Ger J M Stienen

VU University Medical Center, Amsterdamo, North Holland, Netherlands

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Publications (198)1002.74 Total impact

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    ABSTRACT: 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. Copyright ©ERS 2015.
    European Respiratory Journal 06/2015; 46(3). DOI:10.1183/13993003.02052-2014 · 7.64 Impact Factor
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    ABSTRACT: 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 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 cardiac energetics would be a potential ventricular therapeutic target. Copyright © 2015. Published by Elsevier Ltd.
    Journal of Molecular and Cellular Cardiology 06/2015; 86. DOI:10.1016/j.yjmcc.2015.06.016 · 4.66 Impact Factor
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    ABSTRACT: 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 [Ca2+] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca2+-handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca2+] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca2+ in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca2+ 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 [Ca2+] 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 Ca2+-overload. In isolated Langendorff-perfused rat hearts, CK-inhibition increased ventricular stiffness only in the presence of diastolic [Ca2+]. 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 Ca2+ and thereby increase myocardial stiffness. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    The Journal of Physiology 06/2015; 593(17). DOI:10.1113/JP270354 · 5.04 Impact Factor
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    ABSTRACT: 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. Copyright © 2015. Published by Elsevier Ltd.
    Journal of Molecular and Cellular Cardiology 03/2015; 82. DOI:10.1016/j.yjmcc.2015.03.006 · 4.66 Impact Factor
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    ABSTRACT: 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 the diaphragm weakness and its underlying pathophysiological mechanisms are poorly understood. We hypothesized (1) that diaphragm muscle fibers of mechanically ventilated critically ill patients display atrophy and contractile weakness, and (2) that the ubiquitin-proteasome pathway is activated in the diaphragm. We obtained diaphragm muscle biopsies from twenty-two critically ill patients who received mechanical ventilation prior to surgery, and compared these with biopsies obtained from patients during thoracic surgery for resection of a suspected early lung malignancy (controls). In a proof-of-concept study in a MuRF-1 knockout mouse model, we evaluated the role of the ubiquitin-proteasome pathway in the development of contractile weakness during mechanical ventilation. Both slow-twitch and fast-twitch diaphragm muscle fibers of critically ill patients had a ~25% smaller cross sectional area, and a >50% lower contractile force. Markers of the ubiquitin-proteasome pathway were significantly upregulated in the diaphragm of critically ill patients. Finally, MuRF-1 knockout mice were protected against the development of diaphragm contractile weakness during mechanical ventilation. These findings show that (1) diaphragm muscle fibers of critically ill patients display atrophy and severe contractile weakness and (2) 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.
    American Journal of Respiratory and Critical Care Medicine 03/2015; 191(10). DOI:10.1164/rccm.201412-2214OC · 13.00 Impact Factor
  • European Respiratory Journal 03/2015; 45(6). DOI:10.1183/09031936.00205114 · 7.64 Impact Factor
  • Biophysical Journal 01/2015; 108(2):199a-200a. DOI:10.1016/j.bpj.2014.11.1104 · 3.97 Impact Factor
  • Biophysical Journal 01/2015; 108(2):294a. DOI:10.1016/j.bpj.2014.11.1600 · 3.97 Impact Factor
  • Biophysical Journal; 01/2015
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    ABSTRACT: AimsThis study aimed to elucidate the molecular background of increased Ca2+ sensitivity of force production in cardiomyocytes of end-stage human heart failure.Methods and resultsCa2+-activated isometric force and the cross-bridge specific rate of force redevelopment (ktr) were determined in Triton-skinned myocytes from end-stage failing and non-failing donor hearts. Measurements (control: pH 7.2, 0 mM inorganic phosphate (Pi)) were performed under test conditions that probed either the Ca2+-regulatory function of the thin filaments (pH 6.5), the kinetics of the actin-myosin cross-bridge cycle (10 mM Pi), or both (pH 6.5, 10 mM Pi). The control maximal Ca2+-activated force (Fo) and ktrmax did not differ between failing and non-failing myocytes. At submaximal [Ca2+], however, both force and ktr were higher in failing than in donor myocytes. The difference in the Ca2+ 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 Pi) both at pH 7.2 and at pH 6.5. Pi induced a larger reduction in force but a smaller increase in ktr in the failing myocytes than in the non-failing myocytes at submaximal [Ca2+].Conclusion The enhanced Pi sensitivity of the actin-myosin interaction suggests that the Pi release step of the actin-myosin cross-bridge cycle is modified during end-stage human heart failure. This might be of functional importance when Pi 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.
    ESC Heart Failure 12/2014; 1(2). DOI:10.1002/ehf2.12020
  • G J M Stienen
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    ABSTRACT: Heart failure is a multi-factorial progressive disease in which eventually the contractile performance of the heart is insufficient to meet the demands of the body, even at rest. A distinction can be made on the basis of the cause of the disease in genetic and acquired heart failure and at the functional level between systolic and diastolic heart failure. Here the basic determinants of contractile function of myocardial cells will be reviewed and an attempt will be made to elucidate their role in the development of heart failure. The following topics are addressed: the tension generating capacity, passive tension, the rate of tension development, the rate of ATP utilisation, calcium sensitivity of tension development, phosphorylation of contractile proteins, length dependent activation and stretch activation. The reduction in contractile performance during systole can be attributed predominantly to a loss of cardiomyocytes (necrosis), myocyte disarray and a decrease in myofibrillar density all resulting in a reduction in the tension generating capacity and likely also to a mismatch between energy supply and demand of the myocardium. This leads to a decline in the ejection fraction of the heart. Diastolic dysfunction can be attributed to fibrosis and an increase in titin stiffness which result in an increase in stiffness of the ventricular wall and hampers the filling of the heart with blood during diastole. A large number of post translation modifications of regulatory sarcomeric proteins influence myocardial function by altering calcium sensitivity of tension development. It is still unclear whether in concert these influences are adaptive or maladaptive during the disease process.
    Journal of Muscle Research and Cell Motility 11/2014; 36(1). DOI:10.1007/s10974-014-9395-8 · 2.09 Impact Factor
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    P J M Wijnker · A M Murphy · G J M Stienen · J van der Velden
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    ABSTRACT: Cardiac troponin I (cTnI) is well known as a biomarker for the diagnosis of myocardial damage. However, because of its central role in the regulation of contraction and relaxation in heart muscle, cTnI may also be a potential target for the treatment of heart failure. Studies in rodent models of cardiac disease and human heart samples showed altered phosphorylation at various sites on cTnI (i.e. site-specific phosphorylation). This is caused by altered expression and/or activity of kinases and phosphatases during heart failure development. It is not known whether these (transient) alterations in cTnI phosphorylation are beneficial or detrimental. Knowledge of the effects of site-specific cTnI phosphorylation on cardiomyocyte contractility is therefore of utmost importance for the development of new therapeutic strategies in patients with heart failure. In this review we focus on the role of cTnI phosphorylation in the healthy heart upon activation of the beta-adrenergic receptor pathway (as occurs during increased stress and exercise) and as a modulator of the Frank-Starling mechanism. Moreover, we provide an overview of recent studies which aimed to reveal the functional consequences of changes in cTnI phosphorylation in cardiac disease.
    Netherlands heart journal: monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation 09/2014; 22(10). DOI:10.1007/s12471-014-0590-4 · 1.84 Impact Factor
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    ABSTRACT: 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 9 brain-dead organ donors (cases) who had been on MV for 26 ± 5 hours with diaphragm muscle fibers from 9 patients (controls) undergoing surgery for lung cancer who had been on MV less than 2 hours. 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 to controls in slow-twitch or fast-twitch muscle fibers. These observations indicate that 26 hours of MV of humans is not invariably associated with changes in the contractile performance of sarcomeres in the diaphragm.
    AJP Lung Cellular and Molecular Physiology 07/2014; 307(6). DOI:10.1152/ajplung.00076.2014 · 4.08 Impact Factor
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    ABSTRACT: 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 (w30%), indicating LV cardiomyocyte atrophy. The maximal force-generating capacity, normalized to cardiomyocyte CSA, was significantly reduced (w25%). 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 w30% 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. (C) 2014 by the American College of Cardiology Foundation.
    Journal of the American College of Cardiology 07/2014; 64(1):28-37. DOI:10.1016/j.jacc.2014.04.031 · 16.50 Impact Factor
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    ABSTRACT: 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 Ca2+-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 Ca2+-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca2+-sensitivity at physiological Ca2+-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.
    Archives of Biochemistry and Biophysics 07/2014; 554. DOI:10.1016/ · 3.02 Impact Factor
  • Rob C.I. Wüst · H.J. de Vries · H.W.M. Niessen · G.J.M. Stienen
    Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2014; 1837:e50. DOI:10.1016/j.bbabio.2014.05.074 · 5.35 Impact Factor
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    ABSTRACT: 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 3 HCM patients with the R403Q mutation and 9 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) compared to 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 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. This article is protected by copyright. All rights reserved.
    The Journal of Physiology 06/2014; 592(15). DOI:10.1113/jphysiol.2014.274571 · 5.04 Impact Factor
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    ABSTRACT: 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.
    Cardiovascular Research 05/2014; 103(2). DOI:10.1093/cvr/cvu127 · 5.94 Impact Factor
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    ABSTRACT: Background Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca2+ sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH. Methods and Results RV samples from PAH patients undergoing heart‐lung transplantation were compared to non‐failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western‐blot analyses using antibodies to protein‐kinase‐A (PKA), Cα (PKCα) and Ca2+/calmoduling‐dependent‐kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P<0.0001). To test the functional relevance of PKA‐, PKCα‐, and CamKIIδ‐mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca2+ sensitivity is mediated by sarcomeric troponin I (cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P<0.05) and reduced PKA‐mediated cTnI phosphorylation (Ser22/23) (P<0.001). Finally, alterations in Ca2+‐handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca2+ clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P<0.05). Conclusions Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca2+ handling proteins contribute to RV diastolic dysfunction in PAH.
    Journal of the American Heart Association 04/2014; 3(3). DOI:10.1161/JAHA.113.000716 · 4.31 Impact Factor
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    Journal of the American College of Cardiology 04/2014; 63(12):A819. DOI:10.1016/S0735-1097(14)60819-6 · 16.50 Impact Factor

Publication Stats

5k Citations
1,002.74 Total Impact Points


  • 2003–2015
    • VU University Medical Center
      • • Department of Physiology
      • • Institute for Cardiovascular Research (ICaR-VU)
      Amsterdamo, North Holland, Netherlands
    • Erasmus Universiteit Rotterdam
      Rotterdam, South Holland, Netherlands
    • Ruhr-Universität Bochum
      • Institut für Physiologische Chemie
      Bochum, North Rhine-Westphalia, Germany
  • 1989–2015
    • VU University Amsterdam
      • • Department of Physics and Astronomy
      • • Institute for Cardiovascular Research VU
      • • Laboratory for Physiology
      Amsterdamo, North Holland, Netherlands
  • 2011
    • University of Bedfordshire
      Luton, England, United Kingdom
  • 1981–2007
    • University of Amsterdam
      • • Department of Plant Physiology
      • • Laboratory for Physiology
      Amsterdamo, North Holland, Netherlands
  • 2004
    • University of Leuven
      • Division of Experimental Cardiology
      Louvain, Flanders, Belgium
  • 1993
    • La Trobe University
      • Department of Zoology
      Melbourne, Victoria, Australia
    • Karolinska Institutet
      Solna, Stockholm, Sweden
  • 1987
    • University of the Free State
      Bloemfontein, Free State, South Africa