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- Title
Effects of calsequestrin over-expression on excitation–contraction coupling in isolated rabbit cardiomyocytes
- Authors
Miller, Stewart L.W.; Currie, Susan; Loughrey, Christopher M.; Kettlewell, Sarah; Seidler, Tim; Reynolds, Deborah F.; Hasenfuss, Gerd; Smith, Godfrey L.
- Abstract
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 (I Ca,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 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 ∼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 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.
- Subjects
HEART cells; HEART cytology; PROTEINS; BIOMOLECULES; ORGANIC compounds
- Publication
Cardiovascular Research, 2005, Vol 67, Issue 4, p667
- ISSN
0008-6363
- Publication type
Article
- DOI
10.1016/j.cardiores.2005.04.023