Sarcomere-Directed Calcium Reporters in Cardiomyocytes

HomeCirculation ResearchVol. 124, No. 8Sarcomere-Directed Calcium Reporters in Cardiomyocytes Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBSarcomere-Directed Calcium Reporters in CardiomyocytesPutting Sensors Where Sensing Makes Sense Stuart G. Campbell, Yibing Qyang and J. Travis Hinson Stuart G. CampbellStuart G. Campbell Correspondence to Stuart G. Campbell, PhD, Department of Biomedical Engineering, Yale University, 55 Prospect St, New Haven, CT 06511. Email E-mail Address: [email protected] From the Department of Biomedical Engineering (S.G.C.), Yale University, New Haven, CT Department of Cellular and Molecular Physiology (S.G.C.), Yale School of Medicine, New Haven, CT Search for more papers by this author , Yibing QyangYibing Qyang From the Department of Biomedical Engineering (S.G.C.), Yale University, New Haven, CT Yale Stem Cell Center (Y.Q.), Yale University, New Haven, CT Vascular Biology and Therapeutics Program (Y.Q.), Yale University, New Haven, CT Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine (Y.Q.), Yale School of Medicine, New Haven, CT Search for more papers by this author and J. Travis HinsonJ. Travis Hinson The Jackson Laboratory for Genomic Medicine, Farmington, CT (J.T.H.) Department of Cardiology, UConn Health, Farmington, CT (J.T.H.). Search for more papers by this author Originally published11 Apr 2019https://doi.org/10.1161/CIRCRESAHA.119.314877Circulation Research. 2019;124:1151–1153This article is a commentary on the followingMeasurement of Myofilament-Localized Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy MutationsIn this issue of Circulation Research, Sparrow et al1 report the development of genetically encoded Ca2+ indicators that have been conjugated to the thin filament proteins TnI (troponin I) and TnT (troponin T). These constructs, based on RGECO (red genetically encoded Ca2+ indicator for optical imaging), localize and bind to the actin thin filament and do not interfere with the Ca2+-dependence of myosin ATPase activity. This constitutes a new method for probing Ca2+ dynamics in very close proximity to the regulatory Ca2+ binding sites that control muscle contraction. These sensors are ideally positioned to report not only the signals that are driving contraction but also any changes in free Ca2+ induced by myofilament activity.Article, see p 1228TnC contains the low-affinity Ca2+ binding site that senses transient Ca2+ events within the cardiomyocyte, transforming them into contractions of the myofilament system. Because of its high abundance in the myofilament lattice, TnC constitutes a significant cytosolic Ca2+ buffer. What is more, its buffering capacity increases dramatically when actin-myosin complexes are formed.2 This means that myosin activity and even external mechanical loading can feedback to affect the intracellular Ca2+ concentration.3 Hence, the concentration of free Ca2+ in the sarcomere is subject to a complex combination of interacting factors.It is precisely these types of interactions that motivated the painstaking development of thin filament-targeted Ca2+ indicators by Sparrow et al.1 By placing sensors directly in the myofilament compartment, they could precisely distinguish changes in localized sarcomeric Ca2+ handling from bulk Ca2+ handling. The authors virally expressed the probes in isolated guinea pig adult ventricular myocytes and then subjected cells to either drug compounds or mutant myofilament proteins that perturb contractile function. In some cases, the myofilament-targeted sensors and bulk cytosolic sensors did report different signals when subjected to identical perturbations. Essentially all of these differences were observed in the amplitude of the Ca2+ transient, but there were also rare instances of temporal disagreement, in which myofilament-targeted probes registered different rates of change in the Ca2+ transient.One acknowledged drawback of RGECO-TnI or -TnT (RGECO congugated to TnI or TnT, respectively) is that as intensiometric indicators, they cannot be used to report absolute Ca2+ concentration. Similarly, these indicators lack the ability to register changes in resting (diastolic) Ca2+ because signals are normalized to baseline fluorescence. This is a pertinent question in the context of hypertrophic cardiomyopathy (HCM) causing mutations. Induced pluripotent stem cell–derived cardiomyocytes expressing HCM mutations have been shown through ratiometric dye-based methods to exhibit elevated diastolic Ca2+ 4. Confocal imaging with ratiometric dyes is an alternative approach that can give subsarcomeric spatial resolution while also providing data on diastolic changes to Ca2+.5What can be concluded from the new and interesting data provided by Sparrow et al1 using myofilament-targeted Ca2+ indicators? Not surprisingly, they demonstrate that gradients in free Ca2+ can exist between the myofilament lattice volume and other regions of the cytosol. They also suggest that sensing Ca2+ directly in the myofilament compartment can reveal subtle impacts of sarcomeric perturbations that could be missed by whole-cell type measurements. For instance, the HCM mutation R92Q to troponin T showed no change in peak amplitude in the bulk cytosolic Ca2+ transient, but an increase was observed in the transient reported by RGECO-TnI. At the same time, cytosolic and myofilament-targeted sensors were in complete agreement with regards to changes in Ca2+ release and reuptake rates caused by the 2 HCM mutations studied. Overall, this suggests that dye-based measurements of HCM mutation impacts on the Ca2+ transient amplitude should be interpreted with caution, whereas time-course properties are more reliable.This could explain why induced pluripotent stem cell–derived cardiomyocytes expressing HCM mutations do not show increased Ca2+ transient amplitude when examined via dye-based methods.4,6 Resolving these divergent results is important insofar as it relates to one of the hypothesized pathways whereby mutant sarcomeric proteins cause myocardial hypertrophy—hypertrophic signaling can be activated by raised Ca2+ levels via the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway.7,8 It will be essential to test how the relative contributions of myofilament Ca2+ levels regulate hypertrophic signaling.But why do mutations that increase myofilament Ca2+ buffering cause increases in the Ca2+ transient amplitude? This effect is somewhat counterintuitive. The instantaneous effects of an increase in myofilament Ca2+ buffering would be to blunt the peak of the Ca2+ transient while slowing its decay (Figure [A]). However, after many contraction cycles, altered myofilament buffering will shift Ca2+ equilibrium among the various cellular compartments in possibly complex ways. Evidently, it is this type of shift that is observed by Sparrow et al1 and which now remains to be explained.Download figureDownload PowerPointFigure. Possible explanations for increased myofilament Ca2+ transients in the presence of elevated myofilament-based Ca2+ buffering.A, A naive representation of the acute effects of enhanced Ca2+ buffering by TnC (troponin C). B, The steady-state consequences of enhanced myofilament Ca2+ buffering assuming that SERCA (sarco/endoplasmic reticulum ATPase) uptake exceeds extrusion by NCX (Na+/Ca2+ exchanger). C, The reverse scenario, in which NCX flux exceeds that of SERCA. D, Localization of SERCA throughout the sarcoplasmic reticulum (SR), including network and junctional SR, may provide a spatial advantage over NCX in terms of collecting excess Ca2+ buffered by myofilaments containing HCM-related mutations. Derived from the schema of Lipskaia et al.9One possibility concerns the fate of Ca2+ in the myofilament volume after it is released from the sarcoplasmic reticulum (SR). Ca2+ concentration is returned to diastolic rest levels by a combination of reuptake into the SR by the SERCA (sarco/endoplasmic reticulum ATPase) and extrusion from the cell via the NCX (Na+/Ca2+ exchanger). In an environment of increased myofilament Ca2+ buffering, more Ca2+ would be available for both reuptake and extrusion. The steady-state result may, therefore, come down to which of these 2 pathways is dominant: (1) If NCX flux exceeds SERCA uptake, excess myofilament-buffered Ca2+ could result in greater extrusion from the cell and ultimately a drop in SR load and peak Ca2+ concentration (Figure [B]). (2) If SERCA uptake exceeds NCX efflux, the excess myofilament-buffered Ca would wind up in the SR, and peak Ca2+ release will increase (Figure [C]).The experiments of Sparrow et al,1 conducted in isolated adult guinea pig cardiomyocytes, provide evidence of the latter case, at least on the time scale of these experiments (≈48 hours). More than 1 explanation exists for this outcome. Raw SERCA flux could exceed that of NCX by virtue of relative expression. Alternatively, it may come down once again to a matter of location: SERCA is distributed throughout the membrane of the SR, including network SR9, where it has more ready access to the excess Ca2+ buffered by myofilaments (Figure [D]). By contrast, NCX is localized to the vertical transverse tubules at the extreme ends of each sarcomere and in the outer membrane of the cardiomyocyte10 (Figure [D]). Hence, elevated diastolic Ca2+ buffered by the myofilaments may be preferentially directed to the SR simply by virtue of spatial arrangement (Figure 1D). Computational models of cardiomyocyte function have provided valuable insight into such questions11 and are likely to play an important role going forward given the complex spatial and temporal dynamics of intracellular Ca2+ handling.Clearly, much remains to be discovered with respect to the complex changes that take place to Ca2+ signaling in the context of HCM. The work of Sparrow et al1 represents a welcome new tool in elucidating details of this process. It joins several other techniques under intensive development, including induced pluripotent stem cell disease modeling,12,13 improved maturation of engineered human myocardium,14 and computational approaches.15 Together, these tools seem poised to provide important insights into HCM and its associated pathophysiology in the near future.DisclosuresS.G. Campbell is founder of and holds equity in Propria LLC. The other authors report no conflicts.Sources of FundingThis work was supported in part by National Institutes of Health (NIH) HL136590 (to S.G. Campbell), NIH R01HL131940 and DOD 11959515 (to Y. Qyang), and NIH HL142787 (to J.T. Hinson).FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to Stuart G. Campbell, PhD, Department of Biomedical Engineering, Yale University, 55 Prospect St, New Haven, CT 06511. Email stuart.[email protected]eduReferences1. 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A platform for generation of chamber-specific cardiac tissues and disease modeling.Cell. 2019; 176:913.e18–927.e18. doi: 10.1016/j.cell.2018.11.042CrossrefGoogle Scholar15. Sewanan LR, Moore JR, Lehman W, Campbell SG. Predicting effects of tropomyosin mutations on cardiac muscle contraction through myofilament modeling.Front Physiol. 2016; 7:473. doi: 10.3389/fphys.2016.00473CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Solís C and Solaro R (2021) Novel insights into sarcomere regulatory systems control of cardiac thin filament activation, Journal of General Physiology, 10.1085/jgp.202012777, 153:7, Online publication date: 5-Jul-2021. Related articlesMeasurement of Myofilament-Localized Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy MutationsAlexander J. Sparrow, et al. Circulation Research. 2019;124:1228-1239 April 12, 2019Vol 124, Issue 8 Advertisement Article InformationMetrics © 2019 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.119.314877PMID: 30973804 Originally publishedApril 11, 2019 KeywordstroponinEditorialshypertrophic cardiomyopathycalcium signalingsarcomeresPDF download Advertisement SubjectsCalcium Cycling/Excitation-Contraction CouplingCardiomyopathyContractile Function