Cell Calcium 40 (2006) 299–307
Measurement of sub-membrane [Ca
2+
] in adult myofibers and
cytosolic [Ca
2+
] in myotubes from normal and mdx mice
using the Ca
2+
indicator FFP-18
Renzhi Han
a,∗
, Miranda D. Grounds
b
, Anthony J. Bakker
a
a
School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
b
School of Anatomy and Human Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
Received 24 February 2006; received in revised form 4 April 2006; accepted 12 April 2006
Available online 12 June 2006
Abstract
The hypothesis that intracellular Ca
2+
is elevated in dystrophic (mdx) skeletal muscle due to increased Ca
2+
influx is controversial. As
the sub-sarcolemmal Ca
2+
([Ca
2+
]
mem
) should be even higher than the global cytosolic Ca
2+
in the presence of increased Ca
2+
influx, we
investigated [Ca
2+
]
mem
levels in collagenase-isolated adult flexor digitorum brevis (FDB) myofibres and myotubes of mdx and normal mice
with the near-membrane Ca
2+
indicator FFP-18. Confocal imaging showed strong localization of FFP-18 to the sarcolemma only. No significant
difference in [Ca
2+
]
mem
was found in FDB myofibres of normal (77.3 ± 3.8 nM, n = 68) and mdx (79.3 ± 5.6 nM, n = 21, p = 0.89) mice using
FFP-18. Increasing external Ca
2+
to 18 mM did not significantly affect [Ca
2+
]
mem
in either the normal or mdx myofibres. In the myotubes, the
FFP-18 was non-selectively incorporated, distributing throughout the cytoplasm, and FFP-18-derived [Ca
2+
] values were similar to values
obtained with Fura-2. Nevertheless, in the mdx myotubes, the [Ca
2+
] measured with FFP-18 increased linearly to a level ∼2.75 times that of
controls as the time of culture was prolonged. In older mdx myotubes (≥8 days in culture), 18 mM extracellular Ca
2+
increased the steady state
cytosolic [Ca
2+
] to ∼22 times greater level than controls. This study suggests that the sub-sarcolemmal Ca
2+
homeostasis is well maintained
in isolated adult mdx myofibers and also further supports the hypothesis that cytosolic Ca
2+
handling is compromised in mdx myotubes.
© 2006 Elsevier Ltd. All rights reserved.
Keywords: Ca
2+
; Dystrophin; Duchenne muscular dystrophy; Excitation-contraction coupling; Mdx; Skeletal muscle
1. Introduction
Patients with Duchenne muscular dystrophy (DMD) expe-
rience skeletal muscle wasting that results in eventual death
by the third decade of life [1]. DMD results from the absence
of the structural protein dystrophin [2]. Mdx mice also lack
dystrophin and are used as an animal model of DMD [2].
Dystrophin-deficient skeletal muscle cells are thought to be
more prone to abnormal intracellular Ca
2+
accumulation,
∗
Correspondence to: Howard Hughes Medical Institute, University of
Iowa College of Medicine, 4283 Carver Biomedical Research Building, 285
Newton Road, Iowa City, IA 52242, USA. Tel.: +1 319 335 9936;
fax: +1 319 335 6957.
E-mail address: renzhi-han@uiowa.edu (R. Han).
which eventually contributes to muscle necrosis through acti-
vation of Ca
2+
-activated proteases [3]. A number of processes
may be responsible for this phenomenon. The absence of dys-
trophin renders the sarcolemma more fragile and prone to
contraction-induced damage, resulting in the influx of extra-
cellular Ca
2+
and other ions through transient tears in the
sarcolemma during contractile activity [2]. The loss of dys-
trophin is also reported to increase the activity of various
Ca
2+
-permeable ion channels in the plasma membrane of
dystrophic skeletal muscle cells such as mechanosensitive
and transient receptor potential channels [4–8].
Consistent with the hypothesis of altered intracellular
Ca
2+
homeostasis in dystrophic muscle, abnormally high
levels of total intracellular calcium have been reported in
the skeletal muscle of DMD patients [9,10]. Elevated free
0143-4160/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ceca.2006.04.016