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

S.L.W. Miller, S. Currie, C.M. Loughrey, S. Kettlewell, T. Seidler, D.F. Reynolds, G. Hasenfuss, G.L. Smith

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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.
Original languageEnglish
Pages (from-to)667-677
Number of pages11
JournalCardiovascular Research
Volume67
DOIs
Publication statusPublished - May 2005

Keywords

  • E–C coupling SR (function)
  • ion channels
  • calcium (cellular)

Cite this

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