Loss of RET-ROS at complex I induces diastolic dysfunction in mice that is reversed by aerobic exercise

Abstract

Central to the development of heart failure with preserved ejection fraction (HFpEF) is the redox disruption of metabolic processes; however, the underlying mechanisms are not fully understood. This study utilized a murine model (ND6) carrying a homoplasmic mitochondrial DNA point mutation (ND6 G13997A), which maintains functional NADH oxidation but lacks the site-specific reactive oxygen species (ROS) generation via reverse electron transport (RET). We demonstrate that mice with RET-ROS deficiency have reduced exercise capacity despite higher lean body mass, impaired resilience to high-fat/high-sucrose dietary stress, and cardiac hypertrophy with diastolic dysfunction. Importantly, dobutamine-induced stress elevated succinate levels in the heart, accompanied by RET-ROS production in wild-type but not in ND6 mice. Furthermore, ND6 mice showed perturbation in metabolite profiles following dobutamine stress. Mechanistically, the ND6 heart had an upregulated expression of fatty acid transport, oxidation, and synthesis genes (CD36, Cpt1b, Acly, Fas, Elovl6, and Scd1) and increased protein levels of lipid metabolism regulators (acetyl-CoA carboxylase and perilipin 2). Interestingly, 8 wk of forced treadmill running increased acetyl-CoA abundance, alleviated metabolic stress, and improved diastolic function in RET-ROS mutant hearts. In summary, these findings reveal a critical role for RET-ROS in regulating exercise capacity and cardiometabolic health, identifying it as a potentially selective target for modulating cardiac metabolism.NEW & NOTEWORTHY Loss of reverse electron transport (RET)-reactive oxygen species (ROS) impairs diastolic function and exercise capacity, which can be improved by long-term aerobic exercise. RET-ROS may act as a modulator of cardiac metabolism.

Keywords: HFpEF; RET-ROS; exercise; lipid metabolism; mitochondria.

Figure 1. Dobutamine stimulates succinate production in both WT and ND6-P25L hearts, but RET-ROS is only generated in the WT heart.

A) Illustration of wild type (WT) (orange) and ND6-P25L mutation (blue) in mitochondrial complex I under conditions favorable for RET. RET occurs at complex I when the proton motive force (Δp) is large and/or the redox potential difference (ΔE_h) across complex I is low, such that Δ 2E_h <Δ4p. These conditions enable electron movement backward from the CoQ pool, allowing for one electron to pass to oxygen and generate superoxide (O₂^•-). The green arrow in complex I (WT) indicates RET, which does not occur in the ND6-P25L mutation. B) Heart Rate (HR) increased in response to an intravenous bolus of dobutamine (20 μg/kg) (n=3-19). C) Increased succinate production (normalized to heart weight) is detected after dobutamine stimuli in both WT and ND6-P25L (n=4-17). D) However, only WT hearts showed an increase in mitochondrial hydrogen peroxide generation, determined with the mitochondria-targeted probe MitoB (WT: n= 12-17, ND6: n= 4-6). E) Representative image of MitoP/MitoB distribution in the left ventricle in response to dobutamine or vehicle (saline) by mass-spectrometry imaging (MSI) (n =3 hearts per group, 4 tiers per heart). Hue saturation intensity (HSI) mapping of MitoP/MitoB distribution. The color scale ranges from brown (0%) to gold (40 %) in dobutamine-treated WT heart. Scale bar: 4 mm. The results presented are from male mice. Error bars indicate meanα S.E.M., significance was tested with two-way ANOVA and Tukey’s multiple comparisons test.

Figure 2. RET-ROS mutant mice have increased lean body mass, pathological cardiac remodeling, and diastolic dysfunction.

A) ND6-P25L mice showed higher body weights (n=16-18). B) Body mass composition analysis showed increased lean mass but unchanged fat mass in ND6 mice (n=10). C) Heart weight (HW) was increased in ND6 for any given body weight (BW), ANCOVA (WT 143.83; 95 % CI 134.96-152.69 vs. ND6 158.61; 95 % CI 152.11-165.11, P = 0.022) and D) heart weight/tibia length (mg/mm) ratio were increased (n=15-16 per group). E) Left ventricular ejection fraction (EF %) was preserved (>50 %) in both ND6 and controls, with F) increased left ventricular wall thickness in ND6 compared to WT (n= 9-15). G) ND6 mice had elevated E/e ratio (n= 9-14) and H) reduced exercise tolerance, with both reduced distance and speed in the exercise tolerance test (n=5-7). I) No differences were found in the fasted blood glucose levels (mM), however J) the glucose tolerance test (GTT) showed higher insulin levels at 15 minutes in the ND6. K) Fetal genes and Collagen I and III transcripts, were elevated (n= 5-8 per group). The results presented are from male mice. Error bars indicate mean α S.E.M. Statistical significance was determined by Student’s t-test.

Figure 3. ND6-P25L mice gain less lean mass and increase adiposity on a high-fat/high-sucrose diet.

A) Schematic timeline of chow feeding and high-fat/high-sucrose dietary (HFHSD) challenge, glucose tolerance test (GTT), and body mass composition measurements (EchoMRI®) in WT and ND6-P25L male mice. B) There were no differences in body weight between WT and ND6-P25L after 6 weeks of dietary challenge. C) Body mass composition analysis showed a reduced lean mass, and D) increased fat mass only in ND6 mice (n=10). E) Fasted blood glucose levels, and F) higher fasted insulin levels were detected in HFHSD-fed ND6 mice. F) GDF15 levels increased in response to HFHSD, but no differences were found in circulating GDF15 between WT and ND6 mice. The results presented are from male mice (n=10). Error bars indicate mean α S.E.M. Statistical significance was determined by two-way repeated measures ANOVA.

Figure 4. Endurance exercise reverses adverse gene expression and mitigates diastolic dysfunction in ND6-P25L mice.

A) Schematic timeline of the exercise intervention study in male WT and ND6-P25L mice. B) Eight weeks of daily treadmill run, prevented body weight gain in both WT and ND6-P25L mice, presented as a difference in body weight (Δ Body weight) between the start and end of intervention for each mouse (n=7-15). C) Heart weight/tibia length (mg/mm) was increased in exercising WT mice but remained high in ND6-P25L mice (n=11-13). D) Nppa, Nppb, Col1a2, and Col3a1 gene expression was reduced in the ND6-P25L hearts after exercise (n= 5-8). E) Isovolumic Relaxation Time (IVRT) was reduced in sedentary ND6-P25L heart, and exercise did not alter this (n=4-8). F) Diastolic function (E/e’ ratio) was restored to WT levels in exercised ND6-P25L mice (n=4-10). In a new cohort, paired comparison in E/e’ ratio per mouse and genotype at the onset and end of exercise intervention (n=4) showed improved diastolic function in ND6-P25L mice. The results presented are from male mice. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparison test. Data are shown as the mean α S.E.M.

Figure 5. Endurance exercise modifies metabolic gene expression and downregulates lipid metabolism regulators in ND6-P25L heart.

A) Transcript levels of Cd36, Plin2, Acly, Fasn, and Elovl6 were downregulated in both WT and ND6-P25L hearts following 8 weeks of exercise. However, Scd1 expression was upregulated in WT hearts but downregulated in ND6 hearts after exercise (n=4-6). Error bars indicate mean S.E.M. B) Representative immunoblots and C) quantification of Acetyl-CoA-Carboxylase (ACC) (∼280 kDa) and Perilipin 2 (PLIN2) (48 kDa) protein levels normalized to Vinculin (∼110 kDa) in sedentary and exercised WT and ND6-P25L hearts, demonstrate a significant increase in ACC and PLIN2 levels in sedentary ND6 heart (n=3 per group). ACC was significantly reduced in the ND6 heart after exercise. The results presented are from male mice. Error bars indicate mean S.D. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparison test.