Publications by authors named "Ger J M Stienen"

90 Publications

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes.

J Vis Exp 2020 06 22(160). Epub 2020 Jun 22.

Unidade de Investigação Cardiovascular, Departamento de Cirurgia e Fisiologia, Faculdade de Medicina, Universidade do Porto;

In this article, we describe the steps required to isolate a single permeabilized ("skinned") cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca)-containing solutions to determine, amongst others: maximum force development, myofilament Ca-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.
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http://dx.doi.org/10.3791/60427DOI Listing
June 2020

Disturbed cardiac mitochondrial and cytosolic calcium handling in a metabolic risk-related rat model of heart failure with preserved ejection fraction.

Acta Physiol (Oxf) 2020 03 10;228(3):e13378. Epub 2019 Oct 10.

Department of Surgery and Physiology, Cardiovascular R & D center, Faculty of Medicine of the University of Porto, Porto, Portugal.

Aim: Calcium ions play a pivotal role in matching energy supply and demand in cardiac muscle. Mitochondrial calcium concentration is lower in animal models of heart failure with reduced ejection fraction (HFrEF), but limited information is available about mitochondrial calcium handling in heart failure with preserved ejection fraction (HFpEF).

Methods: We assessed mitochondrial Ca handling in intact cardiomyocytes from Zucker/fatty Spontaneously hypertensive F1 hybrid (ZSF1)-lean (control) and ZSF1-obese rats, a metabolic risk-related model of HFpEF. A mitochondrially targeted Ca indicator (MitoCam) was expressed in cultured adult rat cardiomyocytes. Cytosolic and mitochondrial Ca transients were measured at different stimulation frequencies. Mitochondrial respiration and swelling, and expression of key proteins were determined ex vivo.

Results: At rest, mitochondrial Ca concentration in ZSF1-obese was larger than in ZSF1-lean. The diastolic and systolic mitochondrial Ca concentrations increased with stimulation frequency, but the steady-state levels were larger in ZSF1-obese. The half-widths of the contractile responses, the resting cytosolic Ca concentration and the decay half-times of the cytosolic Ca transients were higher in ZSF1-obese, likely because of a lower SERCA2a/phospholamban ratio. Mitochondrial respiration was lower, particularly with nicotinamide adenine dinucleotide (NADH) (complex I) substrates, and mitochondrial swelling was larger in ZSF1-obese.

Conclusion: The free mitochondrial calcium concentration is higher in HFpEF owing to alterations in mitochondrial and cytosolic Ca handling. This coupling between cytosolic and mitochondrial Ca levels may compensate for myocardial ATP supply in vivo under conditions of mild mitochondrial dysfunction. However, if mitochondrial Ca concentration is sustainedly increased, it might trigger mitochondrial permeability transition pore opening.
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http://dx.doi.org/10.1111/apha.13378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064935PMC
March 2020

Inotropic interventions do not change the resting state of myosin motors during cardiac diastole.

J Gen Physiol 2019 01 3;151(1):53-65. Epub 2018 Dec 3.

PhysioLab, University of Florence, Firenze, Italy.

When striated (skeletal and cardiac) muscle is in its relaxed state, myosin motors are packed in helical tracks on the surface of the thick filament, folded toward the center of the sarcomere, and unable to bind actin or hydrolyze ATP (OFF state). This raises the question of whatthe mechanism is that integrates the Ca-dependent thin filament activation, making myosin heads available for interaction with actin. Here we test the interdependency of the thin and thick filament regulatory mechanisms in intact trabeculae from the rat heart. We record the x-ray diffraction signals that mark the state of the thick filament during inotropic interventions (increase in sarcomere length from 1.95 to 2.25 µm and addition of 10 M isoprenaline), which potentiate the twitch force developed by an electrically paced trabecula by up to twofold. During diastole, none of the signals related to the OFF state of the thick filament are significantly affected by these interventions, except the intensity of both myosin-binding protein C- and troponin-related meridional reflections, which reduce by 20% in the presence of isoprenaline. These results indicate that recruitment of myosin motors from their OFF state occurs independently and downstream from thin filament activation. This is in agreement with the recently discovered mechanism based on thick filament mechanosensing in which the number of motors available for interaction with actin rapidly adapts to the stress on the thick filament and thus to the loading conditions of the contraction. The gain of this positive feedback may be modulated by both sarcomere length and the degree of phosphorylation of myosin-binding protein C.
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http://dx.doi.org/10.1085/jgp.201812196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314382PMC
January 2019

Cardiac Disorders and Pathophysiology of Sarcomeric Proteins.

Physiol Rev 2019 01;99(1):381-426

Amsterdam UMC, Vrije Universiteit Amsterdam, Physiology, Amsterdam Cardiovascular Sciences, Amsterdam , The Netherlands ; and Department of Physiology, Kilimanjaro Christian Medical University College, Moshi, Tanzania.

The sarcomeric proteins represent the structural building blocks of heart muscle, which are essential for contraction and relaxation. During recent years, it has become evident that posttranslational modifications of sarcomeric proteins, in particular phosphorylation, tune cardiac pump function at rest and during exercise. This delicate, orchestrated interaction is also influenced by mutations, predominantly in sarcomeric proteins, which cause hypertrophic or dilated cardiomyopathy. In this review, we follow a bottom-up approach starting from a description of the basic components of cardiac muscle at the molecular level up to the various forms of cardiac disorders at the organ level. An overview is given of sarcomere changes in acquired and inherited forms of cardiac disease and the underlying disease mechanisms with particular reference to human tissue. A distinction will be made between the primary defect and maladaptive/adaptive secondary changes. Techniques used to unravel functional consequences of disease-induced protein changes are described, and an overview of current and future treatments targeted at sarcomeric proteins is given. The current evidence presented suggests that sarcomeres not only form the basis of cardiac muscle function but also represent a therapeutic target to combat cardiac disease.
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http://dx.doi.org/10.1152/physrev.00040.2017DOI Listing
January 2019

A 3D diffusional-compartmental model of the calcium dynamics in cytosol, sarcoplasmic reticulum and mitochondria of murine skeletal muscle fibers.

PLoS One 2018 26;13(7):e0201050. Epub 2018 Jul 26.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

Variations of free calcium concentration ([Ca2+]) are powerful intracellular signals, controlling contraction as well as metabolism in muscle cells. To fully understand the role of calcium redistribution upon excitation and contraction in skeletal muscle cells, the local [Ca2+] in different compartments needs to be taken into consideration. Fluorescent probes allow the determination of [Ca2+] in the cytosol where myofibrils are embedded, the lumen of the sarcoplasmic reticulum (SR) and the mitochondrial matrix. Previously, models have been developed describing intracellular calcium handling in skeletal and cardiac muscle cells. However, a comprehensive model describing the kinetics of the changes in free calcium concentration in these three compartments is lacking. We designed a new 3D compartmental model of the half sarcomere with radial symmetry, which accounts for diffusion of Ca2+ into the three compartments and simulates its dynamics at rest and at various rates of stimulation in mice skeletal muscle fibers. This model satisfactorily reproduces both the amplitude and time course of the variations of [Ca2+] in the three compartments in mouse fast fibers. As an illustration of the applicability of the model, we investigated the effects of Calsequestrin (CSQ) ablation. CSQ is the main Ca2+ buffer in the SR, localized in close proximity of its calcium release sites and near to the mitochondria. CSQ knock-out mice muscles still preserve a near-normal contractile behavior, but it is unclear whether this is caused by additional SR calcium buffering or a significant contribution of calcium entry from extracellular space, via stored-operated calcium entry (SOCE). The model enabled quantitative assessment of these two scenarios by comparison to measurements of local calcium in the cytosol, the SR and the mitochondria. In conclusion, the model represents a useful tool to investigate the impact of protein ablation and of pharmacological interventions on intracellular calcium dynamics in mice skeletal muscle.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201050PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062086PMC
January 2019

The force and stiffness of myosin motors in the isometric twitch of a cardiac trabecula and the effect of the extracellular calcium concentration.

J Physiol 2018 07 27;596(13):2581-2596. Epub 2018 May 27.

PhysioLab, University of Florence, Firenze, Italy.

Key Points: Fast sarcomere-level mechanics in intact trabeculae, which allows the definition of the mechano-kinetic properties of cardiac myosin in situ, is a fundamental tool not only for understanding the molecular mechanisms of heart performance and regulation, but also for investigating the mechanisms of the cardiomyopathy-causing mutations in the myosin and testing small molecules for therapeutic interventions. The approach has been applied to measure the stiffness and force of the myosin motor and the fraction of motors attached during isometric twitches of electrically paced trabeculae under different extracellular Ca concentrations. Although the average force of the cardiac myosin motor (∼6 pN) is similar to that of the fast myosin isoform of skeletal muscle, the stiffness (1.07 pN nm ) is 2- to 3-fold smaller. The increase in the twitch force developed in the presence of larger extracellular Ca concentrations is fully accounted for by a proportional increase in the number of attached motors.

Abstract: The mechano-kinetic properties of the cardiac myosin were studied in situ, in trabeculae dissected from the right ventricle of the rat heart, by measuring the stiffness of the half-sarcomere both at the twitch force peak (T ) of an electrically paced intact trabecula at different extracellular Ca concentrations ([Ca ] ), and in the same trabecula after skinning and induction of rigor. Taking into account the contribution of filament compliance to half-sarcomere compliance and the lattice geometry, we found that the stiffness of the cardiac myosin motor is 1.07 ± 0.09 pN nm , which is slightly larger than that of the slow myosin isoform of skeletal muscle (0.6-0.8 pN nm ) and 2- to 3-fold smaller than that of the fast skeletal muscle isoform. The increase in T from 61 ± 4 kPa to 93 ± 9 kPa, induced by raising [Ca ] from 1 to 2.5 mm at sarcomere length ∼2.2 μm, is accompanied by an increase of the half-sarcomere stiffness that is explained by an increase of the fraction of actin-attached motors from 0.08 ± 0.01 to 0.12 ± 0.02, proportional to T . Consequently, each myosin motor bears an average force of 6.14 ± 0.52 pN independently of T and [Ca ] . The application of fast sarcomere-level mechanics to intact trabeculae to define the mechano-kinetic properties of the cardiac myosin in situ represents a powerful tool for investigating cardiomyopathy-causing mutations in the myosin motor and testing specific therapeutic interventions.
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http://dx.doi.org/10.1113/JP275579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023834PMC
July 2018

Successive contractile periods activate mitochondria at the onset of contractions in intact rat cardiac trabeculae.

J Appl Physiol (1985) 2018 04 11;124(4):1003-1011. Epub 2018 Jan 11.

Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center , Amsterdam , The Netherlands.

The rate of oxidative phosphorylation depends on the contractile activity of the heart. Cardiac mitochondrial oxidative phosphorylation is determined by free ADP concentration, mitochondrial Ca accumulation, mitochondrial enzyme activities, and Krebs cycle intermediates. The purpose of the present study was to examine the factors that limit oxidative phosphorylation upon rapid changes in contractile activity in cardiac muscle. We tested the hypotheses that prior contractile performance enhances the changes in NAD(P)H and FAD concentration upon an increase in contractile activity and that this mitochondrial "priming" depends on pyruvate dehydrogenase activity. Intact rat cardiac trabeculae were electrically stimulated at 0.5 Hz for at least 30 min. Thereafter, two equal bouts at elevated stimulation frequency of 1, 2, or 3 Hz were applied for 3 min with 3 min of 0.5-Hz stimulation in between. No discernible time delay was observed in the changes in NAD(P)H and FAD fluorescence upon rapid changes in contractile activity. The amplitudes of the rapid changes in fluorescence upon an increase in stimulation frequency (the on-transients) were smaller than upon a decrease in stimulation frequency (the off-transients). A first bout in glucose-containing superfusion solution resulted, during the second bout, in an increase in the amplitudes of the on-transients, but the off-transients remained the same. No such priming effect was observed after addition of 10 mM pyruvate. These results indicate that mitochondrial priming can be observed in cardiac muscle in situ and that pyruvate dehydrogenase activity is critically involved in the mitochondrial adaptation to increases in contractile performance. NEW & NOTEWORTHY Mitochondrial respiration increases with increased cardiac contractile activity. Similar to mitochondrial "priming" in skeletal muscle, we hypothesized that cardiac mitochondrial activity is altered upon successive bouts of contractions and depends on pyruvate dehydrogenase activity. We found altered bioenergetics upon repeated contractile periods, indicative of mitochondrial priming in rat myocardium. No effect was seen when pyruvate was added to the perfusate. As such, pyruvate dehydrogenase activity is involved in the mitochondrial adaptation to increased contractile performance.
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http://dx.doi.org/10.1152/japplphysiol.01010.2017DOI Listing
April 2018

Dysfunctional sarcomere contractility contributes to muscle weakness in ACTA1-related nemaline myopathy (NEM3).

Ann Neurol 2018 02 6;83(2):269-282. Epub 2018 Feb 6.

Department of Physiology, VU University Medical Center Amsterdam, Amsterdam, the Netherlands.

Objective: Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha-actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients.

Methods: To test this hypothesis, we performed contractility measurements in individual muscle fibers and myofibrils obtained from muscle biopsies of 14 NEM3 patients with different ACTA1 mutations. To identify the structural basis for impaired contractility, low angle X-ray diffraction and stimulated emission-depletion microscopy were applied.

Results: Our findings reveal that muscle fibers of NEM3 patients display a reduced maximal force-generating capacity, which is caused by dysfunctional sarcomere contractility in the majority of patients, as revealed by contractility measurements in myofibrils. Low angle X-ray diffraction and stimulated emission-depletion microscopy indicate that dysfunctional sarcomere contractility in NEM3 patients involves a lower number of myosin heads binding to actin during muscle activation. This lower number is not the result of reduced thin filament length. Interestingly, the calcium sensitivity of force is unaffected in some patients, but decreased in others.

Interpretation: Dysfunctional sarcomere contractility is an important contributor to muscle weakness in the majority of NEM3 patients. This information is crucial for patient stratification in future clinical trials. Ann Neurol 2018;83:269-282.
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http://dx.doi.org/10.1002/ana.25144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821533PMC
February 2018

Diaphragm Atrophy and Weakness in the Absence of Mitochondrial Dysfunction in the Critically Ill.

Am J Respir Crit Care Med 2017 12;196(12):1544-1558

1 Department of Physiology, Amsterdam Cardiovascular Sciences.

Rationale: The clinical significance of diaphragm weakness in critically ill patients is evident: it prolongs ventilator dependency and increases morbidity, duration of hospital stay, and health care costs. The mechanisms underlying diaphragm weakness are unknown, but might include mitochondrial dysfunction and oxidative stress.

Objectives: We hypothesized that weakness of diaphragm muscle fibers in critically ill patients is accompanied by impaired mitochondrial function and structure, and by increased markers of oxidative stress.

Methods: To test these hypotheses, we studied contractile force, mitochondrial function, and mitochondrial structure in diaphragm muscle fibers. Fibers were isolated from diaphragm biopsies of 36 mechanically ventilated critically ill patients and compared with those isolated from biopsies of 27 patients with suspected early-stage lung malignancy (control subjects).

Measurements And Main Results: Diaphragm muscle fibers from critically ill patients displayed significant atrophy and contractile weakness, but lacked impaired mitochondrial respiration and increased levels of oxidative stress markers. Mitochondrial energy status and morphology were not altered, despite a lower content of fusion proteins.

Conclusions: Critically ill patients have manifest diaphragm muscle fiber atrophy and weakness in the absence of mitochondrial dysfunction and oxidative stress. Thus, mitochondrial dysfunction and oxidative stress do not play a causative role in the development of atrophy and contractile weakness of the diaphragm in critically ill patients.
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http://dx.doi.org/10.1164/rccm.201703-0501OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754442PMC
December 2017

Myosin filament activation in the heart is tuned to the mechanical task.

Proc Natl Acad Sci U S A 2017 03 6;114(12):3240-3245. Epub 2017 Mar 6.

PhysioLab, University of Florence, 50019 Florence, Italy;

The mammalian heart pumps blood through the vessels, maintaining the dynamic equilibrium in a circulatory system driven by two pumps in series. This vital function is based on the fine-tuning of cardiac performance by the Frank-Starling mechanism that relates the pressure exerted by the contracting ventricle (end systolic pressure) to its volume (end systolic volume). At the level of the sarcomere, the structural unit of the cardiac myocytes, the Frank-Starling mechanism consists of the increase in active force with the increase of sarcomere length (length-dependent activation). We combine sarcomere mechanics and micrometer-nanometer-scale X-ray diffraction from synchrotron light in intact ventricular trabeculae from the rat to measure the axial movement of the myosin motors during the diastole-systole cycle under sarcomere length control. We find that the number of myosin motors leaving the off, ATP hydrolysis-unavailable state characteristic of the diastole is adjusted to the sarcomere length-dependent systolic force. This mechanosensing-based regulation of the thick filament makes the energetic cost of the systole rapidly tuned to the mechanical task, revealing a prime aspect of the Frank-Starling mechanism. The regulation is putatively impaired by cardiomyopathy-causing mutations that affect the intramolecular and intermolecular interactions controlling the off state of the motors.
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http://dx.doi.org/10.1073/pnas.1619484114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373356PMC
March 2017

Rapid frequency-dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes.

J Physiol 2017 03 22;595(6):2001-2019. Epub 2017 Feb 22.

Department of Physiology, Institute for Cardiovascular Research, VU University Medical Centre, Amsterdam, the Netherlands.

Key Points: Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. The magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiac myocytes are largely unknown. Rapid stimulation frequency-dependent increases but relatively slow decreases in free mitochondrial calcium concentration were observed in rat cardiac myocytes. This asymmetry caused a rise in the mitochondrial calcium concentration with stimulation frequency. These results provide insight into the mechanisms of mitochondrial calcium uptake and release that are important in healthy and diseased myocardium.

Abstract: Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially targeted Förster resonance energy transfer (FRET)-based calcium indicator (4mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca ] ) was measured at different stimulation frequencies (0.1-4 Hz) and external calcium concentrations (1.8-3.6 mm) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura-4AM. The increases in [Ca ] during electrical stimulation at 0.1 Hz were rapid (rise time = 49 ± 2 ms), while the decreases in [Ca ] occurred more slowly (decay half time = 1.17 ± 0.07 s). Model calculations confirmed that this asymmetry caused the rise in [Ca ] during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium-calcium exchanger (mNCE) resulted in a rise in [Ca ] at baseline and, paradoxically, in an acceleration of Ca release.

In Conclusion: rapid increases in [Ca ] allow for fast adjustment of mitochondrial ATP production to increases in myocardial demand on a beat-to-beat basis and mitochondrial calcium release depends on mNCE activity and mitochondrial calcium buffering.
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http://dx.doi.org/10.1113/JP273589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350475PMC
March 2017

Empagliflozin decreases myocardial cytoplasmic Na through inhibition of the cardiac Na/H exchanger in rats and rabbits.

Diabetologia 2017 03 17;60(3):568-573. Epub 2016 Oct 17.

Department of Anesthesiology, Laboratory of Experimental Intensive Care and Anesthesiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, the Netherlands.

Aims/hypothesis: Empagliflozin (EMPA), an inhibitor of the renal sodium-glucose cotransporter (SGLT) 2, reduces the risk of cardiovascular death in patients with type 2 diabetes. The underlying mechanism of this effect is unknown. Elevated cardiac cytoplasmic Na ([Na]) and Ca ([Ca]) concentrations and decreased mitochondrial Ca concentration ([Ca]) are drivers of heart failure and cardiac death. We therefore hypothesised that EMPA would directly modify [Na], [Ca] and [Ca] in cardiomyocytes.

Methods: [Na] [Ca], [Ca ] and Na/H exchanger (NHE) activity were measured fluorometrically in isolated ventricular myocytes from rabbits and rats.

Results: An increase in extracellular glucose, from 5.5 mmol/l to 11 mmol/l, resulted in increased [Na] and [Ca] levels. EMPA treatment directly inhibited NHE flux, caused a reduction in [Na] and [Ca] and increased [Ca]. After pretreatment with the NHE inhibitor, Cariporide, these effects of EMPA were strongly reduced. EMPA also affected [Na] and NHE flux in the absence of extracellular glucose.

Conclusions/interpretation: The glucose lowering kidney-targeted agent, EMPA, demonstrates direct cardiac effects by lowering myocardial [Na] and [Ca] and enhancing [Ca], through impairment of myocardial NHE flux, independent of SGLT2 activity.
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http://dx.doi.org/10.1007/s00125-016-4134-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518059PMC
March 2017

Mitochondrial complex I dysfunction and altered NAD(P)H kinetics in rat myocardium in cardiac right ventricular hypertrophy and failure.

Cardiovasc Res 2016 09 11;111(4):362-72. Epub 2016 Jul 11.

Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, O|2 Building, De Boelelaan 1118, Amsterdam 1081 HV, The Netherlands Faculty of Science, Department of Physics and Astronomy, VU University Amsterdam, Amsterdam, The Netherlands.

Aims: In cardiac hypertrophy (CH) and heart failure (HF), alterations occur in mitochondrial enzyme content and activities but the origin and implications of these changes for mitochondrial function need to be resolved.

Methods And Results: Right ventricular CH or HF was induced by monocrotaline injection, which causes pulmonary artery hypertension, in rats. Results were compared with saline injection (CON). NAD(P)H and FAD autofluorescence were recorded in thin intact cardiac trabeculae during transitions in stimulation frequency, to assess mitochondrial complex I and complex II function, respectively. Oxygen consumption, mitochondrial morphology, protein content, and enzymatic activity were assessed. NAD(P)H autofluorescence upon an increase in stimulation frequency showed a rapid decline followed by a slow recovery. FAD autofluorescence followed a similar time course, but in opposite direction. The amplitude of the early rapid change in NAD(P)H autofluorescence was severely depressed in CH and HF compared with CON. The rapid changes in FAD autofluorescence in CH and HF were reduced to a lesser extent. Complex I-coupled respiration showed an ∼3.5-fold reduction in CH and HF; complex II-coupled respiration was depressed two-fold in HF. Western blot analyses revealed modest reductions in complex I protein content in CH and HF and in complex I activity in supercomplexes in HF. Mitochondrial volume density was similar, but mitochondrial remodelling was evident from changes in ultrastructure and fusion/fission indices in CH and HF.

Conclusion: These results suggest that the alterations in mitochondrial function observed in right ventricular CH and HF can be mainly attributed to complex I dysfunction.
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http://dx.doi.org/10.1093/cvr/cvw176DOI Listing
September 2016

Reduced force of diaphragm muscle fibers in patients with chronic thromboembolic pulmonary hypertension.

Am J Physiol Lung Cell Mol Physiol 2016 07 17;311(1):L20-8. Epub 2016 May 17.

Department of Physiology, VU University Medical Center/Institute for Cardiovascular Research, The Netherlands; Cellular and Molecular Medicine, University of Arizona, Tucson, Arizona

Patients with pulmonary hypertension (PH) suffer from inspiratory muscle weakness. However, the pathophysiology of inspiratory muscle dysfunction in PH is unknown. We hypothesized that weakness of the diaphragm, the main inspiratory muscle, is an important contributor to inspiratory muscle dysfunction in PH patients. Our objective was to combine ex vivo diaphragm muscle fiber contractility measurements with measures of in vivo inspiratory muscle function in chronic thromboembolic pulmonary hypertension (CTEPH) patients. To assess diaphragm muscle contractility, function was studied in vivo by maximum inspiratory pressure (MIP) and ex vivo in diaphragm biopsies of the same CTEPH patients (N = 13) obtained during pulmonary endarterectomy. Patients undergoing elective lung surgery served as controls (N = 15). Muscle fiber cross-sectional area (CSA) was determined in cryosections and contractility in permeabilized muscle fibers. Diaphragm muscle fiber CSA was not significantly different between control and CTEPH patients in both slow-twitch and fast-twitch fibers. Maximal force-generating capacity was significantly lower in slow-twitch muscle fibers of CTEPH patients, whereas no difference was observed in fast-twitch muscle fibers. The maximal force of diaphragm muscle fibers correlated significantly with MIP. The calcium sensitivity of force generation was significantly reduced in fast-twitch muscle fibers of CTEPH patients, resulting in a ∼40% reduction of submaximal force generation. The fast skeletal troponin activator CK-2066260 (5 μM) restored submaximal force generation to levels exceeding those observed in control subjects. In conclusion, diaphragm muscle fiber contractility is hampered in CTEPH patients and contributes to the reduced function of the inspiratory muscles in CTEPH patients.
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http://dx.doi.org/10.1152/ajplung.00113.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967190PMC
July 2016

Mutation-specific effects on thin filament length in thin filament myopathy.

Ann Neurol 2016 06 30;79(6):959-69. Epub 2016 Apr 30.

Department of Physiology, VU University Medical Center, Amsterdam, the Netherlands.

Objective: Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation.

Methods: We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41.

Results: Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force-sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin-thick filament overlap.

Interpretation: These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. Ann Neurol 2016;79:959-969.
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http://dx.doi.org/10.1002/ana.24654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911820PMC
June 2016

Size and speed of the working stroke of cardiac myosin in situ.

Proc Natl Acad Sci U S A 2016 Mar 16;113(13):3675-80. Epub 2016 Mar 16.

Laboratory of Physiology, Department of Biology, Università di Firenze, 50019 Sesto Fiorentino, Florence, Italy;

The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force TP to a value T(0.8-0.2 TP). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s(-1) at high load to ∼8 nm⋅hs(-1) and 6,000 s(-1) at low load. Increases in sarcomere length (1.9-2.2 μm) and external [Ca(2+)]o (1-2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca(2+)-dependent increase of TP and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach.
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http://dx.doi.org/10.1073/pnas.1525057113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822625PMC
March 2016

Decreased creatine kinase is linked to diastolic dysfunction in rats with right heart failure induced by pulmonary artery hypertension.

J Mol Cell Cardiol 2015 Sep 24;86:1-8. Epub 2015 Jun 24.

Multidisciplinary Cardiovascular Research Centre, University of Leeds, UK. Electronic address:

Our objective was to investigate the role of creatine kinase in the contractile dysfunction of right ventricular failure caused by pulmonary artery hypertension. Pulmonary artery hypertension and right ventricular failure were induced in rats by monocrotaline and compared to saline-injected control animals. In vivo right ventricular diastolic pressure-volume relationships were measured in anesthetized animals; diastolic force-length relationships in single enzymatically dissociated myocytes and myocardial creatine kinase levels by Western blot. We observed diastolic dysfunction in right ventricular failure indicated by significantly steeper diastolic pressure-volume relationships in vivo and diastolic force-length relationships in single myocytes. There was a significant reduction in creatine kinase protein expression in failing right ventricle. Dysfunction also manifested as a shorter diastolic sarcomere length in failing myocytes. This was associated with a Ca(2+)-independent mechanism that was sensitive to cross-bridge cycling inhibition. In saponin-skinned failing myocytes, addition of exogenous creatine kinase significantly lengthened sarcomeres, while in intact healthy myocytes, inhibition of creatine kinase significantly shortened sarcomeres. Creatine kinase inhibition also changed the relatively flat contraction amplitude-stimulation frequency relationship of healthy myocytes into a steeply negative, failing phenotype. Decreased creatine kinase expression leads to diastolic dysfunction. We propose that this is via local reduction in ATP:ADP ratio and thus to Ca(2+)-independent force production and diastolic sarcomere shortening. Creatine kinase inhibition also mimics a definitive characteristic of heart failure, the inability to respond to increased demand. Novel therapies for pulmonary artery hypertension are needed. Our data suggest that cardiac energetics would be a potential ventricular therapeutic target.
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http://dx.doi.org/10.1016/j.yjmcc.2015.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564291PMC
September 2015

The striated muscles in pulmonary arterial hypertension: adaptations beyond the right ventricle.

Eur Respir J 2015 Sep 25;46(3):832-42. Epub 2015 Jun 25.

Dept of Pulmonology, VU University Medical Center, Institute for Cardiovascular Research, Amsterdam, The Netherlands

Pulmonary arterial hypertension (PAH) is a fatal lung disease characterised by progressive remodelling of the small pulmonary vessels. The daily-life activities of patients with PAH are severely limited by exertional fatigue and dyspnoea. Typically, these symptoms have been explained by right heart failure. However, an increasing number of studies reveal that the impact of the PAH reaches further than the pulmonary circulation. Striated muscles other than the right ventricle are affected in PAH, such as the left ventricle, the diaphragm and peripheral skeletal muscles. Alterations in these striated muscles are associated with exercise intolerance and reduced quality of life. In this Back to Basics article on striated muscle function in PAH, we provide insight into the pathophysiological mechanisms causing muscle dysfunction in PAH and discuss potential new therapeutic strategies to restore muscle dysfunction.
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http://dx.doi.org/10.1183/13993003.02052-2014DOI Listing
September 2015

Synergistic role of ADP and Ca(2+) in diastolic myocardial stiffness.

J Physiol 2015 Sep 14;593(17):3899-916. Epub 2015 Jul 14.

Department of Physiology, Institute for Cardiovascular Research, VU University Medical Centre, Amsterdam, The Netherlands.

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin-related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca(2+) ] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction. Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca(2+) ] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca(2+) handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca(2+) ] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca(2+) in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca(2+) both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca(2+) ] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca(2+) overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca(2+) ]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca(2+) , and thereby increase myocardial stiffness.
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http://dx.doi.org/10.1113/JP270354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575576PMC
September 2015

A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis.

J Mol Cell Cardiol 2015 May 11;82:93-103. Epub 2015 Mar 11.

Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands; ICIN-Netherlands Heart Institute, Utrecht, The Netherlands.

Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphorylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Ser199 was replaced by aspartic acid (199D) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca(2+)] and at sarcomere lengths of 1.8 and 2.2 μm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca(2+)-sensitivity (pCa50) at 2.2 μm was significantly higher in 199D (pCa50 = 5.79 ± 0.01) compared to 199A (pCa50 = 5.65 ± 0.01) and Wt (pCa50 = 5.66 ± 0.02) at ~63% cTn exchange. Myofilament Ca(2+)-sensitivity was significantly higher even with only 5.9 ± 2.5% 199D exchange compared to 199A, and saturated at 12.3 ± 2.6% 199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (~6%) increase myofilament Ca(2+)-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI.
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http://dx.doi.org/10.1016/j.yjmcc.2015.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405491PMC
May 2015

Diaphragm muscle fiber weakness and ubiquitin-proteasome activation in critically ill patients.

Am J Respir Crit Care Med 2015 May;191(10):1126-38

1 Department of Physiology.

Rationale: The clinical significance of diaphragm weakness in critically ill patients is evident: it prolongs ventilator dependency, and increases morbidity and duration of hospital stay. To date, the nature of diaphragm weakness and its underlying pathophysiologic mechanisms are poorly understood.

Objectives: We hypothesized that diaphragm muscle fibers of mechanically ventilated critically ill patients display atrophy and contractile weakness, and that the ubiquitin-proteasome pathway is activated in the diaphragm.

Methods: We obtained diaphragm muscle biopsies from 22 critically ill patients who received mechanical ventilation before surgery and compared these with biopsies obtained from patients during thoracic surgery for resection of a suspected early lung malignancy (control subjects). In a proof-of-concept study in a muscle-specific ring finger protein-1 (MuRF-1) knockout mouse model, we evaluated the role of the ubiquitin-proteasome pathway in the development of contractile weakness during mechanical ventilation.

Measurements And Main Results: Both slow- and fast-twitch diaphragm muscle fibers of critically ill patients had approximately 25% smaller cross-sectional area, and had contractile force reduced by half or more. Markers of the ubiquitin-proteasome pathway were significantly up-regulated in the diaphragm of critically ill patients. Finally, MuRF-1 knockout mice were protected against the development of diaphragm contractile weakness during mechanical ventilation.

Conclusions: These findings show that diaphragm muscle fibers of critically ill patients display atrophy and severe contractile weakness, and in the diaphragm of critically ill patients the ubiquitin-proteasome pathway is activated. This study provides rationale for the development of treatment strategies that target the contractility of diaphragm fibers to facilitate weaning.
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http://dx.doi.org/10.1164/rccm.201412-2214OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4451621PMC
May 2015

Quadriceps muscle fibre dysfunction in patients with pulmonary arterial hypertension.

Eur Respir J 2015 Jun 5;45(6):1737-40. Epub 2015 Mar 5.

Dept of Physiology, VU University Medical Center, Institute for Cardiovascular Research, Amsterdam, The Netherlands

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http://dx.doi.org/10.1183/09031936.00205114DOI Listing
June 2015

Rapid changes in NADH and flavin autofluorescence in rat cardiac trabeculae reveal large mitochondrial complex II reserve capacity.

J Physiol 2015 Apr 13;593(8):1829-40. Epub 2015 Mar 13.

Department of Physiology, Institute for Cardiovascular Research, VU University Medical Centre, Amsterdam, the Netherlands.

Key Points: A photometry-based technique was developed to measure nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence and contractile properties simultaneously in intact rat trabeculae at a high time resolution. This provides insight into the function of mitochondrial complex I and II. Maximal complex I and complex II activities were determined in saponin-permeabilized right ventricular tissue by respirometry. In trabeculae, complex II function was considerably smaller than the maximal complex II activity, suggesting large complex II reserve capacity. Up-down asymmetry in NADH and FAD kinetics suggests a complex interaction between mitochondrial and contractile function. These data show that simultaneous measurement of contractile properties and NADH and FAD kinetics in cardiac trabeculae provides a mean to study the differences in complex I and II function in intact preparations in health and disease.

Abstract: The functional properties of cardiac mitochondria in intact preparations have been mainly studied by measurements of nicotinamide adenine dinucleotide (NADH) autofluorescence, which reflects mitochondrial complex I function. To assess complex II function, we extended this method by measuring flavin adenine dinucleotide (FAD)-related autofluorescence in electrically stimulated cardiac trabeculae isolated from the right ventricle from the rat at 27°C. NADH and FAD autofluorescence and tension responses were measured when stimulation frequency was increased from 0.5 Hz to 1, 2 or 3 Hz for 3 min, and thereafter decreased to 0.5 Hz. Maximal complex I and complex II activity in vitro were determined in saponin-permeabilized right ventricular tissue by respirometry. NADH responses upon an increase in stimulation frequency showed a rapid decline, followed by a slow recovery towards the initial level. FAD responses followed a similar time course, but in the opposite direction. The amplitudes of early rapid changes in the NADH and FAD concentration correlated well with the change in tension time integral per second (R(2)  = 0.833 and 0.660 for NADH and FAD, respectively), but with different slopes for the up and down transient. Maximal velocity of the increase in FAD concentration (16 ± 4 μm s(-1) ), measured upon an increase in stimulation frequency from 0.5 to 3 Hz was considerably smaller than that of the decrease in NADH (78 ± 13 μm s(-1) ). The respiration measurements indicated that the maximal velocity of NADH utilization (143 ± 14 μm s(-1) ) was 2 times smaller than that of FADH2 (291 ± 19 μm s(-1) ). This indicates that in cardiac mitochondria considerable complex II activity reserve is present.
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http://dx.doi.org/10.1113/jphysiol.2014.286153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405745PMC
April 2015

Altered myocardial force generation in end-stage human heart failure.

ESC Heart Fail 2014 Dec 15;1(2):160-165. Epub 2015 Jan 15.

Laboratory for Physiology, Institute of Cardiovascular Research (ICaR-VU), VU University Medical Center, Amsterdam, Netherlands.

Aims: This study aimed to elucidate the molecular background of increased Ca sensitivity of force production in cardiomyocytes of end-stage human heart failure.

Methods And Results: Ca -activated isometric force and the cross-bridge specific rate of force redevelopment (k ) were determined in Triton-skinned myocytes from end-stage failing and non-failing donor hearts. Measurements (control: pH 7.2, 0 mM inorganic phosphate (P )) were performed under test conditions that probed either the Ca -regulatory function of the thin filaments (pH 6.5), the kinetics of the actin-myosin cross-bridge cycle (10 mM P ), or both (pH 6.5, 10 mM P ). The control maximal Ca -activated force (F ) and k did not differ between failing and non-failing myocytes. At submaximal [Ca ], however, both force and k were higher in failing than in donor myocytes. The difference in the Ca sensitivities of force production was preserved when the thin filament regulatory function was perturbed by acidosis (pH 6.5) but was abolished by cross-bridge modulation (i.e. by P ) both at pH 7.2 and at pH 6.5. P induced a larger reduction in force but a smaller increase in k in the failing myocytes than in the non-failing myocytes at submaximal [Ca ].

Conclusion: The enhanced P sensitivity of the actin-myosin interaction suggests that the P release step of the actin-myosin cross-bridge cycle is modified during end-stage human heart failure. This might be of functional importance when P accumulates (e.g. during cardiac ischaemia). Moreover, this alteration can influence cardiac energetics and the clinical efficacy of sarcomere targeted agents in human heart failure.
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http://dx.doi.org/10.1002/ehf2.12020DOI Listing
December 2014

Unaffected contractility of diaphragm muscle fibers in humans on mechanical ventilation.

Am J Physiol Lung Cell Mol Physiol 2014 Sep 18;307(6):L460-70. Epub 2014 Jul 18.

Departments of Physiology, Department of Cellular and Molecular Medicine, University of Arizona, Tucson, Arizona

Several studies have indicated that diaphragm dysfunction develops in patients on mechanical ventilation (MV). Here, we tested the hypothesis that the contractility of sarcomeres, i.e., the smallest contractile unit in muscle, is affected in humans on MV. To this end, we compared diaphragm muscle fibers of nine brain-dead organ donors (cases) that had been on MV for 26 ± 5 h with diaphragm muscle fibers from nine patients (controls) undergoing surgery for lung cancer that had been on MV for less than 2 h. In each diaphragm specimen we determined 1) muscle fiber cross-sectional area in cryosections by immunohistochemical methods and 2) the contractile performance of permeabilized single muscle fibers by means of maximum specific force, kinetics of cross-bridge cycling by rate of tension redevelopment, myosin heavy chain content and concentration, and calcium sensitivity of force of slow-twitch and fast-twitch muscle fibers. In case subjects, we noted no statistically significant decrease in outcomes compared with controls in slow-twitch or fast-twitch muscle fibers. These observations indicate that 26 h of MV of humans is not invariably associated with changes in the contractile performance of sarcomeres in the diaphragm.
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http://dx.doi.org/10.1152/ajplung.00076.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166782PMC
September 2014

Contractile dysfunction of left ventricular cardiomyocytes in patients with pulmonary arterial hypertension.

J Am Coll Cardiol 2014 Jul;64(1):28-37

Department of Physiology, VU University Medical Center, Amsterdam, the Netherlands. Electronic address:

Background: After lung transplantation, increased left ventricular (LV) filling can lead to LV failure, increasing the risk of post-operative complications and mortality. LV dysfunction in pulmonary arterial hypertension (PAH) is characterized by a reduced LV ejection fraction and impaired diastolic function.

Objectives: The pathophysiology of LV dysfunction in PAH is incompletely understood. This study sought to assess the contribution of atrophy and contractility of cardiomyocytes to LV dysfunction in PAH patients.

Methods: LV function was assessed by cardiac magnetic resonance imaging. In addition, LV biopsies were obtained in 9 PAH patients and 10 donors. The cross-sectional area (CSA) and force-generating capacity of isolated single cardiomyocytes was investigated.

Results: Magnetic resonance imaging analysis revealed a significant reduction in LV ejection fraction in PAH patients, indicating a reduction in LV contractility. The CSA of LV cardiomyocytes of PAH patients was significantly reduced (~30%), indicating LV cardiomyocyte atrophy. The maximal force-generating capacity, normalized to cardiomyocyte CSA, was significantly reduced (~25%). Also, a reduction in the number of available myosin-based cross-bridges was found to cause the contractile weakness of cardiomyocytes. This finding was supported by protein analyses, which showed an ~30% reduction in the myosin/actin ratio in cardiomyocytes from PAH patients. Finally, the phosphorylation level of sarcomeric proteins was reduced in PAH patients, which was accompanied by increased calcium sensitivity of force generation.

Conclusions: The contractile function and the CSA of LV cardiomyocytes is substantially reduced in PAH patients. We propose that these changes contribute to the reduced in vivo contractility of the LV in PAH patients.
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http://dx.doi.org/10.1016/j.jacc.2014.04.031DOI Listing
July 2014

Faster cross-bridge detachment and increased tension cost in human hypertrophic cardiomyopathy with the R403Q MYH7 mutation.

J Physiol 2014 Aug 13;592(15):3257-72. Epub 2014 Jun 13.

Department of Physiology, VU University Medical Centre, Amsterdam, The Netherlands ICIN-Netherlands Heart Institute, Utrecht, The Netherlands.

The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain (β-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCMsmn). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P < 0.0001) than in HCMsmn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s(-1), respectively). Moreover, compared to HCMsmn, tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 μmol l(-1) s(-1) kN(-1) m(-2), respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCMsmn. Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation.
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http://dx.doi.org/10.1113/jphysiol.2014.274571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146374PMC
August 2014

Gene-specific increase in the energetic cost of contraction in hypertrophic cardiomyopathy caused by thick filament mutations.

Cardiovasc Res 2014 Jul 16;103(2):248-57. Epub 2014 May 16.

Department of Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Amsterdam, The Netherlands ICIN Netherlands Heart Institute, Utrecht, The Netherlands.

Aims: Disease mechanisms regarding hypertrophic cardiomyopathy (HCM) are largely unknown and disease onset varies. Sarcomere mutations might induce energy depletion for which until now there is no direct evidence at sarcomere level in human HCM. This study investigated if mutations in genes encoding myosin-binding protein C (MYBPC3) and myosin heavy chain (MYH7) underlie changes in the energetic cost of contraction in the development of human HCM disease.

Methods And Results: Energetic cost of contraction was studied in vitro by measurements of force development and ATPase activity in cardiac muscle strips from 26 manifest HCM patients (11 MYBPC3mut, 9 MYH7mut, and 6 sarcomere mutation-negative, HCMsmn). In addition, in vivo, the ratio between external work (EW) and myocardial oxygen consumption (MVO2) to obtain myocardial external efficiency (MEE) was determined in 28 pre-hypertrophic mutation carriers (14 MYBPC3mut and 14 MYH7mut) and 14 healthy controls using [(11)C]-acetate positron emission tomography and cardiovascular magnetic resonance imaging. Tension cost (TC), i.e. ATPase activity during force development, was higher in MYBPC3mut and MYH7mut compared with HCMsmn at saturating [Ca(2+)]. TC was also significantly higher in MYH7mut at submaximal, more physiological [Ca(2+)]. EW was significantly lower in both mutation carrier groups, while MVO2 did not differ. MEE was significantly lower in both mutation carrier groups compared with controls, showing the lowest efficiency in MYH7 mutation carriers.

Conclusion: We provide direct evidence that sarcomere mutations perturb the energetic cost of cardiac contraction. Gene-specific severity of cardiac abnormalities may underlie differences in disease onset and suggests that early initiation of metabolic treatment may be beneficial, in particular, in MYH7 mutation carriers.
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http://dx.doi.org/10.1093/cvr/cvu127DOI Listing
July 2014

Phosphorylation of protein kinase C sites Ser42/44 decreases Ca(2+)-sensitivity and blunts enhanced length-dependent activation in response to protein kinase A in human cardiomyocytes.

Arch Biochem Biophys 2014 Jul 9;554:11-21. Epub 2014 May 9.

Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands; ICIN-Netherlands Heart Institute, Utrecht, The Netherlands. Electronic address:

Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal force. We investigated whether Ser42/44 pseudo-phosphorylation affects force development and ATPase activity using troponin exchange in human myocardium. Additionally, we studied if pseudo-phosphorylated Ser42/44 modulates length-dependent activation of force, which is regulated by protein kinase A (PKA)-mediated cTnI-Ser23/24 phosphorylation. Isometric force was measured in membrane-permeabilized cardiomyocytes exchanged with human recombinant wild-type troponin or troponin mutated at Ser42/44 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after PKA incubation. ATPase activity was measured in troponin-exchanged cardiac muscle strips. Compared to wild-type, 42D/44D decreased Ca(2+)-sensitivity without affecting maximal force in failing and donor cardiomyocytes. In donor myocardium, 42D/44D did not affect maximal ATPase activity or tension cost. Interestingly, 42D/44D blunted the length-dependent increase in Ca(2+)-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca(2+)-sensitivity at physiological Ca(2+)-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.
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http://dx.doi.org/10.1016/j.abb.2014.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121669PMC
July 2014
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