Karin R Sipido

University of Leuven, Louvain, Flemish, Belgium

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Publications (157)946.3 Total impact

  • 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
  • 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, 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
  • 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
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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
  • Biophysical Journal 01/2015; 108(2):567a. DOI:10.1016/j.bpj.2014.11.3105 · 3.83 Impact Factor
  • Cardiovascular Research 12/2014; 105(1):1-2. DOI:10.1093/cvr/cvu253 · 5.81 Impact Factor
  • Cardiovascular Research 11/2014; DOI:10.1093/cvr/cvu238 · 5.81 Impact Factor
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  • Biophysical Journal 01/2014; 106(2):431a. DOI:10.1016/j.bpj.2013.11.2428 · 3.83 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
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    ABSTRACT: Rationale: In ventricular myocytes of large mammals with low T-tubule (TT) density, a significant number of ryanodine receptors (RyR) are not coupled to the sarcolemma; cardiac remodeling increases non-coupled RyR. Objective: To test the hypothesis that coupled and non-coupled RyRs have distinct microdomain-dependent modulation. Methods and Results: We studied single myocytes from pig left ventricle. The TT network was analyzed in 3-D to measure distance to membrane of release sites. The rising phase of the Ca(2+) transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca(2+) indicator). Ca(2+) sparks following stimulation were thus identified as resulting from coupled or non-coupled RyRs. We used high frequency stimulation as a known activator of CaMKII. Spark frequency increased significantly more in coupled than in non-coupled RyRs. This specific modulation of coupled RyRs was abolished by the CaMKII-blockers AIP and KN-93, but not by KN-92. Colocalization of CaMKII and RyR was not detectably different for coupled and non-coupled sites but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NOX2 inhibition by DPI or apocynin, or global ROS scavenging, also prevented coupled RyR modulation. During stimulated Ca(2+) transients, frequency-dependent increase of the rate of Ca(2+) rise was seen in coupled RyR regions only and abolished by AIP. After myocardial infarction, selective modulation of coupled RyR was lost. Conclusions: Coupled RyRs have a distinct modulation by CaMKII and ROS, dependent on an intact cytoskeleton and consistent with a local Ca(2+)/ROS microdomain, and subject to modification with disease.
    Circulation Research 09/2013; 113(11). DOI:10.1161/CIRCRESAHA.113.301896 · 11.09 Impact Factor
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    ABSTRACT: Diabetic cardiomyopathy is characterized by systolic and early diastolic ventricular dysfunction. In the metabolic syndrome (MS), ventricular stiffness is additionally increased in a later stage. It is unknown whether this is related to intrinsic cardiomyocyte dysfunction, extrinsic factors influencing cardiomyocyte contractility and/or cardiac function, or a combination of both. A first aim was to study cardiomyocyte contractility and Ca2+ handling in vitro in a mouse model of MS. A second aim was to investigate whether in vivo hypocaloric diet or ACE-inhibition (ACE-I) improved cardiomyocyte contractility in vitro, contractile reserve and Ca2+ handling. This study was performed in LDL-receptor (LDLR-/-) and leptin-deficient (ob/ob), double knock-out mice (DKO), featuring obesity, type II diabetes, atherogenic dyslipidemia and hypertension. Single knock-out LDLR-/-, ob/ob and wild type mice were used as controls. Cellular contractility, Ca2+ handling and their response to in vivo treatment with diet or ACE-I were studied in isolated cardiomyocytes at baseline, during beta-adrenergic stimulation or increased extracellular Ca2+, using field stimulation and patch-clamp. In untreated conditions, prolongation of contraction-relaxation cycle and altered Ca2+ handling are observed in MS. Response to increased extracellular Ca2+ and beta-adrenergic stimulation is impaired and could not be rescued by weight loss. ACE-I restored impaired response to beta-adrenergic stimulation in MS, but not the decreased response to increased extracellular Ca2+. Cardiomyocyte contractility and beta-adrenergic response are impaired in MS, due to alterations in cellular Ca2+ handling. ACE-I, but not weight loss, is able to restore cardiomyocyte response to beta-adrenergic stimulation in MS.
    BMC Cardiovascular Disorders 07/2013; 13(1):51. DOI:10.1186/1471-2261-13-51 · 1.50 Impact Factor
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    ABSTRACT: Rationale: Synchronized release of Ca(2+) into the cytosol during each cardiac cycle determines cardiomyocyte contraction. Objective: We investigated synchrony of cytosolic [Ca(2+)] decay during diastole and the impact of cardiac remodeling. Methods and Results: Local cytosolic [Ca(2+)] transients (1 µm intervals) were recorded in murine, porcine and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca(2+)] decay based on the local time constants of decay (TAUlocal). The standard deviation of TAUlocal (SDTAU) as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca(2+) release. Stimulation of SERCA with forskolin or istaroxime accelerated, its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SDTAU and global [Ca(2+)] decay (TAUglobal). NCX-inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca(2+) uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy following trans-aortic constriction (TAC), in pigs with chronic myocardial ischemia and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. Conclusions: In cardiomyocytes, cytosolic [Ca(2+)] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca(2+)] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.
    Circulation Research 07/2013; 113(5). DOI:10.1161/CIRCRESAHA.113.300895 · 11.09 Impact Factor

Publication Stats

4k Citations
946.30 Total Impact Points


  • 1995–2015
    • 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