Background/Aims: Diaphragm dysfunction with increased reactive oxygen species (ROS) occurs within 72 hrs post-myocardial infarction (MI) in mice and may contribute to loss of inspiratory maximal pressure and endurance in patients. Methods: We used wild-type (WT) and whole-body Nox4 knockout (Nox4KO) mice to measure diaphragm bundle force in vitro with a force transducer, mitochondrial respiration in isolated fiber bundles with an O2 sensor, mitochondrial ROS by fluorescence, mRNA (RT-PCR) and protein (immunoblot), and fiber size by histology 72 hrs post-MI. Results: MI decreased diaphragm fiber cross-sectional area (CSA) (~15%, p = 0.015) and maximal specific force (10%, p = 0.005), and increased actin carbonylation (5-10%, p = 0.007) in both WT and Nox4KO. Interestingly, MI did not affect diaphragm mRNA abundance of MAFbx/atrogin-1 and MuRF-1 but Nox4KO decreased it by 20-50% (p < 0.01). Regarding the mitochondria, MI and Nox4KO decreased the protein abundance of citrate synthase and subunits of electron transport system (ETS) complexes and increased mitochondrial O2 flux (JO2) and H2O2 emission (JH2O2) normalized to citrate synthase. Mitochondrial electron leak (JH2O2/JO2) in the presence of ADP was lower in Nox4KO and not changed by MI. Conclusion: Our study shows that the early phase post-MI causes diaphragm atrophy, contractile dysfunction, sarcomeric actin oxidation, and decreases citrate synthase and subunits of mitochondrial ETS complexes. These factors are potential causes of loss of inspiratory muscle strength and endurance in patients, which likely contribute to the pathophysiology in the early phase post-MI. Whole-body Nox4KO did not prevent the diaphragm abnormalities induced 72 hrs post-MI, suggesting that systemic pharmacological inhibition of Nox4 will not benefit patients in the early phase post-MI.

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Heart failure with preserved ejection fraction (HFpEF) accounts for ~50% of all patients with heart failure and frequently affects postmenopausal women. The HFpEF condition is phenotype-specific, with skeletal myopathy that is crucial for disease development and progression. However, most of the current preclinical models of HFpEF have not addressed the postmenopausal phenotype. We sought to advance a rodent model of postmenopausal HFpEF and examine skeletal muscle abnormalities therein. Female, ovariectomized, spontaneously hypertensive rats (SHR) were fed a high fat, high sucrose diet to induce HFpEF. Controls were female sham-operated Wistar-Kyoto rats on a lean diet. In a complementary, longer-term cohort, controls were female sham-operated SHRs on a lean diet to evaluate the effect of strain difference in the model. Our model developed key features of HFpEF that included increased body weight, glucose intolerance, hypertension, cardiac hypertrophy, diastolic dysfunction, exercise intolerance, and elevated plasma cytokines. In limb skeletal muscle, HFpEF decreased specific force by 15-30% (p < 0.05) and maximal mitochondrial respiration by 40-55% (p < 0.05), increased oxidized glutathione by ~2-fold (p < 0.05), and tended to increase mitochondrial H2O2 emission (p = 0.10). Muscle fiber cross-sectional area, markers of mitochondrial content, and indices of capillarity were not different between control and HFpEF in our short-term cohort. Overall, our model of postmenopausal HFpEF recapitulates several key features of the disease. This new model reveals contractile and mitochondrial dysfunction and redox imbalance that are potential contributors to abnormal metabolism, exercise intolerance, and diminished quality of life in patients with postmenopausal HFpEF.

Ferreira LF. 2021. Cardiac and respiratory muscle responses to dietary N-acetylcysteine in rats consuming a high-saturated fat, high-sucrose diet. bioRxiv

PurposeApproximately 50% of heart failure patients have heart failure with preserved ejection fraction (HFpEF), and currently there are no proven therapies for this population. There is an urgent need to develop animal models of HFpEF to understand the mechanisms of this disease and discover treatments. Notably, 70–85% of HFpEF patients are postmenopausal women. We hypothesized that a novel animal model that recapitulated classical risk factors for HFpEF (i.e., female sex, aging/menopause, hypertension, obesity/high fat, high sugar diet) would display cardiac abnormalities, skeletal muscle dysfunction, and exercise intolerance consistent with disease features seen in humans.MethodsFemale, ovariectomized, spontaneously hypertensive rats (SHR) were fed a Western diet (high saturated fat, high sugar) for ~16 weeks to model HFpEF pathophysiology. Female, sham surgery, lean diet, Wistar‐Kyoto rats were used as controls. We evaluated glucose tolerance, tail‐cuff blood pressure, running time to exhaustion, echocardiography, in vitro skeletal muscle force, intact mitochondrial respiration and reactive oxygen species emission, and cardiac and skeletal muscle histology. All researchers were blinded to group assignment during terminal experiments and statistical analyses. Data were compared using Student's t‐tests and are shown as mean ± SD from n = 4–8 rats per group.ResultsHFpEF rats displayed a 20±6% increase in body weight, 20±22% greater area under the glucose response curve, 36±14% higher mean arterial pressure, 70±47% elevation in liver enzymes, and 42±12% decrease in high‐intensity running endurance (p<0.05). Left ventricle (LV) of HFpEF rats exhibited hypertrophy (in mg/mm: Control 17±0.5, HFpEF 21±0.7, p<0.05), fibrosis (in %: Control 2±1.0, HFpEF 4±1.9, p=0.10) and elevated E wave deceleration rate indicative of restrictive LV filling (in m/s2: Control 21±5, HFpEF 35±4, p<0.05). LV fractional shortening, a marker of systolic function, was preserved (in %: Control 47±7, HFpEF 44±7, p>0.05). In limb skeletal muscle, HFpEF caused weakness (force in N/g: Control 17±3, HFpEF 14±1, p<0.05), impaired maximal mitochondrial respiration (in pmol O2/s/mg: Control 71±27, HFpEF 39±11, p<0.05), and a trend toward elevated mitochondrial H2O2 emission (in pmol/min/mg: Control 0.8±0.4, HFpEF 1.7±0.9, p=0.10). In this early stage of HFpEF, there was no limb muscle atrophy in type I (in μm2: Control 1986±526, HFpEF 1752±364), type IIa (in μm2: Control 1595±339, HFpEF 2022±663), or type IIb/x (in μm2: Control 3605±536, HFpEF 4111±653) tibalis anterior fibers (p>0.05), but there was an increase in fibrosis (in %: Control 0.7±0.3, HFpEF 1.5±1, p<0.05).ConclusionOur preclinical model recapitulates key cardiovascular, metabolic, and skeletal muscle features of the disease with a pronounced decrease in high intensity running endurance. The model is clinically relevant for post‐menopausal women with HFpEF ‐ the patient group that represents the majority of the HFpEF population. This model will be valuable for future studies aiming to elucidate mechanisms of disease development and novel treatments.Support or Funding InformationBREATHE T32 HL134621This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.