Thoracentesis to alleviate pleural effusion in acute heart failure: study protocol for the multicentre, open-label, randomised controlled TAP-IT trial.

INTRODUCTION: Pleural effusion is present in half of the patients hospitalised with acute heart failure. The condition is treated with diuretics and/or therapeutic thoracentesis for larger effusions. No evidence from randomised trials or guidelines supports thoracentesis to alleviate pleural effusion due to acute heart failure. The Thoracentesis to Alleviate cardiac Pleural effusion Interventional Trial (TAP-IT) will investigate if a strategy of referring patients with acute heart failure and pleural effusion to up-front thoracentesis by pleural pigtail catheter insertion in addition to pharmacological therapy compared with pharmacological therapy alone can increase the number of days the participants are alive and not hospitalised during the 90 days following randomisation. METHODS AND ANALYSIS: TAP-IT is a pragmatic, multicentre, open-label, randomised controlled trial aiming to include 126 adult patients with left ventricular ejection fraction ≤45% and a non-negligible pleural effusion due to heart failure. Participants will be randomised 1:1, stratified according to site and anticoagulant treatment, and assigned to referral to up-front ultrasound-guided pleural pigtail catheter thoracentesis in addition to standard pharmacological therapy or to standard pharmacological therapy only. Thoracentesis is performed according to local guidelines and can be performed in participants in the pharmacological treatment arm if their condition deteriorates or if no significant improvement is observed within 5 days. The primary endpoint is how many days participants are alive and not hospitalised within 90 days from randomisation and will be analysed in the intention-to-treat population. Key secondary outcomes include 90-day mortality, complications, readmissions, and quality of life. ETHICS AND DISSEMINATION: The study has been approved by the Capital Region of Denmark Scientific Ethical Committee (H-20060817) and Knowledge Center for Data Reviews (P-2021-149). All participants will sign an informed consent form. Enrolment began in August 2021. Regardless of the nature, results will be published in a peer-reviewed medical journal. TRIAL REGISTRATION NUMBER: NCT05017753.

Mitochondrial dysfunction in adults after out-of-hospital cardiac arrest.

BACKGROUND: While preclinical studies suggest that mitochondria play a pivotal role in ischaemia-reperfusion injury, the knowledge of mitochondrial function in human out-of-hospital cardiac arrest remains scarce. The present study sought to compare oxidative phosphorylation capacity in skeletal muscle biopsies from out-of-hospital cardiac arrest patients to healthy controls. METHODS: This was a substudy of a randomised trial comparing targeted temperature management at 33°C versus 36°C for out-of-hospital cardiac arrest patients. Skeletal muscle biopsies were obtained from adult resuscitated comatose out-of-hospital cardiac arrest patients 28 hours after initiation of targeted temperature management, i.e. at target temperature prior to rewarming, and from age-matched healthy controls. Mitochondrial function was analysed by high-resolution respirometry. Maximal sustained respiration through complex I, maximal coupled respiration through complex I and complex II and maximal electron transport system capacity was compared. RESULTS: A total of 20 out-of-hospital cardiac arrest patients and 21 controls were included in the analysis. We found no difference in mitochondrial function between temperature allocations. We found no difference in complex I sustained respiration between out-of-hospital cardiac arrest and controls (23 (18-26) vs. 22 (19-26) pmol O2/mg/s, P=0.76), whereas coupled complex I and complex II respiration was significantly lower in out-of-hospital cardiac arrest patients versus controls (53 (42-59) vs. 64 (54-68) pmol O2/mg/s, P=0.01). Furthermore, electron transport system capacity was lower in out-of-hospital cardiac arrest versus controls (63 (51-69) vs. 73 (66-78) pmol O2/mg/s, P=0.005). CONCLUSIONS: Mitochondrial oxidative phosphorylation capacity in skeletal muscle biopsies was reduced in out-of-hospital cardiac arrest patients undergoing targeted temperature management compared to age-matched, healthy controls. The role of mitochondria as risk markers and potential targets for post-resuscitation care remains unknown.

Oxygen conserving mitochondrial adaptations in the skeletal muscles of breath hold divers.

BACKGROUND: The performance of elite breath hold divers (BHD) includes static breath hold for more than 11 minutes, swimming as far as 300 m, or going below 250 m in depth, all on a single breath of air. Diving mammals are adapted to sustain oxidative metabolism in hypoxic conditions through several metabolic adaptations, including improved capacity for oxygen transport and mitochondrial oxidative phosphorylation in skeletal muscle. It was hypothesized that similar adaptations characterized human BHD. Hence, the purpose of this study was to examine the capacity for oxidative metabolism in skeletal muscle of BHD compared to matched controls. METHODS: Biopsies were obtained from the lateral vastus of the femoral muscle from 8 Danish BHD and 8 non-diving controls (Judo athletes) matched for morphometry and whole body VO2max. High resolution respirometry was used to determine mitochondrial respiratory capacity and leak respiration with simultaneous measurement of mitochondrial H2O2 emission. Maximal citrate synthase (CS) and 3-hydroxyacyl CoA dehydrogenase (HAD) activity were measured in muscle tissue homogenates. Western Blotting was used to determine protein contents of respiratory complex I-V subunits and myoglobin in muscle tissue lysates. RESULTS: Muscle biopsies of BHD revealed lower mitochondrial leak respiration and electron transfer system (ETS) capacity and higher H2O2 emission during leak respiration than controls, with no differences in enzyme activities (CS and HAD) or protein content of mitochondrial complex subunits myoglobin, myosin heavy chain isoforms, markers of glucose metabolism and antioxidant enzymes. CONCLUSION: We demonstrated for the first time in humans, that the skeletal muscles of BHD are characterized by lower mitochondrial oxygen consumption both during low leak and high (ETS) respiration than matched controls. This supports previous observations of diving mammals demonstrating a lower aerobic mitochondrial capacity of the skeletal muscles as an oxygen conserving adaptation during prolonged dives.

The effects of 2 weeks of statin treatment on mitochondrial respiratory capacity in middle-aged males: the LIFESTAT study.

BACKGROUND: Statins are used to lower cholesterol in plasma and are one of the most used drugs in the world. Many statin users experience muscle pain, but the mechanisms are unknown at the moment. Many studies have hypothesized that mitochondrial function could be involved in these side effects. AIM: The aim of the study was to investigate mitochondrial function after 2 weeks of treatment with simvastatin (S; n = 10) or pravastatin (P; n = 10) in healthy middle-aged participants. METHODS: Mitochondrial respiratory capacity and substrate sensitivity were measured in permeabilized muscle fibers by high-resolution respirometry. Mitochondrial content (citrate synthase (CS) activity), antioxidant content, as well as coenzyme Q10 concentration (Q10) were determined. Fasting plasma glucose and insulin concentrations were measured, and whole body maximal oxygen uptake (VO2max) was determined. RESULTS: No differences were seen in mitochondrial respiratory capacity although a tendency was observed for a reduction when complex IV respiration was analyzed in both S (229 (169; 289 (95% confidence interval)) vs. 179 (146; 211) pmol/s/mg, respectively; P = 0.062) and P (214 (143; 285) vs. 162 (104; 220) pmol/s/mg, respectively; P = 0.053) after treatment. A tendency (1.64 (1.28; 2.00) vs. 1.28 (0.99; 1.58) mM, respectively; P = 0.092) for an increased mitochondrial substrate sensitivity (complex I-linked substrate; glutamate) was seen only in S after treatment. No differences were seen in Q10, CS activity, or antioxidant content after treatment. Fasting glucose and insulin as well as VO2max were not changed after treatment. CONCLUSION: Two weeks of statin (S or P) treatment have no major effect on mitochondrial function. The tendency for an increased mitochondrial substrate sensitivity after simvastatin treatment could be an early indication of the negative effects linked to statin treatment.

[Two cases of takotsubo cardiomyopathy in patients treated with high doses of inhaled beta-2-agonists].

Takotsubo cardiomyopathy (TCM) is characterised by reversible left ventricular dysfunction in patients presenting with acute coronary syndrome (ACS). TCM is considered multifactorial, and the repetitive exposure to inhaled beta-2-agonists has been suspected to induce TCM in predisposed individuals. We report two cases of TCM in female patients presenting with ACS both exposed to inhaled beta-2-agonists. Eccocardiography revealed apical ballooning and reduced left ventricular function. Coronary angiography was with no significant stenosis. Both patients recovered by anticongestive treatment.

[Fistula-producing giant coronary aneurysm in a 62-year-old woman].

Giant coronary aneurysms (GCA with a diameter > 20 mm) are rare with a prevalence < 0.02%. A 62-year-old woman with no history of ischaemic heart disease was admitted to hospital with acute chest pain. A coronary angiography revealed a left an-terior descendent-associated GCA. A cardiac computed tomo-g-raphy demonstrated a "snake-pit"-like fistula connecting the GCA and the pulmonary artery. Atherosclerosis, connective tissue dis-orders, and previous coronary intervention will predispose to GCA. No evidence-based treatment regimen exists, but coiling, excision or a conservative approach, as in this case, is possible strategies.

Impaired mitochondrial function in chronically ischemic human heart.

Chronic ischemic heart disease is associated with myocardial hypoperfusion. The resulting hypoxia potentially inflicts damage upon the mitochondria, leading to a compromised energetic state. Furthermore, ischemic damage may cause excessive production of reactive oxygen species (ROS), producing mitochondrial damage, hereby reinforcing a vicious circle. Ischemic preconditioning has been proven protective in acute ischemia, but the subject of chronic ischemic preconditioning has not been explored in humans. We hypothesized that mitochondrial respiratory capacity would be diminished in chronic ischemic regions of human myocardium but that these mitochondria would be more resistant to ex vivo ischemia and, second, that ROS generation would be higher in ischemic myocardium. The aim of this study was to test mitochondrial respiratory capacity during hyperoxia and hypoxia, to investigate ROS production, and finally to assess myocardial antioxidant levels. Mitochondrial respiration in biopsies from ischemic and nonischemic regions from the left ventricle of the same heart was compared in nine human subjects. Maximal oxidative phosphorylation capacity in fresh muscle fibers was lower in ischemic compared with nonischemic myocardium (P < 0.05), but the degree of coupling (respiratory control ratio) did not differ (P > 0.05). The presence of ex vivo hypoxia did not reveal any chronic ischemic preconditioning of the ischemic myocardial regions (P > 0.05). ROS production was higher in ischemic myocardium (P < 0.05), and the levels of antioxidant protein expression was lower. Diminished mitochondrial respiration capacity and excessive ROS production demonstrate an impaired mitochondrial function in ischemic human heart muscle. No chronic ischemic preconditioning effect was found.

Simvastatin effects on skeletal muscle: relation to decreased mitochondrial function and glucose intolerance.

OBJECTIVES: Glucose tolerance and skeletal muscle coenzyme Q(10) (Q(10)) content, mitochondrial density, and mitochondrial oxidative phosphorylation (OXPHOS) capacity were measured in simvastatin-treated patients (n = 10) and in well-matched control subjects (n = 9). BACKGROUND: A prevalent side effect of statin therapy is muscle pain, and yet the basic mechanism behind it remains unknown. We hypothesize that a statin-induced reduction in muscle Q(10) may attenuate mitochondrial OXPHOS capacity, which may be an underlying mechanism. METHODS: Plasma glucose and insulin concentrations were measured during an oral glucose tolerance test. Mitochondrial OXPHOS capacity was measured in permeabilized muscle fibers by high-resolution respirometry in a cross-sectional design. Mitochondrial content (estimated by citrate synthase [CS] activity, cardiolipin content, and voltage-dependent anion channel [VDAC] content) as well as Q(10) content was determined. RESULTS: Simvastatin-treated patients had an impaired glucose tolerance and displayed a decreased insulin sensitivity index. Regarding mitochondrial studies, Q(10) content was reduced (p = 0.05), whereas mitochondrial content was similar between the groups. OXPHOS capacity was comparable between groups when complex I- and complex II-linked substrates were used alone, but when complex I + II-linked substrates were used (eliciting convergent electron input into the Q intersection [maximal ex vivo OXPHOS capacity]), a decreased (p < 0.01) capacity was observed in the patients compared with the control subjects. CONCLUSIONS: These simvastatin-treated patients were glucose intolerant. A decreased Q(10) content was accompanied by a decreased maximal OXPHOS capacity in the simvastatin-treated patients. It is plausible that this finding partly explains the muscle pain and exercise intolerance that many patients experience with their statin treatment.

Decreased mitochondrial oxidative phosphorylation capacity in the human heart with left ventricular systolic dysfunction.

AIMS: Heart failure (HF) with left ventricular systolic dysfunction (LVSD) is associated with a shift in substrate utilization and a compromised energetic state. Whether these changes are connected with mitochondrial dysfunction is not known. We hypothesized that the cardiac phenotype in LVSD could be caused by reduced mitochondrial oxidative phosphorylation (OXPHOS) capacity and reduced mitochondrial creatine kinase (miCK) capacity. The study aim was to test mitochondrial OXPHOS capacity in LVSD myocardium compared with OXPHOS capacity in a comparable patient group without LVSD. METHODS AND RESULTS: Myocardial biopsies were obtained from the left ventricle during cardiac valve or left ventricular assist device (LVAD) surgery. Patients were stratified according to left ventricular ejection fraction (LVEF) into LVSD (LVEF <45%, n = 14) or CONTROL (LVEF >45%, n = 15). Mitochondrial respiration was measured in muscle fibres with addition of non-fatty acid substrates or octanoyl-l-carnitine, a medium chain fatty acid (MCFA). The in situ enzyme capacity of miCK was determined from APD titrations in the presence or absence of creatine. Maximal OXPHOS capacity with non-fatty acid substrates was lower in the LVSD group compared with the CONTROL group (P ≤ 0.05). ADP sensitivity always increased significantly (P ≤ 0.05) with the addition of creatine, after which the sensitivity was highest (P ≤ 0.05) in LVSD compared with CONTROL. The stimulation of OXPHOS from octanoyl-l-carnitine titrations elicited ∼40% lower respiration in LVSD compared with CONTROL (P ≤ 0.05). CONCLUSION: Human LVSD is associated with markedly diminished OXPHOS capacity, particularly in MCFA oxidation. This offers a candidate mechanism for a compromised energetic state and decreased reliance on fatty acid utilization in HF.

Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects.

Skeletal muscle mitochondrial content varies extensively between human subjects. Biochemical measures of mitochondrial proteins, enzyme activities and lipids are often used as markers of mitochondrial content and muscle oxidative capacity (OXPHOS). The purpose of this study was to determine how closely associated these commonly used biochemical measures are to muscle mitochondrial content and OXPHOS. Sixteen young healthy male subjects were recruited for this study. Subjects completed a graded exercise test to determine maximal oxygen uptake (VO2peak) and muscle biopsies were obtained from the vastus lateralis. Mitochondrial content was determined using transmission electron microscopy imaging and OXPHOS was determined as the maximal coupled respiration in permeabilized fibres. Biomarkers of interest were citrate synthase (CS) activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I­V protein content, and complex I­IV activity. Spearman correlation coefficient tests and Lin's concordance tests were applied to assess the absolute and relative association between the markers and mitochondrial content or OXPHOS. Subjects had a large range of VO2peak (range 29.9­71.6ml min−1 kg−1) and mitochondrial content (4­15% of cell volume).Cardiolipin content showed the strongest association with mitochondrial content followed by CS and complex I activities. mtDNA was not related to mitochondrial content. Complex IV activity showed the strongest association with muscle oxidative capacity followed by complex II activity.We conclude that cardiolipin content, and CS and complex I activities are the biomarkers that exhibit the strongest association with mitochondrial content, while complex IV activity is strongly associated with OXPHOS capacity in human skeletal muscle.

Erythropoietin treatment enhances muscle mitochondrial capacity in humans.

Erythropoietin (Epo) treatment has been shown to induce mitochondrial biogenesis in cardiac muscle along with enhanced mitochondrial capacity in mice. We hypothesized that recombinant human Epo (rhEpo) treatment enhances skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity in humans. In six healthy volunteers rhEpo was administered by sub-cutaneous injection over 8 weeks with oral iron (100 mg) supplementation taken daily. Mitochondrial OXPHOS was quantified by high-resolution respirometry in saponin-permeabilized muscle fibers obtained from biopsies of the vastus lateralis before and after rhEpo treatment. OXPHOS was determined with the mitochondrial complex I substrates malate, glutamate, pyruvate, and complex II substrate succinate in the presence of saturating ADP concentrations, while maximal electron transport capacity (ETS) was assessed by addition of an uncoupler. rhEpo treatment increased OXPHOS (from 92 ± 5 to 113 ± 7 pmol·s(-1)·mg(-1)) and ETS (107 ± 4 to 143 ± 14 pmol·s(-1)·mg(-1), p < 0.05), demonstrating that Epo treatment induces an upregulation of OXPHOS and ETS in human skeletal muscle.

5'-AMP Activated Protein Kinase is Involved in the Regulation of Myocardial ß-Oxidative Capacity in Mice.

5'-adenosine monophosphate-activated protein kinase (AMPK) is considered central in regulation of energy status and substrate utilization within cells. In heart failure the energetic state is compromised and substrate metabolism is altered. We hypothesized that this could be linked to changes in AMPK activity and we therefore investigated mitochondrial oxidative phosphorylation capacity from the oxidation of long- and medium-chain fatty acids (LCFA and MCFA) in cardiomyocytes from young and old mice expressing a dominant negative AMPKα2 (AMPKα2-KD) construct and their wildtype (WT) littermates. We found a 35-45% (P < 0.05) lower mitochondrial capacity for oxidizing MCFA in AMPKα2-KD of both age-groups, compared to WT. This coincided with marked decreases in protein expression (19/29%, P < 0.05) and activity (14/21%, P < 0.05) of 3-hydroxyacyl-CoA-dehydrogenase (HAD), in young and old AMPKα2-KD mice, respectively, compared to WT. Maximal LCFA oxidation capacity was similar in AMPKα2-KD and WT mice independently of age implying that LCFA-transport into the mitochondria was unaffected by loss of AMPK activity or progressing age. Expression of regulatory proteins of glycolysis and glycogen breakdown showed equivocal effects of age and genotype. These results illustrate that AMPK is necessary for normal mitochondrial function in the heart and that decreased AMPK activity may lead to an altered energetic state as a consequence of reduced capacity to oxidize MCFA. We did not identify any clear aging effects on mitochondrial function.