Mysterious Ca(2+)-independent muscular contraction: déjà vu.

Abstract

The permeabilized cells and muscle fibres technique allows one to study the functional properties of mitochondria without their isolation, thus preserving all of the contacts with cellular structures, mostly the cytoskeleton, to study the whole mitochondrial population in the cell in their natural surroundings and it is increasingly being used in both experimental and clinical studies. The functional parameters (affinity for ADP in regulation of respiration) of mitochondria in permeabilized myocytes or myocardial fibres are very different from those in isolated mitochondria in vitro. In the present study, we have analysed the data showing the dependence of this parameter upon the muscle contractile state. Most remarkable is the effect of recently described Ca(2+)-independent contraction of permeabilized muscle fibres induced by elevated temperatures (30-37°C). We show that very similar strong spontaneous Ca(2+)-independent contraction can be produced by proteolytic treatment of permeabilized muscle fibres that result in a disorganization of mitochondrial arrangement, leading to a significant increase in affinity for ADP. These data show that Ca(2+)-insensitive contraction may be related to the destruction of cytoskeleton structures by intracellular proteases. Therefore the use of their inhibitors is strongly advised at the permeabilization step with careful washing of fibres or cells afterwards. A possible physiologically relevant relationship between Ca(2+)-regulated ATP-dependent contraction and mitochondrial functional parameters is also discussed.

Full text

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MYSTERIOUS Ca 2+- INDEPENDENT MUSCULAR CONTRACTION : déjà vu

Andrey V. Kuznetsov*, Rita Guzun†, François Boucher†, Rafaela Bagur†,
Accepted Manuscript
Tuuli Kaambre‡ and Valdur Saks†‡







* Cardiac Surgery Research Laboratory, Department of Heart Surgery,
Innsbruck Medical University, Innsbruck, Austria

† INSERM U1055
Laboratory of Fundamental and Applied Bioenergetics,
Joseph Fourier University, Grenoble, France

‡ Laboratory of Bioenergetics,
National Institute of Chemical Physics and Biophysics, Tallinn, Estonia




Address for Correspondence: V.A.Saks
Laboratory of Bioenergetics
Joseph Fourier University
2280, Rue de la Piscine
BP53X – 38041
Grenoble Cedex 9, France
e-mail : Valdur.Saks@ujf-grenoble.fr

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ABSTRACT
The permeabilized cells and muscle fibers technique allows to study the functional
properties of mitochondria without their isolation, thus preserving intact all contacts with
cellular structures, mostly cytoskeleton, to study the whole population of mitochondria in the
cell in their natural surrounding and is increasingly used both in experimental and clinical
studies. The functional parameters (affinity for ADP in regulation of respiration) of
mitochondria in permeabilized myocytes or myocardial fibers are very different from those in
isolated mitochondria in vitro. In this article we analyze the data showing the dependence of
this parameter upon muscle contractile state. Most remarkable is the effect of recently
described calcium- independent contraction of permeabilized muscle fibers induced by
elevated temperatures (30-37°C). We show that very similar strong spontaneous calcium-
independent contraction can be produced by proteolytic treatment of permeabilized muscle
fibers that resulting in a disorganization of mitochondrial arrangement leading to significant
increase of affinity for ADP. These data show that calcium-insensitive contraction may be
related to the destruction of cytoskeleton structures by intracellular proteases. Therefore, use
of their inhibitors is strongly advised at the step of permeabilization with careful washing of
fibers or cells afterwards. Possible physiologically relevant relationship between calcium-
regulated, ATP-dependent contraction and mitochondrial functional parameters is also
discussed.


Key words: skeletal muscle, heart, permeabilized myocytes, contraction, respiration,
mitochondria
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INTRODUCTION.
Accepted Manuscript
With great interest we have read recent paper by Christopher G. R. Perry et al.
entitled: « Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory
control in skeletal muscle » published in Biochemical Journal 2011: 437, 215–222. In the
paper of Perry et al. the authors used permeabilized fiber bundles (PmFBs) technique for
studies of regulation of respiration both in skeletal muscle of rat (either red or white
gastrocnemius) and biopsy samples taken from human skeletal muscle (vastus lateralis) [1].
Since we were among the first who suggested and widely used permeabilized muscle fibers to
study delicate mechanisms of energy metabolism and energy transfer in various types of
muscles [2] and have collected a lot of experience in this field, we would like to make several
comments to this paper. Importantly, the permeabilized cells and fibers technique allows to
study the functional properties of mitochondria without their isolation, thus preserving all
interactions with other cellular structures intact (mostly with cytoskeleton and ER) and to
study the whole population of mitochondria in the cell in their natural surrounding [2,3].
Another advantage is that this approach allows analyzing very small, limited tissue samples
(few mg) like human biopsies. This is why the PmFBs technique is increasingly used in many
experimental and clinical studies [1-5]. Perry et al. have made a detailed and interesting study
of the influence of several factors, such as myosin ATPase inhibition, temperature,
oxygenation and contraction on the functional parameters of mitochondrial oxidative
phosphorylation – apparent Km for ADP in regulation of respiration and maximal respiration
rates [1]. The most exiting finding of this paper is the important role of fibers contraction in
all these processes, which was found as rather mysterious both for authors and for readers.
Very strong contraction was induced simply by placing fibers into solution which contained
high concentration of EGTA and neither calcium nor added ATP, at 30°C and at higher
temperature [1]. Thus, the muscle contraction was independent of calcium even if there were
trace amounts of adenine nucleotides left in the cells. This strong shortening, a
supercontraction, was rather rapid and significantly decreased the apparent Km values for
ADP [1].
It is most interesting to understand what is behind this unusual but very strong muscle
shortening excluding calcium-regulated actomyosin contraction cycle. In our work we have
made one observation that could help to solve the mystery of calcium-insensitive contraction
seen by Perry et al. [1] and even explain it, as it is described below.
MATERIALS AND METHODS.
Cardiomyocytes were isolated as described in [6, 7]. The permeabilized fibers were
prepared from rat soleus muscle as described in [2]. The dissection of the muscle strips
immersed in ice-cold solution A [2] was carried out under microscope or magnifying glasses,
through diverse smooth horizontal moves in order to obtain thin muscle-fibers bundles. It is
important to take in account that this procedure has to be done at 4oC and under a microscope
using a cold light source (optical fibers, LED) to avoid damage the muscle fibers due to heat.
Another important point is dissection time, because of spending more than 20 min for
obtaining the fibers bundles could trigger hypoxia effects, damaging the mitochondrial
oxidative-phosphorylation. The permeabilization of the fibers bundles was performed in a vial
containing 3mL of solution A (supplemented with 2mg·mL-1 BSA) and 50 µg·mL-1 saponin
(in some experiments 100 µg·mL-1 were used) and shaking gently at 4oC for 30 min. After this
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step permeabilized fibers were transfered to a tube containing Mitomed [2] (supplemented
with 2 mg·mL-1 BSA and leupeptin 1 µM) and shaked gently at 4oC for 10 min to wash out
saponin, other metabolites, especially traces of ADP or ATP, and proteases released during
fiber preparation. This washing procedure was repeated 3 times. Mitochondrial respiration of
permeabilized cells was measured in Mitomed solution in the presence of leupeptin [2].
Accepted Manuscript
Mitochondria were visualized with MitoTracker Green or with TMRM as described in
[7]. The digital images of TMRM and MitoTracker® Green fluorescence were acquired with
inverted confocal microscope (Leica DM IRE2) with a 63-x water immersion lens. The
MitoTracker® Green fluorescence was excited with the 488 nm line of argon laser, using 510
to 550 nm for emission. TMRM fluorescence was measured using 543 nm for excitation
(Helium-Neon laser) and greater than 580 nm for emission.
RESULTS AND DISCUSSION
We have been using the permeabilized cell technique for several decades [2,4,5], and
even directly studied the effect of contraction on the parameters of respiration regulation, but
that contraction was regulated by calcium, dependent on ATP and totally reversible, as normal
contraction should be [6]. Supercontraction of permeabilized cardiomyocytes, when their
length was shortened by factor of about 3 (1-3 µM of calcium and 1 mM of ATP) resulted in a
decrease of apparent Km for ADP from 320 ± 20 µM to 17 ± 3 µM, and the ordered crystal-
like arrangement of mitochondria was lost [6]. Return to 0.1 µM calcium restored both the
initial length and high apparent Km for ADP (in average to 300 µM) [6]. No effect of calcium
on high value of apparent Km for ADP (around 350 µM) was seen in ghost cardiomyocytes
from which myosin was removed by treatment with KCl [6]. Thus, the high value of apparent
Km for ADP is a parameter characteristic for relaxed cardiomyocytes. In the regular, relaxing
solution without calcium, which is traditionally used in multiple studies, the apparent Km for
ADP was always equally high both in permeabilized cardiomyocytes and fibers [4, 5]. This is
in agreement with the fact that no shortening was usually observed, when very many
preparations of cardiomyocytes permeabilized by saponin were visualized by confocal
microscopy (one example is shown in Figures 1A and B) at room temperature (22-25°C) even
for long time [4-11]. Similar results were obtained in many other laboratories (see references
in [3]).
Nevertheless, in our work we have made one observation that could help to solve the
mystery of calcium-insensitive contraction seen by Perry et al. [1] and even explain it. Most
probably, it is the collapse of cytoskeleton that makes fibers to become rapidly shorter in their
study. We have taken an advantage of the situation to present again our data recorded 7 years
ago and even published (Figures 2 and 4 in reference [7]), they are also shortly mentioned in
another of our publications [8]. This surprising phenomenon – Ca 2+ independent contraction
– has already been seen in experiments with the proteolytic treatment of permeabilized
cardiomyocytes and is reproduced in Figure 1C. In the Supplement to this commentaries one
can see the video of this spectacular phenomenon: in the absence of Ca and ATP, addition of
trypsin in 1 µM concentration results in sudden shortening of permeabilized cardiomyocytes
to about one tenth of the initial length (!), and then surprising spontaneous increase again in
size but with chaotic mitochondrial arrangement, but the initial regular rod-like cell shape and
the intracellular structural organization are lost (Figure 1C). Interestingly, mitochondria stay
still attached to some cellular structures. This amazing effect shows that the rod like shape of
cardiomyocytes and very regular crystal-like arrangement of mitochondria are maintained by
very multiple interactions and forces within cellular cytoskeletal and sarcomere structures
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which are destroyed by proteolysis. Very sensitive to proteolytic enzymes’ action are tubulins,
microtubular system, also plectin, titin and Z-line associated proteins and finally myosin itself
[9-11].
Accepted Manuscript
Interestingly, the calcium induced ATP dependent supercontraction decreased also the
effect of trypsin on the apparent Km for ADP [7], obviously by hindering the access of trypsin
molecules to some important cytoskeletal proteins. Decrease in apparent Km for ADP may
mean that strong contraction of myofibrils evidently deforms the intermyofibrillar
mitochondria, probably resulting in the dissociation of some important cytoskeletal proteins
(for example tubulin) from mitochondrial outer membrane and increasing the VDAC
permeability [4, 5]. The influence of myofibrillar contraction on the shape of mitochondria
has also been recorded by Yaniv et al. [12].
Connection between contractile state and mitochondrial functional parameters may be
indeed important, as supposed both by Yaniv et al. and Perry et al. [1, 12]. Under
physiological conditions, an increase of the heart muscle contractile force is caused by the
increased ventricle filling, stretch of cardiomyocytes, increase of sarcomere length and
decrease in the lattice spacing in myofibrils (length-dependent activation, the Frank-Starling
law of the heart) [13, 14]. The data described above allow us to suppose that this may even
increase the apparent Km for ADP and thus increase the flux of phosphocreatine into
cytoplasm due to increased recycling of ADP in mitochondria coupled to creatine
phosphorylation [4, 5]. In skeletal muscle the effect of contraction on mitochondria may
depend whether the contraction is isotonic or isometric.
The effects of proteolytic enzymes described above made it necessary to use their
inhibitors during cell permeabilization to protect the cell cytoskeleton from the eventual
action of intracellular proteases which may be released or activated by detergents, and to
perform multiple steps of washing before using the permeabilized cells for functional
measurements. According to our experience in testing various inhibitors, the combination of
soybeen trypsin inhibitor (STI) and leupeptin gave the best results. “Double headed” [15, 16]
STI could simultaneously inhibit both trypsin- and chymotrypsin-type serine proteinases
whereas leupeptin efficiently inhibits a number of serine proteinases, threonine proteinases as
well as thiol proteinases [13], which originate from lysosomes or proteasome (cf. [17], [18]
and [19]).
Not only biochemical, but also mechanical damages can be the reasons of variable Km
and contracted fibers with collapsed cytoskeleton. The scissors or forceps must be extremely
sharp and antimagnetic to prevent mechanical damage of the fibers. Uncorrect dissection of
fibers, too hard-handed treatment and vigorous stirring during washing and permeabilization
are frequently the causes of damaging the cytoskeletal structures, intracellular position,
organization and surrounding of mitochondria within the cell. Moreover, insufficient
mechanical dissection and permeabilization can lead to insufficient quality of the preparation,
and thus to low maximal state 3 mitochondrial respiration, low respiratory control ratio
(RCR), low accessibility of mitochondria to various agents, inhibitors, etc [2]. To prevent this
situation, it is recommended to use about 10 mm long and 100 to maximally 250 μm thick
bundles (containing each between 2–5 single fibers). The quality of fibers prepared could be
evaluated before the experiments by using oxygraphic quality test [11]. The respiration
control ratio (RCR, the ratio of respiration rates in States 3 and 2) with ADP (2 mM) was
higher than 5 and cytochrome c effect [2] was not seen. Among these tests the most sensitive
is the pyruvate kinase (PK)-phosphoenolpyruvate (PEP) –creatine test shown in Figure 2.
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These necessary steps are included into all last versions of experimental protocols we
recommend to use [2, 11]. Also, we recommend using for oxygraphic measurements the
Mitomed solution of the following composition: 110 mM sucrose, 60 mM K-lactobionate, 0.5
mM EGTA, 3 mM MgCl2, 0.5 mM dithiothreitol, 20 mM taurine, 3 mM KH2PO4, 20 mM K-
HEPES, pH 7.1, with 5 mM glutamate, 2 mM malate, 2 mg/ml essentially fatty acid free
bovine serum albumin; this solution contains lactobionate and taurin known to have
membrane stabilizing action [2, 7]. In this solution, the permeabilized cells and fibers are
more stable than in salt solutions previously used.
Accepted Manuscript
Also, all reagents have to be of high degree of purity. The use of Mitomed solution
allows avoiding using high EGTA concentrations and serious problems related to the
contaminations in the commercial preparations of EGTA [7].
Accounting for all these important details, the collapse of cytoskeleton, mysterious
contraction and fibers inactivation should be avoided.


AKNOWLEDGEMENTS
This work was supported by project SYBECAR from Agence de la Recherche Nationale,
France, by grant No. 7823 and 8987 from the Estonian Science Foundation, SF0180114Bs08
from Estonia Ministry of Education and Science and by a research grant from the Austrian
Science Fund (FWF): [P 22080-B20].

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12.Yaniv Y., Juhaszova M., Wang S., Fishbein K.W., Zorov D.B., Sollott S.J. (2011)
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LEGEND TO THE FIGURE
Figure 1. A. No changes in mitochondrial arrangement and cardiomyocyte shape
(morphology) during cell permeabilization with saponin (50 µg/ml) in Mitomed solution.
Mitochondria were visualized with MitoTracker Green as described in [7]. Scale bar, 10 µm.
Accepted Manuscript
B. Increased zoom of the last image in Fig. 1A shows unchanged, very regular mitochondrial
arrangement in saponin-permeabilized rat heart cardiomyocyte in calcium free medium (for
ref. see [8]).
C. Dramatic changes in cardiomyocyte morphology and regular arrangement of mitochondria
during cell incubation with saponin (50 µg/ml) + Trypsin (1 µM). Mitochondria were
visualized with TMRM as described in [7]. Confocal images of the same cell were taken
successively at time points shown in seconds, s. Scale bar, 10 µm. Adapted from [7].
Figure 2. The quality test by measuring the effects of PK-PEP system on the mitochondrial
respiration of permeabilized fibers of rat soleus muscle, (test called the Gellerich-Guzun
protocol [4, 5,11]). The respiration rates were measured as described [2, 7, 11] after addition
of substrates (S) to the permeabilized fibers (PF); addition of ATP (2 mM) activates the
intracellular ATPases producing ADP and activating respiration. If the fibers are well
dissected and permeabilized, successively added PK-PEP system removes significant part of
the extramitochondrial endogenous ADP produced by intracellular ATP-consuming reactions
and continuously regenerates extramitochondrial ATP, thus decreasing the respiration rate
(usually by 30 - 40 %). Subsequent addition of creatine (Cr, 20 mM) induces ADP production
in the mitochondrial creatine kinase reaction and maximally activates respiration. This ADP,
produced in the intermembrane space by the mitochondrial creatine kinase, is not trapped by
PK-PEP due to low permeability of the VDAC in mitochondrial outer membrane for adenine
nucleotides in situ [4, 5, 11]. Atractyloside (Atr, 30 µM) completely suppresses this
respiration, that showing the intactness of mitochondrial inner membrane [11]. The acceptor
control ratio (ACR, the ratio of respiration rates in the presence of Cr, 20 mM + ATP, 2 mM
and State 2) with creatine activation was usually in the range of 5-8, the same as RCR with
activation by ADP. However, if the fibers are not enough dissected, the permeabilization is
inadequate and the PK-PEP system cannot enter inside the intracellular space, does not
change the respiration rate and usually the effect of creatine is very small or not detectable.
This Figure is representative of about 70 experiments by using rat soleus fibers.


LEGEND TO SUPPLEMENTARY MATERIAL
Dynamics of changes in cardiomyocyte morphology and regular arrangement of
mitochondria in time during cell incubation with saponin (50 µg/ml) + Trypsin (1 µM).
Mitochondria were visualized with TMRM as described in [7]. The video shows successive
changes in cell morphology and mitochondrial arrangement at moments of time t indicated.
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208 s0 s52 s
104 s
300 s
300 s
A

Accepted Manuscript




B


C













Figure 1
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Figure 2
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