Pressure overload-induced heart failure is characterized by a substantial defect in cardiac oxidative capacity, at least in part due to a mitochondrial defect downstream of substrate-specific pathways. Numerous changes in mitochondrial protein levels have been detected, and the contribution of these to oxidative defects and impaired cardiac energetics in failing hearts is discussed.

Pressure overload-induced impairment in fatty acid oxidation precedes the onset of congestive heart failure but mitochondrial respiratory capacity is maintained until the EF decreases in vivo. These temporal relations suggest a tight link between impaired substrate oxidation capacity in the development of heart failure and contractile dysfunction and may imply therapeutic and prognostic value.

Mitochondrial oxidative phosphorylation (OXPHOS) and cellular workload are tightly balanced by the key cellular regulator, calcium (Ca2+). Current models assume that cytosolic Ca2+ regulates workload and that mitochondrial Ca2+ uptake precedes activation of matrix dehydrogenases, thereby matching OXPHOS substrate supply to ATP demand. Surprisingly, knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice results in only minimal phenotypic changes and does not alter OXPHOS. This implies that adaptive activation of mitochondrial dehydrogenases by intramitochondrial Ca2+ cannot be the exclusive mechanism for OXPHOS control. We hypothesized that cytosolic Ca2+, but not mitochondrial matrix Ca2+, may adapt OXPHOS to workload by adjusting the rate of pyruvate supply from the cytosol to the mitochondria. Here, we studied the role of malate-aspartate shuttle (MAS)-dependent substrate supply in OXPHOS responses to changing Ca2+ concentrations in isolated brain and heart mitochondria, synaptosomes, fibroblasts, and thymocytes from WT and MCU KO mice and the isolated working rat heart. Our results indicate that extramitochondrial Ca2+ controls up to 85% of maximal pyruvate-driven OXPHOS rates, mediated by the activity of the complete MAS, and that intramitochondrial Ca2+ accounts for the remaining 15%. Of note, the complete MAS, as applied here, included besides its classical NADH oxidation reaction the generation of cytosolic pyruvate. Part of this largely neglected mechanism has previously been described as the “mitochondrial gas pedal.” Its implementation into OXPHOS control models integrates seemingly contradictory results and warrants a critical reappraisal of metabolic control mechanisms in health and disease.

Rationale: Diaphragm weakness impairs quality-of-life, exercise capacity, and survival in patients with chronic heart failure (CHF) and reduced left ventricular ejection fraction. However, the underlying cellular mechanisms responsible in humans remain poorly resolved. Objective: We prospectively evaluated clinical, functional (in vivo/in vitro), histological/ultrastructural and molecular alterations of the diaphragm from CHF patients receiving a left ventricular assist device compared to patients without CHF undergoing elective coronary bypass grafting (control) in the observational LIpsia DiaPhrAgm and MUScle Heart Failure Trial (LIPAMUS-HF). Methods and Results: Participants (Controls=21, CHF=18) underwent cardiopulmonary exercise and spirometry/respiratory muscle testing alongside diaphragm and cardiac imaging. Diaphragm biopsies were phenotyped for mitochondrial respiration, muscle fiber function, histology/ultrastructure, and protein expression. In vivo respiratory muscle function and diaphragm thickness were reduced in CHF by 38% and 23%. Diaphragm biopsies revealed a fiber-type shift and severe fiber atrophy in CHF alongside elevated proteasome-dependent proteolysis (i.e., MuRF1 expression, ubiquitination, ubiquitin proteasome activity) and myofibrillar protein oxidation, which corresponded to upregulated NADPH oxidase (Nox2/Nox4) signaling. Mitochondria demonstrated severe intrinsic functional and ultrastructural abnormalities in CHF characterized by accumulation of small mitochondria and inhibited autophagy/mitophagy. Single muscle fiber contractile function revealed reduced Ca2+ sensitivity in CHF and there was evidence of ryanodine receptor 1 (RyR1) dysfunction indicating Ca2+ leak from the sarcoplasmatic reticulum. Mitochondrial and Ca2+ measures corresponded to upregulated Nox4 isoform NADPH oxidase expression. Molecular markers correlated to whole-body exercise intolerance and diaphragm dysfunction/wasting. Conclusions: CHF patients demonstrate an obvious diaphragm myopathy independent of disuse or other confounding factors such as ageing, obesity, or hypertension. Diaphragm weakness in CHF was associated with intracellular abnormalities characterized by fiber atrophy, oxidative stress, mitochondrial dysfunction, impaired Ca2+ homeostasis, elevated proteasome dependent proteolysis, but inhibited autophagy/mitophagy, which we speculate offers a novel therapeutic molecular target regulated by a Nox-MuRF1/ubiquitin proteasome-mitochondria-RyR1/Ca2+ signaling axis.

Key pointsr Pressure overload induces cardiac hypertrophy developing into heart failure. r During pressure overload-induced heart failure development in the rat, mitochondrial capacity to produce reactive oxygen species (ROS) increased significantly with the onset of diastolic functional changes.r Treatment to reduce ROS production was able to diminish mitochondrial ROS production but was not able to prevent or delay heart failure development.r The results question a primary role of ROS in the mechanism causing contractile dysfunction under pressure overload. AbstractWe investigated the impact of cardiac reactive oxygen species (ROS) during the development of pressure overload-induced heart failure. We used our previously described rat model where transverse aortic constriction (TAC) induces compensated hypertrophy after 2 weeks, heart failure with preserved ejection fraction at 6 and 10 weeks, and heart failure with systolic dysfunction after 20 weeks. We measured mitochondrial ROS production rates, ROS damage and assessed the therapeutic potential of in vivo antioxidant therapies. In compensated hypertrophy (2 weeks of TAC) ROS production rates were normal at both mitochondrial ROS production sites (complexes I and III). Complex I ROS production rates increased with the appearance of diastolic dysfunction (6 weeks of TAC) and remained high thereafter. Surprisingly, maximal ROS production at complex III peaked at 6 weeks of pressure overload. Mitochondrial respiratory capacity (state 3 respiration) was elevated 2 and 6 weeks after TAC, decreased after this point and was significantly impaired at 20 weeks, when contractile function was also impaired and ROS damage was found with increased hydroxynonenal. Treatment with the ROS scavenger α-phenyl-N-tert-butyl nitrone or the uncoupling agent dinitrophenol significantly reduced ROS production rates at 6 weeks. Despite the decline in ROS production capacity, no differences in contractile function between treated and untreated animals were observed. Increased ROS production occurs early in the development of heart failure with a peak at the onset of diastolic dysfunction. However, ROS production may not be related to the onset of contractile dysfunction.M. Schwarzer, M. Osterholt and A. Lunkenbein contributed equally to this work.