Iron overload and apoptosis of HL-1 cardiomyocytes: effects of calcium channel blockade.
BACKGROUND
Iron overload cardiomyopathy that prevails in some forms of hemosiderosis is caused by excessive deposition of iron into the heart tissue and ensuing damage caused by a raise in labile cell iron. The underlying mechanisms of iron uptake into cardiomyocytes in iron overload condition are still under investigation. Both L-type calcium channels (LTCC) and T-type calcium channels (TTCC) have been proposed to be the main portals of non-transferrinic iron into heart cells, but controversies remain. Here, we investigated the roles of LTCC and TTCC as mediators of cardiac iron overload and cellular damage by using specific Calcium channel blockers as potential suppressors of labile Fe(II) and Fe(III) ingress in cultured cardiomyocytes and ensuing apoptosis.


METHODS
Fe(II) and Fe(III) uptake was assessed by exposing HL-1 cardiomyocytes to iron sources and quantitative real-time fluorescence imaging of cytosolic labile iron with the fluorescent iron sensor calcein while iron-induced apoptosis was quantitatively measured by flow cytometry analysis with Annexin V. The role of calcium channels as routes of iron uptake was assessed by cell pretreatment with specific blockers of LTCC and TTCC.


RESULTS
Iron entered HL-1 cardiomyocytes in a time- and dose-dependent manner and induced cardiac apoptosis via mitochondria-mediated caspase-3 dependent pathways. Blockade of LTCC but not of TTCC demonstrably inhibited the uptake of ferric but not of ferrous iron. However, neither channel blocker conferred cardiomyocytes with protection from iron-induced apoptosis.


CONCLUSIONS
Our study implicates LTCC as major mediators of Fe(III) uptake into cardiomyocytes exposed to ferric salts but not necessarily as contributors to ensuing apoptosis. Thus, to the extent that apoptosis can be considered a biological indicator of damage, the etiopathology of cardiosiderotic damage that accompanies some forms of hemosiderosis would seem to be unrelated to LTCC or TTCC, but rather to other routes of iron ingress present in heart cells.
Department of Pediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, China.
Mei-pian
MP
Chen
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Safra Campus at Givat Ram, Jerusalem, Israel.
Z Ioav
ZI
Cabantchik
Department of Pediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, China.
Shing
S
Chan
Department of Pediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, China.
Godfrey Chi-fung
GC
Chan
Department of Pediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, China.
Yiu-fai
YF
Cheung
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D020747:Calcium Channels, T-Type / Q000378:metabolism*; D009202:Cardiomyopathies / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D005290:Ferric Compounds / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D007501:Iron / Q000378:metabolism*; D019190:Iron Overload / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D020747:Calcium Channels, T-Type; D005290:Ferric Compounds; D007501:Iron; D053148:Caspase 3; D002118:Calcium