Andrea Schrepper

Proteomic remodelling of mitochondrial oxidative pathways in pressure overload-induced heart failure

AIMS Impairment in mitochondrial energetics is a common observation in animal models of heart failure, the underlying mechanisms of which remain incompletely understood. It was our objective to investigate whether changes in mitochondrial protein levels may explain impairment in mitochondrial oxidative capacity in pressure overload-induced heart failure. METHODS AND RESULTS Twenty weeks following aortic constriction, Sprague-Dawley rats developed contractile dysfunction with clinical signs of heart failure. Comparative mitochondrial proteomics using label-free proteome expression analysis (LC-MS/MS) revealed decreased mitochondrial abundance of fatty acid oxidation proteins (six of 11 proteins detected), increased levels of pyruvate dehydrogenase subunits, and upregulation of two tricarboxylic acid cycle proteins. Regulation of mitochondrial electron transport chain subunits was variable, with downregulation of 53% of proteins and upregulation of 25% of proteins. Mitochondrial state 3 respiration was markedly decreased independent of the substrate used (palmitoyl-carnitine -65%, pyruvate -75%, glutamate -75%, dinitrophenol -82%; all P < 0.05), associated with impaired mitochondrial cristae morphology in failing hearts. Perfusion of isolated working failing hearts showed markedly reduced oleate (-68%; P < 0.05) and glucose oxidation (-64%; P < 0.05). CONCLUSION 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.

Decreased rates of substrate oxidation ex vivo predict the onset of heart failure and contractile dysfunction in rats with pressure overload

AIMS Left ventricular hypertrophy is a risk factor for heart failure. However, it also is a compensatory response to pressure overload, accommodating for increased workload. We tested whether the changes in energy substrate metabolism may be predictive for the development of contractile dysfunction. METHODS AND RESULTS Chronic pressure overload was induced in Sprague-Dawley rats by aortic arch constriction for 2, 6, 10, or 20 weeks. Contractile function in vivo was assessed by echocardiography and by invasive pressure measurement. Glucose and fatty acid oxidation as well as contractile function ex vivo were assessed in the isolated working heart, and respiratory capacity was measured in isolated cardiac mitochondria. Pressure overload caused progressive hypertrophy with normal ejection fraction (EF) at 2, 6, and 10 weeks, and hypertrophy with dilation and impaired EF at 20 weeks. The lung-to-body weight ratio, as marker for pulmonary congestion, was normal at 2 weeks (indicative of compensated hypertrophy) but significantly increased already after 6 and up to 20 weeks, suggesting the presence of heart failure with normal EF at 6 and 10 weeks and impaired EF at 20 weeks. Invasive pressure measurements showed evidence for contractile dysfunction already after 6 weeks and ex vivo cardiac power was reduced even at 2 weeks. Importantly, there was impairment in fatty acid oxidation beginning at 2 weeks, which was associated with a progressive decrease in glucose oxidation. In contrast, respiratory capacity of isolated mitochondria was normal until 10 weeks and decreased only in hearts with impaired EF. CONCLUSION 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.

Induction of heart failure by minimally invasive aortic constriction in mice: reduced peroxisome proliferator-activated receptor γ coactivator levels and mitochondrial dysfunction.

OBJECTIVE Mitochondrial dysfunction has been suggested as a potential cause for heart failure. Pressure overload is a common cause for heart failure. However, implementing pressure overload in mice is considered a model for compensated hypertrophy but not for heart failure. We assessed the suitability of minimally invasive transverse aortic constriction to induce heart failure in C57BL/6 mice and assessed mitochondrial biogenesis and function. METHODS Minimally invasive transverse aortic constriction was performed through a ministernotomy without intubation (minimally invasive transverse aortic constriction, n = 68; sham operation, n = 43). Hypertrophy was assessed based on heart weight/body weight ratios and histologic analyses, and contractile function was assessed based on intracardiac Millar pressure measurements. Expression of selected metabolic genes was assessed with reverse transcription-polymerase chain reaction and Western blotting. Maximal respiratory capacity (state 3) of isolated mitochondria was measured with a Clark-type electrode. RESULTS Survival was 62%. Within 7 weeks, minimally invasive transverse aortic constriction induced significant hypertrophy (heart weight/body weight ratio: 10.08±0.28 mg/g for minimally invasive transverse aortic constriction vs 4.66±0.07 mg/g for sham operation; n=68; P<.01). Fifty-seven percent of mice undergoing minimally invasive transverse aortic constriction displayed signs of heart failure (pleural effusions, dyspnea, weight loss, and dp/dtmax of 3114±422 mm Hg/s, P<.05). All of them had heart weight/body weight ratios of greater than 10. Mice undergoing minimally invasive transverse aortic constriction with heart weight/body weight ratios of less than 10 had normal contractile function (dp/dtmax of 6471±292 mm Hg/s vs dp/dtmax of 6933±205 mmHg/s in sham mice) and no clinical signs of heart failure. The mitochondrial coactivator peroxisome proliferator-activated receptor γ coactivator alpha (PGC-1α) was downregulated in failing hearts only. PGC-1α and fatty acid oxidation gene expression were also decreased in failing hearts. State 3 respiration of isolated mitochondria was significantly reduced in all hearts subjected to pressure overload. CONCLUSIONS Contractile dysfunction and heart failure can be induced in wild-type mice by means of minimally invasive aortic constriction. Pressure overload-induced heart failure in mice is associated with mitochondrial dysfunction, as characterized by downregulation of PGC-1α and reduced oxidative capacity.

Pressure overload differentially affects respiratory capacity in interfibrillar and subsarcolemmal mitochondria

Years ago a debate arose as to whether two functionally different mitochondrial subpopulations, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), exist in heart muscle. Nowadays potential differences are often ignored. Presumably, SSM are providing ATP for basic cell function, whereas IFM provide energy for the contractile apparatus. We speculated that two distinguishable subpopulations exist that are differentially affected by pressure overload. Male Sprague-Dawley rats were subjected to transverse aortic constriction for 20 wk or sham operation. Contractile function was assessed by echocardiography. Heart tissue was analyzed by electron microscopy. Mitochondria were isolated by differential centrifugation, and respiratory capacity was analyzed using a Clark electrode. Pressure overload induced left ventricular hypertrophy with increased posterior wall diameter and impaired contractile function. Mitochondrial state 3 respiration in control was 50% higher in IFM than in SSM. Pressure overload significantly impaired respiratory rates in both IFM and SSM, but in SSM to a lower extent. As a result, there were no differences between SSM and IFM after 20 wk of pressure overload. Pressure overload reduced total citrate synthase activity, suggesting reduced total mitochondrial content. Electron microscopy revealed normal morphology of mitochondria but reduced total mitochondrial volume density. In conclusion, IFM show greater respiratory capacity in the healthy rat heart and a greater depression of respiratory capacity by pressure overload than SSM. The differences in respiratory capacity of cardiac IFM and SSM in healthy hearts are eliminated with pressure overload-induced heart failure. The strong effect of pressure overload on IFM together with the simultaneous appearance of mitochondrial and contractile dysfunction may support the notion of IFM primarily producing ATP for contractile function.

Mitochondrial reactive oxygen species production and respiratory complex activity in rats with pressure overload‐induced heart failure

Pressure overload induces cardiac hypertrophy developing into heart failure. 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. Treatment to reduce ROS production was able to diminish mitochondrial ROS production but was not able to prevent or delay heart failure development. The results question a primary role of ROS in the mechanism causing contractile dysfunction under pressure overload.

Adrenergic Repression of the Epigenetic Reader MeCP2 Facilitates Cardiac Adaptation in Chronic Heart Failure.

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Diaphragm muscle weakness in mice is early-onset post-myocardial infarction and associated with elevated protein oxidation

Heart failure induced by myocardial infarction (MI) causes diaphragm muscle weakness, with elevated oxidants implicated. We aimed to determine whether diaphragm muscle weakness is 1) early-onset post-MI (i.e., within the early left ventricular remodeling phase of 72 h); and 2) associated with elevated protein oxidation. Ligation of the left coronary artery to induce MI (n = 10) or sham operation (n = 10) was performed on C57BL6 mice. In vitro contractile function of diaphragm muscle fiber bundles was assessed 72 h later. Diaphragm mRNA and protein expression, enzyme activity, and individual carbonylated proteins (by two-dimensional differential in-gel electrophoresis and mass spectrometry) were subsequently assessed. Infarct size averaged 57 ± 1%. Maximal diaphragm function was reduced (P < 0.01) by 20% post-MI, with the force-frequency relationship depressed (P < 0.01) between 80 and 300 Hz. The mRNA expression of inflammation, atrophy, and regulatory Ca(2+) proteins remained unchanged post-MI, as did the protein expression of key contractile proteins. However, enzyme activity of the oxidative sources NADPH oxidase and xanthine oxidase was increased (P < 0.01) by 45 and 33%, respectively. Compared with sham, a 57 and 45% increase (P < 0.05) was observed in the carbonylation of sarcomeric actin and creatine kinase post-MI, respectively. In conclusion, diaphragm muscle weakness was rapidly induced in mice during the early left ventricular remodeling phase of 72 h post-MI, which was associated with increased oxidation of contractile and energetic proteins. Collectively, these findings suggest diaphragm muscle weakness may be early onset in heart failure, which is likely mediated in part by posttranslational oxidative modifications at the myofibrillar level.

Biphasic response of skeletal muscle mitochondria to chronic cardiac pressure overload — Role of respiratory chain complex activity

Pressure overload induced heart failure affects cardiac mitochondrial function and leads to decreased respiratory capacity during contractile dysfunction. A similar cardiac mitochondrial dysfunction has been demonstrated by studies which induce heart failure through myocardial infarction or pacing. These heart failure models differ in their loading conditions to the heart and show nevertheless the same cardiac mitochondrial changes. Based on these observations we speculated that a workload independent mechanism may be responsible for the impairment in mitochondrial function after pressure overload, which may then also affect the skeletal muscle. We aimed to characterize changes in mitochondrial function of skeletal muscle during the transition from pressure overload (PO) induced cardiac hypertrophy to chronic heart failure. PO by transverse aortic constriction caused compensated hypertrophy at 2 weeks, HF with normal ejection fraction (EF) at 6 and 10 weeks, and hypertrophy with reduced EF at 20 weeks. Cardiac output was normal at all investigated time points. PO did not cause skeletal muscle atrophy. Mitochondrial respiratory capacity in soleus and gastrocnemius muscles showed an early increase (up to 6 weeks) and a later decline (significant at 20 weeks). Respiratory chain complex activities responded to PO in a biphasic manner. At 2 weeks, activity of complexes I and II was increased. These changes pseudo-normalized within the 6-10 week interval. At 20 weeks, all complexes showed reduced activities which coincided with clinical heart failure symptoms. However, both protein expression and supercomplex assembly (Blue-Native gel) remained normal. There were also no relevant changes in mRNA expression of genes involved in mitochondrial biogenesis. This temporal analysis reveals that mitochondrial function of skeletal muscle is changed early in the development of pressure overload induced heart failure without being directly influenced by an increased loading condition. The observed early increase and the later decline in respiratory capacity can be explained by concomitant activity changes of complex I and complex II and is not due to differences in gene expression or supercomplex assembly.

Myocardial mitochondrial dysfunction in mice lacking adiponectin receptor 1

Hypoadiponectinemia is an independent predictor of cardiovascular disease, impairs mitochondrial function in skeletal muscle, and has been linked to the pathogenesis of Type 2 diabetes. In models of Type 2 diabetes, myocardial mitochondrial function is impaired, which is improved by increasing serum adiponectin levels. We aimed to define the roles of adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2) in adiponectin-evoked regulation of mitochondrial function in the heart. In isolated working hearts in mice lacking AdipoR1, myocardial oxygen consumption was increased without a concomitant increase in cardiac work, resulting in reduced cardiac efficiency. Activities of mitochondrial oxidative phosphorylation (OXPHOS) complexes were reduced, accompanied by reduced OXPHOS protein levels, phosphorylation of AMP-activated protein kinase, sirtuin 1 activity, and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling. Decreased ATP/O ratios suggested myocardial mitochondrial uncoupling in AdipoR1-deficient mice, which was normalized by lowering increased mitochondrial 4-hydroxynonenal levels following treatment with the mitochondria-targeted antioxidant Mn (III) tetrakis (4-benzoic acid) porphyrin. Lack of AdipoR2 did not impair mitochondrial function and coupling in the heart. Thus, lack of AdipoR1 impairs myocardial mitochondrial function and coupling, suggesting that impaired AdipoR1 signaling may contribute to mitochondrial dysfunction and mitochondrial uncoupling in Type 2 diabetic hearts.

The Influence of a High Fat Diet and Metformin Treatment on the Activity of Respiratory Chain Complexes

High fat diet (HFD) is one cause of obesity and type 2 Diabetes and Diabetes is associated with mitochondrial dysfunction. Metformin the most common drug to treat T2D reduces blood glucose levels and improves insulin sensitivity and has recently been linked to beneficial effects in cardiac surgery. Metformin has been suggested to act on complex I of the respiratory chain of mitochondria. Our aim was to analyze how high fat diet and metformin treatment influence the activities of the mitochondrial complexes of the respiratory chain. Male SD-rats were fed normal chow or a high fat diet. Animals were treated with or without Metformin. At 23 weeks of age mitochondria of heart and skeletal muscle were isolated. We analyzed individual complex activities of the respiratory chain in both, interfibrillar (IFM) and subsarcolemmal mitochondria (SSM). HFD led to increased body weight and reduced glucose tolerance. Metformin treatment improved glucose tolerance. HFD led to an increase in complex I, III, IV and combined complex I and III activity in both heart and skeletal muscle (complex I, heart IFM NC versus HFD: 28,4 ± 7,59 versus 153 ± 54,5)but did not affect complex II. With HFD, Metformin treatment led to a reduction in complex I, III and in the combination of I and III activities in all tissues (complex I + III skeletal muscle IFM HFD versus HFD-MET: 626 ± 156 versus 143 ± 71,9). Influence of Metformin was strongest in skeletal muscle IFM. Metformin had fewer effects on skeletal muscle SSM or cardiac mitochondria. With normal chow Metformin reduced activity of complex I, III and combined complex I and III respiratory chain complexes (muscle SSM, complex I, NC vs NC-MET: 373 ± 30,1 versus 244 ± 41,5). Contrary to expectations we found no reduction of complex activities with HFD. Metformin treatment slightly decreased activities of complex I in heart and skeletal muscle with HFD. However, metformin treatment in healthy animals impaired complex activities significantly. Thus, Metformin may be detrimental without proper pathological stimulus such as HFD or diabetes. These findings should be considered if general use of metformin in cardiac surgery is discussed.