Karin R Sipido

University of Leuven, Louvain, Flemish, Belgium

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Publications (161)954.93 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: An acute increase in blood pressure is associated with the occurrence of premature ventricular complexes (PVCs). To study the timing of these PVCs in respect to afterload-induced changes in myocardial deformation in a controlled, pre-clinical relevant, novel closed-chest pig model. An acute left ventricular (LV) afterload challenge was applied by partial balloon inflation in the descending aorta, lasting 5 to 10 heartbeats (8 pigs;396 inflations). The balloon inflation enhanced the reflected wave (augmentation index 30±8% vs 59±6%; p<0.001), increasing systolic central blood pressure by 35±4%. This challenge resulted in a more abrupt LV pressure decline which was delayed beyond ventricular repolarization (rate of pressure decline 0.16±0.01 vs 0.27±0.04 mmHg/ms; p<0.001 and interval T-wave to peak-pressure 1±12ms vs. 36±9ms; p=0.008), during which the velocity of shortening at the basal septum abruptly increased (i.e. post-systolic shortening) (peak strain rate of -0.6±0.5s(-1) vs -2.5±0.8s(-1); p<0.001). It is exactly at this time of LV pressure decline, with increased post-systolic shortening, and not at peak pressure, that PVCs arose (22% of inflations). These PVCs preferentially occurred at the basal and apical segments. In the same regions, monophasic action potentials demonstrated the appearance of DAD-like transient depolarizations as origin for the PVCs. An acute blood pressure increase results in a more abrupt LV pressure decline, delayed after ventricular repolarization, which has a profound effect on myocardial mechanics, with enhanced post-systolic shortening. Coincidence with induced transient depolarizations and PVCs provides support for the mechano-electrical origin of pressure induced premature beats. Copyright © 2015. Published by Elsevier Inc.
    Heart rhythm: the official journal of the Heart Rhythm Society 06/2015; DOI:10.1016/j.hrthm.2015.06.037 · 4.92 Impact Factor
  • Karin R Sipido · Niall Macquaide · Virginie Bito
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    ABSTRACT: In cardiac myocytes, Ca(2+) release from the sarcoplasmic reticulum (SR) Ca(2+) store through the opening of ryanodine receptors (RyRs) is the major source of Ca(2+) for activation of myofilaments and contraction. Over the past 20 years, tools have become available to study this release process in detail, allowing new insights into the regulation of SR Ca(2+) release and RyR function. To assess these processes, we recommend and here review a systematic approach that evaluates the essential transport mechanisms and Ca(2+) fluxes in isolated single cardiac myocytes by using fluorescent Ca(2+) indicators and whole-cell recording of membrane voltage and ionic currents under voltage clamp. The approach includes an assessment of the L-type Ca(2+) current as a trigger for opening of RyRs and release of SR Ca(2+), of the SR Ca(2+) content, of intrinsic properties of RyRs, and of Ca(2+)-removal systems. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 05/2015; 2015(5):pdb.top066142. DOI:10.1101/pdb.top066142 · 4.63 Impact Factor
  • Virginie Bito · Karin R Sipido · Niall Macquaide
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    ABSTRACT: The decline of an intracellular calcium ([Ca(2+)]i) transient during a single excitation-contraction coupling (ECC) cycle reflects the combined activity of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) pump and the sarcolemmal Na(+)-Ca(2+) exchanger (NCX), along with minor contributions of the plasma membrane Ca(2+)-ATPase and mitochondrial Ca(2+) uniporter, in removing Ca(2+) from the cytosol. A traditional approach for assessing the individual components is to fit the decline of the [Ca(2+)]i transient evoked during electrical stimulation with an exponential. This reflects mostly the SERCA-dependent rate of uptake, which can be properly deduced after correcting for a component of NCX removal. As NCX function is an important determinant of the membrane potential as well as the Ca(2+) balance, we present here several detailed protocols for assessing NCX function. As the reversal potential and the amplitudes of the current are highly dependent on the prevailing concentrations of Na(+) and Ca(2+), we show how NCX function can be assessed under highly controlled conditions, with Ca(2+) and Na(+) clamped, as well as under more physiological conditions, with freely changing Ca(2+) and Na(+). © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 05/2015; 2015(5):pdb.prot076992. DOI:10.1101/pdb.prot076992 · 4.63 Impact Factor
  • Niall Macquaide · Virginie Bito · Karin R Sipido
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    ABSTRACT: This protocol describes the measurement of Ca(2+) sparks in intact myocytes by using a Ca(2+)-sensitive dye and imaging using laser scanning confocal microscopy. It takes advantage of spontaneous Ca(2+)-release events-sparks-using them as a measure of the activity of ryanodine receptors (RyRs). Two methodologies are described: One requires that cardiomyocytes be stimulated, preferably under voltage clamp by depolarizing pulses, until steady-state is reached, and then stimulation is stopped and Ca(2+) sparks are recorded. The second requires that cells be permeabilized and bathed in a solution to load the cell with Ca(2+) sufficient to elicit Ca(2+) sparks, but not Ca(2+) waves. These are then analyzed offline to quantify spark frequency and morphology. The advantages and disadvantages of each approach are discussed. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 05/2015; 2015(5):pdb.prot076984. DOI:10.1101/pdb.prot076984 · 4.63 Impact Factor
  • Virginie Bito · Karin R Sipido · Niall Macquaide
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    ABSTRACT: In cardiac myocytes, the physiological increase of intracellular calcium, the [Ca(2+)]i transient, elicited during excitation-contraction coupling typically reaches a peak amplitude of up to 1 µm, from a resting value of ∼100 nm, within 50-100 msec, depending on the species. Various conditions will affect the amplitude and rise time of the [Ca(2+)]i transient and, depending on the nature of the Ca(2+) signals under study, a variety of different probes are available for monitoring changes in intracellular Ca(2+). In this protocol, we focus on Fluo-3, which exists in the cytosol in its salt form K5Fluo-3. This form is practically nonfluorescent in the absence of Ca(2+), but the fluorescence increases dramatically on Ca(2+) binding. Although Fluo-3 is a single excitation-emission dye, it has a number of advantages for investigators, including an ideal dissociation constant (Kd) value and high quantum yield, meaning that it can be used at low concentrations that introduce minimal buffering. Here, we describe the basic setup and methodology for recording the global cytosolic [Ca(2+)]i transient with this probe during simultaneous patch-clamp and whole-cell recording of membrane voltage or of ionic currents under voltage clamp. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 04/2015; 2015(4):pdb.prot076950. DOI:10.1101/pdb.prot076950 · 4.63 Impact Factor
  • Niall Macquaide · Virginie Bito · Karin R Sipido
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    ABSTRACT: Here, we describe a protocol for the reliable measurement of the amount of Ca(2+) in the sarcoplasmic reticulum (SR) Ca(2+) store of cardiac myocytes. The whole-cell patch-clamp method is used to provide controlled loading of the SR during conditioning depolarizing pulses, followed by rapid application of a high dose of caffeine to release all SR Ca(2+) and to prevent Ca(2+) reuptake by the SR. Simultaneous measurement of membrane currents records Ca(2+) extruded through the Na(+)-Ca(2+) exchanger. The integral of the caffeine-induced Na(+)-Ca(2+) exchange current is then used as a measure of the SR Ca(2+). Derived measurements include the Ca(2+) buffering capacity and measurement of fractional release as an indicator of coupling gain. Caveats, advantages, and disadvantages of this method and alternative methods are discussed. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 04/2015; 2015(4):pdb.prot076976. DOI:10.1101/pdb.prot076976 · 4.63 Impact Factor
  • Virginie Bito · Niall Macquaide · Karin R Sipido
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    ABSTRACT: Here, we describe a method for characterizing the L-type Ca(2+) current, ICaL, which is a major trigger for Ca(2+) release from the sarcoplasmic reticulum (SR). The protocol includes measuring ICaL amplitude and voltage dependence and the elicited SR Ca(2+) release. The procedure for measuring ICaL activity is performed using solutions (internal and external) and voltage control such that other ionic currents are eliminated. The resultant relationship between the Ca(2+) current and the associated internal [Ca(2+)]i transient is a first approach for evaluating coupling gain. We discuss which parameters are most appropriate for this analysis and how an evaluation of gain needs to be further explored by measuring the SR Ca(2+) content. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 04/2015; 2015(4):pdb.prot076968. DOI:10.1101/pdb.prot076968 · 4.63 Impact Factor
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    ABSTRACT: This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation–contraction coupling and arrhythmias: Na+ channel and Na+ transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na+/Ca2+ exchange (NCX) and Na+/K+-ATPase (NKA). While the relevance of Ca2+ homeostasis in cardiac function has been extensively investigated, the role of Na+ regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na+ content have multiple effects on the heart by influencing intracellular Ca2+ and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na+ homeostasis. Among the proteins that accomplish this task are the Na+/Ca2+ exchanger (NCX) and the Na+/K+ pump (NKA). By transporting three Na+ ions into the cytoplasm in exchange for one Ca2+ moved out, NCX is one of the main Na+ influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na+ ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na+ and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na+ homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na+/Ca2+ exchanger (NCX1) and Na+/K+ pump and the controversies that still persist in the field.
    The Journal of Physiology 03/2015; 593(6). DOI:10.1113/jphysiol.2014.282319 · 4.54 Impact Factor
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    ABSTRACT: Postconditioning and cyclosporine A prevent mitochondrial permeability transition pore opening providing cardioprotection during ischemia/reperfusion. Whether microvascular obstruction is affected by these interventions is largely unknown. Pigs subjected to coronary occlusion for 1 h followed by 3 h of reperfusion were assigned to control (n = 8), postconditioning (n = 9) or cyclosporine A intravenous infusion 10–15 min before the end of ischemia (n = 8). Postconditioning was induced by 8 cycles of repeated 30-s balloon inflation and deflation. After 3 h of reperfusion magnetic resonance imaging, triphenyltetrazolium chloride/Evans blue staining and histopathology were performed. Microvascular obstruction (MVO, percentage of gadolinium-hyperenhanced area) was measured early (3 min) and late (12 min) after contrast injection. Infarct size with double staining was smaller in cyclosporine (46.2 ± 3.1 %, P = 0.016) and postconditioning pigs (47.6 ± 3.9 %, P = 0.008) versus controls (53.8 ± 4.1 %). Late MVO was significantly reduced by cyclosporine (13.9 ± 9.6 %, P = 0.047) but not postconditioning (23.6 ± 11.7 %, P = 0.66) when compared with controls (32.0 ± 16.9 %). Myocardial blood flow in the late MVO was improved with cyclosporine versus controls (0.30 ± 0.06 vs 0.21 ± 0.03 ml/g/min, P = 0.002) and was inversely correlated with late-MVO extent (R 2 = 0.93, P
    Archiv für Kreislaufforschung 03/2015; 110(2):475. DOI:10.1007/s00395-015-0475-8 · 5.96 Impact Factor
  • Demetrio J. Santiago · Eef Dries · Ilse Lenaerts · Karin R. Sipido
    Biophysical Journal 01/2015; 108(2):567a. DOI:10.1016/j.bpj.2014.11.3105 · 3.97 Impact Factor
  • Cardiovascular Research 12/2014; 105(1):1-2. DOI:10.1093/cvr/cvu253 · 5.81 Impact Factor
  • Diane Gal · Wouter Vandevelde · Heping Cheng · Karin R Sipido
    Cardiovascular Research 11/2014; 104(3). DOI:10.1093/cvr/cvu238 · 5.81 Impact Factor
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    ABSTRACT: Rationale: In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs. Objective: Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation. Methods and Results: We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca2+ transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca2+ indicator). Ca2+ sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca2+/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca2+/calmodulin-dependent kinase II blockers autocamtide-2–related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca2+/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca2+ transients, frequency-dependent increase of the rate of Ca2+ rise was seen in coupled RyR regions only and abolished by autocamtide-2–related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost. Conclusions: Coupled RyRs have a distinct modulation by Ca2+/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca2+/reactive oxygen species microdomain, and subject to modification with disease.
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    Biophysical Journal 01/2014; 106(2):431a. DOI:10.1016/j.bpj.2013.11.2428 · 3.97 Impact Factor
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    Cardiovascular Research 01/2014; 101(1):1-3. DOI:10.1093/cvr/cvt272 · 5.81 Impact Factor
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    ABSTRACT: Differentiation of cardiac fibroblasts (Fb) into myofibroblasts (MyoFb) is responsible for connective tissue buildup in myocardial remodeling. We examined MyoFb differentiation and reversibility.Methods and ResultsAdult rat cardiac Fb were cultured on a plastic substratum providing mechanical stress, with conditions to obtain different levels of Fb differentiation. Fb spontaneously differentiated to proliferating MyoFb (p-MyoFb) with stress fiber formation decorated with alpha-smooth muscle actin (α-SMA). Transforming growth factor-β1 (TGF-β1) promoted differentiation into α-SMA positive MyoFb showing near absence of proliferation i.e. non-p-MyoFb. SD-208, a TGF-β-receptor-I kinase blocker, inhibited p-MyoFb differentiation as shown by stress fiber absence, low α-SMA expression, and high proliferation levels. Fb seeded in collagen matrices induced no contraction, whereas p-MyoFb and non-p-MyoFb induced 2.5- and 4-fold contraction. Fb produced little collagen but high levels of IL-10. Non-p-MyoFb had high collagen production and high MCP-1 and TIMP-1 levels. Transcriptome analysis indicated differential activation of gene networks related to differentiation of MyoFb (e.g. paxilin, PAK) and reduced proliferation of non-p-MyoFb (e.g. cyclins and cell cycle regulation). Dedifferentiation of p-MyoFb with stress fiber de-polymerization, but not of non-p-MyoFb, was induced by SD-208 despite maintained stress. Stress fiber de-polymerization could also be induced by mechanical strain release in p-MyoFb and non-p-MyoFb (2 day culture in unrestrained 3-D collagen matrices). Only p-MyoFb showed true dedifferentiation after long-term 3-D culture. Fb, p-MyoFb and non-p-MyoFb have a distinct gene expression, ultrastructural and functional profile. Both reduction in mechanical strain and TGF-β-receptor-I kinase inhibition can reverse p-MyoFb differentiation but not of non-p-MyoFb.
    Cardiovascular Research 12/2013; 101(3). DOI:10.1093/cvr/cvt338 · 5.81 Impact Factor
  • Europace 10/2013; 15(11):1684-1684. DOI:10.1093/europace/eut342 · 3.05 Impact Factor
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    ABSTRACT: Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hypertrophy response to pressure overload. Here we evaluated the consequences of melusin overexpression in the setting of myocardial infarction (MI) using a comprehensive multicenter approach.Methods and ResultsMice overexpressing melusin in the heart (TG) and wild type controls (WT) were subjected to permanent LAD-ligation and both the acute response (day 3) and subsequent remodelling (2 weeks) were examined. Mortality in wild type mice was significant between day 3 and 7, primarily due to cardiac rupture, but melusin overexpression strongly reduced mortality (43.2% in wild type vs. 27.3% in melusin-TG, p=0.005). At day 3 after MI, a time point preceding the mortality peak, TG hearts had increased HSP70 expression, increased ERK1/2 signalling, reduced cardiomyocyte hyper-contractility and reduced inflammatory cell infiltrates and increased matricellular protein expression in the infarcted area.At 2 weeks after MI melusin overexpression conferred a favorable adaptive remodelling characterized by reduced left ventricle dilatation and better preserved contractility in presence of a comparable degree of hypertrophy. Adaptive remodelling in melusin TG mice was characterized by reduced apoptosis and fibrosis as well as increased cardiomyocyte contractility. Consistent with its function as chaperone protein, Melusin overexpression exerts a dual protective action following MI reducing an array of maladaptive processes. In the early phase after MI, reduced inflammation and myocyte remodelling protect against cardiac rupture. Chronically, reduced myocyte loss and matrix remodelling, with preserved myocyte contractility, confer adaptive LV remodelling.
    Cardiovascular Research 10/2013; 101(1). DOI:10.1093/cvr/cvt235 · 5.81 Impact Factor

Publication Stats

4k Citations
954.93 Total Impact Points


  • 1997–2015
    • University of Leuven
      • • Department of Cardiovascular Sciences
      • • Division of Experimental Cardiology
      Louvain, Flemish, Belgium
  • 2003–2013
    • Catholic University of Louvain
      Лувен-ла-Нев, Walloon, Belgium
  • 2011
    • Sahlgrenska University Hospital
      Goeteborg, Västra Götaland, Sweden
  • 2007
    • Universiteit Utrecht
      • Division of Heart and Lungs
      Utrecht, Provincie Utrecht, Netherlands
  • 2005
    • The University of Manchester
      Manchester, England, United Kingdom
  • 1992
    • Johns Hopkins University
      • Department of Medicine
      Baltimore, Maryland, United States