Graded Maxium Exercise Test for Laboratory Mice (GXTₘ)

Simulating Various Human Disease Characteristics to Support Drug Development

Introduction

This article introduces a Graded Maximal Exercise Test (GXTₘ) method for assessing cardiovascular metabolic phenotypes in mice, using an animal metabolic treadmill to evaluate cardiovascular health. The method involves progressively increasing the exercise load to simulate human maximal exercise tests (e.g., treadmill or cycling ergometry), enabling a comprehensive evaluation of cardiovascular function in mice.

1. Research Objective:

Cardiovascular Disease Research: Rising obesity rates increase the risk of cardiovascular diseases. Human studies often use graded maximal exercise testing (GXT) to assess cardiovascular function (CVF) and diagnose diseases, but there is a lack of standardized exercise tests and sufficient parameters for mice.

Cardiovascular Function Studies: Mouse models are crucial for cardiovascular disease research, and reliable methods to assess CVF in mice are necessary.

Establishing Animal Models: Models such as obese mice, atherosclerotic mice (e.g., APOE knockout and LDLR knockout mice) are vital for studying the mechanisms of human-related diseases.

Simulating Human Disease Characteristics: For example, LDLR knockout mice model the clinical features of familial hypercholesterolemia, including damage to the aortic valve and aortic root, which helps explore the relationship between atherosclerosis and diabetes.

Detecting Potential Heart Disease: GXTₘ can help detect latent heart problems in mice, similar to how human treadmill tests detect potential cardiovascular diseases.

Evaluating Drug Efficacy: This method provides a way to assess the effectiveness of drugs for treating cardiovascular diseases. For instance, gene therapy using AAV9 virus for liver-specific expression of LDLR in mice shows partial restoration of LDLR function, validating the therapy's effect.

Guiding Treatment Decisions: Based on test results, more personalized treatment plans can be devised, including drug therapy, exercise rehabilitation, or other interventions.

Understanding Disease Progression: Observing various parameter changes during exercise tests in different mouse models helps understand disease progression, offering insights for human disease research.

Screening Treatment Targets: Gene-modified mouse models reveal potential therapeutic targets, such as genes linked to obesity and other diseases, which can be further validated in later studies.

Metabolic Disease Research: GXTₘ helps study the mechanisms and treatment methods of metabolic cardiovascular diseases like obesity, diabetes, heart metabolic disorders, hyperlipidemia, atherosclerosis, and hypertension.

2. Research Methods:

Graded Maximal Exercise Testing (GXTₘ): The treadmill speed and incline are progressively increased, with parameters such as speed, duration, and incline (e.g., speed 0m/min for 3 minutes at 0° incline, then 6m/min for 2 minutes at 0°, and 9m/min for 2 minutes at 5°) until exhaustion (defined as the mouse making continuous contact with the electrified grid for 5 seconds). At exhaustion, VO₂ reaches its peak (VO₂max), and parameters such as RER, maximum running speed, and lactate levels before and after the test are measured.

Progressive Maximal Exercise Testing (PXTₘ): The treadmill incline is fixed at 0°, and speed is progressively increased until exhaustion. Lactate is measured before and after the test.

Subjects: Male C57BL/6J (WT), FVBN/J, obese C57BL/6J (diet-induced), and Casq2 (cardiac troponin C deficiency) mice, 4-6 months old, housed under a 12-hour light/dark cycle and fed a standard diet.

Equipment Calibration: The energy metabolism treadmill software and gas analysis system are calibrated before the test, adjusting parameters like pressure and flow, and calibrating gas concentrations to ensure accurate measurements of oxygen consumption (VO₂), carbon dioxide production (VCO₂), and respiratory exchange ratio (RER).

3. Research Results:

GXTₘ Testing Characteristics: By comparing with human GXT tests, the GXTₘ method is designed to account for mouse physiological differences, inducing VO₂max and generating comparable variables such as anaerobic threshold (AT), metabolic crossover point, and lactate changes. The GXTₘ lasts 8-12.5 minutes, while PXTₘ lasts 20-29 minutes (excluding warm-up).

Sensitivity to Cardiovascular Dysfunction: GXTₘ detects changes in VO₂max, exhaustion time, maximum running speed, and AT in dysfunctional mouse models, indicating cardiovascular impairment, while PXTₘ is less sensitive in these aspects. For example, Casq2 and obese mice showed significant decreases in VO₂max, exhaustion time, and running speed in GXTₘ, but no significant changes in PXTₘ.

Substrate Utilization Changes: GXTₘ identifies the crossover point for fuel utilization from fat to carbohydrates, and in dysfunctional mouse models, the timing and rates of carbohydrate oxidation differ from healthy mice. PXTₘ is less effective at determining the crossover point.

4. Discussion:

Design Considerations for GXTₘ: The design of GXTₘ follows the principles of human GXT, adjusted for mouse equipment and physiological differences. Despite some limitations in mouse testing, this method successfully induces VO₂max and generates comparable data.

Value of Determining Anaerobic Threshold: PXTₘ is too long to effectively stimulate the cardiovascular system and cannot determine AT. In contrast, GXTₘ can identify AT through RER, providing a sensitive marker for evaluating CVF in mice, with significant clinical and testing applications.

Metabolic Crossover Point: GXTₘ helps identify the metabolic crossover point, reflecting changes in fuel substrate utilization with exercise intensity, and can serve as a supplementary parameter for assessing CVF in cardiovascular dysfunction models.

Standardized Cardiovascular Function Assessment: GXTₘ provides a non-invasive, cost-effective method for assessing cardiovascular metabolic phenotypes in mice and is advantageous compared to other cardiac testing methods.

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Tow-Int Animal Metabolic Treadmill:

Tow-Int animal metabolic treadmill is designed specifically for animal respiratory metabolism measurement. This closed-system treadmill monitors parameters such as oxygen consumption (VO₂), VO₂max, CO₂ production, and respiratory exchange rate during exercise. The treadmill is essential for studying animal endurance, exercise-induced injuries, nutritional status, drug effects, exercise physiology, cardiovascular diseases, and other pathological mechanisms. Currently, Tow-Int Tech is conducting a trial activity for the energy metabolism treadmill. For more details, please contact us or scan the QR code in the image.