Effects of calsequestrin over-expression on excitation-contraction coupling in isolated rabbit cardiomyocytes.
OBJECTIVE
This study investigated the role of calsequestrin (CSQ) in the control of excitation-contraction (E-C) coupling in the heart.


METHODS
CSQ over-expression was induced in isolated rabbit ventricular cardiomyocytes using an adenovirus coding for rabbit CSQ (Ad-CSQ). After 24 h of culture, CSQ protein expression was increased by 58+/-18% (n=10). An adenovirus coding for beta-galactosidase (Ad-LacZ) was used as a control.


RESULTS
In voltage-clamped, Fura-2-loaded cardiomyocytes, L-type Ca2+ current (I(Ca,L)) and Ca2+ transient amplitude were both increased in the Ad-CSQ group by approximately 78%. Doubling the external Ca2+ concentration in the control group (Ad-LacZ) increased the LTCC amplitude to a similar degree (85+/-6%), but increased the Ca2+ transient amplitude by 149+/-13%. This suggests that SR Ca2+ release may be inhibited upon CSQ over-expression. Alternatively, nifedipine (0.5 microM) was used to reduce I(Ca,L) in Ad-CSQ-transfected cells to values comparable to control (Ad-LacZ). Under these conditions, Ca2+ transient amplitude was not different from Ad-LacZ, but the SR Ca2+ content was approximately 60% higher as assessed by both the caffeine-induced Ca2+ release and the accompanying Na+/Ca2+ exchanger current (I(NCX)). The cause of the increased I(Ca,L) is unknown. No change in the expression level of the alpha1-subunit of the L-type Ca channel was observed. beta-Escin-permeabilized cardiomyocytes were used to study Ca2+ sparks imaged with Fluo-3 at 145-155 nmol/L [Ca2+]. Spontaneous Ca2+ spark frequency, duration, width, and amplitude were unchanged in the Ad-CSQ group, but SR Ca2+ content was 48% higher than Ad-LacZ.


CONCLUSIONS
CSQ over-expression increased SR Ca2+ content but reduced the gain of E-C coupling in rabbit cardiomyocytes.
Miller
Stewart L W
SL
Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
Currie
Susan
S
Loughrey
Christopher M
CM
Kettlewell
Sarah
S
Seidler
Tim
T
Reynolds
Deborah F
DF
Hasenfuss
Gerd
G
Smith
Godfrey L
GL
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism; D002155:Calsequestrin / Q000235:genetics / Q000378:metabolism*; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D004594:Electrophysiology; D015870:Gene Expression; D005822:Genetic Vectors / Q000008:administration & dosage; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000502:physiology; D009543:Nifedipine / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D002155:Calsequestrin; D000697:Central Nervous System Stimulants; D019831:Sodium-Calcium Exchanger; D002110:Caffeine; D009543:Nifedipine; D002118:Calcium