title: Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes.
abstract: Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes.
  
  
  
  To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes.
  
  
  
  Freshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy.
  
  
  
  Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Samir
authors.initials: S
authors.lastname: Awasthi
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Leighton T
authors.initials: LT
authors.lastname: Izu
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Ziliang
authors.initials: Z
authors.lastname: Mao
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Jian
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Trevor
authors.initials: T
authors.lastname: Landas
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Aaron
authors.initials: A
authors.lastname: Lerner
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Rafael
authors.initials: R
authors.lastname: Shimkunas
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Rahwa
authors.initials: R
authors.lastname: Woldeyesus
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Julie
authors.initials: J
authors.lastname: Bossuyt
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Brent M
authors.initials: BM
authors.lastname: Wood
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Yi-Je
authors.initials: YJ
authors.lastname: Chen
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Dennis L
authors.initials: DL
authors.lastname: Matthews
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Deborah K
authors.initials: DK
authors.lastname: Lieu
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Nipavan
authors.initials: N
authors.lastname: Chiamvimonvat
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis.
authors.forename: Kit S
authors.initials: KS
authors.lastname: Lam
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis. jwjchan@ucdavis.edu ychenizu@ucdavis.edu.
authors.forename: Ye
authors.initials: Y
authors.lastname: Chen-Izu
authors.affiliation: From the Center for Biophotonics (S.A., Z.M., A.L., D.L.M., J.W.C.), Division of Cardiology (D.K.L., N.C., Y.C.-I.), Division of Hematology/Oncology, Department of Internal Medicine (K.S.L.), and Department of Pathology and Laboratory Medicine (J.W.C.), UC Davis School of Medicine, University of California, Davis, Sacramento; and Departments of Pharmacology (L.T.I., Z.J., T.L., J.B., B.W., Y.-J.C., Y.C.-I.), Biomedical Engineering (S.A., R.S., R.W., Y.C.-I.), Biochemistry and Molecular Medicine (K.S.L.), and Microsurgery Core (Y.-J.C.), University of California, Davis. jwjchan@ucdavis.edu ychenizu@ucdavis.edu.
authors.forename: James W
authors.initials: JW
authors.lastname: Chan
mesh_terms: D000814:Aniline Compounds; D000818:Animals; D009202:Cardiomyopathies / Q000378:metabolism* / Q000503:physiopathology; D004195:Disease Models, Animal; D056966:Excitation Contraction Coupling; D005456:Fluorescent Dyes; D007700:Kinetics; D008297:Male; D040542:Mechanotransduction, Cellular; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton; D064847:Multimodal Imaging / Q000379:methods*; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D012518:Sarcomeres / Q000378:metabolism*; D013314:Stress, Mechanical; D014966:Xanthenes
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D005456:Fluorescent Dyes; D014966:Xanthenes
keywords: calcium signaling; cardiomyopathies; mechanotransduction, cellular; microscopy, fluorescence, multiphoton; multimodal imaging; myocardial contraction; sarcomeres