title: A novel Z-groove index characterizing myocardial surface structure.
abstract: OBJECTIVE
  The role of t-tubule structures in excitation-contraction coupling of ventricular myocytes has been investigated by disruption using prolonged culture, or osmotic shock with formamide. We have used a new method, the Scanning Ion Conductance Microscope (SICM), to investigate in more detail the changes in surface structure of live myocytes during these interventions and to relate them to contractile effects.
  
  
  METHODS
  Freshly isolated ventricular myocytes from adult rat hearts were either incubated with formamide, then washed to produce osmotic shock, or put into culture for 2, 4 and 7 days. Contractile characteristics of single myocytes were then measured using the IonOptix system, and in parallel imaged using the SICM which produces a 3-dimensional topographical representation of the cell surface. Loss of t-tubules was quantitated with confocal microscopy after staining with the membrane dye di-8-ANNEPS, and sarcomere structure revealed by immunocytochemical detection of alpha-actinin.
  
  
  RESULTS
  Detubulation was produced by either method, with formamide equivalent to 4 day culture in quantitative measures of ANNEPS t-tubule/membrane ratio. SICM images confirmed the loss of t-tubule indentations. Disruption of the Z-groove structure and flattening of the surface were also noted with formamide and, to a lesser extent, culture. A novel Z-groove index was introduced to describe this effect more quantitatively. Contraction and relaxation were impaired by the detubulation methods, but formamide had a markedly greater depressant effect on contraction amplitude than equivalent detubulation by culture.
  
  
  CONCLUSIONS
  Changes in contraction amplitude after detubulation with formamide were more closely related to the alteration in Z-groove structure than loss of t-tubules alone. As well as disrupting t-tubule-induced excitation and calcium movements, formamide may alter the transmission of contraction in the myocyte by interference with sarcomere attachment at the Z-line.
authors.affiliation: National Heart and Lung Institute, Imperial College, London SW3 6LY, UK.
authors.forename: Julia
authors.initials: J
authors.lastname: Gorelik
authors.forename: Lin Q
authors.initials: LQ
authors.lastname: Yang
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Max
authors.initials: M
authors.lastname: Lab
authors.forename: Yuri
authors.initials: Y
authors.lastname: Korchev
authors.forename: Sian E
authors.initials: SE
authors.lastname: Harding
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure; D002478:Cells, Cultured; D004553:Electric Conductivity; D004594:Electrophysiology; D005456:Fluorescent Dyes; D005559:Formamides / Q000494:pharmacology; D008297:Male; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning / Q000379:methods; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000502:physiology / Q000648:ultrastructure*; D009997:Osmotic Pressure; D011726:Pyridinium Compounds; D051381:Rats; D017207:Rats, Sprague-Dawley; D012518:Sarcomeres / Q000648:ultrastructure; D013997:Time Factors
chemical_list: C089669:1-(3-sulfonatopropyl)-4-(beta-(2-(di-n-octylamino)-6-naphthyl)vinyl)pyridinium betaine; D005456:Fluorescent Dyes; D005559:Formamides; D011726:Pyridinium Compounds; C031066:formamide