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Adrenergic Stimulation in Cardiac Myocytes

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Adrenergic Stimulation in Cardiac Myocytes Cardiovascular Research (2014) 104, 347–354 doi:10.1093/cvr/cvu201 Balanced changes in Ca buffering by SERCA and troponin contribute to Ca handling during b-adrenergic stimulation in cardiac myocytes Sarah J. Briston1, Katharine M. Dibb1, R. John Solaro2, David A. Eisner1, and Andrew W. Trafford1* 1Unit of Cardiac Physiology, Manchester Academic Health Science Centre, Core Technology Facility, 46 Grafton St, Manchester M13 9NT, UK; and 2Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, USA Received 24 January 2014; revised 24 July 2014; accepted 25 August 2014; online publish-ahead-of-print 2 September 2014 Time for primary review: 33 days Aims During activation of cardiac myocytes, less than 1% of cytosolic Ca is free; the rest is bound to buffers, largely SERCA, and troponin C. Signalling by phosphorylation, as occurs during b-adrenergic stimulation, changes the Ca-binding affinity of these proteins and may affect the systolic Ca transient. Our aim was to determine the effects of b-adrenergic stimulation on Ca buffering and to differentiate between the roles of SERCA and troponin. ..................................................................................................................................................................................... Methods Ca buffering was studied in cardiac myocytes from mice: wild-type (WT), phospholamban-knockout (PLN-KO), and mice and results expressing slow skeletal troponin I (ssTnI) that is not protein kinase A phosphorylatable. WT cells showed no change in Ca buffering in response to the b-adrenoceptor agonist isoproterenol (ISO). However, ISO decreased Ca buffering in PLN-KO myocytes, presumably unmasking the role of troponin. This effect was confirmed in WT cells in which SERCA activity was blocked with the application of thapsigargin. In contrast, ISO increased Ca buffering in ssTnI cells, presumably revealing the effect of an increase in Ca binding to SERCA. ..................................................................................................................................................................................... Conclusions These data indicate the individual roles played by SERCAand troponininCa buffering during b-adrenergic stimulationand that these two buffers effectively counterbalance each other so that Ca buffering remains constant during b-adrenergic stimulation, a factor which may be physiologically important. This study also emphasizes the importance of taking into account Ca buffering, particularly in disease states where Ca binding to myofilaments or SERCA may be altered. ----------------------------------------------------------------------------------------------------------------------------------------------------------- Keywords Calcium † Buffering † SERCA † Troponin † Phospholamban 1. Introduction therefore possible that, under some circumstances, changes in the prop- erties of the systolic Ca transient result from changes in buffering prop- Cardiac contraction is initiated by the systolic Ca transient. The entry of erties rather than of the underlying Ca fluxes. Experimentally induced Ca into the cell via the L-type Ca current triggers the release of more Ca changes in Ca buffering do, indeed, have large effects on the systolic from the sarcoplasmic reticulum (SR) through a specialized release Ca transient.5 Recent work has found that Ca binding to cardiac myofi- channel known as the ryanodine receptor. This process, known as brils is altered in hypertrophic cardiomyopathy.6– 8 Changes in intracel- Ca-induced Ca release, is responsible for delivery of Ca to troponin C lular pH may also affect Ca binding to buffers.9 The major fast (TnC) and triggering of contraction. Much work has been devoted to in- cytoplasmic buffers are thought to be TnC and the SR Ca-ATPase vestigating how changes in the properties of the various Ca transporting (SERCA, predominantly SERCA2a in the myocardium).3 Ca binding to proteins result in changes in the size and kinetics of the Ca transient and these proteins is regulated by b-adrenergic stimulation with phosphor- thence of contraction (for reviews see Bers1 and Eisner et al.2). ylation of TnI decreasing Ca binding to TnC10 and phosphorylation of What is often overlooked is that less than 1% of the Ca that enters the phospholamban increasing Ca binding to SERCA.11 It is therefore cytoplasm is free with the vast majority being bound to Ca buffers.3,4 It is likely that the Ca buffering ability of TnC and SERCA will change * Corresponding author. Tel: +44 161 275 7969; fax: +44 161 275 2703, Email: [email protected], [email protected] & The Author 2014. Published by Oxford University Press on behalf of the European Society of Cardiology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 348 S.J. Briston et al. during b-adrenergic stimulation. To the best of our knowledge, For the PLN-KO studies, the Ca indicator Fluo-5F, pentapotassium salt however, no study has attempted to assess whether changes in Ca buf- (Invitrogen, UK; 100 mM) was loaded via the patch pipette and a 475 nm fering contribute to alterations of Ca cycling under these conditions. light-emitting diode (Cairn Research Instruments, UK) used to excite the 2+ One complication in assessing the contribution of changes in buffering fluorophore. Fluorescence was converted to [Ca ]i using a published method.4 Briefly, at the end of the experiment, the cell was damaged with to Ca handling during b-adrenergic stimulation is that there is an accom- + the patch pipette. This resulted in an abrupt increase of [Ca2 ] to levels panying increase in the L-type Ca current and SERCA activity, which will i that saturate the indicator, and this was taken as the maximum fluorescence also affect the systolic Ca transient. In this study, therefore, we have 2+ (Fmax). Assuming that fluorescence is zero in the absence of Ca then [Ca ]i investigated the effects of b-adrenergic stimulation on Ca buffering, can be calculated from the level of fluorescence (F) as follows: and used transgenic and knockout mice to distinguish the role played [ 2+] = · ( − ) by buffering due to SERCA and troponin. The results show that, in wild- Ca i Kd F/ Fmax F type (WT) mouse ventricular myocytes, b-adrenergic stimulation has 15,16 The value of Kd was taken to be 1035 nM. no effect on buffering and this reflects a combination of increased buffer- For the ssTnI and thapsigargin experiments, the Ca indicator Fura-2 pen- ing by SERCA and decreased buffering by troponin. This constancy of Ca tapotassium salt (Invitrogen, UK, 100 mM) was used but we were unable to buffering power in the face of b-adrenergic stimulation is likely to be calibrate for Ca and, instead (after subtracting background fluorescence) physiologically important. measured the ratio of the emitted fluorescence excited at 360 nm (F360) and 380 nm (F380): R ¼ F365: F380. The ratio values were normalized to the diastolic value and are expressed as R/Rrest, where Rrest is the ratio at diastolic 2. Methods Ca. In cells where an Fmax measurement was attained, this was found to be 1.98 + 0.17-fold higher than the caffeine response in isoproterenol (ISO; All procedures accord to the UK Animals (Scientific Procedures) Act, 1986 n ¼ 6 cells), showing that the indicator was not saturated. and the University of Manchester Ethical Review Process. 2.5 Calculation of SR content and cytoplasmic 2.1 Mice Ca buffering Phospholamban-knockout mice12 (PLN-KO) were obtained from the SR Ca content was measured by applying 5 mM caffeine together with Mutant Mouse Regional Resource Centers (US, strain Pln Tm1 Egk). Since 20 mM 2,3-butanedione monoxime (BDM) to release Ca from the SR. the PLN-KO mice are homozygous, C57Bl/6J (Charles River, UK) were BDM was used to prevent excessive cell contraction and also release Ca used as controls. The ssTnI mice are CD-1 strain background in which from the SR.17 We also confirmed that BDM application had no effect on cardiac troponin I (TnI) has been replaced by the non-protein kinase A Ca buffering (see Supplementary material online, Figure S1). For simplicity, (PKA) phosphorylatable slow skeletal troponin (ssTnI).13 The colony is het- in the figures, this caffeine plus BDM solution is referred to as ‘caff’. Measure- erozygous and therefore their respective wild-type littermates were used as ments were made of both the amplitude of the resulting cytoplasmic Catran- controls. C57Bl/6J mice were used for the thapsigargin study. sient and the integral of the accompanying Na-Ca exchange (NCX) current. The integral of this current gives a measure of the amount of Ca released 2.2 Isolation of cardiac myocytes from the SR that is pumped out of the cell by NCX.18 In order to estimate Mice were killed by cervical dislocation and hearts excised and placed in the total amount of Ca pumped out of the cell, this integral must be cor- ice-cold isolation solution. The aorta was cannulated and retrogradely per- rected for Ca removed by the electroneutral plasma membrane Ca-ATPase fused with isolation solution for 10 min at 378C. Collagenase (0.25–0.5 mg/ (PMCA). As in previous work19 we have estimated the fractional contribu- mL, type I, Sigma, UK) and protease (0.05 mg/mL, type XIV, Sigma, UK) were tion of PMCA as follows. The rate constant of decay of the caffeine response then added, and the heart digested for 6–7 min. The heart wasthen perfused (kcaff) gives a measure of the total activity of NCX and PMCA. We then with a taurine-containing solution for a further 20 min, and the ventricles applied Ni (10 mM) to inhibit NCX and then reapplied caffeine. The resulting were finely minced and filtered through 200 mm gauze to obtain intact rate constant (kNi) represents PMCA activity. The amount of Ca removed by cardiac myocytes. Cells were stored in an experimental solution and kept NCX must be multiplied by kcaff/(kcaff 2 kNi) to calculate the total amount of at room temperature.
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