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  • Review Article
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Electron microscopy of cardiac 3D nanodynamics: form, function, future

Nature Reviews Cardiology volume 19, pages 607–619 (2022)Cite this article

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Abstract

The 3D nanostructure of the heart, its dynamic deformation during cycles of contraction and relaxation, and the effects of this deformation on cell function remain largely uncharted territory. Over the past decade, the first inroads have been made towards 3D reconstruction of heart cells, with a native resolution of around 1 nm3, and of individual molecules relevant to heart function at a near-atomic scale. These advances have provided access to a new generation of data and have driven the development of increasingly smart, artificial intelligence-based, deep-learning image-analysis algorithms. By high-pressure freezing of cardiomyocytes with millisecond accuracy after initiation of an action potential, pseudodynamic snapshots of contraction-induced deformation of intracellular organelles can now be captured. In combination with functional studies, such as fluorescence imaging, exciting insights into cardiac autoregulatory processes at nano-to-micro scales are starting to emerge. In this Review, we discuss the progress in this fascinating new field to highlight the fundamental scientific insight that has emerged, based on technological breakthroughs in biological sample preparation, 3D imaging and data analysis; to illustrate the potential clinical relevance of understanding 3D cardiac nanodynamics; and to predict further progress that we can reasonably expect to see over the next 10 years.

Key points

  • Electron microscopy (EM) methods are currently the only means of obtaining (sub-)nanometre-scale information on most biological structures.

  • After decades of dwindling interest, seminal developments in sample preparation, imaging and analysis have led to a renaissance of EM.

  • However, data acquisition and processing remain time-consuming and laborious, and the inability to observe dynamic events in live cells limits the uptake and utility of EM.

  • Recent developments promise the advent of temporally resolved, structure–function-correlative, large-volume, 3D EM.

  • Initial applications of these new developments show EM to be a powerful driver of modern fundamental and translational heart research.

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Fig. 1: Imaging modalities for the investigation of 3D cardiac structure and function across scales.
Fig. 2: Comparison of ‘conventional’ (chemical fixation/dehydration) and ‘native’ (HPF) sample preservation.
Fig. 3: Advantages of 3D analysis.

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References

  1. Palade, G. E. The fine structure of mitochondria. Anat. Rec. 114, 427–451 (1952).

    Article  CAS  PubMed  Google Scholar 

  2. Beams, H. W. & Evans, T. C. Electron microscope studies on the structure of cardiac muscle. Anat. Rec. 105, 59–81 (1949).

    Article  CAS  PubMed  Google Scholar 

  3. Kisch, B., Grey, C. E. & Kelsch, J. J. Electron histology of the heart. Exp. Med. Surg. 6, 346–365 (1948).

    CAS  PubMed  Google Scholar 

  4. Hill, A. V. The abrupt transition from rest to activity in muscle. Proc. R. Soc. B 136, 399–420 (1949).

    CAS  Google Scholar 

  5. Lindner, E. Die submikroskopische Morphologie des Herzmuskels. Z. Zellforsch. Mikrosk. Anat. 45, 702–746 (1957).

    Article  CAS  PubMed  Google Scholar 

  6. Moore, D. H. & Ruska, H. Electron microscope study of mammalian cardiac muscle cells. J. Biophys. Biochem. Cytol. 3, 261–268 (1957).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hanson, J. & Huxley, H. E. Structural basis of the cross-striations in muscle. Nature 172, 530–532 (1953).

    Article  CAS  PubMed  Google Scholar 

  8. Costantin, L. L., Franzini-Armstrong, C. & Podolsky, R. J. Localization of calcium-accumulating structures in striated muscle fibers. Science 147, 158–160 (1965).

    Article  CAS  PubMed  Google Scholar 

  9. Porter, K. R. & Palade, G. E. Studies on the endoplasmic reticulum. Its form and distribution in striated muscle cells. J. Biophys. Biochem. Cytol. 3, 269–300 (1957).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Fawcett, D. W. & McNutt, N. S. The ultrastructure of the cat myocardium. I. Ventricular papillary muscle. J. Cell Biol. 42, 1–45 (1969).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Franzini-Armstrong, C. Studies of the triad: structure of the junction in frog twitch fibers. J. Cell Biol. 47, 488–499 (1970).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Block, B. A., Imagawa, T., Campbell, K. P. & Franzini-Armstrong, C. Structural evidence for direct interaction between the molecular components of the transverse tubule/sarcoplasmic reticulum junction in skeletal muscle. J. Cell Biol. 107, 2587–2600 (1988).

    Article  CAS  PubMed  Google Scholar 

  13. Fleischer, S., Ogunbunmi, E. M., Dixon, M. C. & Fleer, E. A. Localization of Ca2+ release channels with ryanodine in junctional terminal cisternae of sarcoplasmic reticulum of fast skeletal muscle. Proc. Natl Acad. Sci. USA 82, 7256–7259 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lai, F. A., Erickson, H. P., Rousseau, E., Liu, Q. Y. & Meissner, G. Purification and reconstitution of the calcium release channel from skeletal muscle. Nature 331, 315–319 (1988).

    Article  CAS  PubMed  Google Scholar 

  15. Franzini-Armstrong, C., Protasi, F. & Ramesh, V. Shape, size, and distribution of Ca2+ release units and couplons in skeletal and cardiac muscles. Biophys. J. 77, 1528–1539 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Revel, J. P. & Karnovsky, M. J. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J. Cell Biol. 33, 7–12 (1967).

    Article  Google Scholar 

  17. Legato, M. J. The correlation of ultrastructure and function in the mammalian myocardial cell. Prog. Cardiovasc. Dis. 11, 391–409 (1969).

    Article  CAS  PubMed  Google Scholar 

  18. Johnson, E. A. & Sommer, J. R. A strand of cardiac muscle. Its ultrastructure and the electrophysiological implications of its geometry. J. Cell Biol. 33, 103–129 (1967).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Levin, K. R. & Page, E. Quantitative studies on plasmalemmal folds and caveolae of rabbit ventricular myocardial cells. Circ. Res. 46, 244–255 (1980).

    Article  CAS  PubMed  Google Scholar 

  20. Schaper, J. & Schaper, W. Ultrastructural correlates of reduced cardiac function in human heart disease. Eur. Heart J. 4, 35–42 (1983).

    Article  PubMed  Google Scholar 

  21. Heggtveit, H. A. Contributions of electron microscopy to the study of myocardial ischaemia. Bull. World Health Organ. 41, 865–872 (1969).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Lichtig, C. & Brooks, H. Myocardial ultrastructure and function during progressive early ischemia in the intact heart. J. Thorac. Cardiovasc. Surg. 70, 309–315 (1975).

    Article  CAS  PubMed  Google Scholar 

  23. Klar, T. A., Jakobs, S., Dyba, M., Egner, A. & Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kohl, T., Westphal, V., Hell, S. W. & Lehnart, S. E. Superresolution microscopy in heart – cardiac nanoscopy. J. Mol. Cell. Cardiol. 58, 13–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Gwosch, K. C. et al. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 17, 217–224 (2020).

    Article  CAS  PubMed  Google Scholar 

  26. M’Saad, O. & Bewersdorf, J. Light microscopy of proteins in their ultrastructural context. Nat. Commun. 11, 3850 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. De Mazière, A. M. G. L., van Ginneken, A. C. G., Wilders, R., Jongsma, H. J. & Bouman, L. N. Spatial and functional relationship between myocytes and fibroblasts in the rabbit sinoatrial node. J. Mol. Cell. Cardiol. 24, 567–578 (1992).

    Article  PubMed  Google Scholar 

  28. Pinali, C., Bennett, H., Davenport, J. B., Trafford, A. W. & Kitmitto, A. Three-dimensional reconstruction of cardiac sarcoplasmic reticulum reveals a continuous network linking transverse-tubules: this organization is perturbed in heart failure. Circ. Res. 113, 1219–1230 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Rog-Zielinska, E. A. et al. Electron tomography of rabbit cardiomyocyte three-dimensional ultrastructure. Prog. Biophys. Mol. Biol. 121, 77–84 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Dubochet, J. Cryo-EM–the first thirty years. J. Microsc. 245, 221–224 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Dubochet, J., Lepault, J., Freeman, R., Berriman, J. A. & Homo, J. C. Electron microscopy of frozen water and aqueous solutions. J. Microsc. 128, 219–237 (1982).

    Article  Google Scholar 

  32. Bers, D. M. Cardiac excitation–contraction coupling. Nature 415, 198–205 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Maleckar, M. M., Edwards, A. G., Louch, W. E. & Lines, G. T. Studying dyadic structure-function relationships: a review of current modeling approaches and new insights into Ca2+ (mis)handling. Clin. Med. Insights Cardiol. 11, 1179546817698602 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Knollmann, B. C. et al. Casq2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+ release, and catecholaminergic polymorphic ventricular tachycardia. J. Clin. Invest. 116, 2510–2520 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Swift, F. et al. Extreme sarcoplasmic reticulum volume loss and compensatory T-tubule remodeling after SERCA knockout. Proc. Natl Acad. Sci. USA 109, 3997–4001 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Jones, P. P., MacQuaide, N. & Louch, W. E. Dyadic plasticity in cardiomyocytes. Front. Physiol. 9, 1773 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Lugo, C. A., Cantalapiedra, I. R., Peñaranda, A., Hove-Madsen, L. & Echebarria, B. Are SR Ca2+ content fluctuations or SR refractoriness the key to atrial cardiac alternans? Insights from a human atrial model. Am. J. Physiol. Heart Circ. Physiol. 306, 1540–1552 (2014).

    Article  CAS  Google Scholar 

  38. Wu, H.-D. et al. Ultrastructural remodelling of Ca2+ signalling apparatus in failing heart cells. Cardiovasc. Res. 95, 430–438 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. van Oort, R. J. et al. Disrupted junctional membrane complexes and hyperactive ryanodine receptors after acute junctophilin knockdown in mice. Circulation 123, 979–988 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Langer, G. A. & Peskoff, A. Calcium concentration and movement in the diadic cleft space of the cardiac ventricular cell. Biophys. J. 70, 1169–1182 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Frank, J. S. & Langer, G. A. The myocardial interstitium: its structure and its role in ionic exchange. J. Cell Biol. 60, 586–601 (1974).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Novotová, M. et al. Structural variability of dyads relates to calcium release in rat ventricular myocytes. Sci. Rep. 10, 8076 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Scriven, D. R. L., Asghari, P. & Moore, E. D. W. Microarchitecture of the dyad. Cardiovasc. Res. 98, 169–176 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Colman, M. A., Pinali, C., Trafford, A. W., Zhang, H. & Kitmitto, A. A computational model of spatio-temporal cardiac intracellular calcium handling with realistic structure and spatial flux distribution from sarcoplasmic reticulum and t-tubule reconstructions. PLoS Comp. Biol. 13, e1005714 (2017).

    Article  CAS  Google Scholar 

  45. Sun, X. H. et al. Molecular architecture of membranes involved in excitation-contraction coupling of cardiac muscle. J. Cell Biol. 129, 659–671 (1995).

    Article  CAS  PubMed  Google Scholar 

  46. Hopwood, D. Cell and tissue fixation. Histochem. J. 17, 389–442 (1985).

    Article  CAS  PubMed  Google Scholar 

  47. Gerdes, A. M., Kriseman, J. & Bishop, S. P. Morphometric study of cardiac muscle: the problem of tissue shrinkage. Lab. Invest. 46, 271–274 (1982).

    CAS  PubMed  Google Scholar 

  48. Dobro, M. J., Melanson, L. A., Jensen, G. J. & McDowall, A. W. Plunge freezing for electron cryomicroscopy. Meth. Enzymol. 481, 63–82 (2010).

    Article  CAS  Google Scholar 

  49. Padrón, R., Alamo, L., Craig, R. & Caputo, C. A method for quick-freezing live muscles at known instants during contraction with simultaneous recording of mechanical tension. J. Microsc. 151, 81–102 (1988).

    Article  PubMed  Google Scholar 

  50. Moor, H. & Riehle, U. Snap freezing under high pressure: a new fixation technique for freeze-etching. In Proc. 4th European Regional Conference on Electron Microscopy (ed. Bocciarelli, S.) 2, 33–34 (Rome, 1968).

  51. Gilkey, J. C. & Staehelin, L. A. Advances in ultrarapid freezing for the preservation of cellular ultrastructure. Microsc. Res. Tech. 3, 177–210 (1986).

    Article  Google Scholar 

  52. Rog-Zielinska, E. A. et al. Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography. J. Mol. Cell. Cardiol. 153, 86–92 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Cannell, M. B., Kong, C. H., Imtiaz, M. S. & Laver, D. R. Control of sarcoplasmic reticulum Ca2+ release by stochastic RyR gating within a 3D model of the cardiac dyad and importance of induction decay for CICR termination. Biophys. J. 104, 2149–2159 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Koh, X., Srinivasan, B., Ching, H. S. & Levchenko, A. A 3D Monte Carlo analysis of the role of dyadic space geometry in spark generation. Biophys. J. 90, 1999–2014 (2006).

    Article  CAS  PubMed  Google Scholar 

  55. Dulhunty, A. F. & Franzini-Armstrong, C. The relative contributions of the folds and caveolae to the surface membrane of frog skeletal muscle fibres at different sarcomere lengths. J. Physiol. 250, 513–539 (1975).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ingber, D. E. Tensegrity-based mechanosensing from macro to micro. Prog. Biophys. Mol. Biol. 97, 163–179 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Irving, T. C., Konhilas, J., Perry, D., Fischetti, R. & de Tombe, P. P. Myofilament lattice spacing as a function of sarcomere length in isolated rat myocardium. Am. J. Physiol. Heart Circ. Physiol. 279, 2568–2573 (2000).

    Article  Google Scholar 

  58. Talmon, Y., Burns, J. L., Chestnut, M. H. & Siegel, D. P. Time-resolved cryotransmission electron microscopy. J. Electron. Microsc. Tech. 14, 6–12 (1990).

    Article  CAS  PubMed  Google Scholar 

  59. Kohl, P., Cooper, P. J. & Holloway, H. Effects of acute ventricular volume manipulation on in situ cardiomyocyte cell membrane configuration. Prog. Biophys. 82, 221–227 (2003).

    Article  Google Scholar 

  60. MacDonald, E. A. et al. Sinoatrial node structure, mechanics, electrophysiology and the chronotropic response to stretch in rabbit and mouse. Front. Physiol. 11, 809 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Oda, T. & Yanagisawa, H. Cryo-electron tomography of cardiac myofibrils reveals a 3D lattice spring within the Z-discs. Commun. Biol. 3, 585 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Risi, C. et al. Ca2+-induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy. Proc. Natl Acad. Sci. USA 114, 6782–6787 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Sharma, M. R., Jeyakumar, L. H., Fleischer, S. & Wagenknecht, T. Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy. J. Biol. Chem. 275, 9485–9491 (2000).

    Article  CAS  PubMed  Google Scholar 

  64. Efremov, R. G., Leitner, A., Aebersold, R. & Raunser, S. Architecture and conformational switch mechanism of the ryanodine receptor. Nature 517, 39–43 (2015).

    Article  CAS  PubMed  Google Scholar 

  65. Dhindwal, S. et al. A cryo-EM-based model of phosphorylation- and FKBP12.6-mediated allosterism of the cardiac ryanodine receptor. Sci. Signal. 10, eaai8842 (2017).

    Article  PubMed  CAS  Google Scholar 

  66. des Georges, A. et al. Structural basis for gating and activation of RyR1. Cell 167, 145–157 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Chi, X. et al. Molecular basis for allosteric regulation of the type 2 ryanodine receptor channel gating by key modulators. Proc. Natl Acad. Sci. USA 116, 25575–25582 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. McNary, T. G. et al. Mechanical modulation of the transverse tubular system of ventricular cardiomyocytes. Prog. Biophys. Mol. Biol. 110, 218–225 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Rog-Zielinska, E. A. et al. Beat-by-beat cardiomyocyte T-tubule deformation drives tubular content exchange. Circ. Res. 128, 203–215 (2021).

    Article  CAS  PubMed  Google Scholar 

  70. Rog-Zielinska, E. A., O’Toole, E. T., Hoenger, A. & Kohl, P. Mitochondrial deformation during the cardiac mechanical cycle. Anat. Rec. 302, 146–152 (2019).

    Article  CAS  Google Scholar 

  71. Sinha, B. et al. Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 144, 402–413 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Wendt-Gallitelli, M. F. & Isenberg, G. X-ray microanalysis of single cardiac myocytes frozen under voltage-clamp conditions. Am. J. Physiol. 256, 574–583 (1989).

    Google Scholar 

  73. Brette, F. & Orchard, C. T-tubule function in mammalian cardiac myocytes. Circ. Res. 92, 1182–1192 (2003).

    Article  CAS  PubMed  Google Scholar 

  74. Burton, R. A. B. et al. Caveolae in rabbit ventricular myocytes: distribution and dynamic diminution after cell isolation. Biophys. J. 113, 1047–1059 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Scriven, D. R., Klimek, A., Asghari, P., Bellve, K. & Moore, E. D. Caveolin-3 is adjacent to a group of extradyadic ryanodine receptors. Biophys. J. 89, 1893–1901 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Page, E. Quantitative ultrastructural analysis in cardiac membrane physiology. Am. J. Physiol. 235, 147–158 (1978).

    Article  Google Scholar 

  77. Wong, J. et al. Nanoscale distribution of ryanodine receptors and caveolin-3 in mouse ventricular myocytes: dilation of t-tubules near junctions. Biophys. J. 104, 22–24 (2013).

    Article  CAS  Google Scholar 

  78. Bang, B. H. & Bang, F. B. Graphic reconstruction of the third dimension from serial electron microphotographs. J. Ultrastruct. Res. 1, 138–139 (1957).

    Article  CAS  PubMed  Google Scholar 

  79. Pinali, C. & Kitmitto, A. Serial block face scanning electron microscopy for the study of cardiac muscle ultrastructure at nanoscale resolutions. J. Mol. Cell. Cardiol. 76, 1–11 (2014).

    Article  CAS  PubMed  Google Scholar 

  80. Hayashi, T. et al. Three-dimensional electron microscopy reveals new details of membrane systems for Ca2+ signaling in the heart. J. Cell Sci. 122, 1005–1013 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. McIntosh, R., Nicastro, D. & Mastronarde, D. New views of cells in 3D: an introduction to electron tomography. Trends Cell. Biol. 15, 43–51 (2005).

    Article  CAS  PubMed  Google Scholar 

  82. Asghari, P. et al. Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification. eLife 9, e51602 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Asghari, P. et al. Nonuniform and variable arrangements of ryanodine receptors within mammalian ventricular couplons. Circ. Res. 115, 252–262 (2014).

    Article  CAS  PubMed  Google Scholar 

  84. Kong, C. H. T., Rog-Zielinska, E. A., Kohl, P., Orchard, C. H. & Cannell, M. B. Solute movement in the t-tubule system of rabbit and mouse cardiomyocytes. Proc. Natl Acad. Sci. USA 115, 7073–7080 (2018).

    Article  Google Scholar 

  85. Rog-Zielinska, E. A. et al. Species differences in the morphology of transverse tubule openings in cardiomyocytes. Europace 20, 120–124 (2018).

    Article  Google Scholar 

  86. Tsushima, K. et al. Mitochondrial reactive oxygen species in lipotoxic hearts induce post-translational modifications of AKAP121, DRP1, and OPA1 that promote mitochondrial fission. Circ. Res. 122, 58–73 (2018).

    Article  CAS  PubMed  Google Scholar 

  87. Lavorato, M. et al. Increased mitochondrial nanotunneling activity, induced by calcium imbalance, affects intermitochondrial matrix exchanges. Proc. Natl Acad. Sci. USA 114, 849–858 (2017).

    Article  CAS  Google Scholar 

  88. Huang, X. et al. Kissing and nanotunneling mediate intermitochondrial communication in the heart. Proc. Natl Acad. Sci. USA 110, 2846–2851 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Glancy, B. et al. Mitochondrial reticulum for cellular energy distribution in muscle. Nature 523, 617–620 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Aston, D. et al. High resolution structural evidence suggests the sarcoplasmic reticulum forms microdomains with acidic stores (lysosomes) in the heart. Sci. Rep. 7, 40620 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Iribe, G. et al. Axial stretch of rat single ventricular cardiomyocytes causes an acute and transient increase in Ca2+ spark rate. Circ. Res. 104, 787–795 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Peper, J. et al. Caveolin3 stabilizes McT1-mediated lactate/proton transport in cardiomyocytes. Circ. Res. 128, 102–120 (2021).

    Article  CAS  Google Scholar 

  93. Gherghiceanu, M. & Popescu, L. M. Heterocellular communication in the heart: electron tomography of telocyte–myocyte junctions. J. Cell. Mol. Med. 15, 1005–1011 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Quinn, T. A. et al. Electrotonic coupling of excitable and nonexcitable cells in the heart revealed by optogenetics. Proc. Natl Acad. Sci. USA 113, 14852–14857 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Wang, Z. et al. The molecular basis for sarcomere organization in vertebrate skeletal muscle. Cell 184, 2135–2150 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Chen, W. & Kudryashev, M. Structure of RyR1 in native membranes. EMBO Rep. 21, 49891 (2020).

    Article  Google Scholar 

  97. Wagenknecht, T., Hsieh, C. & Marko, M. Skeletal muscle triad junction ultrastructure by focused-ion-beam milling of muscle and cryo-electron tomography. Eur. J. Transl. Myol. 25, 49–56 (2015).

    Article  PubMed  Google Scholar 

  98. De Winter, D. A. M., Hsieh, C., Marko, M. & Hayles, M. F. Cryo-FIB preparation of whole cells and tissue for cryo-TEM: use of high-pressure frozen specimens in tubes and planchets. J. Microsc. 281, 125–137 (2021).

    Article  CAS  Google Scholar 

  99. Weber, M. S., Wojtynek, M. & Medalia, O. Cellular and structural studies of eukaryotic cells by cryo-electron tomography. Cells 8, 57 (2019).

    Article  CAS  PubMed Central  Google Scholar 

  100. Kitmitto, A. Applications of electron cryo-microscopy to cardiovascular research. Methods Mol. Med. 129, 315–327 (2006).

    CAS  PubMed  Google Scholar 

  101. Agip, A. N. A. et al. Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states. Nat. Struct. Mol. Biol. 25, 548–556 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Risi, C. et al. High-resolution cryo-EM structure of the cardiac actomyosin complex. Structure 29, 50–60 (2021).

    Article  CAS  PubMed  Google Scholar 

  103. von der Ecken, J., Heissler, S. M., Pathan-Chhatbar, S., Manstein, D. J. & Raunser, S. Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution. Nature 534, 724–728 (2016).

    Article  CAS  PubMed  Google Scholar 

  104. Elad, N. et al. The role of integrin-linked kinase in the molecular architecture of focal adhesions. J. Cell Sci. 126, 4099–4107 (2013).

    CAS  PubMed  Google Scholar 

  105. Samsó, M. A guide to the 3D structure of the ryanodine receptor type 1 by cryo-EM. Protein Sci. 26, 52–68 (2017).

    Article  PubMed  CAS  Google Scholar 

  106. Dulhunty, A. F., Beard, N. A. & Casarotto, M. G. Recent advances in understanding the ryanodine receptor calcium release channels and their role in calcium signalling. F1000Res. 7, 1851 (2018).

    Article  CAS  Google Scholar 

  107. Lee, C. H. & MacKinnon, R. Structures of the human HCN1 hyperpolarization-activated channel. Cell 168, 111–120 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Noreng, S., Li, T. & Payandeh, J. Structural pharmacology of voltage-gated sodium channels. J. Mol. Biol. 433, 166967 (2021).

    Article  CAS  PubMed  Google Scholar 

  109. Merino, F. et al. Structural transitions of F-actin upon ATP hydrolysis at near-atomic resolution revealed by cryo-EM. Nat. Struct. Mol. Biol. 25, 528–537 (2018).

    Article  CAS  PubMed  Google Scholar 

  110. Wu, M., Gu, J., Guo, R., Huang, Y. & Yang, M. Structure of mammalian respiratory supercomplex I1III2IV1. Cell 167, 1598–1609 (2016).

    Article  CAS  PubMed  Google Scholar 

  111. Zhu, J., Vinothkumar, K. R. & Hirst, J. Structure of mammalian respiratory complex I. Nature 536, 354–358 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Daghistani, H. M., Rajab, B. S. & Kitmitto, A. Three-dimensional electron microscopy techniques for unravelling mitochondrial dysfunction in heart failure and identification of new pharmacological targets. Br. J. Pharmacol. 176, 4340–4359 (2019).

    Article  CAS  PubMed  Google Scholar 

  113. Turk, M. & Baumeister, W. The promise and the challenges of cryo-electron tomography. FEBS Lett. 594, 3243–3261 (2020).

    Article  CAS  PubMed  Google Scholar 

  114. Renken, C. et al. Structure of frozen–hydrated triad junctions: a case study in motif searching inside tomograms. J. Struct. Biol. 165, 53–63 (2009).

    Article  CAS  PubMed  Google Scholar 

  115. Schorb, M., Haberbosch, I., Hagen, W. J. H., Schwab, Y. & Mastronarde, D. N. Software tools for automated transmission electron microscopy. Nat. Methods 16, 471–477 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  PubMed  Google Scholar 

  117. Buchholz, T. O. et al. Content-aware image restoration for electron microscopy. Methods Cell. Biol. 152, 277–289 (2019).

    Article  PubMed  Google Scholar 

  118. Fang, L. et al. Deep learning-based point-scanning super-resolution imaging. Nat. Methods 18, 406–416 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Bepler, T., Kelley, K., Noble, A. J. & Berger, B. Topaz-Denoise: general deep denoising models for cryoEM and cryoET. Nat. Commun. 11, 5208 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Heinrich, L. et al. Whole-cell organelle segmentation in volume electron microscopy. Nature 599, 141–146 (2021).

    Article  CAS  PubMed  Google Scholar 

  121. Berg, S. et al. ilastik: interactive machine learning for (bio)image analysis. Nat. Methods 16, 1226–1232 (2019).

    Article  CAS  PubMed  Google Scholar 

  122. Belevich, I., Joensuu, M., Kumar, D., Vihinen, H. & Jokitalo, E. Microscopy Image Browser: a platform for segmentation and analysis of multidimensional datasets. PLoS Biol. 14, 1002340 (2016).

    Article  CAS  Google Scholar 

  123. Vergara, H. M. et al. Whole-body integration of gene expression and single-cell morphology. Cell 184, 4819–4837 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Perez, A. J. et al. A workflow for the automatic segmentation of organelles in electron microscopy image stacks. Front. Neuroanat. 8, 126 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  125. Martinez-Sanchez, A., Garcia, I., Asano, S., Lucic, V. & Fernandez, J. J. Robust membrane detection based on tensor voting for electron tomography. J. Struct. Biol. 186, 49–61 (2014).

    Article  PubMed  Google Scholar 

  126. Hussain, A. et al. An automated workflow for segmenting single adult cardiac cells from large-volume serial block-face scanning electron microscopy data. J. Struct. Biol. 202, 275–285 (2018).

    Article  PubMed  Google Scholar 

  127. Khadangi, A., Boudier, T. & Rajagopal, V. EM-net: deep learning for electron microscopy image segmentation. bioRxiv https://doi.org/10.1101/2020.02.03.933127 (2020).

    Article  Google Scholar 

  128. Hatano, A. et al. Isolation and reconstruction of cardiac mitochondria from SBEM images using a deep learning-based method. J. Struct. Biol. 214, 107806 (2021).

    Article  PubMed  CAS  Google Scholar 

  129. Xu, C. S. et al. An open-access volume electron microscopy atlas of whole cells and tissues. Nature 599, 147–151 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Lucchi, A., Smith, K., Achanta, R., Knott, G. & Fua, P. Supervoxel-based segmentation of mitochondria in EM image stacks with learned shape features. IEEE Trans. Med. Imaging 31, 474–486 (2012).

    Article  PubMed  Google Scholar 

  131. Wei, D. et al. MitoEM dataset: large-scale 3D mitochondria instance segmentation from EM images. Med. Image Comput. Comput. Assist. Interv. 12265, 66–76 (2020).

    PubMed  PubMed Central  Google Scholar 

  132. Maron, B. J., Ferrans, V. J. & Roberts, W. C. Ultrastructural features of degenerated cardiac muscle cells in patients with cardiac hypertrophy. Am. J. Pathol. 79, 387–434 (1975).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. Maron, B. J., Ferrans, V. J. & Jones, M. The spectrum of degenerative changes in hypertrophied human cardiac muscle cells: an ultrastructural study. Recent. Adv. Stud. Card. Struct. Metab. 8, 447–466 (1975).

    CAS  Google Scholar 

  134. Burch, G. E. Ultrastructural myocardial changes produced by viruses. Recent. Adv. Stud. Card. Struct. Metab. 6, 501–523 (1975).

    CAS  Google Scholar 

  135. Brandenburg, S. et al. Junctophilin-2 expression rescues atrial dysfunction through polyadic junctional membrane complex biogenesis. JCI Insight 4, e127116 (2019).

    Article  PubMed Central  Google Scholar 

  136. Pinali, C. et al. Post-myocardial infarction T-tubules form enlarged branched structures with dysregulation of junctophilin-2 and bridging integrator 1 (BIN-1). J. Am. Heart Assoc. 6, e004834 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  137. Collins, H. E. et al. Mitochondrial morphology and mitophagy in heart diseases: qualitative and quantitative analyses using transmission electron microscopy. Front. Aging 2, 670267 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  138. Lavorato, M. et al. Dyad content is reduced in cardiac myocytes of mice with impaired calmodulin regulation of RyR2. J. Muscle Res. Cell Motil. 36, 205–214 (2015).

    Article  CAS  PubMed  Google Scholar 

  139. Takemura, G. et al. Electron microscopic findings are an important aid for diagnosing mitochondrial cardiomyopathy with mitochondrial DNA mutation 3243A>G. Circ. Heart Fail. 9, e003283 (2016).

    Article  PubMed  Google Scholar 

  140. Erlandson, R. A. Role of electron microscopy in modern diagnostic surgical pathology. Mod. Surg. Pathol. 1, 71–84 (2009).

    Article  Google Scholar 

  141. Beikoghli Kalkhoran, S. et al. Assessing the effects of mitofusin 2 deficiency in the adult heart using 3D electron tomography. Physiol. Rep. 5, e13437 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  142. Pinali, C. et al. Three-dimensional structure of the intercalated disc reveals plicate domain and gap junction remodeling in heart failure. Biophys. J. 108, 498–507 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Toomer, K. A. et al. Primary cilia defects causing mitral valve prolapse. Sci. Transl. Med. 11, eaax0290 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Ceska, T., Chung, C.-W., Cooke, R., Phillips, C. & Williams, P. A. Cryo-EM in drug discovery. Biochem. Soc. Trans. 47, 281–293 (2019).

    Article  CAS  PubMed  Google Scholar 

  145. Scapin, G., Potter, C. S. & Carragher, B. Cryo-EM for small molecules discovery, design, understanding, and application. Cell. Chem. Biol. 25, 1318–1325 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  146. Jiang, D. et al. Structure of the cardiac sodium channel. Cell 180, 122–134 (2020).

    Article  CAS  PubMed  Google Scholar 

  147. Jiang, D. et al. Structural basis for voltage-sensor trapping of the cardiac sodium channel by a deathstalker scorpion toxin. Nat. Commun. 12, 128 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. Wang, W. & MacKinnon, R. Cryo-EM structure of the open human ether-à-go-go-related K+ channel hERG. Cell 169, 422–430 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. Wang, M.-C. et al. The three-dimensional structure of the cardiac L-type voltage-gated calcium channel: comparison with the skeletal muscle form reveals a common architectural motif. J. Biol. Chem. 279, 7159–7168 (2004).

    Article  CAS  PubMed  Google Scholar 

  150. Zhao, Y. et al. Molecular basis for ligand modulation of a mammalian voltage-gated Ca2+ channel. Cell 177, 1495–1506 (2019).

    Article  CAS  PubMed  Google Scholar 

  151. Iyer, K. A. et al. Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM. Sci. Adv. 6, eabb2964 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Feng, X. et al. A fast and effective microfluidic spraying-plunging method for high-resolution single-particle cryo-EM. Structure 25, 663–670.e3 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  153. Unwin, N. & Fujiyoshi, Y. Gating movement of acetylcholine receptor caught by plunge-freezing. J. Mol. Biol. 422, 617–634 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  154. Lu, Z. et al. Gas-assisted annular microsprayer for sample preparation for time-resolved cryo-electron microscopy. J. Micromech. Microeng. 24, 115001 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  155. Mäeots, M.-E. et al. Modular microfluidics enables kinetic insight from time-resolved cryo-EM. Nat. Commun. 11, 3465 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  156. Jorgensen, A. O. & Campbell, K. P. Evidence for the presence of calsequestrin in two structurally different regions of myocardial sarcoplasmic reticulum. J. Cell Biol. 98, 1597–1602 (1984).

    Article  CAS  PubMed  Google Scholar 

  157. Thomas, M. J. et al. Localization and function of the Na+/Ca2+-exchanger in normal and detubulated rat cardiomyocytes. J. Mol. Cell. Cardiol. 35, 1325–1337 (2003).

    Article  CAS  PubMed  Google Scholar 

  158. Darkow, E. et al. The lectin LecA sensitizes the human stretch-activated channel TREK-1 but not Piezo1 and binds selectively to cardiac non-myocytes. Front. Physiol. 11, 457 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  159. Sartori-Rupp, A. et al. Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells. Nat. Commun. 10, 342 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  160. Shu, X. et al. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9, e1001041 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  161. Micheva, K. D. & Smith, S. J. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55, 25–36 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank the staff at the Electron Microscopy Core Facility EMBL Heidelberg for many years of on-site support and advice, as well as A. Vlachos, J. Madl and J. O’Reilly, all at the University of Freiburg, for helpful comments on the manuscript. E.A.R.-Z. is a German Research Foundation Emmy Noether Fellow (DFG #396913060). The authors are members of the German Research Foundation Collaborative Research Centre SFB1425 (DFG #422681845).

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Authors and Affiliations

  1. Institute for Experimental Cardiovascular Medicine, University Heart Center and Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Peter Kohl, Joachim Greiner & Eva A. Rog-Zielinska

  2. Faculty of Engineering, University of Freiburg, Freiburg, Germany

    Peter Kohl

  3. Centre for Integrative Biological Signalling Studies (CIBSS), University of Freiburg, Freiburg, Germany

    Peter Kohl

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  1. Peter Kohl
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Correspondence to Eva A. Rog-Zielinska.

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IMOD: https://bio3d.colorado.edu/imod/

Glossary

Electron microscopy

(EM). A microscopy method in which the sample is exposed to a beam of accelerated electrons, while images are obtained either by trans-illumination of samples with spatially variable permeability for electrons, or reconstructed from electrons reflected by the sample surface in scanning mode; this allows in-plane linear resolutions of <1 nm (102–103 times higher than conventional photon-based microscopy).

Electron tomography

(ET). A 3D EM method in which a sample (typically 200–300 nm thick) is tilted relative to the imaging plane of the electron beam, and individual transmitted images are captured (usually between −60o and +60o tilt levels, and often along two mutually perpendicular tilt axes); using these images, the sample volume can be reconstructed with a native voxel size <1 nm3.

Cryo-EM

A method of imaging samples while frozen and hydrated; by omitting fixatives, solvent substitution, resin embedding and heavy metal staining, researchers can visualize native nanostructural details down to the level of single molecules.

Serial block-face or focused ion beam SEM

3D EM methods in which, between individual runs of SEM image acquisition, a thin layer of the sample surface is removed, either mechanically or using a focused ion beam; the sample volume is reconstructed from voxels whose resolution is limited by the surface removal technique, usually yielding voxels of ≥10 nm3.

Anisotropic

The property of an object, here the unitary 3D imaging readouts (voxels), that does not have edge lengths of equal size; usually, the edge lengths in the imaging plane are identical, while that between imaging planes is larger (the voxel shape is a square cuboid).

Isotropic

The property of an object, here the unitary 3D imaging readouts (voxels), that has edge lengths of equal size (the voxel shape is a cube).

Single-particle analysis

(SPA). A variant of cryo-EM that uses post-acquisition methods to three-dimensionally reconstruct individual molecules (such as proteins) by combining multiple images (usually thousands) of a population of molecules at random angular orientations; this approach allows researchers to achieve near-atomic-scale structural resolution.

Correlative light and electron microscopy

(CLEM). An approach in which a single sample is first imaged using fluorescence microscopy (to visualize the presence or dynamics of suitable reporters in live or fixed cells) and then, aided by meticulous transfer of coordinates, re-imaged using EM; correlation of the resulting data sets allows researchers to interrelate nanoscale to mesoscale structural and functional information on cells and subcellular structures.

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Kohl, P., Greiner, J. & Rog-Zielinska, E.A. Electron microscopy of cardiac 3D nanodynamics: form, function, future. Nat Rev Cardiol 19, 607–619 (2022). https://doi.org/10.1038/s41569-022-00677-x

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