High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle

Krajčová, Adéla; Urban, Tomáš; Megvinet, David; Waldauf, Petr; Borggrefe, Martin; Hlavicka, Jan; Budera, Petr; Janoušek, L; Pokorná, Eva; Duška, František

doi:10.1371/journal.pone.0226142

Cited by 15 publications

(15 citation statements)

References 51 publications

(87 reference statements)

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“…The compromised mitochondrial membrane integrity can cause misleading results and thus should be excluded from the data analysis. A common practice (also adopted in our experiments) is to accept up to a 10% increase in respiration rate following the addition of cytochrome c [43]; however, this acceptable threshold has not been properly validated, and some research suggests that a 10 to 20% increase in respiration could be acceptable [48]. Based on our results, the maximal respiration rate was significantly reduced only when the increase in respiration rate observed after adding cytochrome c exceeded 20%.…”

Section: Discussionmentioning

confidence: 73%

Methodological considerations when assessing mitochondrial respiration and biomarkers for mitochondrial content in human skeletal muscle

Kuang

Saner

Botella

et al. 2021

Preprint

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Background: The assessment of mitochondrial respiration and mitochondrial content are two common measurements in the fields of skeletal muscle research and exercise science. However, to verify the validity of the observed changes in both mitochondrial respiration and mitochondrial content following an intervention such as exercise training, it is important to determine the reliability and reproducibility of the experimental design and/or techniques employed. We examined the repeatability of widely used methodologies for assessing mitochondrial respiration and mitochondrial content, respectively; the measurement of maximal mitochondrial oxidative phosphorylation in permeabilized muscle fibres using high-resolution respirometry, and the measurement of citrate synthase activity as a biomarker for mitochondrial content in a microplate with spectrophotometer. Result: For mitochondrial respiration, the coefficient of variation for repeated measurements using muscle sampled from same biopsy decreased from 12.7% to 11% when measured in triplicate with outliers excluded, rather than in duplicate. The coefficient of variation was 9.7% for repeated muscle biopsies sampled across two separated days. For measurements of citrate synthase activity, the coefficient of variation was 3.5% of three technical repeats on the same plate, 10.2% for duplicate analyses using the same muscle lysate when conducted in the same day, and 30.5% when conducted four weeks apart. Conclusion: We have provided evidence for important technical considerations when measuring mitochondrial respiration with human skeletal muscle: 1) the relatively large technical variability can be reduced by increasing technical repeats and excluding outliers; 2) the biological variability and absolute mitochondrial respiration value of the participants should be considered when estimating the required sample size; 3) a new threshold of 15% for the increase in respiration rate after the addition of cytochrome c test for testing mitochondrial outer membrane integrity. When analysing citrate synthase activity, our evidence suggests it is important to consider the following: 1) all samples from the same study should be homogenized and measured at the same time using the same batch of freshly made chemical reagents; 2) biological variability should be considered when detecting small change in mitochondrial content; 3) the relative change should be used to compare the outcomes from different studies.

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Section: Discussionmentioning

confidence: 73%

Methodological considerations when assessing mitochondrial respiration and biomarkers for mitochondrial content in human skeletal muscle

Kuang

Saner

Botella

et al. 2021

Preprint

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“…Heart muscle homogenates were prepared similarly[19]. After removal of connective tissue and fat, fresh myocardium fragments were diluted in medium MiR05: 0.5 mM EGTA, 3 mM MgCl 2 x 6 H 2 O, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH 2 PO 4 , 20 mM HEPES, 110 mM sucrose and 1 g/L of BSA (pH adjusted to 7.1 at 24°C) to obtain 2.5 and 1% tissue solution of human and rat heart muscle, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…After removal of connective tissue and fat, fresh myocardium fragments were diluted in medium MiR05: 0.5 mM EGTA, 3 mM MgCl 2 x 6 H 2 O, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH 2 PO 4 , 20 mM HEPES, 110 mM sucrose and 1 g/L of BSA (pH adjusted to 7.1 at 24°C) to obtain 2.5 and 1% tissue solution of human and rat heart muscle, respectively. Then the process of homogenization was two-step: tissue fragments were firstly manually homogenized by 10–12 initial strokes up and down with a glass loose pestle (large clearance 0.114 ± 0.025 mm; Wheaton ™ , Millville, USA) in the glass Dounce grinder[19] subsequently homogenized with electro motor homogenizer, with final technique as for skeletal muscle above. Finally, crude homogenate was filtered through polyamide mesh (SILK & PROGRESS s.r.o., Czech Republic) in order to remove the remaining connective tissue.…”

Section: Methodsmentioning

confidence: 99%

Kinetic characteristics of propofol-induced inhibition of electron-transfer chain and fatty acid oxidation in human and rodent skeletal and cardiac muscles

et al. 2019

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IntroductionPropofol causes a profound inhibition of fatty acid oxidation and reduces spare electron transfer chain capacity in a range of human and rodent cells and tissues–a feature that might be related to the pathogenesis of Propofol Infusion Syndrome. We aimed to explore the mechanism of propofol-induced alteration of bioenergetic pathways by describing its kinetic characteristics.MethodsWe obtained samples of skeletal and cardiac muscle from Wistar rat (n = 3) and human subjects: vastus lateralis from hip surgery patients (n = 11) and myocardium from brain-dead organ donors (n = 10). We assessed mitochondrial functional indices using standard SUIT protocol and high resolution respirometry in fresh tissue homogenates with or without short-term exposure to a range of propofol concentration (2.5–100 μg/ml). After finding concentrations of propofol causing partial inhibition of a particular pathways, we used that concentration to construct kinetic curves by plotting oxygen flux against substrate concentration during its stepwise titration in the presence or absence of propofol. By spectrophotometry we also measured the influence of the same propofol concentrations on the activity of isolated respiratory complexes.ResultsWe found that human muscle and cardiac tissues are more sensitive to propofol-mediated inhibition of bioenergetic pathways than rat’s tissue. In human homogenates, palmitoyl carnitine-driven respiration was inhibited at much lower concentrations of propofol than that required for a reduction of electron transfer chain capacity, suggesting FAO inhibition mechanism different from downstream limitation or carnitine-palmitoyl transferase-1 inhibition. Inhibition of Complex I was characterised by more marked reduction of Vmax, in keeping with non-competitive nature of the inhibition and the pattern was similar to the inhibition of Complex II or electron transfer chain capacity. There was neither inhibition of Complex IV nor increased leak through inner mitochondrial membrane with up to 100 μg/ml of propofol. If measured in isolation by spectrophotometry, propofol 10 μg/ml did not affect the activity of any respiratory complexes.ConclusionIn human skeletal and heart muscle homogenates, propofol in concentrations that are achieved in propofol-anaesthetized patients, causes a direct inhibition of fatty acid oxidation, in addition to inhibiting flux of electrons through inner mitochondrial membrane. The inhibition is more marked in human as compared to rodent tissues.

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“…Tissue fragments were then homogenized by 5-6 slow strokes up and down with 2 mL Potter-Elvehjam teflon/glass homogenizer (clearance 0.25 mm; Wheaton TM , Millville, USA) driven by electric motor homogenizer (750 rpmi; HEi-Torque Value 100, Heidolph, Germany) [17]. Heart muscle homogenates were prepared similarly [19]. After removal of connective tissue and fat, fresh myocardium was dissected into fine fragments and 2.5 and 1% tissue solution ( 25 mg of human and 10 mg of rat heart muscle tissue /per 1 mL) was obtained when diluting in a respiration medium of different composition (MiR05): 0.5 mM EGTA, 3 mM MgCl 2 x 6 H 2 O, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH 2 PO 4 , 20 mM HEPES, 110 mM sucrose and 1 g/L of BSA (pH adjusted to 7.1 at 24°C).…”

Section: Preparation Of Tissue Homogenatesmentioning

confidence: 99%

“…After removal of connective tissue and fat, fresh myocardium was dissected into fine fragments and 2.5 and 1% tissue solution ( 25 mg of human and 10 mg of rat heart muscle tissue /per 1 mL) was obtained when diluting in a respiration medium of different composition (MiR05): 0.5 mM EGTA, 3 mM MgCl 2 x 6 H 2 O, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH 2 PO 4 , 20 mM HEPES, 110 mM sucrose and 1 g/L of BSA (pH adjusted to 7.1 at 24°C). Then the process of homogenization was two-step: tissue fragments were firstly manually homogenized by 10-12 initial strokes up and down with a glass loose pestle (large clearance 0.114 ± 0.025 mm; Wheaton TM , Millville, USA) in the glass Dounce grinder and subsequently homogenized with 5-6 slow strokes by a motor-driven PTFE pestle of 2 mL Potter-Elvehjam homogenizer (750 rpmi; Wheaton TM , Millville, USA) [19]. Finally, crude homogenate was filtered through polyamide mesh (SILK & PROGRESS s.r.o., Czech Republic) in order to remove the remaining connective tissue.…”

Section: Preparation Of Tissue Homogenatesmentioning

confidence: 99%

Kinetic characteristics of propofol-induced inhibition of electron-transfer chain and fatty acid oxidation in human and rodent skeletal and cardiac muscles

Duška¹,

Urban²,

Waldauf³

et al. 2019

Preprint

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Introduction: Propofol causes a profound inhibition of fatty acid oxidation (FAO) and reduces spare electron transfer chain (ETC) capacity in a range of human and rodent cells and tissues -a feature that might be related to the pathogenesis of Propofol Infusion Syndrome. We aimed to explore the mechanism of propofol-induced alteration of bioenergetic pathways by describing its kinetic characteristics.Methods: We obtained samples of skeletal and cardiac muscle from Wistar rat (n=3) and human subjects: vastus lateralis from hip surgery patients (n=11) and myocardium from brain-dead organ donors (n=10). We assessed mitochondrial functional indices using standard SUIT protocol and high resolution respirometry in fresh tissue homogenates with or without short-term exposure to a range of propofol concentration (2.5-100 µg/ml). After finding concentrations of propofol causing partial inhibition of a particular pathways, we used that concentration to construct kinetic curves by plotting oxygen flux against substrate concentration during its stepwise titration in the presence or absence of propofol. By spectrophotometry we also measured the influence of the same propofol concentrations on the activity of isolated respiratory complexes.2 Results: We found that human muscle and cardiac tissues are more sensitive to propofolmediated inhibition of bioenergetic pathways than rats tissue. In human homogenates, palmitoyl carnitine-driven respiration was inhibited at much lower concentrations of propofol than that required for a reduction of ETC capacity, suggesting FAO inhibition mechanism different from downstream limitation or carnitine-palmitoyl transferase-1 inhibition. Inhibition of Complex I was characterised by more marked reduction of Vmax, in keeping with non-competitive nature of the inhibition and the pattern was similar to the inhibition of Complex II or ETC capacity. There was no inhibition of Complex IV nor increased leak through inner mitochondrial membrane with up to 100 µg/ml of propofol. If measured in isolation by spectrophotometry, propofol 10 µg/ml did not affect the activity of any respiratory complexes.Conclusion: In human skeletal and heart muscle homogenates, propofol in concentrations that are achieved in propofol-anaesthetized patients, causes a direct inhibition of fatty acid oxidation, in addition to inhibiting flux of electrons through inner mitochondrial membrane. The inhibition is more marked in human as compared to rodent tissues.

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High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle

Cited by 15 publications

References 51 publications

Methodological considerations when assessing mitochondrial respiration and biomarkers for mitochondrial content in human skeletal muscle

Methodological considerations when assessing mitochondrial respiration and biomarkers for mitochondrial content in human skeletal muscle

Kinetic characteristics of propofol-induced inhibition of electron-transfer chain and fatty acid oxidation in human and rodent skeletal and cardiac muscles

Kinetic characteristics of propofol-induced inhibition of electron-transfer chain and fatty acid oxidation in human and rodent skeletal and cardiac muscles

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