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Mechanical Load Regulates Excitation-Ca 2+ Signaling-Contraction in Cardiomyocyte

2021; Lippincott Williams & Wilkins; Volume: 128; Issue: 6 Linguagem: Inglês

10.1161/circresaha.120.318570

1524-4571

Rafael Shimkunas, Bence Hegyi, Zhong Jian, John A. Shaw, Mohammad A. Kazemi-Lari, Debika Mitra, J. Kent Leach, Xiaocen Li, Mark Jaradeh, Nicholas Balardi, Yi-Je Chen, Ariel L. Escobar, Anthony J. Baker, Julie Bossuyt, Tamás Bányász, Nipavan Chiamvimonvat, Kit S. Lam, Donald M. Bers, Leighton T. Izu, Ye Chen‐Izu,

Ion channel regulation and function

HomeCirculation ResearchVol. 128, No. 6Mechanical Load Regulates Excitation-Ca2+ Signaling-Contraction in Cardiomyocyte Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBMechanical Load Regulates Excitation-Ca2+ Signaling-Contraction in Cardiomyocyte Rafael Shimkunas, Bence Hegyi, Zhong Jian, John A. Shaw, Mohammad A. Kazemi-Lari, Debika Mitra, J. Kent Leach, Xiaocen Li, Mark Jaradeh, Nicholas Balardi, Yi-Je Chen, Ariel L. Escobar, Anthony J. Baker, Julie Bossuyt, Tamas Banyasz, Nipavan Chiamvimonvat, Kit S. Lam, Donald M. Bers, Leighton T. Izu and Ye Chen-Izu Rafael ShimkunasRafael Shimkunas Department of Pharmacology (R.S., B.H., Z.J., M.J., N.B., Y.-J.C., J.B., T.B., D.M.B., L.T.I., Y.C.-I.), University of California, Davis. Department of Biomedical Engineering (R.S., D.M., J.K.L., Y.C.-I.), University of California, Davis. , Bence HegyiBence Hegyi https://orcid.org/0000-0003-3113-221X Department of Pharmacology (R.S., B.H., Z.J., M.J., N.B., Y.-J.C., J.B., T.B., D.M.B., L.T.I., Y.C.-I.), University of California, Davis. , Zhong JianZhong Jian https://orcid.org/0000-0003-1072-5788 Department of Pharmacology (R.S., B.H., Z.J., M.J., N.B., Y.-J.C., J.B., T.B., D.M.B., L.T.I., Y.C.-I.), University of California, Davis. , John A. ShawJohn A. Shaw Department of Aerospace Engineering, University of Michigan, Ann Arbor (J.A.W., M.A.K.-L.). , Mohammad A. Kazemi-LariMohammad A. Kazemi-Lari https://orcid.org/0000-0003-3964-5933 Department of Aerospace Engineering, University of Michigan, Ann Arbor (J.A.W., M.A.K.-L.). , Debika MitraDebika Mitra Department of Biomedical Engineering (R.S., D.M., J.K.L., Y.C.-I.), University of California, Davis. , J. Kent LeachJ. Kent Leach Department of Biomedical Engineering (R.S., D.M., J.K.L., Y.C.-I.), University of California, Davis. , Xiaocen LiXiaocen Li Department of Biochemistry and Molecular Medicine (X.L., K.S.L.), University of California, Davis. , Mark JaradehMark Jaradeh https://orcid.org/0000-0001-8916-7533 Department of Pharmacology (R.S., B.H., Z.J., M.J., N.B., Y.-J.C., J.B., T.B., D.M.B., L.T.I., Y.C.-I.), University of California, Davis. , Nicholas BalardiNicholas Balardi https://orcid.org/0000-0002-0743-6611 Department of Pharmacology (R.S., B.H., Z.J., M.J., N.B., Y.-J.C., J.B., T.B., D.M.B., L.T.I., Y.C.-I.), University of California, Davis. , Yi-Je ChenYi-Je Chen Department of Pharmacology (R.S., B.H., Z.J., M.J., N.B., Y.-J.C., J.B., T.B., D.M.B., L.T.I., Y.C.-I.), University of California, Davis. , Ariel L. EscobarAriel L. Escobar Department of Bioengineering, University of California, Merced (A.L.E.). , Anthony J. BakerAnthony J. Baker Department of Medicine, University of California, San Francisco (A.J.B.). , Julie BossuytJulie Bossuyt Department of Pharmacology (R.S., B.H., Z.J., M.J., N.B., Y.-J.C., J.B., T.B., D.M.B., L.T.I., Y.C.-I.), University of California, Davis. , Tamas BanyaszTamas Banyasz https://orcid.org/0000-0003-3894-0738 Department of Pharmacology (R.S., B.H., Z.J., M.J., N.B., Y.-J.C., J.B., T.B., D.M.B., L.T.I., Y.C.-I.), University of California, Davis. Department of Physiology, University of Debrecen, Hungary (T.B.). , Nipavan ChiamvimonvatNipavan Chiamvimonvat https://orcid.org/0000-0001-9499-8817 Department of Internal Medicine (N.C., Y.C.-I.), University of California, Davis. , Kit S. LamKit S. Lam Department of Biochemistry and Molecular Medicine (X.L., K.S.L.), University of California, Davis. , Donald M. BersDonald M. Bers https://orcid.org/0000-0002-2237-9483 Department of Pharmacology (R.S., B.H., Z.J., M.J., N.B., Y.-J.C., J.B., T.B., D.M.B., L.T.I., Y.C.-I.), University of California, Davis. , Leighton T. IzuLeighton T. Izu Department of Pharmacology (R.S., B.H., Z.J., M.J., N.B., Y.-J.C., J.B., T.B., D.M.B., L.T.I., Y.C.-I.), University of California, Davis. and Ye Chen-IzuYe Chen-Izu Correspondence to: Ye Chen-Izu, PhD, Department of Pharmacology, School of Medicine (SOM), Department of Biomedical Engineering, College of Engineering (COE), Department of Internal Medicine, Division of Cardiovascular Medicine, SOM, University of California, Davis, 451 Health Science Dr, Davis, CA 95616. Email E-mail Address: [email protected] https://orcid.org/0000-0003-4818-9570 Department of Pharmacology (R.S., B.H., Z.J., M.J., N.B., Y.-J.C., J.B., T.B., D.M.B., L.T.I., Y.C.-I.), University of California, Davis. Department of Biomedical Engineering (R.S., D.M., J.K.L., Y.C.-I.), University of California, Davis. Department of Internal Medicine (N.C., Y.C.-I.), University of California, Davis. Originally published19 Feb 2021https://doi.org/10.1161/CIRCRESAHA.120.318570Circulation Research. 2021;128:772–774Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: February 19, 2021: Ahead of Print The heart is a smart pump, automatically adjusting its contractile strength in response to mechanical loads placed upon it, via intrinsic adaptations discovered over a century ago.1,2 When the cardiomyocyte encounters an increase in preload (larger end-diastolic volume), the Frank-Starling effect enhances contractile force and stroke volume, mainly by instantaneous sarcomere-length and myofilament-based effects, independent of Ca2+ transient changes. In contrast, when the heart pumps blood against an increase in afterload (larger resistance), the Anrep effect develops over minutes to increase Ca2+ transients and enhance contractility. Most prior studies have used stretching methods to control preload on cardiomyocytes; it has been difficult to independently control afterload, and the mechanisms underlying the Anrep effect remain unresolved.2 We have developed a new methodology to control afterload at the single-myocyte level using our Cell-in-Gel system,3,4 and studies how afterload affects the myocyte excitation-Ca2+ signaling-contraction (E-C) coupling (Figure [A]).Download figureDownload PowerPointFigure. Cell-in-Gel Technology for Controlling Mechanical Load on Cells.A, Schematic of cardiomyocyte excitation-contraction coupling with mechano-transduction feedback. B, Cardiomyocytes were embedded in the polymer matrix made of polyvinyl alcohol (PVA) and 4B-PEG crosslinker. C, Mathematical modeling of the Cell-in-Gel system show that the single cardiomyocyte experiences 3-dimensional mechanical stresses during auxotonic contraction in the hydrogel.3D, Patch-Clamp-in-Gel was performed by embedding the cell-electrode in the gel (Da). Action potentials (APs) were recorded first when the cell was contracting load-free (LF, black trace in Db), during the gel-forming protocol by adding 10% crosslinker to polymerize PVA (Dc), and after gel formation (Gel, red trace in Db). Bar charts compare cells under load-free vs in-gel, showing steady-state APD95 (Dd, P=1.9×10−10) and AP amplitude (De, P=0.45). E, Simultaneous measurements of [Ca2+]i and contraction using the gel-dissolve protocol (Ea). Bar charts compare the cells in load-free vs in-gel, showing diastolic [Ca2+]i (Eb, P=0.17), systolic peak [Ca2+]i (Ec, P=1.2×10−12), contraction amplitude (Ed, P=3.7×10−3), and relaxation time (Ee, P=2.12×10−4). F, Cells were embedded in the gels of different stiffness by mixing 10% PVA with crosslinker of indicated concentrations (CL%). Upper shows [Ca2+]i transient peak, and lower shows contraction amplitude. One-way ANOVA P<0.0001, and Tukey test for pair-wise comparison of neighboring groups.Statistical tests: the bars show mean and SEM of each group with indicated number of cells/animals. All groups passed D'Agostino-Pearson normality test. One-way ANOVA test was used for multiple groups comparison; t test for 2 groups comparison. ns indicates not significant. P: *P<0.05, **P<0.01, ***P<0.001.We embedded freshly isolated rabbit ventricular myocytes in a 3-dimensional viscoelastic hydrogel comprising polyvinyl alcohol (PVA) and 4-boronate-polyethylene glycol crosslinker (Figure [B]).4 Since the myocytes are embedded at slack length without preload, the Cell-in-Gel system is well suited for studying afterload effects. Mechanical analyses show that the myocyte contracting in-gel experiences 3-dimensional mechanical stresses including longitudinal tension due to cell shortening, transverse compression due to cell broadening, and surface traction with normal and shear stress (Figure [C]).3We further developed a Patch-Clamp-in-Gel technique (Figure [D]a) using a gel-forming protocol. First, we establish patch-clamp of the cell under load-free condition with the myocyte bathed in a modified Tyrode solution containing PVA. Next, add 4-boronate-polyethylene glycol to crosslink PVA, which embeds the cell-electrode assembly in-gel. Finally, repeat the electrophysiology recordings on the same cell now contracting under afterload in-gel. Figure [D]b shows the action potentials recorded first in load-free and then under afterload. Upon adding 10% 4-boronate-polyethylene glycol to 10% PVA, polymerization occurred in only minutes. As the hydrogel became stiffer, the increases of afterload during cell contraction caused progressive increases in action potential duration (Figure [D]c). After reaching steady state, afterload significantly prolonged APD95 (Figure [D]d), with unchanged action potential amplitude (Figure [D]e).We studied afterload effects on Ca2+ signaling and cell contraction using a gel-dissolve protocol (Figure [E]). First, we measured cytosolic Ca2+ concentration ([Ca2+]i) and sarcomere length shortening simultaneously while the myocyte was embedded in-gel and paced to perform E-C coupling under afterload. Next, sorbitol (1% w/v) was added to the perfusion solution to dissolve the hydrogel. Finally, we repeated experiments on the same cell, now contracting in load-free condition. These self-control experiments show that afterload did not alter diastolic [Ca2+]i (Figure [E]b) but increased systolic Ca2+ transients (Figure [E]c), reduced sarcomere shortening magnitude (Figure [E]c), and slowed relaxation (Figure [E]e). Thus, afterload-induced mechano-chemo-transduction regulates the Ca2+ signaling system, causing mechano-chemo-transduction-Ca2+ gain to enhance contractility.The afterload effects on electrophysiology and Ca2+ signaling provide feedback loops in the dynamic system of E-C coupling, which may enable autoregulation. To test this hypothesis, we systematically tuned afterload levels using hydrogels of different stiffness (mixing 10% PVA with different crosslinker concentrations, CL%). Cardiomyocytes showed progressively larger Ca2+ transients under higher afterload in stiffer gels (Figure [F], CL 5%–10%, gel elastic shear modulus 1–10 kPa). Remarkably, the increases of Ca2+ transients enabled cardiomyocytes to maintain relatively stable contraction amplitude despite load increases (bottom). It was not until very high afterload (CL 11%–15%, gel elastic shear modulus 11–15 Pa) that myocyte contraction declined with reduced mechano-chemo-transduction-Ca2+ gain. In conclusion, our studies reveal mechano-chemo-electro-transduction feedbacks in the dynamic system of cardiac excitation-Ca2+ signaling-contraction coupling, which enable autoregulation of contractility at the single-myocyte level independent of neurohormonal influences. The Cell-in-Gel methodology provides a powerful tool for further dissection of mechano-chemo-electro-transduction molecular pathways that underlie the heart's intrinsic adaptive responses to mechanical loading in health and diseases.Nonstandard Abbreviation and AcronymsPVApolyvinyl alcoholData AvailabilityThe methods, data, and materials of this study are available upon request to the corresponding author and through the university material transfer agreement. New Zealand White rabbits, 4 to 6 months old male, were purchased from Charles River Laboratories (Wilmington, MA) and used to isolate cardiomyocytes using standard enzymatic technique. All animal procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health and the protocols approved by the University of California Davis Institutional Animal Care and Use Committee (IACUC).Sources of FundingThis work was supported by the grants from National Institutes of Health R01HL123526 and R01HL141460 (Y. Chen-Izu), R01HL149431 and R01HL90880 (L.T. Izu), P01HL141084 (D.M. Bers), T32HL0863500 (N. Chiamvimonvat), and F31HL129746 (R. Shimkunas).Disclosures None.FootnotesFor Sources of Funding and Disclosures, see page 773.Correspondence to: Ye Chen-Izu, PhD, Department of Pharmacology, School of Medicine (SOM), Department of Biomedical Engineering, College of Engineering (COE), Department of Internal Medicine, Division of Cardiovascular Medicine, SOM, University of California, Davis, 451 Health Science Dr, Davis, CA 95616. Email [email protected]eduReferences1. Cingolani HE, Pérez NG, Cingolani OH, Ennis IL. The Anrep effect: 100 years later.Am J Physiol Heart Circ Physiol. 2013; 304:H175–H182. doi: 10.1152/ajpheart.00508.2012CrossrefMedlineGoogle Scholar2. Izu LT, Kohl P, Boyden PA, Miura M, Banyasz T, Chiamvimonvat N, Trayanova N, Bers DM, Chen-Izu Y. Mechano-electric and mechano-chemo-transduction in cardiomyocytes.J Physiol. 2020; 598:1285–1305. doi: 10.1113/JP276494CrossrefMedlineGoogle Scholar3. Shaw J, Izu L, Chen-Izu Y. Mechanical analysis of single myocyte contraction in a 3-D elastic matrix.PLoS One. 2013; 8:e75492. doi: 10.1371/journal.pone.0075492CrossrefMedlineGoogle Scholar4. Jian Z, Han H, Zhang T, Puglisi J, Izu LT, Shaw JA, Onofiok E, Erickson JR, Chen YJ, Horvath B, et al.. Mechanochemotransduction during cardiomyocyte contraction is mediated by localized nitric oxide signaling.Sci Signal. 2014; 7:ra27. doi: 10.1126/scisignal.2005046CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Khokhlova A, Solovyova O, Kohl P and Peyronnet R (2022) Single cardiomyocytes from papillary muscles show lower preload-dependent activation of force compared to cardiomyocytes from the left ventricular free wall, Journal of Molecular and Cellular Cardiology, 10.1016/j.yjmcc.2022.02.008, 166, (127-136), Online publication date: 1-May-2022. Hegyi B, Shimkunas R, Jian Z, Izu L, Bers D and Chen-Izu Y (2021) Mechanoelectric coupling and arrhythmogenesis in cardiomyocytes contracting under mechanical afterload in a 3D viscoelastic hydrogel, Proceedings of the National Academy of Sciences, 10.1073/pnas.2108484118, 118:31, Online publication date: 3-Aug-2021. Kazemi-Lari M, Shaw J, Wineman A, Shimkunas R, Jian Z, Hegyi B, Izu L and Chen-Izu Y (2021) A viscoelastic Eshelby inclusion model and analysis of the Cell-in-Gel system, International Journal of Engineering Science, 10.1016/j.ijengsci.2021.103489, 165, (103489), Online publication date: 1-Aug-2021. Crocini C and Gotthardt M (2021) Cardiac sarcomere mechanics in health and disease, Biophysical Reviews, 10.1007/s12551-021-00840-7, 13:5, (637-652), Online publication date: 1-Oct-2021. Izu L, Shimkunas R, Jian Z, Hegyi B, Kazemi-Lari M, Baker A, Shaw J, Banyasz T and Chen-Izu Y (2021) Emergence of Mechano-Sensitive Contraction Autoregulation in Cardiomyocytes, Life, 10.3390/life11060503, 11:6, (503) March 19, 2021Vol 128, Issue 6Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.120.318570PMID: 33601939 Originally publishedFebruary 19, 2021 Keywordscalcium signalingaction potentialshydrogelsmuscle contractionmyocytes, cardiacstress, mechanicalpolythene glycolmechanotransduction, cellularsarcomerePDF download Advertisement SubjectsBasic Science Research