Characterization of Spatial-Temporal Heterogeneity Induced by Flecainide Using Clinically Valid Concentrations

Scientists’   growing   suspicion   regarding   the   predictive   capacity   of   single   cell   measurements   was   triggered   by   the   counterintuitive   results   of   CAST   I   in   1989⁴⁷⁴.   Despite   the   fact   that   the   antiarrhythmic   activity   of   NaV1.5-­‐blockers   has   been   demonstrated   quite   robustly   and   redundantly458,  511-­‐513  over  more  than  three  decades  of  experiments,  the  increased  mortality  rates  of   CAST  I  disproved  this  predictive  potential.  Flecainide,  being  an  antiarrhythmic  drug  known  for  its   strong   modulation   of   NaV1.5⁴⁶⁶,   has   been   shown   to   evoke   proarrhythmic   responses   in   cardiac  

tissues  exposed  to  the  drug^{474,  482}.  Since  then,  several  hypotheses  have  been  proposed  to  clarify  the   mechanisms  behind  the  electrical  instabilities  that  collectively  develop  in  the  intact  tissue495,  497,  499.     We  investigated  the  effects  of  this  drug  using  clinically  valid  concentrations,  to  observe  and  measure   the   different   biophysical   parameters   at   play   that   could   give   us   some   insights   on   the   electrical   instabilities   emanating   from   modifying   the   channel   in   three   different   cardiac   substrates,   with   a   gradient   in   NaV1.5   functionality:   one   with   lower   NaV1.5   availability   (mdx,  section   3.2),   the   second   one  with  a  normally  functioning  channel  (WT,  section  3.2.2)  and  the  last  one  with  a  hyper-­‐functional   channel  (ΔKPQ,  section  3.3).  In  this  regard,  optical  mapping  is  a  fundamental  experimental  tool  in   the  study  of  spatially  extended  electrophysiological  heterogeneity  of  the  cardiac  substrate,  without   which  appreciation  of  the  complexity  of  these  models  wouldn’t  have  been  possible.  

This  Thesis          49  

(This  page  is  intentionally  left  blank)    

Chapter 2

Experimental Methods

The  present  chapter  details  the  methods  used  to  obtain  the  experimental  results  of  this  thesis.  The   main   experimental   protocol   used   is   optical   mapping   of   the   propagating   electrical   wave   on   the   surface   of   the   murine   left   ventricular   (LV)   free   wall   epicardium,   using   Di-­‐4-­‐Anepps,   a   voltage   sensitive  dye  (VSD).  Section  2.1  outlines  the  details  the  optical  mapping  setup  in  terms  of  hardware   and   software   used:   vECG   recordings   and   pacing   electrodes   (2.1.1),   the   details   of   murine   heart   isolation   (2.1.2).   Optical   mapping   of   membrane   voltage   measurements   were   performed   using   an   electromechanical   decoupler   and   a   VSD   (2.1.3)   and   details   of   the   setup   are   elaborated   in   section   2.1.4.   Section   2.2   outlines   the   details   concerning   the   optimization   of   signal   quality   for   further   analysis  and  the  mathematical  background  of  the  strategies  used  in  this  thesis  to  evaluate  the  CV  in   the   medium   of   propagation.   Starting   with   processing   the   raw   optical   signal   and   the   creation   of   activation  maps  (2.2.1),  to  dispersion  maps  (2.2.3),  we  also  show  the  methodological  approaches  in   the  evaluation  of  CV  2.2.4,  as  well  as  the  numerical  data  used  to  simulate  propagation  in  the  murine   heart,   which   are   detailed   in   section   2.2.5.   Results   of   the   optical   mapping   data   are   presented   in   Chapter  3  and  are  further  discussed  in  Chapter  4.  

The  study  of  electrophysiological  properties  of  the  healthy  and  diseased  heart  provides  important   insights  into  the  understanding  of  the  complex  electrical  activity,  which  requires  the  development  of   mapping   techniques   that   simultaneously   record   spatial   and   temporal   information.   Traditionally,   surface  electrodes  have  been,  and  continue  to  be,  used  measure  extracellular  cardiac  potentials⁵¹⁴   (Figure  5b).  However,  this  type  of  surface  mapping  actually  suffers  from  several  drawbacks,  such  as   low  spatial  resolution  and  reduced  flexibility  (since  only  a  finite  number  of  electrodes  can  be  placed   on  the  surface  of  one  heart  with  a  fixed  spacing  between  electrodes  once  the  electrode  array  is  set),   low  depth  of  field  and  electrical  (interference)  artifacts  from  stimulating  electrodes⁵¹⁴.    The  mouse   heart  is  a  widespread  model  for  cardiovascular  studies,  due  to  factors  like  the  existence  of  low  cost   technology  for  genetic  engineering  in  this  species⁵¹⁵,  the  relatively  fast  reproduction  capacity  of  the   animal  and  the  ease  of  handling  the  animal  for  experimental  work.  Nevertheless,  the  use  of  murine   hearts  for  gathering  electrophysiological  data  is  a  particular  challenging  task.  It’s  faced  with  several   technical   difficulties   that   start   with   the   rapid   heart   rate⁵⁰¹,   the   undersized   heart   (the   apico-­‐basal   distance   is   ~6mm)   and   the   restricted   time   for   the   spread   of   any   activating   wave   front   (a   propagating   electrical   wave   can   traverse   the   entire   epicardium   in   less   than   6-­‐8ms)⁵⁰⁴.   For   such   a   small   heart,   the   number   of   extracellular   electrodes   is   limited   by   spatial   constraints   and   a   lower   number  of  detection  sites  makes  the  electrode  array  a  poor  choice  for  detecting  complex  electrical   patterns.  

Optical  mapping  with  VSDs  has  made  it  possible  to  record  cardiac  APs  with  high  spatial-­‐temporal   resolution  that  is  otherwise  not  attainable  using  electrode  arrays⁵⁰⁵.  Optical  techniques  use  changes   in  transmitted  light  from  the  prep  to  map  the  electrical  activity.  VSDs  are  compounds  that  bind  to   cell   membranes   and   fluoresce   with   an   intensity   proportional   to   the   local   membrane   potential⁵¹⁶,  

and  with  a  response  time  which  is  several  orders  of  magnitude  faster  than  the  most  rapid  changes  in   the  cardiac  membrane  potential⁵¹⁷.    VSDs  can  produce  phototoxic  effects.  For  instance,  Di-­‐4-­‐Anepps,   a  widespread  used  VSD  in  cardiac  mapping,  is  reported  not  to  be  toxic  at  low  concentrations  in  the   absence  of  light,  but  degrades  membranes  in  the  presence  of  increased  levels  of  light,  where  light   absorption   at   high   intensities   can   cause   tissue   heating   and   alterations   in   electrophysiological   parameters⁵¹⁸.  Therefore,  a  major  advantage  of  electrode  arrays  is  that  the  prep  is  not  subjected  to   potentially  phototoxic  effects  of  potentiometric  dyes⁵¹⁹.  In  addition,  these  electrodes  record  are  very   fast   and   capable   of   measuring   the   AP   upstroke   at   a   temporal   rate   close   to   0.1ms520,   521.   Another   fundamental  difference  between  electrodes  and  optical  AP  (OAP)  recordings  is  related  to  the  source   of   these   signals⁵¹⁶.   Using   extracellular   electrodes,   the   source   is   a   single   cell,   whereas   an   OAP   originates  from  a  small  lump  of  cells,  where  the  overall  volume  depends  on  several  factors  such  as   optical   magnification,   detector   size,   light   transmission   properties   of   the   cardiac   tissue⁵¹⁶.   For   comprehensive  reviews  regarding  optical  mapping,  refer  to  the  reviews  by  Girouard  et  al.⁵¹⁶  (1996),   Efimov⁵⁰⁵  (2004),  Herron  and  Jalife⁵¹⁴  (2012).