Effects of calsequestrin over-expression on excitation-contraction coupling in isolated rabbit cardiomyocytes

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Miller, S.L.W. and Currie, S. and Loughrey, C.M. and Kettlewell, S. and Seidler, T. and Reynolds, D.F. and Hasenfuss, G. and Smith, G.L. (2005) Effects of calsequestrin over-expression on excitation-contraction coupling in isolated rabbit cardiomyocytes. Cardiovascular Research, 67. pp. 667-677. ISSN 0008-6363 (http://dx.doi.org/10.1016/j.cardiores.2005.04.023)

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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 β-galactosidase (Ad-LacZ) was used as a control. Results: In voltage-clamped, Fura-2-loaded cardiomyocytes, L-type Ca2+ current (ICa,L) and Ca2+ transient amplitude were both increased in the Ad-CSQ group by ∼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 μM) was used to reduce ICa,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 ∼60% higher as assessed by both the caffeine-induced Ca2+ release and the accompanying Na+/Ca2+ exchanger current (INCX). The cause of the increased ICa,L is unknown. No change in the expression level of the α1-subunit of the L-type Ca channel was observed. β-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.

ORCID iDs

Miller, S.L.W., Currie, S. ORCID logoORCID: https://orcid.org/0000-0002-4237-4428, Loughrey, C.M., Kettlewell, S., Seidler, T., Reynolds, D.F., Hasenfuss, G. and Smith, G.L.;
CORE (COnnecting REpositories)