Download Water content and its intracellular distribution in intact and saline

Cardiovascular Research 53 (2002) 48–58
www.elsevier.com / locate / cardiores
Review
Water content and its intracellular distribution in intact and saline
perfused rat hearts revisited
Mayis K. Aliev a , *, Pierre Dos Santos b , Jacqueline A. Hoerter c , Sybille Soboll d ,
Alexander N. Tikhonov e , Valdur A. Saks f
a
Institute of Experimental Cardiology, Cardiology Research Center, 3 rd Cherepkovskaya Street 15 A, 121552 Moscow, Russia
b
INSERM U-441, Pessac, France
c
INSERM U-446, Chatenay-Malabry, France
d
Institut fur Physiologische Chemie I, Duesseldorf, Germany
e
Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow, Russia
f
University of Joseph Fourier, Grenoble, France and National Institute of Chemical Physics and Biophysics, Tallinn, Estonia
Received 4 May 2001; accepted 3 August 2001
Abstract
Precise estimation of cellular water content is a necessary basis for quantitative studies of metabolic control in the heart; however,
marked discrepancies in water spaces of heart tissue are found in the literature. Reasons for this wide diversity are analyzed, and the
conclusion is that the most probable value of total intracellular water content is 615 ml H 2 O / kg of wet mass (wm) and intracellular
content of dry substance is 189 g / kg wm in intact in vivo rat heart. An extracellular water of 174 ml per kg wm and 22 g of dry mass per
kg wm in vascular and interstitium spaces account for the rest of the tissue mass. These values can be directly related to normoosmotic
saline perfused hydrated hearts, characterized by water accumulation in the extracellular spaces. Due to essentially intact heart cells, the
experimentally determined dry mass, water and metabolite contents of these hydrated hearts can be extrapolated to the original
morphological configuration of an intact heart muscle before the onset of edema. Such an ‘extrapolated’ heart is defined as a standardized
perfused heart (SPH). SPH is the heart in its original morphological configuration, characterized by cell density and cellular water
contents of the intact heart, but with perfusate in the extracellular spaces. The total cellular water is distributed in the cell compartments of
SPH and intact hearts according to volumes of particular compartments and density of their dry mass. The volumes of bulk water phases
in different organelles, accessible to diffusion of low molecular metabolites, were obtained after corrections for the fraction of ‘bound’
water of 0.3 g per g of compartmental dry mass content. The diffusible water spaces are proposed to be 321, 55, 153, 21 and 8 ml / kg wm
for myofibrils, sarcoplasm, mitochondria, sarcoplasmic reticulum and nuclei, respectively. The SPH model allows direct comparison of
metabolic data for intact and perfused hearts. We used this model to analyze the penetration of extracellular marker into cells of intact and
hydrated perfused rat hearts.  2002 Elsevier Science B.V. All rights reserved.
Keywords: Interstitial space; Intra / extracellular ions; Mitochondria; Myocytes; NMR
1. Introduction
A precise estimation of water content in tissue and in
different subcellular compartments is the basis of in vivo
calculations of the rates of biochemical reactions [1], the
Abbreviations: wm, wet mass; dm, dry mass; SPH, standardized
perfused heart; swm, wet mass of SPH; sdm, dry mass of SPH
*Corresponding author. Tel.: 17-095-414-67-55; fax: 17-095-414-6699.
E-mail address: aliev m [email protected] (M.K. Aliev).
] ]
energy fluxes between cellular compartments [2], the
concentration gradients of ions [3], the membrane potential
of mitochondria in situ [4], as well as of quantitative
studies of metabolic control in different organs, including
hearts.
In pathological situations, such as early stages of cardiac
hypertrophy [5] and cardiac ischemia [6–10], cardiomyocytes undergo significant cellular edema due to
increase in cellular osmolarity. Cell swelling causes stretch
Time for primary review 27 days.
0008-6363 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 01 )00474-6
M.K. Aliev et al. / Cardiovascular Research 53 (2002) 48 – 58
and / or deformation of cell membranes and of the underlying cytoskeletal network [11]. These events lead to essential changes in the activities of cellular transporters and ion
channels, cell membrane potential, protein and carbohydrate metabolism, lipogenesis and gene expression. As a
consequence, these alterations of cellular hydration have
been suggested in hepatic cells to be a new important
mechanism for metabolic control, linking cell function to
hormonal and environmental alterations (for reviews see
[12,13]). The relevance of this mechanism for the metabolic control in cardiac cells is currently under consideration
(for reviews see [11,14]). However, exploration of this
topic requires an accurate estimation of the cardiac cell
water contents.
Osmotic effects, underlying the metabolic control by
cell volume regulation, were first studied by Overton [15].
According to his works, 65% of total cellular water is
osmotically active in cell volume regulation, while the
remaining 35% of water molecules are bound to biopolymers and cellular membranes. Functional significance of
bound water in cellular metabolism was clearly demonstrated in resting cysts of Artemia salina [16]. When the
humidity level drops below 30% (30 g of water per 100 g
of their dry mass), the cyst metabolism completely stops.
At a humidity level of 35%, metabolic pathways of
carbohydrates, amino acids and Krebs cycle are activated,
while further increase in humidity, up to 63%, activates the
pathways of protein and nucleic acid synthesis, and cells
can perform all the essential metabolic reactions necessary
for their development.
Despite the great number of publications, the Overton’s
concept of cellular bound and osmotically inactive water is
still a matter of discussions (for references see reviews
[1,17–21]). The detailed analysis of conflicting results
should be based on reliable data on water contents in
muscle cells and its intracellular distribution. However,
until now, there is no final agreement even on the total
water contents in normal cardiac cells. Table 1 lists the
main published data on the water and dry mass contents in
saline perfused and intact hearts. This table demonstrates
(column 4) a wide variability of water estimates, from 1.81
[24] up to 3.15 ml / g dm [23].
There is also wide range of estimations of water content
in mitochondria: from 1.0 ml / mg protein according to [27]
to 1.8 ml / mg protein [28], corresponding to 9.2 or 16.5%
of total cellular water, respectively [29]. The total volume
occupied by mitochondria in the normal cardiac cell equals
to 33–35% [30,31]. A correct estimation of mitochondrial
water is fundamental for our understanding of the role of
mitochondria in pathological situation. For example, ischemia, besides increasing the total cell hydration, causes
pathological dehydration of mitochondrial matrix and
reciprocal swelling of mitochondrial intermembrane space
[32]. Preventing these mitochondrial alterations by opening
the mitochondrial ATP-sensitive K 1 channel [33] or by
ischemic preconditioning of the organ [34] protects the
49
heart against ischemia–reperfusion damage. Besides, ischemic preconditioning also prevents the cell hydration [7]
or may even lead to cardiomyocyte dehydration [35].
In this communication, we analyze the results of different published studies on total intracellular water contents
in saline perfused hearts and in normal hearts in vivo, and
evaluate possible artifacts leading to current underestimation of cardiac cellular water contents and discrepancies in
the data. Based on these results, we suggest that the most
probable value of total cellular water content is 3.25 ml / kg
dm, and propose its intracellular distribution, corrected for
a fraction of bound water of 0.3 g per 1 g of the
compartmental dry mass.
2. Water in intact hearts
2.1. Total cellular water
Recently Dobson and Cieslar [26] and Cieslar et al. [36]
performed tracer estimation of intracellular, interstitial and
blood spaces in the intact rat myocardium in vivo. Extracellular tracers, 14 C-inulin or 14 C-mannitol, were injected intravenously 30 min before taking samples of heart
tissue and blood [26]. Various parameters were measured:
(i) tracer contents in hearts, blood and blood plasma; (ii)
total water contents in tissue determined by gravimetric
heart drying procedure; (iii) perfusion bed volumes (vascular plus capillary volumes) obtained by enzymatic determination of 2,3-diphosphoglycerate (specific marker of
red blood cells) contents in blood and tissue. Their
estimation of cellular water contents in intact rat hearts,
2.75 and 2.64 ml / g dm (Table 1, column 4), appears very
high when compared with recent direct estimation of
Clarke et al. [24], 1.81 ml / g dm, or Dobson [3], 2.4 ml / g
dm (Table 1).
Because of the importance of this topic, let us consider
in more detail the experimental results of Dobson and
Cieslar [26]. Two misuses of their data should be noted.
First, with 14 C-mannitol, which completely distributes in
erythrocytes [26], the plasma space (total tissue counts
divided by specific activity of tracer in blood plasma)
corresponds to the relative mass of extracellular space but
not to its water content (Table 1, column 2). This is
because the mass of plasma is a sum of water and dry
matter masses of plasma. Second, with 14 C-inulin which is
completely excluded from erythrocytes [26], the plasma
space outlines the volume of extracellular space without
erythrocytes (Fig. 1A). Therefore, we reevaluated these
data and compared them with the results of morphometry
measurements performed under the same conditions
[30,37]. Morphometry estimates are most reliable due to
the advantage of direct measurements and corrections for
compression factors [30,37]. We considered only the
measurements with inulin because they are commonly
considered as more reliable [38–40].
50
Table 1
Main published data on the water and dry mass contents in saline perfused and in vivo hearts
Reference
Protocol
Experimental data
Dry mass
(g / kg wm)
Perfused hearts, working model of Neely (WH) or Langendorff perfusion (LP)
Morgan et al. [22]
Rat, WH
18965
(20 min perfusion, n56)
a
Guinea-pig, WH
(15 min perfusion, n59)
13963
Cell. H 2 O
in SPH
model
(ml / kg swm)
513
Extracellular H 2 O
(ml / kg wm)
Cellular H 2 O,
(ml / kg wm)
1000 g – (112)
33167
by 3 H-sorbitol
480612
2.5460.13
259617
by 3 H-inulin
602620
4.3260.24
439648
by 14 C-urea
3.1560.23
a,b
Masuda et al. [3]
Rat, WH
(50 min perfusion, n59)
14763
508615
by 14 C-mannitol
34868
2.3760.10
486
Clarke et al. [24]
Rat, LP
(30 min perfusion, n56)
11364
682655
by 31 P-PPA (NMR)
205659
by 31 P-DMMP (NMR)
1.8160.59
367
Askenazy and Navon [10]
Rat, LP
(20 min perfusion, n536)
by 1 H-H 2 O (NMR)
2.5060.06
507
by
59
Co-cobaltcyanide (NMR)
In vivo hearts
Clarke et al. [24]
Rat (tracer
equilibration 0.5–3 h, n56)
22364
15666
by 3 H-sucrose in blood
621610
2.7860.09
Polimeni et al. [25]
Rat (tracer equilibration
10–160 min, n58)
21467
20967
by 3 H-inulin in plasma
57767
2.7660.12
Dobson and
Cieslar [26]
Rat (tracer
equilibration 30 min, n55)
210610
212610
by 14 C-inulin in plasma
236612
by 14 C-mannitol in plasma
578610
2.7560.18
615
554612
2.6460.18
615
c
210610
Dry mass contents were estimated by gravimetry of dried tissue sample; extracellular water contents were estimated from the radioactivity of indicated tracer in the tissue sample or by NMR
spectroscopy (NMR) of indicated compound in the tissue. PPA and DMMP are phenylphosphonate and dimethyl methylphosphonate, respectively. Cellular water contents were estimated as total tissue
water minus its extracellular content, 10002(columns 112). For perfused hearts, the total cellular water content by the SPH model was obtained by multiplying the data of column 4 by the dry mass
content in SPH, 202.6 g / kg swm (see 2.1).
Throughout this paper we assume that the mass of 1 ml of water equals 1 g.
a
Exposure time to 3 H-inulin, 5–8 min, was too short in this protocol to attain complete equilibration; this leads to underestimation of extracellular space size and respective overestimation of
cellular water content per dry mass unit.
b 14
C-urea at the given time exposure, 13–15 min, labels only part of total tissue water [23].
c
For total cellular water content by SPH model see Section 2.1.
M.K. Aliev et al. / Cardiovascular Research 53 (2002) 48 – 58
Bunger [23]
Cell. H 2 O
ml per g
dry mass,
3/1
M.K. Aliev et al. / Cardiovascular Research 53 (2002) 48 – 58
51
Fig. 1. Schematic presentation of the main tissue compartments: cellular (Cell), interstitial (IS) and vascular perfusion bed (PB) in blood perfused intact
hearts (A), saline perfused hydrated hearts (B) and assumed standardized perfused hearts (C). In intact hearts (A) extracellular spaces (IS1PB) are filled
with plasma and blood cells in PB. In saline perfused hearts (B) the washout of plasma proteins leads to saline accumulation in the extracellular spaces due
to more filtration at a given hydrostatic pressure. Cellular water contents in these hearts could be the same as in the intact ones because the saline is
isoosmotic to cardiomyocytes. The SPH model (C) defines the heart in the original morphological configuration of the intact heart in which saline replaces
plasma and blood cells in the extracellular space.
We found that the values obtained by Dobson and
Cieslar [26] for blood (106611 g / kg wm, n510) and
interstitial space (153611 g / kg wm, n55) sizes were
calculated correctly. These data were evaluated by comparison with the direct morphometry estimates of Anversa
et al. [30] for intact rat hearts in comparable units (g / kg
wm), presented in Table 2. It is evident that the estimates
of Dobson and Cieslar are certainly higher than corresponding values obtained from morphometry measurements: 80 g / kg wm for capillary bed size and 116 g / kg
wm for interstitium plus T-system sizes (Table 2). The
weight difference of 26 g (106 g / kg280 g / kg) for
perfusion bed size can be accounted for by the persistence
of blood remnants in heart chambers after the freeze
clamping procedure [26]. For this reason, we chose to use
the morphometry value (75.5 ml / kg wm, Table 2) as a
reliable measure of heart perfusion bed volume. The
difference of 37 g for interstitium sizes (153 g / kg2116
g / kg) may be due to some artifacts. For example, extracellular plasma space for inulin determined by Dobson and
Cieslar, 212610 g / kg wm, is identical to that determined
by Polimeni et al. [25], 20967 g / kg wm, under similar
conditions (Table 1). However, Polimeni et al. [25] found
the extrapolated time-zero inulin plasma space, obtained
from a series of eight measurements performed every 30
min after tracer bolus injection, to be lower by 34 g / kg
wm, leading to an actual value of 17565 g / kg wm. Taking
this correction into account, the interstitial space determined by morphometry methods and tracer methods can be
considered to be similar, 116 ml / kg wm and 119 ml / kg
wm (153234), respectively. The artifacts in tracer methods will be considered below (Section 3.3).
This analysis gives an important conclusion: with appropriate corrections for artifacts the data from tracer measurements in the intact hearts are similar to morphometry
estimations, substantiating each other. As a consequence, it
Table 2
General parameters of main tissue compartments of in vivo blood-perfused rat heart related to 1 kg of tissue wet mass
Extracellular space
a
Volume (ml)
Weight (g)b
Dry mass (g)c
Water content (ml)c
Vascular bed
Interstitium
75.562.8
8063
1360.6
6764
109.466.3
11666
960.6
10767
Cell
Total
(tissue)
758.5612.8
80469
189611
615621
943.465.3
1000
211610
789610
a
The original morphometry data of Anversa et al. [30], 8063 ml / l for capillary bed volume and 11666 ml / l for interstitium plus T-system volumes,
were converted into ml / kg wm units using the value of 1.0660.006 g / ml wm [41] for heart tissue density.
b
The compartmental weights were obtained by back multiplying the volumes by the mean density of tissue, 1.06 g / ml.
c
See Section 2.1 for explanation.
M.K. Aliev et al. / Cardiovascular Research 53 (2002) 48 – 58
52
seems reasonable to use the morphometry estimates of
extracellular space sizes of Table 2 as a reliable basis for
considering water contents in the in vivo rat heart.
Taking into account the dry mass contents measured in
the blood (16%) [26] and blood plasma (8%) [26], the dry
mass contents in vascular bed and interstitium compartments of intact rat heart can be estimated as 13 g / kg wm
(80 g / kg wm30.16) and 9 g / kg wm (116 g / kg wm3
0.08), respectively (Table 2). The water contents in
vascular bed and interstitium, 67 ml / kg wm and 107
ml / kg wm, respectively (Table 2), are calculated as the
difference between weights and dry mass contents.
Total tissue water content measured by Dobson and
Cieslar [26] is 790610 ml / kg wm. With minor correction
for the water content in the assumed blood remnants in
heart chambers (26 g / kg wm30.84521.8 ml / kg wm) we
obtain a value of 768.2 ml of tissue water (790 ml–21.8
ml) in 974 g (1000–26 g) of heart tissue, that is 788.7 ml
of tissue water per kg wm (Table 2). The corrected total
dry mass content in heart tissue is (1000–788.7)5211.3
g / kg wm (Table 2).
Thus, the total cellular water space of intact heart can be
calculated as the difference between tissue and extracellular water contents that is 614.8 ml / kg wm [(788.7– 67.2–
106.7) ml / kg wm] (Table 2). In the same way the
intracellular dry mass content was calculated as 189.2
g / kg wm (Table 2). These final values of 615 ml H 2 O / kg
wm and 189 g dm / kg wm, obtained from a revaluation of
data from the literature, can be treated as the most probable
estimates of total cellular water and dry mass contents in
intact rat hearts. Based on these estimates, we may
consider the distribution of water and dry mass in main
cellular compartments of intact rat heart.
2.2. Intracellular water distribution
Table 3 presents the water contents in different cellular
compartments calculated on the basis of the morphometry
determination of Anversa et al. [31] for rat left ventricular
myocardium, and the measured densities of dry masses in
cellular organelles [43]. In our calculations we assumed
that the total cellular space of 758.5 ml / kg wm (Table 2)
is distributed among cellular compartments according to
their contribution to total cellular volume and density of
dry mass in particular compartments.
The dry mass contents in these compartments (third
column in Table 3) were obtained by multiplying rD,X (the
density of the dry mass in the compartment X in g / ml) by
VV,X (the volume of this compartment, related to kg wm,
ml / kg wm, column 2 of Table 3).
The values of rD,0 (the density of dry mass in the
reference compartment (myofibrils) in g / ml) were obtained
by solving Eq. (1) in [43] under the assumption that the
relative densities of dry masses ( rD,X / rD,0 ) are equal to
0.76 in the sarcoplasm and nuclei, 1.0 in the myofibrils,
1.04 in the sarcoplasmic reticulum, and 1.78 in mitochondria [43]
O( r
rD,cell 5 rD,0 3
x
D,X /
rD,0 ) 3VV,X
(1)
In this equation, rD,cell is the cellular dry mass related to
kg wm (189.2 g in Table 3). Finally, we obtain
rD,X 5 rD,0 3 ( rD,X / rD,0 )
(1a)
The water contents in cellular compartments (MH 2 O , 4th
column in Table 3) were calculated according to modified
Eq. (2) from [43]
MH 2 O 5VV,X 3 (1 1 (1 2 1 /rS ) 3 rD,X ) 2 MD
(2)
where VV,X is the volume (2nd column in Table 3), MD is a
dry mass content (3rd column in Table 3), rD,X is a density
of the dry mass in the compartment X determined according to Eq. (1a), and rS is the density of solid cellular dry
mass ( rS 51.3166 g / cm 3 ). The value of rS was estimated
by dividing the cellular dry mass (189.2 g / kg wm, Table
3) by its volume (758.5 ml of cell volume2614.8 ml of
cellular water, Table 3).
The data for mitochondria presented in Table 3 need
special consideration. First, the in vivo mitochondria are
characterized by a high relative density of dry mass (up to
47.2% of cellular dry mass) and a high relative amount of
protein. For comparison, the protein content in the matrix
of perfused rat hearts was determined by non-polar fractionation technique as 40.5% of total cellular protein [37].
Taking into account that the protein content in mitochondrial matrix is about 88% of mitochondrial protein
Table 3
Most probable intracellular water and dry mass distribution in intact rat heart related to 1 kg of tissue wet mass
Cellular
compartment
Myofibrils
Sarcoplasm
Mitochondria
Sarcoplasmic reticulum
Nucleus
Total
% of cell
volume [31]
53.9
8.7
32.7
3.5 [42]
1.2
100.0
Volume of
compartment (ml)
Dry mass in
compartment (g)
Total water in
compartment (ml)
Water space for
diffusion (ml)
408.8
66.0
248.0
26.6
9.1
82.7
10.1
89.4
5.6
1.4
346.0
58.3
180.2
22.3
8.0
321
55
153
20
8
758.5
189.2
614.8
558
M.K. Aliev et al. / Cardiovascular Research 53 (2002) 48 – 58
[44], the protein content in intact mitochondria should be
46% (40.5% / 0.88). This value is comparable with the dry
mass content of in vivo mitochondria. Second, the predicted water content of in vivo mitochondria, 2.02 ml / mg
dm (180.2 / 89.4, Table 3), practically coincides with value
of 1.93 ml / mg dm, determined for in situ rat heart
mitochondria by X-ray microanalysis [43]. Taking into
consideration the mitochondrial protein content, which
comprises in rat heart mitochondria about 75% of their dry
mass [45–47], the values of 2.69 and 2.58 ml / mg protein
are obtained. These values are considerably higher than
data reported for isolated rat liver mitochondria (1.96622
ml / mg protein (n59), measured with 14 C-polyethylene
glycol) [48], and for isolated rat heart mitochondria
(1.8060.27 ml / mg protein, measured with inulin) [28].
To explain these discrepancies, it should be noted that
isolated and native mitochondria are characterized by
different architecture. The architecture of in vivo mitochondria is stabilized by cytoskeleton [17]. This may
preclude the shrinkage of mitochondrial matrix detected in
isolated mitochondria [49]. The shrinkage of mitochondrial
matrix in isolated mitochondria depends on the composition of the isolation media [50], of the ionic composition and pH of incubation media and of the metabolic
state of mitochondria [48]. This means that the isolated
mitochondria cannot be used as a decisive reference for the
estimation of in vivo mitochondrial water volumes. Underestimation of water contents in isolated mitochondria may
arise also from partial permeability of their outer membranes to inulin [50].
Thus, it seems reasonable to consider the data of Table 3
as the most probable ones for distribution of water and dry
mass in the cellular compartments of intact rat heart. These
values, mainly based on the analysis of data for rat hearts,
can be directly applied to hearts of other species characterized by the same density of cellular dry mass in vivo.
Cellular water distribution in this case can be predicted (as
in Table 3) from morphometry data on cellular compartments in the hearts of ten different animal species,
including humans [51].
2.3. Volume of intracellular aqueous domains accessible
for low molecular metabolites
Table 3 shows in column 4 the contents of total water
(MH 2 O ) in heart cell and its compartments. However, to
calculate the real concentrations of low molecular metabolites (ATP, PCr, Cr, Pi, glucose, etc.) in cell compartments,
we need to know the volumes of bulk aqueous domains
accessible to these metabolites (Wa ). Thus, we have to
correct the content of total water inside the cell for the
fraction of water molecules that drop out of the aqueous
bulk phase. These are the ‘immobilized’ osmotically
inactive water molecules bound to biopolymers and cellular membranes.
53
The concept of cellular bound and osmotically inactive
water, first proposed by Overton [15] in 1902, is still a
matter of controversy (for references see reviews [1,17–
21]). An average amount of water bound to proteins was
evaluated as 0.3 g H 2 O / g dm [52,53]. To function,
globular proteins require a threshold level of hydration,
about 0.4 g H 2 O per g dry protein [54]. In lipid membranes, about 14–18 water molecules are bound to one
lipid molecule [55]. Nucleic acids contain 10–11 molecules of water per pair of bases (for references see review
[56]).
The fraction of bound water was evaluated by different
methods. As early as in 1930, Hill [57] measured the water
vapor tensions for frog’s blood and skeletal muscles by the
thermoelectric method. He concluded that the dominant
portion of cell water (94%) behaves as a usual solvent,
while the osmotically ‘inert’ fraction of water was about
6%. Similar results were obtained later by other methods.
Belton et al., studying the proton relaxation in frog skeletal
muscles by the NMR method, concluded that at a temperature ranging from 28 to 208C approximately 94% of total
water in the tissues corresponded to bulk water while the
remaining fraction (6%) was strongly immobilized [58].
Outhred and George [59] confirmed the limited mobility of
several percent of cellular water.
To discriminate between bulk and immobilized water,
other workers used osmotic effects. For instance, Garlid
[60] squeezed the water out of the matrix of rat liver
mitochondria by a progressive increase in osmolality by
non-permeant sucrose in the incubation media. Extrapolating the matrix water contents to the limit of infinite
concentration of sucrose, he obtained a fraction of osmotically inactive water of 0.28 ml of H 2 O / mg dm. Unlike
water, the content of DMSO (dimethylsulphoxide) in the
matrix was extrapolated to zero, indicating the complete
removal of bulk water from the matrix [60]. Thus, DMSO
molecules do not penetrate into the osmotically inactive
cell water domains [61].
Based on these data, we could accept 0.3 g H 2 O / g dm
as the most probable content of bound water in the cell.
This value corresponds to 56.8 g of cellular water / kg wm
in intact hearts (189.230.3, Table 3) or 9% of total
cellular water (56.8 / 614.83100%, Table 3). This is
osmotically inert fraction of cell water [57] characterized
by strongly immobilized water protons [58]. The main
portion of cell water (about 91%) belongs to osmotically
active water from the bulk phase aqueous domains where
the low molecular metabolites and water molecules can
freely diffuse.
Garlid [60,62] considered the fraction of osmotically
inactive water as an aqueous phase with abnormal solvent
properties for low molecular permeant nonelectrolytes,
such as glycerol, ethanol, urea and antipyrin. It is important to note that the amount of bound water, 0.3 g
H 2 O / g dm, is not sufficient to form complete monomolecular water layer around each macromolecule [63].
54
M.K. Aliev et al. / Cardiovascular Research 53 (2002) 48 – 58
Therefore, in such abnormal solvent phase, the ‘solution’
of nonelectrolytes might be considered as the replacement
of bound water molecules by nonelectrolyte ones or as the
inclusion of nonelectrolyte molecules between the immobilized water ones. In both cases, the nonelectrolytes
are expected to be immobilized and osmotically inactive.
The real interaction of low molecular nonelectrolytes with
macromolecules is a rather complex event [64–67].
Based on accepted value of bound water space, 0.3 g
H 2 O / g dm, we have calculated the probable sizes of
compartmental water spaces, accessible for the diffusion of
low molecular metabolites (Table 3, last column). The data
were calculated as the difference between total water
content in the given compartment and 0.3 g of bound water
per gram of the compartmental dry mass.
The relevance of the data presented in Table 3 to saline
perfused hydrated hearts is considered below.
most precise because these authors dried the finely powdered frozen tissue instead of using the conventional
procedure using the whole heart. The dry mass of intact
hearts obtained by conventional procedures is higher:
226614 g / kg wm (data from 33 references with n $ 8,
reviewed by Polimeni [68]). Standardized dry mass for
such hearts, dried by conventional procedures, can be
estimated as 202.6 g sdm / kg wm (189.2 g dm3(226 /
211.3)10.25 g dm).
The concept of SPH gives the opportunity for a direct
comparison of metabolic control data in the intact and
hydrated perfused hearts, as the cellular water contents and
its intracellular distribution are assumed similar in both
cases. Using this model, the measured water or metabolite
contents must be expressed per assumed 189.4 or 202.6 g
sdm of SPH (see above) and then distributed in the water
of cellular compartments. An example of using this model
of SPH is given below.
3. Water in saline perfused hearts
3.2. First exploration of standardized perfused heart
model
3.1. Standardization problem
In normoosmotic saline perfused hearts, cardiomyocytes
essentially preserve their functional capabilities, dry mass
and metabolite contents. Assuming that due to isoosmolarity of perfusing solution the water content per cell volume
remains the same as in intact heart, we can evaluate the
changes in the morphological configuration (defined as the
respective contribution of perfusion bed, interstitium and
cellular space to total tissue volume) occurring upon saline
perfusion. These changes are mainly characterized by a
decreased cardiomyocyte density due to water accumulation in the extracellular spaces (Fig. 1B, columns 1 and 2
of Table 1), caused by the replacement of blood and
plasma by saline perfusate in the vascular space and
interstitium, respectively. Because cells are assumed to be
essentially intact, the measured dry mass, water and
metabolite contents of hydrated hearts can be extrapolated
to the original morphological configuration of heart muscle
before the onset of edema. We suggest defining such an
extrapolated heart as a standardized perfused heart (SPH).
SPH is the heart in its original morphological configuration
but with perfusate in the extracellular spaces (Fig. 1C).
The dry mass of such a standardized heart (standard dry
mass (sdm)) is the sum of cellular dry mass (DM,cell 5189.2
g, Table 3) and the dry mass of perfusate in the vascular
and interstitium spaces. The dry mass of perfusate, calculated as product of dry mass density of perfusate (Dperf 5
0.00134 g / ml) and volumes of vascular and interstitium
spaces (Table 2), is low, 0.25 g (Dperf 3(75.51109.4) ml).
So, the dry mass of standardized heart is 189.4 g sdm / kg
wm.
The dry mass value of standardized heart is based on the
corrected dry mass estimate of Dobson and Cieslar [26]
(211 g / kg wm, Table 2). This value can be taken as the
Assuming that standardized dry mass of conventionally
dried heart is equal to 202.6 g sdm / kg wm, we can
estimate the maximal amount of cellular water in saline
perfused hearts from the reported data on their cellular
water and dry mass contents. These values, obtained by
simple multiplication of column 4 data in Table 1 by the
dry mass contents in SPHs, 202.6 g sdm / kg wm, are listed
in column 5 of Table 1. The mean value of four estimations of the literature data, 468634 ml / kg wm of standardized heart, is lower than the predicted maximal water
content of 615 ml / kg wm.
Considering these data, we can point out two important
topics.
First, the value of total water content for SPH obtained
from data of Askenazy and Navon [10] (507 ml / kg swm)
is the same as that obtained from data of Morgan et al. [22]
(513 ml / kg swm). This confirms that the new multinuclear
NMR method for continuous monitoring of 59 Co
(cobaltcyanide as extracellular marker) and 1 H (H 2 O as
total water marker) signal intensities [10] is as reliable as
the classic method based on the measurements of total
water contents by gravimetry of dried tissue using radioactivity of 3 H sorbitol as extracellular tracer [22]. Because of
non-invasiveness and possibility of continuous monitoring
of cellular water contents, the NMR method of Askenazy
and Navon [10]can be considered as more advantageous.
Second, the value for standardized water spaces obtained from data of Clarke et al. [24] (367 ml / kg swm) is
lower than the averaged value (468634 ml / kg wm). These
authors used 31 P NMR spectroscopy to monitor dimethyl
methylphosphonate (DMPP) and phenylphosphonate
(PPA) contents in perfused hearts. DMPP was used as a
marker of total water spaces, and PPA as a pH-sensitive
marker of extracellular spaces. Careful examination of
M.K. Aliev et al. / Cardiovascular Research 53 (2002) 48 – 58
NMR spectrum in Fig. 1 of this paper reveals asymmetry
of the PPA resonance with some shoulder-like left-side
directed enlargement. Taking into account the usual pH
difference about 0.4 unit between intra- and extracellular
spaces in myocardium and pH-sensitivity of PPA, this
shoulder-like enlargement may be caused by penetration of
PPA into more acidic environment of heart cells. Indeed,
this shoulder appears quite clearly in the 31 P NMR spectra
after 28 min of heart ischemia, when intracellular pH
decreases to 6.1 (Fig. 2 in Ref. [24]). These data can be
considered as a first direct manifestation of intracellular
penetration of extracellular markers, suggested previously
by Polimeni [68] and Polimeni and Buraczewski [69] for
all low-molecular ‘extracellular’ markers. The penetration
of PPA into cardiomyocytes may be the reason for low
standardized cellular water contents obtained by Clarke et
al. [24] as compared with other estimations (Table 1).
Taken together, the data of Clarke et al. [24] and
Polimeni [25,68,69] suggest that the origin of differences
between calculated (468634 ml / kg swm, n54) and
predicted (615 ml / kg wm) standardized cellular water
contents could be the penetration of the ‘extracellular
markers’ into cardiomyocytes. The maximum percentage
of penetration can be evaluated as 23.865.6% (n54), that
is the average of four normalized differences between
predicted standardized (column 5 in Table 1) and calculated (column 3 in Table 1) water spaces. For example, for
data of Masuda et al. [3], this normalized difference is
(615–486) / 6153100520.9%.
This example clearly shows how, using the SPH model,
we can compare the absolute estimates of water contents
obtained by different methods and authors.
3.3. Extracellular marker penetration into heart cells
Recently we attempted to quantify the penetration of
extracellular markers into cells of saline perfused isolated
guinea pig hearts [38]. Kinetic curves for low-molecularweight markers (LMM: 35 SO 4 or 14 C-sucrose) or 3 H-inulin
washout from the perfused hearts were analyzed by
mathematical model of tracer exchange in the extracellular
spaces of perfused myocardium [70]. This kinetic model
permits the estimation of the volumes of vascular perfusion
bed and interstitium and the extent of penetration of
extracellular tracer into heart cells. Extracellular marker
penetration into cells was evaluated as 28.1% for LMM
and 18.2% for inulin. LMM penetration into cardiomyocytes was 1.54-fold higher than inulin. The percentage of low-molecular tracer leakage into cells, 28.1%,
is comparable with presumed percentage of tracer penetration for chosen published data (23.865.6% (n54), see
Section 3.2).
These data indicate that the actual in vitro tracer
penetration into cellular space may be high enough to
account for aforesaid difference between calculated
55
(468634 ml / kg wm) and predicted (615 ml / kg wm, Table
2) standardized cellular water contents.
Possible leakage of ‘extracellular tracer’ into cardiomyocytes in vivo can be estimated from the data of
Dobson and Cieslar [26]. With inulin, the plasma excess in
tracer-determined compared to morphometry-determined
interstitium spaces, is 37 g (see Section 2.1). This aliquot
will contain 34 ml of water per kg wm of tissue (37
g / kg30.92). The 34 ml of plasma water, when actually
distributed in cellular water, will give 5.5% tracer penetration into cells (34 / 6153100%). In the same way, based on
the 14 C-mannitol-determined value of 17368 g / kg wm for
interstitium space [26], we obtain a 57 g / kg wm overestimation of the real interstitium space (173–116). With
water contents of 52.4 ml in these aliquots (57 g / kg3
0.92), the possible in vivo percentage of 14 C-mannitol
penetration into cardiomyocytes would equal 8.5% (52.4 /
6153100). Again, as in vitro, penetration of low-molecular tracers into cardiomyocytes is increased by a factor of
1.54 (8.5% / 5.5%) when compared to inulin. Lower percentages of tracer penetrations in vivo may indicate the
better conservation of cardiomyocytes under blood perfusion conditions.
From these calculations and the basic assumption that
under normoosmotic saline perfusion the heart cells essentially retain their original water contents, we conclude that
the value of 615 ml cellular H 2 O / kg wm of intact heart or
615 ml / 189.4 g sdm53.25 ml cellular H 2 O / g sdm (see
Section 3.1) will represent an actual measure of total
cellular water contents in standardized normoosmotic
perfused hydrated rat hearts.
To use this value, the dry mass of perfused hearts should
be determined very carefully, as in paper of Dobson and
Cieslar [26] (see Section 3.1). For the conventionally dried
hearts the proposed cellular water contents will be 615
ml / 202.6 g sdm53.03 ml / g sdm.
3.4. Content of intracellular water in the hearts with
cellular edema
Standardized heart model implies that normal cardiomyocytes preserve their in vivo water content per cell
volume unit. However, it has been reported that, compared
to intact hearts, cardiomyocytes of saline perfused hearts
accumulate 33.5% additional amount of water, even in
normoosmotic perfusion medium [69]. This high value is
likely overestimated. Indeed, the basic isoosmotic perfusion formula theoretically supposes complete preservation
of initial (in vivo) water contents in the well-oxygenated
cardiomyocytes of saline perfused hearts. This consideration is reinforced by recent direct demonstration that in
saline perfused hearts the cardiomyocytes quickly (in a
few minutes) respond to any changes in osmolarity of
perfusate by accumulation or loss of total cellular water
[10].
56
M.K. Aliev et al. / Cardiovascular Research 53 (2002) 48 – 58
Therefore, we reanalyzed the data of Polimeni and
Buraczewski [69]. They compared the morphometrically
measured in vitro cardiomyocyte water content of 3.34
ml / g dm with in vivo cellular water contents of 2.50 ml / g
dm previously measured by Polimeni [25] (see Fig. 2 in
Ref. [69]). Polimeni used low-molecular extracellular
tracers to determine the contents of cellular water in intact
hearts [37]. Taking into account the ratio of dry mass
contents in standardized (189.4 g / kg swm, see Section
2.2) and intact (211 g / kg wm, Table 2) hearts, as well as
possible 8.5% penetration of low-molecular tracer into
intact cardiomyocytes (see Section 3.3), the actual content
of cellular water in vivo in the experiments of Polimeni
[37] can be calculated as 3.04 ml / g sdm (2.503(211 /
189.4) /(120.085)). Thus, the cell hydration for data of
Polimeni and Buraczewski [69] can be estimated as 9.9%
(3.34 / 3.043100). This value, lower than the 33.5%
reported by Polimeni and Buraczewski [69], appears more
acceptable.
A predicted slight cell hydration in saline perfused
hearts suggests an experimental re-evaluation of osmolarity
values in all used ‘normoosmotic’ crystalloid perfusion
media. The actually isoosmotic well oxygenated perfusion
solution should guarantee 10061% conservation of initial
(in vivo) water contents in cardiomyocytes of otherwise
hydrated hearts. Only with actually isoosmotic (normoosmotic) perfusion media the model of standardized saline
perfused heart can be successively used for metabolic
analysis. This example also shows the usefulness of the
SPH model for the analysis of data in hydrated hearts.
explained by the underestimation of the volume of total
cellular water (see details in Section 3.3).
The cellular contents of charged ATP and PCr molecules, determined by chemical and NMR techniques, are
similar in the majority of experiments (see review of Saks
[1]). This means that the vast majority of these molecules
belong to free (unbound) molecules. When we cannot
neglect the bound metabolites, special corrections should
be taken into account to determine the concentrations of
free metabolites.
Our analysis based on the SPH model demonstrates that
this approach can be used to evaluate the most probable
values of cellular water contents and metabolite concentrations in intact and hydrated hearts. The SPH model
supposes normalization of values per dry mass of tissue,
since the determination of tissue dry mass is rather simple
and essentially free from severe artifacts. Such a normalization has an advantage over the conventional practice of
normalizing the measured values to wet mass or protein
mass of hydrated hearts, since the determination of actual
tissue wet mass of hydrated heart can be severely affected
by postperfusional manipulations [38,40]. The measured
protein contents in hydrated hearts are sometimes very
high, up to 155 mg / g wm [71], while even in the SPH
hearts the protein content, taken as 70% of dry mass
content [29,44], must be lower, about 133 mg / g wm
(189.4 g dm / kg swm30.7). Potentially, the SPH model
may be the basis for unified treatment of data in heart
bioenergetics, allowing the direct comparison of metabolic
data from different works.
4. Concentrations of intracellular metabolites in
intact and ‘standardized’ perfused hearts
Acknowledgements
Having evaluated volumes of the bulk water domains
where metabolites can diffuse, one can calculate the
concentration, Ci , of a certain metabolite i as the ratio of
the total amount of this metabolite, Mi , to the diffusion
accessible water space of the compartment, Vi , (Table 3,
last column).
For example, for a chemically measured ATP content of
23.862.6 mmol per g dm of perfused rat heart [24], the
calculations of ATP concentration will be performed in
two steps. First, the ATP content will be expressed per
202.6 g dm of standardized heart, yielding 4822 mmol
ATP/ kg swm (23.83202.6). Second, assuming for simplicity (for details see Ref. [2]) a uniform distribution of
ATP in the diffusion accessible water domains of myofibril, sarcoplasm and mitochondria with a pooled volume of
529 ml / kg swm (3211551153, Table 3), we obtain an
ATP concentration in all these compartments of about 9.12
mM. The original estimate by Clarke et al. [24] of the
cellular ATP, 10.1 mM, is higher, despite the fact that they
normalized the ATP content per whole fraction of estimated total cellular water. This discrepancy could be
The authors wish to thank Professors V.I. Lobyshev,
Moscow State University, Russia, and V.I. Kapelko, Cardiology Research Center, Moscow, Russia, for valuable
discussions; Dr. K.D. Garlid, Oregon Graduate Institute of
Science and Technology, USA, for constructive advice and
critical analysis of the text and an anonymous reviewer for
valuable comments.
Part of this work was supported by grant 00-04-48330
from the Russian Foundation for Basic Researches (MKA
and ANT) and INTAS grant 99-1086 (ANT). MKA thanks
INSERM for a 3-month support in France.
References
[1] Saks VA, Khuchua ZA, Vasilyeva EV, Belikova YuO, Kuznetsov AV.
Metabolic compartmentation and substrate channeling in muscle
cells. Role of coupled creatine kinases in in vivo regulation of
cellular respiration – a synthesis. Mol Cell Biochem 1994;133–
134:155–192.
[2] Aliev MK, Saks VA. Compartmentalized energy transfer in cardiomyocytes: use of mathematical modeling for analysis of in vivo
regulation of respiration. Biophys J 1997;73:428–445.
M.K. Aliev et al. / Cardiovascular Research 53 (2002) 48 – 58
[3] Masuda T, Dobson GP, Veech RL. The Gibbs–Donnan near-equilibrium system of heart. J Biol Chem 1990;265:20321–20334.
[4] Wan B, Doumen C, Duszynski J, Salama G, LaNoue KF. A method
of determining electrical potential gradient across mitochondrial
membrane in perfused rat heart. Am J Physiol 1993;265:H445–
H452.
[5] Meerson FZ. The myocardium in hyperfunction, hypertrophy and
heart failure. Circ Res 1969;25(Suppl II):88.
[6] Amano J, Sunamori M, Kameda T, Okamura T, Suzuki A. Correlation among water content, left ventricular function, coronary blood
flow, and myocardial metabolism after hypothermic ischemic cardiac arrest. Adv Myocardiol 1983;4:465–471.
[7] Murry CE, Richard VJ, Reimer KA, Jennings RB. Ischemic preconditioning slows energy metabolism and delays ultrastructural
damage during a sustained ischemic episode. Circ Res 1990;66:913–
931.
[8] Qui Y, Hearse DJ. Comparison of ischemic vulnerability and
responsiveness to cardioplegic protection in crystalloid-perfused
versus blood-perfused hearts. J Thoracic Cardiovasc Surg
1992;103:960–968.
[9] Garcia-Dorado D, Oliveras J, Gili G et al. Analysis of myocardial
oedema by magnetic resonance imaging early after coronary artery
occlusion with or without reperfusion. Cardiovasc Res
1993;27:1462–1469.
[10] Askenazy A, Navon G. Continuous monitoring of intracellular
volumes in isolated rat hearts during normothermic perfusion and
ischemia. J Mag Res 1997;124:42–50.
[11] Vandenberg JI, Rees SA, Wright AR, Powell T. Cell swelling and
ion transport pathways in cardiac myocytes. Cardiovasc Res
1996;32:85–97.
[12] Haussinger D, Lang F, Gerok W. Regulation of cell function by the
cellular hydration state. Am J Physiol 1994;267:E343–E355.
[13] Weiergraber O, Haussinger D. Hepatocellular hydration: signal
transduction and functional implications. Cell Physiol Biochem
2000;10:409–416.
[14] Deaton LE. Comparative aspects of cellular-volume regulation in
cardiomyocytes. Physiol Zool 1997;70:379–390.
[15] Overton E. Beitrage zur allgemeinen Muskel-und Nervenphisiologie
II. Pflugers Arch 1902;92:346–386.
[16] Clegg JS. Interrelationships between water and cellular matabolism
in Artemia cysta. VI. RNA and protein synthesis. J Cell Physiol
1977;91:143–154.
[17] Saks VA, Dos Santos P, Gellerich FN, Diolez P. Quantitative studies
on enzyme–substrate compartmentation, functional coupling and
metabolic channeling in muscle cells. Mol Cell Biochem
1998;184:291–307.
[18] Ling GN. The polarized multilayer theory of cell water. In: Keith
AD, editor, The aqueous cytoplasm, New York: Marcel Dekker,
1979, pp. 23–60.
[19] Ling GN. In search of the physical basis of life, New York: Plenum
Press, 1985.
[20] Belton PS, Ratcliffe RG. NMR and compartmentation in biological
tissues. Progr NMR Spectrosc 1985;17:241–279.
[21] Garlid KD. The state of water in biological systems. Int Rev Cytol
2000;192:281–302.
[22] Morgan HE, Regen DM, Park CR. Identification of a mobile
carrier-mediated sugar transport system in muscle. J Biol Chem
1964;239:369–374.
[23] Bunger R. Compartmented pyruvate in perfused working heart. Am
J Physiol 1985;249:H439–H449.
[24] Clarke K, Anderson R, Nedelec JF, Foster D, Ally A. Intracellular
and extracellular spaces and the direct quantification of molar
intracellular concentrations of phosphorus metabolites in the isolated
rat heart using 31 P NMR spectroscopy and phosphonate markers.
Mag Res Med 1994;32:181–188.
[25] Polimeni PP, Cutilletta AF, Otten MD. Cation distributions in the
hypertrophic myocardium (aortic construction) of the rat. Cardiovasc
Res 1983;17:170–176.
57
[26] Dobson PF, Cieslar JH. Intracellular, interstitial and plasma spaces
in the rat myocardium in vivo. J Mol Cell Cardiol 1997;29:3357–
3363.
[27] LaNoue KF, Walajtys EJ, Williamson JR. Regulation of glutamate
metabolism and interactions with the citric acid cycle in rat heart
mitochondria. J Biol Chem 1973;248:7171–7183.
[28] Kauppinen RA, Hiltunen JK, Hassinen I. Subcellular distribution of
phosphagens in isolated perfused rat heart. FEBS Lett
1980;112:273–276.
[29] Bunger R, Soboll S. Cytosolic adenylates and adenosine release in
perfused working rat heart. Comparison of whole tissue with
cytosolic non-aqueous fractionation analysis. Eur J Biochem
1986;159:203–212.
[30] Anversa P, Olivetti G, Melissari M, Loud AV. Morphometric study
of myocardial hypertrophy induced by abdominal aortic stenosis.
Lab Invest 1979;40:341–349.
[31] Anversa P, Levicky V, Beghi C, McDonald SL, Kikkawa Y.
Morphometry of exercise-induced right ventricular hypertrophy in
the rat. Circ Res 1983;52:57–64.
[32] Kowaltowski AJ, Seetharaman S, Paucek P, Garlid KD. Bioenergetic
consequences of opening the mitochondrial ATP-sensitive K 1
channel of heart mitochondria. Am J Physiol 2001;280:H649–H657.
[33] Garlid KD, Paucek P, Yarov-Yarovoj V et al. Cardioprotective effect
of diazoxide and its interaction with mitochondrial ATP-sensitive
K 1 channels: possible mechanism of cardioprotection. Circ Res
1997;81:1072–1082.
[34] Laclau MN, Boudina S, Thambo JB et al. Cardioprotection by
ischemic preconditioning preserves mitochondrial function and
functional coupling between adenine nucleotide translocase and
creatine kinase. J Mol Cell Cardiol 2001;33:947–956.
[35] Askenazy N, Navon G. Intermittent ischemia: energy metabolism,
cellular volume regulation, adenosine and insights into preconditioning. J Mol Cell Cardiol 1997;29:1715–1730.
[36] Cieslar J, Huang MT, Dobson GP. Tissue spaces in rat heart, liver
and skeletal muscle in vivo. Am J Physiol 1998;275:R1530–R1536.
[37] Polimeni PI. Extracellular space and ionic distribution in rat
ventricle. Am J Physiol 1974;227:676–683.
[38] Aliev MK, Khatkevich AN, Tsyplenkova VG, Meertsuk FE,
Kapelko VI. Tracer kinetics analysis of the extracellular spaces in
saline perfused hearts. Exp Clin Cardiol 2001, in press.
[39] Fisher RB, Lindsay DB. The action of insulin on the penetration of
sugars into the perfused rat heart. J Physiol 1956;131:526–541.
[40] Taylor IM, Huffines WD, Young DT. Tissue water and electrolytes
in an isolated perfused rat’s heart preparation. J Appl Physiol
1961;16:95–102.
[41] Cellarius YuG, Eriskovskaya NK. Stereological study of absolute
total volumes of structural components of the myocardium in
hypertrophy. Bull Exper Biol Med 1979;87:627–630, [Russian].
[42] Page E, McCallister LP. Quantitative electron microscopic description of heart muscle cells. Application to normal, hypertrophied and
thyroxin-stimulated hearts. Am J Cardiol 1973;31:172–181.
[43] von Zgliniski T, Bimmer M. The intracellular distribution of ions
and water in rat liver and heart cells. J Microsc 1987;146:77–85.
[44] Albers B, Bray D, Lewis J et al. In: Molecular biology of the cell,
New York, London: Garland, 1983, p. 487.
[45] Clejan S, Jonas E, Collipp PJ, Fugler L, Maddaiah VT. Influence of
growth hormone and thyroxine on thermotropic effects of respiration
and 1-anilino-8-naphthalene sulphonate fluorescence and on lipid
composition of cardiac membranes. Biochim Biophys Acta
1981;678:250–256.
[46] Daum G. Lipids of mitochondria. Biochim Biophys Acta
1985;822:1–42.
[47] Horikawa Y, Goel A, Somlyo AP, Somlyo AV. Mitochondrial
calcium in relaxed and tetanized myocardium. Biophys J
1998;74:1579–1590.
[48] Holian A, Wilson DF. Relationship of transmembrane pH and
electrical gradients with respiration and adenosine 59-triphosphate
synthesis in mitochondria. Biochemistry 1980;19:4213–4221.
58
M.K. Aliev et al. / Cardiovascular Research 53 (2002) 48 – 58
[49] Beavis AD, Brannan RD, Garlid KD. Swelling and contraction of
the mitochondrial matrix. J Biol Chem 1985;260:13424–13433.
[50] Werkheiser WC, Bartley W. The study of steady-state concentrations
of internal solutes of mitochondria by rapid centrifugal transfer to a
fixation medium. Biochem J 1957;66:79–91.
[51] Barth E, Stammler G, Speiser B, Schaper J. Ultrastructural quantitation of mitochondria and myofibrils in cardiac muscle from 10
different animal species including man. J Mol Cell Cardiol
1992;24:669–681.
[52] Bull H, Breese K. Protein hydration. I. Binding sites. Arch Biochem
Biophys 1968;128:488–496.
[53] Kuntz JD. Hydration of macromolecules. III. Hydration of polypeptides. J Am Chem Soc 1971;93:514–516.
[54] Rupley JA, Careri G. Protein hydration and function. Adv Protein
Chem 1991;41:37–172.
[55] Luzardo MC, Amalfa F, Nunez AM et al. Effect of trehalose and
sucrose on the hydration and dipole potential of lipid bilayers.
Biophys J 2000;78:2452–2458.
[56] Yevdokimov YM, Golo VL, Salyanov VI, Lortkipanidze GB, Kats
EI. The ‘phantom’ structure of solvent and the packing of doublestranded molecules of nucleic acids in particles of mesomorphic
dispersions. Biofizika (Biophys Russia) 2000;45:1029–1038.
[57] Hill AV. The state of water in muscle and blood and the osmotic
behavior of muscle. Proc Roy Soc 1930;B106:477–505.
[58] Belton PS, Jackson RR, Packer KJ. Pulsed NMR studies of water in
striated muscle. I. Transverse nuclear spin relaxation times and
freezing effects. Biochim Biophys Acta 1972;286:16–25.
[59] Outhred RK, George EP. Water and ions in muscle and model
systems. Biophys J 1973;13:97–103.
[60] Garlid KD. Overview of our understanding of intracellular water in
hydrated cells. In: Crowe JH, Clegg JS, editors, Dry biological
systems, New York: Academic, 1978, pp. 3–19.
[61] Elford BC. Non-solvent water in muscle. Nature 1970;227:282–283.
[62] Garlid KD. Aqueous phase structure in cells and organelles. In:
Drost-Hansen W, Clegg JS, editors, Cell-associated water, New
York: Academic, 1979, pp. 293–361.
[63] Khurgin YI, Sherman FB, Tusupkaliev U. Izotermy gidratatsii
globulyarnyh belkov v dinamicheskom rezhime (The hydration
isotherms of globular proteins in the dynamic mode). Biokhimia
(Biochemistry USSR) 1977;42:490–497.
[64] Kuntz JD. Hydration of macromolecules. II. Effects of urea on
protein hydration. J Am Chem Soc 1971;93:514–516.
[65] Priev A, Almagor A, Yedgar S, Gavish B. Glycerol decreases the
volume and compressibility of protein interior. Biochemistry
1996;35:2061–2066.
[66] Avdulov NA, Chochina SV, Daragan VA et al. Direct binding of
ethanol to bovine serum albumin: a fluorescent and 13 C NMR
multiplet relaxation study. Biochemistry 1996;35:340–347.
[67] Tsai AM, Neumann DA, Bell LN. Molecular dynamics of solid-state
lysozyme as affected by glycerol and water: a neutron scattering
study. Biophys J 2000;79:2728–2732.
[68] Polimeni PI, Measurement of myocardial electrolyte distributions.
In: Linden RJ (ed). Techniques in the Life Sciences, P3 / II.
Cardiovascular Physiology. Elsevier, Shannon, 1984;P317:1–34.
[69] Polimeni PI, Buraczewski SI. Expansion of extracellular tracer space
in the isolated heart perfused with crystalloid solutions: extracellular
space, trans-sarcolemmal leakage, or both? J Mol Cell Cardiol
1988;20:15–22.
[70] Aliev MK. The analysis of extracellular calcium exchange in
perfused myocardium using mathematical modeling. J Mol Cell
Cardiol 1989;21:849–863.
[71] Dos Santos P, Aliev MK, Diolez P et al. Metabolic control of
contractile performance in isolated perfused rat heart. Analysis of
experimental data by reaction: diffusion mathematical model. J Mol
Cell Cardiol 2000;32:1703–1734.