Graded Maximal Exercise Testing to Assess Mouse Cardio-Metabolic Phenotypes - PubMed (original) (raw)

Graded Maximal Exercise Testing to Assess Mouse Cardio-Metabolic Phenotypes

Jennifer M Petrosino et al. PLoS One. 2016.

Abstract

Functional assessments of cardiovascular fitness (CVF) are needed to establish animal models of dysfunction, test the effects of novel therapeutics, and establish the cardio-metabolic phenotype of mice. In humans, the graded maximal exercise test (GXT) is a standardized diagnostic for assessing CVF and mortality risk. These tests, which consist of concurrent staged increases in running speed and inclination, provide diagnostic cardio-metabolic parameters, such as, VO2max, anaerobic threshold, and metabolic crossover. Unlike the human-GXT, published mouse treadmill tests have set, not staged, increases in inclination as speed progress until exhaustion (PXT). Additionally, they often lack multiple cardio-metabolic parameters. Here, we developed a mouse-GXT with the intent of improving mouse-exercise testing sensitivity and developing translatable parameters to assess CVF in healthy and dysfunctional mice. The mouse-GXT, like the human-GXT, incorporated staged increases in inclination, speed, and intensity; and, was designed by considering imitations of the PXT and differences between human and mouse physiology. The mouse-GXT and PXTs were both tested in healthy mice (C57BL/6J, FVBN/J) to determine their ability to identify cardio-metabolic parameters (anaerobic threshold, VO2max, metabolic crossover) observed in human-GXTs. Next, theses assays were tested on established diet-induced (obese-C57BL/6J) and genetic (cardiac isoform Casq2-/-) models of cardiovascular dysfunction. Results showed that both tests reported VO2max and provided reproducible data about performance. Only the mouse-GXT reproducibly identified anaerobic threshold, metabolic crossover, and detected impaired CVF in dysfunctional models. Our findings demonstrated that the mouse-GXT is a sensitive, non-invasive, and cost-effective method for assessing CVF in mice. This new test can be used as a functional assessment to determine the cardio-metabolic phenotype of various animal models or the effects of novel therapeutics.

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Conflict of interest statement

Competing Interests: The authors state that their commercial funding does not alter their adherence to PLOS ONE Editorial policies and criteria.

Figures

Fig 1. Description of exercise testing in mice and men.

(A) Schematic description of exercise testing in mice and men. The PXTm maintains fixed inclination (0°) while speed increases until the test is terminated (Table 1). Training in mice was done on a chamber-enclosed treadmill that allowed it to function as an open circuit indirect calorimeter; and thus, allowed for derivation of VO2 and VCO2 values. With the GXTm (middle) and GXTh (right), speed and incline simultaneously increased as stages progressed (Table 1). (B) Human and mouse tests used indirect calorimetry to measure VO2 (solid line) and record VO2max as well as measure CO2 (dashed line). Mouse and man tests were randomly selected from WT males (n = 7) and healthy men (n = 6) and used for derivation of all parameters (all data are shown in S1 and S2 Figs). During maximal exercise testing both species have similar responses (RER, lactic acid formation, fuel utilization, O2 use, heart rate, speed, exhaustion). In the GXTm, and GXTh. at VO2max, VCO2 intersected or surpassed VO2, and was a parameter of a positive test (as RER >1.0, middle and right panel). In the PXTm, VO2max did not fulfill this criterion (left panel).

Fig 2. Kinetics and parameters from PXTm, GXTm, and GXTh using single test analysis.

The same single mouse and man tests were randomly selected from WT males (n = 7) and healthy men (n = 6) and used for derivation of all parameters. (A) RER (VCO2/ VO2) represents fuel substrate utilization during PXTm, GXTm, and GXTh. An RER of .85 (solid horizontal arrow) indicates 50% carbohydrate and 50% fat oxidation. In a single test, the point in which there is an abrupt increase in RER is known as anaerobic threshold (AT, dashed arrow). (B) Carbohydrate (dashed line) and fat (solid line) oxidation was determined from RER values (S6 Table) in the PXTm, GXTm, and GXTh. Dotted vertical arrow indicates the crossover time point in the test where there is a shift from predominant lipid oxidation to predominate carbohydrate oxidation.

Fig 3. VO2max achieved with the GXTm, but not the PXTm, identifies impaired cardiovascular fitness in mouse models of cardiovascular dysfunction.

(A) Averaged VO2 kinetics obtained from WT (dashed line), _Casq2_-/- (solid thick line), and obese (solid line) mice that performed the PXTm and GXTm. VO2 is indicated for each group from the beginning of the test until the end (Minute 2, after the 2 minute warm, to point of exhaustion). Of note, during the PXTm, the largest increase in VO2 occurred with the first stage, and leveled off as the test continued in dysfunctional models. (B) Relative VO2max values and (C) change from baseline to maximal oxygen consumption (VO2delta) are compared (mean±SD, MANOVA, Tukey HSD Multiple Comparisons, alpha = .007) in WT-healthy (white bar), WT-obese (hashed bar), and _Casq2_-/- (black bar) mice. Asterisks indicate significance at the alpha = .007 level (MANOVA, multiple comparisons Tukey HSD for the PXTm and GXTm of WT v. obese and WT v. _Casq2_-/-), hash indicates significant difference at the alpha = .05 level between tests for the same genotypes (Student’s t-Test), and diamond indicates significance at the alpha = .007 level (MANOVA, multiple comparisons Tukey HSD for the PXTm and GXTm of obese v. _Casq2_-/-).

Fig 4. Time until exhaustion at true VO2max is confirmed by increases in lactate concentrations and demonstrates impaired cardiovascular fitness with the GXTm, but not the PXTm.

(A) Time until exhaustion derived from VO2 kinetics in same groups of mice described in Fig 3. The dashed line shows the time used for warm up. (B) Change from pre to post test lactate concentration (LAdelta) in same group of mice. Bar graphs represent mean ± SD for all but lactate, which is mean ± SEM. Student’s t-Test; p < .05, WT v. _Casq2_-/-.

Fig 5. Anaerobic threshold and maximum speed assess dysfunction in mice with the GXTm, but not the PXTm.

(A) Average RER kinetics from same from WT (dashed line), _Casq2_-/- (solid thick line), and obese (solid line) mice performed the PXTm and GXTm. (B) AT was reported as %AT, a time point where AT occurred/total test time in mouse groups (Fig 3). (C) Maximum speed achieved on test (m/m). For all measures, asterisks indicates significance at the alpha = .007 level (MANOVA, multiple comparisons Tukey HSD for the PXTm and GXTm of WT v. obese and WT v. _Casq2_-/-), hash indicates significant difference at the alpha = .05 level between tests for the same genotype (Student’s t-Test), and diamond indicates significance at the alpha = .007 level (mean ± SD, MANOVA, multiple comparisons Tukey HSD for the PXTm and GXTm of obese v. _Casq2_-/-).

Fig 6. Carbohydrate and fat oxidation kinetics can be used to identify the crossover point in the GXTm, but not the PXTm.

Averaged fuel utilization kinetics in WT (n = 7), obese (n = 11), and _Casq2_-/- (n = 4) mice. Fat (dashed line) and carbohydrate (Carb, solid line)) oxidation were derived from RER as described in S3 Table during the PXTm (A) and GXTm tests (B). In GXTm tests, the arrow indicates crossover, the point at which carbohydrate and fat oxidation intersect (dashed arrows).

Fig 7. Carbohydrate and fat oxidation parameters from the GXTm, but not the PXTm, can be used to identify impaired cardiovascular fitness in dysfunctional models.

(A) Fuel utilization kinetics (Fig 6B) from the GXTm were used to quantify the percent of the test at which the crossover point was achieved relative to total time of test (% of test time at which crossover is achieved is the minute crossover occurred divided by total test time and multiplied by 100). Asterisk indicates significance at the alpha = .007 level (MANOVA, multiple comparisons Tukey HSD for the GXTm). (B) Time until 100% carbohydrate oxidation. Asterisks show significant difference between dysfunctional v. WT groups, (Student’s T-test, GXTm for WT v. obese and WT v. _Casq2_-/-). (C) Rate of carbohydrate oxidation after crossover in all mouse groups. This rate is determined by dividing time after crossover by total time of test (100%) (Student’s T-test for the GXTm for WT v. obese and WT v. _Casq2_-/-. Bar graphs represent mean ± SD).

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Graded Maximal Exercise Testing to Assess Mouse Cardio-Metabolic Phenotypes - PubMed (original) (raw)