart tissue. In this study, we extended the HFD feeding period to 24 weeks and used a lower STZ concentration (30 mg/kg) to mimic DbCM pathology. C57BL/6J mice, which carry a gene predisposing them to T2DM, were used as the model animal41,42. Our observations revealed that after 12 weeks, the mice in the HFD/STZ group began to lose weight. It is important to note that weight loss in diabetes can be a complex issue. While insulin insufficiency and hyperglycemia can lead to weight loss, other factors such as decreased appetite, dietary changes, and increased physical activity may also play a role. Additionally, not all diabetic patients experience weight loss; some may even experience weight gain, particularly if they have insulin resistance and consume a high-calorie diet.

This method has some limitations. First, it takes a relatively long time to develop DbCM, 24 weeks in total. Additionally, STZ may exhibit toxicity toward organs and tissues beyond pancreatic islets, making the HFD/STZ model less accurate in replicating human disease. Furthermore, citrate buffer, used for STZ, has a pH of 4.5. Administering acidic solutions intraperitoneally can harm animals by causing red blood cell hemolysis, as well as pain and discomfort43, which may limit the model's applicability. Finally, the cardiac manifestations in T2DM patients are varied and often accompanied by hypertension, coronary artery disease, and other cardiovascular diseases, complicating clinical management. In this model, cardiac manifestations include LV dilation and hypertrophy, as well as systolic and diastolic dysfunction, but these do not fully represent the spectrum of DbCM seen in clinical practice. DbCM also includes features such as electrophysiologic alterations44 or atrial changes45, whereas the focus here is primarily on LV structure and pump function.

Consistent with previous studies, the method described here of combining HFD feeding with STZ injections aims to induce diabetes more rapidly by partially destroying pancreatic tissue and inducing insulin resistance to model DbCM. Enlarged cardiomyocytes with disorganized arrangement, myocardial fiber rupture, dissolution, reduced EF, FS, etc., indicate myocardial dysfunction and reduced cardiac function. In summary, we have presented an approach for establishing DbCM in a cost-effective and convenient manner. Utilizing noninvasive echocardiographic techniques and pathological staining, we assessed the efficacy of this method.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant numbers: U23A20395, 81900258, and 82170375); the Key Research and Development Project of Science & Technology Department of Sichuan Province (2022ZDZX0020); Chinese Medical Association Cardiovascular Branch (CSC) Clinical Research Special Fund Project (CSCF2020B04); 1· 3· 5 project of West China Hospital, Sichuan University (ZYGD23021). Thanks to Qing Yang (Animal Imaging Core Facilities, West China Hospital, Sichuan University) for their help in small animal ultrasonography.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Animal ultrosound systemFujifilm Visual SonicsVEVO 3100Echocardiography
Blood glucometerYuwellGU100Assess blood glucose level
Citric acidSigma-Aldrich251275
IsofluraneRWD life scienceR510-22Anesthesia
Isoflurane vaporizerRWD life scienceR500Anesthesia
Mouse insulin (INS) ELISA KitWuhan Feiyue Biotechnology Co.,LtdFY-EM14029Assess serum insulin level
Nair hair removal creamNair255gRemove the fur of mouse
Rodent diet with 60% kcal fatResearch Diets IncD12492High fat diet feeding
Sodium citrateSigma-AldrichS4641
Sterile filterMerck MilliporeSLHV033N
StreptozocinSolarbioS8050
Ultrasound gelKepplerKL-250Echocardiography
Workstation DongleFujifilm Visual SonicsVevo LABEchocardiographic data analysis

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Murine ModelHigh-Fat DietStreptozotocin InjectionInsulin ResistanceOral Glucose ToleranceTransthoracic EchocardiographyCardiac DysfunctionMasson's Trichrome StainingSerum Insulin
Modeling and Evaluation of Murine Diabetic Cardiomyopathy Model
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