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  • Title: Effects of calsequestrin over-expression on excitation-contraction coupling in isolated rabbit cardiomyocytes.
    Author: Miller SL, Currie S, Loughrey CM, Kettlewell S, Seidler T, Reynolds DF, Hasenfuss G, Smith GL.
    Journal: Cardiovasc Res; 2005 Sep 01; 67(4):667-77. PubMed ID: 15913577.
    Abstract:
    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.
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