Imaging Sarcoplasmic Reticulum Ca 2+ Signaling in Intact Cardiac Myocytes
2020; Lippincott Williams & Wilkins; Volume: 142; Issue: 15 Linguagem: Inglês
10.1161/circulationaha.120.047784
ISSN1524-4539
AutoresFujian Lu, Yan Zhao, Wenjun Xie, Qianjin Guo, Shi‐Qiang Wang, Xianhua Wang, Heping Cheng,
Tópico(s)Plant Stress Responses and Tolerance
ResumoHomeCirculationVol. 142, No. 15Imaging Sarcoplasmic Reticulum Ca2+ Signaling in Intact Cardiac Myocytes Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBImaging Sarcoplasmic Reticulum Ca2+ Signaling in Intact Cardiac Myocytes Fujian Lu, PhD Yan Zhao, BS Wenjun Xie, PhD Qianjin Guo, PhD Shi-Qiang Wang, PhD Xianhua Wang, PhD Heping ChengPhD Fujian LuFujian Lu Fujian Lu, PhD, 5 Yiheyuan Road, Haidian District, Beijing 100871, China. Email E-mail Address: [email protected] State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China (F.L., Y.Z., Q.G., X.W., H.C.) , Yan ZhaoYan Zhao State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China (F.L., Y.Z., Q.G., X.W., H.C.) Academy for Advanced Interdisciplinary Studies (Y.Z.), Peking University, Beijing, China. , Wenjun XieWenjun Xie https://orcid.org/0000-0003-0493-062X The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, China (W.X.). , Qianjin GuoQianjin Guo State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China (F.L., Y.Z., Q.G., X.W., H.C.) , Shi-Qiang WangShi-Qiang Wang College of Life Sciences(S-Q.W.), Peking University, Beijing, China. , Xianhua WangXianhua Wang State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China (F.L., Y.Z., Q.G., X.W., H.C.) Research Unit of Mitochondria in Brain Diseases, Chinese Academy of Medical Sciences, PKU-Nanjing Institute of Translational Medicine, Nanjing, China (X.W., H.C.). , Heping ChengHeping Cheng Heping Cheng, PhD, 5 Yiheyuan Road, Haidian District, Beijing 100871, China. Email E-mail Address: [email protected] https://orcid.org/0000-0002-9604-6702 State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China (F.L., Y.Z., Q.G., X.W., H.C.) Research Unit of Mitochondria in Brain Diseases, Chinese Academy of Medical Sciences, PKU-Nanjing Institute of Translational Medicine, Nanjing, China (X.W., H.C.). Originally published12 Oct 2020https://doi.org/10.1161/CIRCULATIONAHA.120.047784Circulation. 2020;142:1503–1505In cardiac myocytes (CMs), endoplasmic/sarcoplasmic reticulum (SR) Ca2+ signaling is essential for excitation–contraction (EC) coupling and many other physiological and pathophysiological processes. For instance, SR Ca2+ overloading leads to hyperactivity of ryanodine receptors and spontaneous electrogenic Ca2+ waves that trigger arrhythmias as seen in catecholaminergic polymorphic ventricular tachycardia models.1 Conversely, SR Ca2+ insufficiency results in endoplasmic/SR stress–mediated cell death.2 However, quantitative measurement of intra-SR Ca2+ dynamics has been met with great challenges. In CMs from small rodents, the most commonly used species with hundreds of genetic and disease models, loading low-affinity indicators tends to cause significant cytosolic retention that confounds SR Ca2+ measurement. Saponin permeabilization of the plasma membrane to release the cytosolic species inevitably disrupts the integrity of Ca2+ signaling and EC coupling machinery. Until now, no direct and nondisruptive means have been available for visualization of intra-SR Ca2+ dynamics in rat and mouse CMs.Here we adopted an endoplasmic/SR–targeted expression of carboxylesterase 2 (srCES)3 as a strategy to achieve selective and efficient indicator loading into the SR in CMs. Specifically, srCES provides high esterase activity for rapid cleavage of acetoxymethyl ester–based indicators to convert them into the hydrophilic, Ca2+-sensitive forms and trap them inside the SR (Figure A). All animal protocols were approved by the Institutional Animal Care and Use Committee. Immunofluorescence analysis revealed that 24 to 36 hours after srCES-adenovirus (Figure A) infection, cultured rat CMs displayed a striated pattern of srCES expression along with enrichment in perinuclear regions (data not shown), suggesting successful targeting of srCES to SR lumen. When incubated with Fluo5N-AM for 10 minutes, nearly all srCES-expressing myocytes displayed a clear striated distribution of Fluo5N fluorescence (Figure B), with periodic peaks approximately every 2 μm (Figure C) and brightened perinuclear envelopes (Figure B). Moreover, Fluo5N fluorescent peaks clearly overlapped the t-tubular structures revealed by CellMask Deep Red (Figure C) as expected for its enrichment in the junctional SR cistern. To extend the applicability to freshly isolated CMs, we adopted an adeno-associated virus serotype 9-based strategy for in vivo heart-specific expression of srCES under cardiac troponin T promoter. Low-dose adeno-associated virus was subcutaneously injected into P1 mice (Figure D). Histological analysis confirmed a mosaic srCES expression in adult hearts (Figure E), and SR loading of Fluo5N in single freshly isolated cells was robust and highly reproducible (Figure F).Download figureDownload PowerPointFigure. Imaging SR Ca2+ dynamics by srCES-aided indicator loading in intact cardiac myocytes.A, Strategy for SR-targeted indicator loading. In the srCES adenoviral vector, Carboxylesterase 2 (CES2) is fused with a SR signal peptide (sig) at the N terminus and a myc tag at the C terminus, followed by an endoplasmic reticulum retention motif (KDEL). Expression was under the control of the cytomegalovirus promoter (CMV). Luminal srCES hydrolyzes the membrane-permeable, AM-linked Ca2+ indicator (CI-AM), resulting in retention of the charged, Ca2+ responsive form (CI/ Ca2+) in the lumen of the SR. B, Confocal images showing that nearly all rat cardiac myocytes with adenoviral infection were successfully stained with Fluo5N at comparable intensity. C, Expanded views of staining patterns of Fluo5N and the T-tubule surface membrane marker CellMask Deep Red (left) and their spatial profiles (right) for the boxed region in a srCES-expressing rat cardiac myocyte. D, Strategy for in vivo srCES expression in mouse hearts. (Upper) Map of the cTnT-promoter driven, srCES adeno-associated viral construct; (lower) the experimental workflow. On the first day after birth, mouse pups were injected intravenously with adeno-associated virus (AAV)-cTnT-srCES-myc and imaging experiments were performed about 1 month later. E, Immunohistochemistry micrographs of myocardial sections from a control and an AAV-cTnT-srCES-myc injected mouse. F, Representative Fluo5N staining pattern in a freshly isolated mouse cardiac myocyte expressing srCES. Plot shows the profile of Fluo5N signal in the boxed region. Dual-color line-scan images of local and global cytosolic and SR Ca2+ dynamics elicited by caffeine application (10 mmol/L; G) or by electric field stimulation (H) or during a spontaneous spark-blink event (average of 17 events) in a rat cardiac myocyte (I). Corresponding time course plots are shown below. Inset in I shows junctional SR (J) at 3 adjacent sarcomeres. J, Statistics for the amplitudes of spontaneous sparks, blinks, and electrical pacing- or caffeine (10 mmol/L)-induced cytosolic transients and SR depletion. Data are from rat (upper panel: n=17, 23, 23 events, respectively) and mouse cardiac myocytes (lower panel: n=25, 17, 22 events, respectively). K, Cytosolic (upper) and SR (lower) views of the initiation, propagation and termination of a spontaneous Ca2+ wave (left). Wavefront-aligned images of the boxed regions are shown in the middle. Traces to the right show time courses of local cytosolic and SR Ca2+ signals at the time points of (1) wave ignition, (2) quasi-steady propagation, and (3) abortion. Marker 'a' indicates the onset of cytosolic Ca2+ rise, and marker 'b' the onset of SR Ca2+ release.To track cytosolic and SR Ca2+ dynamics simultaneously, Rhod2-AM was subsequently loaded for 8 minutes. On caffeine application, Fluo5N signal steeply declined from baseline by 66.7±2.5% and 45.3±1.8% in rat and mouse CMs, respectively (Figure G and J). Using this value as a reference, we directly examined fractional SR release during EC coupling. On average, the nadir of the Fluo5N signal decline was 26.5±1.7% and 18.3±1.5% in rat and mouse CMs, respectively (Figure H and J). Thus, only a minor fraction of SR Ca2+ is released by electric field stimulation, consistent with notion that SR store is capable of supporting 2 to 4 full-fledged Ca2+ transients during EC coupling, measured indirectly using Na+–Ca2+ exchange currents.4The high sensitivity of the SR Ca2+ measurement also enabled us to investigate elementary SR Ca2+ signaling events, Ca2+ blinks,5 for the first time in intact rodent CMs. Blinks arose spontaneously within 20.7±1.4 ms, reaching a nadir of −0.13±0.01 ΔF/F0, and recovered in 27.8±1.5 ms, accompanying Rhod2-reported sparks in the cytosol (Figure I). Blinks were more sharply confined (≈0.74 μm) than sparks (≈2.19 μm), indicating that Ca2+ handling is differentially regulated in the SR and the cytosol. Furthermore, these blinks are considerably smaller, narrower, and of shorter duration than their counterparts reported in rabbits.5 This result suggests that elementary Ca2+ signaling units are functionally organized in a species-specific manner.With a dual view of SR and cytosolic Ca2+ dynamics, we sought to discriminate between different mechanisms of Ca2+ wave propagation: regenerative Ca2+-induced Ca2+ release on the cytosolic side or frontal local SR overload-induced release on the SR side. Figure K shows that sudden SR release, reflected by downward deflection of Fluo5N signal, coincided with the onset of cytosolic Ca2+ rise with no detectable delay at initiation site. As the wave propagated at a quasi-steady velocity of 87 μm/s, a 73-ms delay developed, with cytosolic Ca2+ rise preceding the SR depletion. The delay was increased, and the amplitude in either the cytosolic or SR compartment diminished at termination site. Importantly, our results revealed no discernible frontal local overloading of the SR, thus rejecting the frontal SR overloading hypothesis and favoring the Ca2+-induced Ca2+ release model of wave propagation. Further, the average cytosolic Ca2+ level at which sudden SR release took place was 0.25±0.02 ΔF/F0, reflecting a threshold for Ca2+-induced Ca2+ release to regenerate at the front of a propagating wave.In summary, we have developed a robust and efficient method for imaging SR Ca2+ with high sensitivity and spatiotemporal resolution. We anticipate that it will be broadly applicable to a large panel of genetic and disease models of the rodent and hence empower the mechanistic investigation of SR Ca2+ signaling in physiology and its dysregulation in heart diseases.AcknowledgmentsWe thank Dr Robert Blum, University Hospital of Würzburg, for providing us the srCES cDNA.Sources of FundingThis work was supported by the National Key R&D Program of China (2017YFA0504000 to XW; 2016YFA0500403 to HC) and the National Science Foundation of China (91854209 and 31630035 to SW; 31670039 and 8182780030 to XW; 31821091 to HC).DisclosuresNone.Footnotes*Dr Lu and Y. Zhao contributed equally.Data sharing: The data that support the findings of this study and research materials, as well as experimental procedures and protocols, are available from the corresponding author upon reasonable request.https://www.ahajournals.org/journal/circFujian Lu, PhD, 5 Yiheyuan Road, Haidian District, Beijing 100871, China. Email [email protected]edu.cnHeping Cheng, PhD, 5 Yiheyuan Road, Haidian District, Beijing 100871, China. Email [email protected]edu.cnReferences1. Priori SG, Chen SR. Inherited dysfunction of sarcoplasmic reticulum Ca2+ handling and arrhythmogenesis.Circ Res. 2011; 108:871–883. doi: 10.1161/CIRCRESAHA.110.226845LinkGoogle Scholar2. Krebs J, Agellon LB, Michalak M. Ca2+ homeostasis and endoplasmic reticulum (ER) stress: an integrated view of calcium signaling.Biochem Biophys Res Commun. 2015; 460:114–121. doi: 10.1016/j.bbrc.2015.02.004CrossrefMedlineGoogle Scholar3. Rehberg M, Lepier A, Solchenberger B, Osten P, Blum R. A new non-disruptive strategy to target calcium indicator dyes to the endoplasmic reticulum.Cell Calcium. 2008; 44:386–399. doi: 10.1016/j.ceca.2008.02.002CrossrefMedlineGoogle Scholar4. Shannon TR, Ginsburg KS, Bers DM. Potentiation of fractional sarcoplasmic reticulum calcium release by total and free intra-sarcoplasmic reticulum calcium concentration.Biophys J. 2000; 78:334–343. doi: 10.1016/S0006-3495(00)76596-9CrossrefMedlineGoogle Scholar5. Brochet DX, Yang D, Di Maio A, Lederer WJ, Franzini-Armstrong C, Cheng H. Ca2+ blinks: rapid nanoscopic store calcium signaling.Proc Natl Acad Sci U S A. 2005; 102:3099–3104. doi: 10.1073/pnas.0500059102CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails October 13, 2020Vol 142, Issue 15Article InformationMetrics Download: 984 © 2020 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.120.047784PMID: 33044861 Originally publishedOctober 12, 2020 Keywordscalcium signalingcarboxylesteraseexcitation-contraction couplingcardiac myocytesarcoplasmic reticulumPDF download SubjectsIon Channels/Membrane TransportCalcium Cycling/Excitation-Contraction Coupling
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