| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Health and Human Performance, Auburn University and 2 Department of Anatomy, Physiology, and Pharmacology, Auburn University College of Veterinary Medicine, Auburn, Alabama 36849; and 3 Department of Human Nutrition, Foods, and Exercise Science, Virginia Tech, Blacksburg, Virginia 24061
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
This study sought to determine
the effect of a myocardial volume overload (MVO) on sarcolemmal (SL)
lactate (La
) transport and the aerobic profile of
skeletal muscle. SL vesicles were obtained from female rats 10 wk after
either a MVO was induced by creation of an infrarenal fistula
(n = 10), or sham surgeries were performed
(n = 11). Influx of 14C-labeled
L(+)-La was measured at various unlabeled
La concentrations under zero-trans conditions.
La transport kinetics were determined using a
Michaelis-Menten equation with an added linear component to
discriminate between carrier-mediated and diffusional transport.
Although heart and lung weights were significantly increased
(P < 0.0001) in the MVO group, left ventricular function was only modestly altered (P < 0.05). A
significant reduction in type I myosin heavy chain (MHC) in the soleus
and a strong trend (P = 0.06) for a reduced type IIx
MHC in the plantaris were observed in MVO rats, but no differences in
citrate synthase activity or monocarboxylate transporter proteins
(MCT)-1 expression were noted in any muscle. Carrier-mediated
La influx into SL vesicles was similar between sham and
MVO (Km = 12 ± 1 and 18 ± 3 mM;
apparent Vmax = 772 ± 99 and 827 ± 80 nmol · mg1 · min1,
respectively). Total influx at 100 mM was lower in MVO, and this was due to a 30% reduction in membrane diffusion. In conclusion, a 10-wk MVO did not alter MCT-mediated La transport or
protein expression but was associated with modest changes in
myofibrillar proteins and impaired SL diffusive properties.
congestive heart failure; monocarboxylate transporter proteins; monocarboxylate; membrane diffusion
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
CONGESTIVE HEART FAILURE (CHF) may be characterized by a low functional capacity that is due to a rapid onset of exertional fatigue. It is now well known that changes in skeletal muscle structure, function, and metabolism are concomitant with the progression of CHF. Some of these changes include reduced concentrations of ATP and phosphocreatine (4), metabolic abnormalities (10, 22), a reduced activity of mitochondrial enzymes, regardless of muscle fiber composition (12), and a general transition of fiber types toward those that possess a "glycolytic" profile (37). Accordingly, some investigators have reported that experimental and human forms of CHF are associated with a greater reliance on glycolytic metabolism (10) and a more profound development of acidosis (H+ accumulation) at a given work rate (23). These observations clearly demonstrate that the progression of CHF is associated with changes in the oxidative profile of skeletal muscle that may contribute to exertional intolerance.
In agreement with a diminished aerobic capacity, Weber and Janicki
(43) reported that the exertional threshold required to
reach a "lactate threshold" in a group of CHF patients was inversely related to the severity of the disease. They attributed this
observation to a greater rate of lactic acid (HLa) production due to
insufficient peripheral oxygen delivery in CHF patients. The large
majority of HLa formed during activity dissociates to a lactate
(La) anion and H+ within the physiological pH
range. Because accumulation of these metabolites in the myoplasm has
been shown to impair function of the sarcoplasmic reticulum
(16), key metabolic enzymes (40), Ca2+ sensitivity of myofilaments (15), and the
rates of force production and relaxation (1), an elevated
La concentration {[La]; and associated
H+ concentration ([H+])} in muscle and
blood during activity may explain some of the functional impairment
associated with CHF.
The role of skeletal muscle in systemic La dynamics is
the cornerstone of the "lactate shuttle hypothesis"
(7). Briefly, this hypothesis holds that La
production and distribution throughout the body are a major mechanism by which intermediary metabolism is coordinated in different tissues and cells within those tissues. With respect to skeletal muscle, it is
now known that substantial La exchange occurs between
muscle and blood, between active and inactive muscles, between active
muscles and fibers within muscles (7), and even between
myocellular compartments as part of an intracellular La
shuttle (8). In fact, skeletal muscle represents the
greatest source of La production and consumption in the
body due to its relative mass. Therefore, whole body La
dynamics are ultimately regulated in, and by, skeletal muscle (32).
Movement of La and H+ across the sarcolemma
(SL) of striated muscle occurs primarily via two isoforms of SL-bound
monocarboxylate transporter proteins (MCT1 and MCT4). La
transport (19) and uptake (3, 28), as well as
MCT expression (3, 31), have been shown to be strongly
related to the oxidative capacity of skeletal muscle. Furthermore,
La transport and expression of MCT proteins in skeletal
muscle have also been shown to be increased by contractile activity and
training (25, 26, 29), whereas La transport
is decreased by hypodynamia (14).
Given the close dynamic relationship between La and
H+ transport and the oxidative properties of skeletal
muscle and that oxidative properties are altered during the development
of CHF, it is plausible that SL La transport may be
influenced as well. In the context of the "lactate shuttle"
(7), the acidosis observed in skeletal muscle of
individuals with CHF during activity may not only be explained by an
accelerated HLa production, but a reduced capacity for La
extrusion from active muscle fibers due to alterations in SL transport
properties. Similarly, blood [La] may also remain
elevated for longer periods of time because of impaired uptake into
consuming tissues (e.g., active and inactive skeletal muscle) due to a
reduced capacity for La oxidation and SL transport.
Combined, these effects would result in a rapid, exaggerated
development of systemic lactacidosis during activity and a prolonged
recovery of acid-base status on cessation. This could partly explain
the low exertional threshold observed in individuals afflicted with CHF.
The purpose of this investigation was to examine the effect of a
myocardial volume overload (MVO), which is a commonly used model to
study the progression of CHF, on skeletal muscle La
transport characteristics. Purified SL vesicles were chosen for this
end in an attempt to circumvent confounding variables such as blood
flow, multiple membrane barriers, and cellular metabolism and to
control the environments on both sides of the membrane. It was our
hypothesis that a chronic (10 wk) MVO would have deleterious effects on
La transport in vesicles isolated from mixed rat skeletal
muscle. Furthermore, we hypothesized that this would be associated with changes in metabolic and myofibrillar properties of various hindlimb muscles.
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Animals, Housing, and Care
All procedures in the current investigation involving animals were reviewed and approved by the Auburn University Institutional Animal Care and Use Committee. Twenty-one female Sprague-Dawley rats were obtained at 3 mo of age and were randomly assigned to either an MVO (n = 10) or sham-operated (n = 11) group. All animals were housed in pairs (1 MVO + 1 sham-operated per cage), maintained on a 12:12-h light-dark cycle (6:00 AM to 6:00 PM light), and received food and water ad libitum.Induction of MVO
A MVO was surgically induced in rats by creation of an aortocaval fistula as described by Garcia and Diebold (17). Briefly, animals were anesthetized with a mixture of ketamine and xylazine (60 and 7 mg/kg, respectively), and an abdominal laparotomy was performed to expose the inferior vena cava and descending aorta. Circulation was impeded via light finger pressure, and a beveled 18-gauge needle was used to create a fistula between both vessels just below the renal arteries. The resulting puncture was sealed with a few drops of cyanoacrylate, and patency of the fistula was confirmed by visualization of pulsatile flow of oxygenated blood into the vena cava. The abdomen was flushed with saline, and the musculature and skin were closed with sutures and autoclips. This technique greatly increases venous return and results in a progressive, biventricular eccentric hypertrophy of the myocardium (9). Sham surgeries, consisting of an abdominal laparotomy without creation of the fistula, were also performed on the remaining rats. All animals were kept in a warm, dry room after surgery until they were ambulatory and showed no signs of discomfort. Rats were killed 10 wk postsurgery for experimental procedures.Activity Monitoring
To ascertain whether skeletal muscle properties may have been influenced by changes in activity levels, gross locomotor activity was monitored during the middle (week 5) and final (week 10) weeks of the experimental period after surgery. This was achieved by placing the rats on an enclosed activity wheel that was interfaced with a revolution counter. Activity was monitored during two consecutive 12-h dark cycles of each week, and data were recorded as a 2-day average number of revolutions per 12-h cycle.Myocardial Function Measurements
All animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). A tracheotomy was performed, and the rats were placed on mechanical ventilation (40 breaths/min) for the remainder of the surgical procedure and functional measurements. The thoracic cavity was exposed, and an ultrasonic flow probe (Transonic T-101) was placed around the ascending aorta for measurement of cardiac output (Q). A 16-gauge beveled needle was passed through the apex of the heart, and a 3-Fr microtip pressure transducer (Millar SPR-249A) was advanced into the left ventricle (LV) for measurement of end-diastolic (LVEDP) and ventricular pressures throughout the cardiac cycle. The analog signals from the flow probe and pressure transducer were digitized and interfaced with a chart recorder and computer for calculation of the derivative of LV pressures (±dp/dt).Both hindlimbs were quickly skinned after functional measurements, and the musculature was removed. The soleus, plantaris, and extensor digitorum longus (EDL) were bilaterally excised and snap-frozen in liquid N2 for subsequent biochemical and morphological analyses. The remaining hindlimb muscle groups were removed for preparation of SL vesicles.
Skeletal Muscle Biochemistry, Morphology, and MCT Expression
Citrate synthase.
Frozen muscle samples were thawed, trimmed free of connective tissue,
weighed, and minced in a 20:1 volume of a 100 mM phosphate buffer with
0.05% bovine serum albumin, pH 7.4 at 25°C. The minced muscle was
homogenized in a glass-glass tissue grinder, brought to a final
dilution of 101:1, and spun at 400 g for 10 min at 4°C.
The supernatant was removed and stored at 
80°C until used for
protein (6) and enzymatic analysis.
1 · min1).
Separation of myosin heavy chains. Remaining soleus, plantaris, and EDL samples were homogenized with a glass-glass tissue grinder in a 10:1 volume of buffer consisting of (in mM) 250 sucrose, 100 KCl, 5 EDTA, 20 Tris, pH 6.8. Protein concentrations of homogenates were determined by the method of Bradford (6) using bovine serum albumin as a standard. Electrophoretic separation of myosin heavy chains (MHC) was performed according to the detailed methodology of Talmadge and Roy (41). Homogenates were diluted in 2× sample buffer (21) to a final protein concentration of 0.125 µg/ml, boiled for 2 min, and 1.25-1.50 µg of protein was loaded into each well of an 8% SDS-PAGE gel. The electrophoresis system (BioRad Mini Protean II) was run at a constant 80 V for 24 h at 4°C. Gels were stained in a solution containing 40% methanol, 10% acetic acid, 0.1% Coomassie brilliant blue R-250, and destained in 40% methanol and 10% acetic acid. The resulting bands were quantified with a scanning densitometer (Alpha Innotech IS-2000), and the proportion of each MHC isoform was taken from the relative area under each peak.
Western immunoblotting of MCT1.
A portion of each MHC homogenate was spun at 600 g (4°C)
to pellet erythrocytes, and the resulting supernatants were removed for
determination of MCT1 expression. Fifty micrograms of supernatant proteins were separated on 12% SDS-PAGE gels and transferred to nitrocellulose membranes (60 min at 100 V). Fifty micrograms of supernatants prepared from ventricular myocardium were also run on each
gel as a standard. Membranes were first blocked for 4 h in
Tris-buffered saline with 0.05% Tween-20 and 5% nonfat milk, incubated overnight on a rocker (4°C) with a chicken 
-MCT1
polyclonal antibody (Chemicon, 1:2,500), then for 1 h at room
temperature with a horseradish peroxidase-conjugated -chicken
secondary antibody (1:2,500). MCT1 bands were detected via enhanced
chemiluminescence and quantitated using computerized densitometry.
Preparation of Purified SL Vesicles
Skeletal muscle SL vesicles were purified by the method of Grimditch et al. (18), with modifications. Twelve to fifteen grams of skeletal muscle (gastrocnemius, quadriceps, and gluteus maximus groups) were quickly excised from rat hindlimbs and placed into an ice-cold homogenizing medium (HM) consisting of 250 mM sucrose and 20 mM HEPES, pH 7.4 at 4°C. The tissue was trimmed free of visible connective tissue, fat, nerve, and blood vessels, minced with blunt scissors, and subjected to mechanical homogenization at 80% of maximal power (Fisher Powergen 700). The sample was transferred to a Teflon-glass tissue grinder for 10 passes with the pestle under a mechanical stirrer (Caframo RZR1) at 50-75% of maximal power. The sample was brought to a volume of 40 ml with HM, transferred to ultracentrifuge tubes, and termed crude homogenate (CH). A 200-µl sample of CH was removed, diluted 1:2 with HM, snap-frozen in liquid N2, and stored at80°C until determination of protein
concentration (6) and SL marker enzyme analysis. A volume
of KCl medium (3 M KCl, 250 mM Na-pyrophosphate) equal to 10% of the
final CH volume was added and vigorously mixed to salt out contractile proteins.
The CH was spun in a Beckman L8-70 ultracentrifuge with a 60-Ti
fixed-angle rotor at 222,000 g for 90 min (4°C) in a
vacuum. The supernatant from this first spin was discarded, and the
inner walls of the ultracentrifuge tubes were wiped clean of any excess fat and KCl medium. The remaining pellets were suspended in HM by
Teflon-glass homogenization as previously described, brought to a final
volume of 40 ml, and transferred to an Erlenmeyer flask. Forty-thousand
Kunitz units (1,000 Kunitz units/ml) of deoxyribonuclease I
(Worthington Biochemical, Lakewood, NJ) were mixed with the prepurified
membrane suspension, which was then incubated in a shaking water bath
at 30°C for 60 min. The sample was spun after incubation, as
previously described (222,000 g for 90 min). Each resulting
pellet was suspended by Teflon-glass homogenization in 14 ml of 34%
sucrose, placed in the bottom of a 36-ml open-top ultracentrifuge tube,
and underlayed with 2 ml of 45% sucrose. A discontinuous sucrose
density gradient was formed by careful addition of 3.5 ml each of 32, 30, 27, and 12% sucrose on top of the membranes. Gradients were
subjected to isopycnic centrifugation in a Beckman SW-27
swinging-bucket rotor at 68,000 g for 16 h, and SL
vesicles were harvested from the 27 and 30% layers. The membranes were
diluted with Krebs-Ringer-HEPES buffer (KRH; in mM: 135 NaCl, 5 KCl,
1.2 MgSO4, and 50 HEPES, pH 7.4 at 25°C) and pelleted in
a fixed-angle rotor for 90 min at 222,000 g. The resulting
purified vesicles were suspended in KRH buffer to a protein
concentration of 1-1.5 mg/ml. A 50-µl aliquot was partitioned for subsequent determination of protein concentration (6)
and SL marker enzyme analysis. After protein analysis and final
suspension, vesicles were snap-frozen in liquid N2 and
stored at 80°C until used for transport experiments.
Purity of the vesicle preparation vs. CH was determined by measuring
the activity of the SL marker enzyme, K+-stimulated
p-nitrophenolphosphatase (KpNPPase).
KpNPPase is a domain of the Na+-K+
adenosinetriphosphatase that is located on the extracellular surface of
an intact SL and is regarded as a good indicator of vesicle purity
(14, 18). Activity was assayed in a medium consisting of
(in mM) 40 HEPES, 4 MgCl2, 0.8 EGTA, and 20 KCl, pH 7.4, 37°C. Basic phosphatase activity was also assayed in the same medium
but without KCl. The reaction was initiated by addition of
p-nitrophenolphosphate as a substrate (final
concentration = 5 mM) and stopped with 1 ml of 1 N NaOH. The
difference between the K+-stimulated and basic phosphatase
activity (expressed as µmol p-nitrophenol
formed · mg protein1 · h1)
represents the KpNPPase. A purification index, defined as
the ratio of SL- to CH-specific activities, was used to estimate
quality of the final preparation. SL yield was expressed as milligrams of SL protein obtained per gram of trimmed starting tissue.
La Transport Assays
transport in SL vesicles was studied under
zero-trans conditions at 25°C, as previously described
(33, 34). Sixty microliters of an isosmotic KRH solution
containing various concentrations (2, 5, 10, 20, and 100 mM, pH 7.4 at
25°C) of unlabeled L(+)-La and 0.5 µCi of uniformly
labeled 14C-labeled L(+)-La tracer were
placed on the bottom of a 1.5-ml Eppendorf tube. The concentration of
NaCl was decreased accordingly with the addition of
L(+)-La to maintain isosmolarity of the intra- and
extravesicular milieu. Twenty microliters of vesicle suspension were
carefully pipetted onto the side of the tube. Transport was initiated
by vortexing the tube and halted at various times (2, 3, 4, 5, 10, 15, and 20 s) by addition of 1 ml of ice-cold KRH containing 3 mM
HgCl2. A metronome and stopwatch were used for time
measurement during all transport experiments. In an attempt to validate
the mathematical determination of the linear diffusive component of
influx, assays were also performed at 20 mM extravesicular
[La] on SL vesicles preincubated for 1 h at
room temperature with 1 mM p-chloromercuriphenylsulfonic
acid (pCMBS), a sulfhydryl group reagent. Background binding
in vesicles was assessed by incubating vesicles in the various
tracer-containing media that were diluted with ice-cold
KRH/HgCl2 stop solution. The radioactivity from these
background experiments was subtracted from that obtained in each influx
assay. The stopped, diluted vesicles were immediately vacuum-filtered
through a protein-binding cellulose-ester membrane (Millipore,
0.45-µm average pore size) and washed twice with 3 ml of
KRH/HgCl2 stop solution. The vesicle-containing filters and
standards were placed in vials with 5 ml Scintiverse BD (Fisher Scientific) cocktail, and radioactivity was counted in a Wallac 1515 Winspectral liquid-scintillation counter.
Specific influx of La (defined as nmol/mg SL protein)
during the 0- to 20-s time interval was fitted to a monoexponential equation of the type yt = C Aekt, where yt is
La influx at time t, C is the asymptote of
influx with increasing time, A is a constant, e
is the base of natural logarithms, and k is the rate
constant for the approach of La influx toward the
asymptote. Initial rates of influx were determined as the solution to
the first derivative of the exponential equation at
time 0 (C · k). These initial rates
from total influx trials were then plotted as a function of
extravesicular [La] and applied to a Michaelis-Menten
plus linear equation V = (Vmax · [S])/([S] + Km) + ([S] · M); where
V is the rate of influx, Vmax is the
maximal rate of carrier-mediated influx, [S] is the extravesicular
[La], Km is the Michaelis-Menten
affinity constant, and M is the linear slope to determine the relative
contributions of carrier-mediated transport and passive diffusion at
each [S]. Specific diffusion into SL vesicles was analyzed by
comparing both the rates of transport at each individual
[La] and the Ms for each animal. The rates of
carrier-mediated transport were used for determination of maximal
velocity of transport (apparent Vmax) and
transporter-substrate affinity (Km). All kinetic
analyses were performed using the Kaleidagraph 3.09 for Windows
software package (Synergy software).
Statistical Analysis
Differences between sham-operated and MVO rats were analyzed with an independent Student's t-test. However, activity measurements and La influx rates in saturation
experiments were analyzed with a two-way repeated-measures ANOVA and a
Student-Newman-Keuls post hoc test. A significance level of 0.05 was
selected for all analyses in this investigation.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Morphological Parameters
Mean body and organ weight data from sham-operated and MVO rats are provided in Table 1. Although initial body weights did not differ between groups, MVO rats demonstrated a 67% greater increase in body weight during the 10 wk after surgery (P < 0.01). Mean heart and lung weights were also significantly higher in the MVO group, even when expressed relative to body weight (P < 0.0001 for all comparisons).
|
Hemodynamic Measurements
Hemodynamic measurements in situ are presented in Table 2. As expected, creation of an arteriovenous fistula resulted in a 2.5-fold increase in total
after 10 wk (P < 0.0001). This, taken together with
the increase in heart weight, indicates that the fistula surgery
successfully induced a chronic MVO and cardiac hypertrophy. LVEDPs were
significantly greater in MVO rats (P < 0.05), although
these values (mean = 6.4 mmHg) were not outside normal
physiological ranges. Additionally, although the rates of LV pressure
changes (±dp/dt) tended to be lower in the MVO group, the
differences in means were not statistically significant (P > 0.05). Thus it appears that the MVO rats in this
investigation were still in a state of myocardial compensation at the
time of death.
|
Activity Measurements
Gross locomotor activity levels, reported as the average number of activity wheel revolutions achieved during two consecutive 12-h dark cycles, are provided in Table 3. A two-way repeated-measures ANOVA revealed a significant main effect of group (MVO vs. sham), with activity levels being ~30% lower in MVO rats during both measurement periods. No differences were observed between the trials at the 5th and 10th wk (P > 0.05 for trial and group by trial interaction).
|
Skeletal Muscle Aerobic Profile
CS.
Specific activities of the mitochondrial enzyme CS (µmoles citrate
formed · 100 mg
protein1 · min1) are reported for
soleus, plantaris, and EDL supernatants in Table
4. No significant differences in CS
activities were observed between groups in any of the muscle
supernatants assayed (P > 0.05 for all comparisons).
|
MHC analysis.
A scanned image of a representative polyacrylamide gel is provided in
Fig. 1, and the proportions of MHC
isoforms I, IIa, IIx, and IIb in various hindlimb muscles are presented
in Table 5. Soleus samples from MVO rats
demonstrated a significantly lower (5%) proportion of type I MHC
with a concomitant increase in type IIa MHC (P < 0.05). Although no additional significant differences in MHC expression
were observed in the other muscles examined, there was a strong trend
for a lower percentage of type IIx isoforms in plantaris samples from
MVO rats (P = 0.06). This effect was associated with
minute, nonsignificant increases in both IIa and IIb isoforms.
|
|
MCT1 expression.
Results of the Western immunoblot analysis of MCT1 content of soleus,
plantaris, and EDL samples are shown in Fig.
2. When MCT1 expression levels were
normalized as a percentage of the heart value, no significant
differences were observed between groups in any of the muscles studied
(P > 0.05).
|
SL Characterization and La Transport
SL characterization.
Isopycnic ultracentrifugation of sucrose density gradients resulted in
a purified SL membrane fraction between the 27 and 30% layers. This
was indicated by high specific activities of the SL marker enzyme
KpNPPase, compared with those of the CH (Table 6). The resulting purity indexes (SL vs.
CH KpNPPase ratios) are comparable with some previously
published results (33, 35) and are approximately twofold
higher than other recently reported results (13). No
differences were observed between sham-operated and MVO groups with
respect to CH and SL KpNPPase activities, purity indexes, or
SL yields (P > 0.05). An electron micrograph from a
representative SL preparation is provided in Fig.
3 and confirms the existence of vesicles
that are primarily spherical in nature and are free of contamination by
mitochondria or other subcellular organelles.
|
|
La transport in SL vesicles.
Incubation of SL vesicles in tracer-containing media resulted in an
influx of La that demonstrated an exponential
relationship with time between 0 and 20 s (Fig.
4). The slopes of the ensuing
curves increased in a manner accordant with extravesicular
[La]. These curves were fitted by monoexponential
equations (r2 = 0.93-0.99) for
determination of initial rates of influx at various
[La].
|
transported per milligram of SL protein per minute, are plotted as a function of extravesicular [La] in Fig.
5. Total influx rates
(carrier-mediated + diffusion; Fig. 5A) rose
progressively with [La] and were significantly lower in
the MVO rats at 100 mM (P < 0.05). A trend for reduced
influx in the MVO group was also observed at 20 mM (P = 0.08). The data for total influx were closely fit (r2 = 0.96-0.99) by an equation
containing both Michaelis-Menten and linear components. The specific
rates of lactic acid diffusion (nmol · mg1 · min1;
5B) increased in a linear manner with
[La] and were consistently lower in vesicles
from MVO rats. In fact, the slope of diffusion was reduced by 30% in
the MVO group (8.9 ± 0.9 vs. 12.9 ± 1.4, P < 0.05). Similar to what was observed for total influx, the differences
in diffusion became greater with increasing [La] and
reached statistical significance at 100 mM (P < 0.05). The diffusive component, when expressed relative to total influx (Fig. 6), increased from ~20 to 60% between
2 and 100 mM extravesicular [La], respectively, and did
not differ between sham-operated and MVO rats at any concentration
(P > 0.05). In an attempt to validate the addition of
a linear diffusive component to the Michaelis-Menten equation, the
equation-derived percent diffusion was compared with results from
influx assays performed at 20 mM [La] on SL vesicles
that were preincubated with pCMBS, a sulfhydryl group
reagent. Because no differences in the relative percent diffusion were
observed between groups, the data from sham-operated and MVO groups
were pooled for this comparison. At 20 mM extravesicular [La], the equation-derived diffusive contribution of
35 ± 3% was very similar to the 33 ± 5% determined from
the pCMBS trials.
|
|
influx and diffusion of
lactic acid at each concentration was taken to represent the
MCT-mediated component of transport and is illustrated in Fig.
7. This estimated carrier-mediated
transport displayed saturation kinetics with increasing
[La] and affinity constants (Km)
of 12 ± 1 and 18 ± 3 mM and apparent Vmax of 772 ± 99 and 827 ± 80 nmol · mg SL
protein1 · min1 in the
sham-operated and MVO groups, respectively. In contrast to
total influx and specific diffusion, no significant differences between
groups were observed at any [La] or in either kinetic
parameter (Km, Vmax)
examined (P > 0.05 for all comparisons).
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The primary finding of this investigation is that a 10-wk MVO did
not alter the rates of MCT-mediated SL La transport at
any concentration examined, the apparent Vmax of transport, or the MCT1 content of various hindlimb muscles. Although there appeared to be a slight decrease in carrier-substrate affinity (indicated by an increased Km), the difference
between groups was not statistically significant. Therefore, a 10-wk
MVO in this investigation did not appear to alter the SL-bound MCT
La-H+ cotransport system. Surprisingly,
however, SL diffusional properties were impaired in animals with an MVO.
The Km values obtained in SL vesicles from
sham-operated and MVO rats in this study (12 and 18 mM, respectively)
are consistent with those reported by McDermott and Bonen
(27) using the SL purification method of Klip et al.
(20) and with values obtained in intact soleus strips
(3). However, they are considerably lower than values of
~40 mM reported by others (33-35) using the method
of Grimditch et al. (18) that was modified for this study. We attribute this to the fact that the Km values
reported here are specific to MCT-mediated transport, whereas those
reported by Roth and Brooks (33-35) appear to be for
total La influx (MCT mediated + diffusion). In fact,
the Km values for total La influx
in the current investigation typically ranged between 30 and 40 mM,
which is consistent with their (33-35) results.
The Vmax values obtained in this investigation
are the highest reported for any "classic" SL preparation. In fact,
the Vmax of zero-trans MCT-mediated
transport was six- to eightfold greater than those of total influx
reported by Roth and Brooks (33-35) and others
(13, 14). This discrepancy can be explained primarily by
the calculation of initial rates of transport and is presented graphically in Fig. 8. In other
investigations (13, 14, 33-35), initial rates were
typically determined by linear regression analysis of 5- to 20-s influx
measurements. In the present study, La influx was also
measured before 5 s and the data were fitted by a monoexponential
equation (see MATERIALS AND METHODS). As Fig. 8 clearly
demonstrates, influx during zero-trans conditions is
substantially more rapid during the first 5 s, and calculation of
initial velocities with the current model yields rates that are more
than 10-fold greater than those derived from a 5- to 20-s linear slope.
Therefore, we propose that the values reported in this study are more
representative of true initial rates of transport.
|
To our knowledge, this is the first investigation to appropriately add
a linear function to a Michaelis-Menten equation when analyzing total
La influx kinetics. The linear function was added to
discriminate between carrier-mediated transport and diffusion of lactic
acid and was in agreement with assays performed at 20 mM
[La] after incubation of vesicles with
pCMBS. With the use of this model, we observed that total
influx was reduced at 20 and 100 mM and that this was explained by an
alteration in diffusive properties of the SL vesicles. Analysis of the
linear slope of diffusion revealed a 30% deficit in MVO rats,
suggesting that diffusional properties of the SL were impaired over a
wide range of concentrations (2-100 mM) in these animals. At the
present time, we have no direct explanation for this observation. It
has been shown that the progression of CHF is associated with an
accumulation of interstitial collagen in skeletal muscle and that this
may be secondary to hypoperfusion, deconditioning, and/or increased
concentrations of ANG II (36). Additionally, alterations
in the cholesterol-to-phospholipid and/or lecithin-to-sphingomyelin
ratios of biological membranes are associated with the pathogenesis of
many diseases. Both of these phenomena could certainly have profound
effects on membrane fluidity and could possibly explain the change in
diffusive properties that were observed in this study
(11).
The progression of CHF is associated with deleterious effects in
skeletal muscle that seem to be related to the severity of the disease
(36). With the use of a rodent model of myocardial infarction, Delp et al. (12) observed that reductions in
skeletal muscle mitochondrial enzyme activity and alterations in
fiber-type distribution were only present in rats with severe LV
dysfunction (LVEDP >20 mmHg and an ~50% reduction in
±dp/dt). Similar results were obtained by Simonini et al.
(37), who observed that skeletal muscle alterations were
only present in a group of rats with an LVEDP >15 mmHg. The MVO rats
in the present study demonstrated significant elevations in heart,
lung, and body weights, suggesting the development of both pulmonary
and peripheral edema and progression to a state of CHF. However,
measurements of ± dp/dt and LVEDP indicated a very
moderate change in LV function in this group. In fact, the highest
individual LVEDP measured in the MVO group was 13 mmHg, which is in the
"moderate LV dysfunction" range reported by Delp et al.
(12). Analysis of La influx in vesicles
obtained from a single rat with evidence of decompensated CHF (e.g.,
rapid weight gain, severe generalized edema, heavily labored breathing,
extreme lethargy) demonstrated a Vmax of 110.1 nmol · mg1 · min1, which is
only 22% of the lowest value observed in the 10-wk MVO rats.
Additionally, a trend for a negative correlation was observed between
the Vmax of total La influx and
heart weight in MVO rats (r = 0.64, P = 0.07). Therefore, a likely reason for the lack of effect on skeletal
muscle with respect to MCT-mediated La transport and CS
activity in this investigation was that the 10-wk MVO period did not
induce a sufficiently severe disease state. Nevertheless, our results
do make the important point that MVO animals in a compensated state
with only mild myocardial dysfunction are generally similar to control
animals with regard to skeletal muscle-facilitated La
transport and oxidative capacity.
Another noteworthy observation was that activity levels were nearly
30% lower in MVO rats and that this was associated with a significant
reduction in type I MHC expression in soleus muscles. Additionally,
there was a strong trend for a lower percentage of type IIx MHC in the
plantaris. These results are congruent with other investigations that
demonstrated alterations in skeletal muscle MHC expression and
fiber-type distributions in humans (39, 42) and animal
models of CHF (12) as well as reduced contractile activity
(5, 14, 24). Consequently, these results raise questions
as to 1) whether the changes in MHC expression in this study
were primarily due to the MVO or reduced activity levels and
2) why CS activity, MCT1 expression, and MCT-mediated
La transport activities were unaffected given the
observed changes in MHC isoforms.
Although reduced activity levels may play some role in skeletal muscle
alterations in CHF, several lines of evidence argue against this as the
primary stimulus for peripheral abnormalities. Simonini et al.
(37) reported that although hindlimb muscle samples from
rats with large myocardial infarctions had reductions in the activity
and mRNA levels of mitochondrial enzymes as well as type I fibers,
physical activity levels were not different from those of sham-operated
rats. Furthermore, indexes of disease states such as LVEDP, infarction
size, and ventricular hypertrophy were poorly correlated with activity
levels (r ~ 0.16 for each relationship) but were
significantly related to the observed changes in skeletal muscle. Delp
et al. (12) noted that enzymatic perturbations occurred in
muscles composed of primarily glycolytic type IIb fibers that would not
be recruited to a great extent during normal cage activity
(2). Finally, Vescovo et al. (42)
demonstrated that whereas gastrocnemius samples from CHF patients
exhibited a typical slow-to-fast transition in MHC profile, those
obtained from chronically bed-ridden stroke patients actually showed a profound increase in the proportion of type I MHC isoforms. In the
present study, a marked reduction in activity (30%) was associated with modest changes in types I and IIa isoforms in the soleus and type
IIx isoforms in the plantaris. Therefore, it is tempting to speculate
that these effects are primarily due to hypodynamia. However, this is
unlikely given the evidence cited above, the fact that MHC isoforms
throughout the slow-to-fast continuum appeared to be affected, and that
all animals were confined in cages that limited physical activity.
Additionally, it is uncertain how accurately intermittent measurement
periods on a locomotor wheel or in a photoelectric cell
(37) reflect chronic activity levels under these conditions.
It is well documented that changes in La transport
characteristics and MCT expression are closely related to changes in
oxidative enzyme activities and fiber characteristics of skeletal
muscle (3, 14, 30). Therefore, it was interesting to note
in the current study that although MHC profiles were altered, CS
activity, MCT1 expression, and MCT-mediated transport were unchanged.
Because the observed changes in MHC compositions were relatively small, a possible explanation is that the primary stimuli (e.g., MVO and/or
hypodynamia) were not great enough to elicit changes in La transport. This is supported by the fact that only
types I and IIa MHC isoforms were detected in soleus samples from MVO
rats, which is a typical observation in normal, healthy rats.
Dubouchaud et al. (14) observed that a reduction of influx
into classic SL vesicles, at 1 mM La, after hindlimb
suspension was associated with expression of type IIb MHC in the
soleus. Therefore, it is possible that had more profound changes
occurred (e.g., expression of IIx and/or IIb isoforms), MCT-mediated
transport would have been altered as well.
Another possible explanation for the lack of difference in
carrier-mediated transport was the use of SL vesicles from mixed skeletal muscle. Roth and Brooks (35) noted that
La influx kinetics were nearly identical in mixed muscle
SL vesicles from control, sprint-, and endurance-trained rats, despite
increases in CS activity with both training paradigms. Although
subsequent efforts using mixed muscle SL preparations have noted
changes in La transport in response to chronic stimuli
(14, 24), the contributions of carrier-mediated transport
and diffusion were not identified. Because expression and distribution
of both MCT1 and MCT4 in striated muscle appear to be fiber-type
specific (31, 44), it is possible that discrete changes in
La transport may have been confounded by the use of mixed
muscles in the present study. However, MCT1 expression levels, as
indicated by Western immunoblotting, were not different in the various
muscles examined. Therefore, it seems unlikely that discreet changes in MCT-mediated transport were masked in this investigation.
This is not the first study to report a dissociation between changes in
biochemical, structural, and functional properties in skeletal muscle.
Pilegaard et al. (29) observed in humans that total
La transport and MCT1 and MCT4 expression were increased
in biopsies from trained legs after 8 wk of high-intensity one-legged
cycling. However, no differences were present between trained and
untrained legs with respect to muscle CS activity, fiber-type
distribution, or capillarity. Similarly, hindlimb unweighting in rats
has been shown to induce a slow-to-fast transition in cytosolic enzymes and myofibrillar proteins in postural muscles but had no effect on
mitochondrial properties (5). Therefore, although there is
a strong correlation between La transport, biochemical,
and myofibrillar characteristics of skeletal muscle, a cause-and-effect
relationship has not been established. It seems evident that regulation
of these properties by contractile activity and/or chronic disease
states is complex but not entirely interdependent. In fact, Pilegaard
et al. (29) suggest that even individual La
transport proteins MCT1 and MCT4 are subject to differential regulation
in skeletal muscle.
In conclusion, the results of this investigation demonstrate that a
10-wk MVO in rats did not alter carrier-mediated La
transport in mixed skeletal muscle SL vesicles. However, diffusive properties of the membranes appeared to be reduced by an MVO, and this
observation was associated with a modest slow-to-fast transition in
myofibrillar proteins. Additional research should be conducted to fully
elucidate possible alterations in systemic La dynamics
that are associated with CHF. Subsequent endeavors should use a longer
MVO period or an alternate model of CHF to allow the development of a
more severe state of LV dysfunction and disease.
Perspectives
In the context of the La shuttle hypothesis
(7), changes in either metabolic regulation (e.g., enzyme
activities) or La transport across the SL are important.
More specifically, exaggerated systemic lactacidosis in individuals
with CHF is likely to be explained eventually by alterations in one or
more components of the La shuttle. In the current study,
changes in oxidative capacity, MHC isoforms, MCT1 expression, and
saturable La transport across the SL were minor. However,
significant changes were noted in SL La diffusion, which
suggests that the SL itself may represent an important component of the
La shuttle. This observation may also suggest that an
alteration in La diffusion is an early indicator of
membrane changes and other perturbations in skeletal muscle during the
progression of CHF. It should be emphasized that as transmembrane
La concentration gradients increase, a greater proportion
of total La flux is due to diffusion (see Fig. 6). To
this point, no studies have directly addressed this phenomenon, which
needs to be considered in future research with respect to exercise
training, disuse, and possibly a variety of chronic disease states.
| |
ACKNOWLEDGEMENTS |
|---|
The authors thank J. Stewart, M. F. Hirshman, M. Tovio-Kinnucan, E. Spangenburg, H. Demirel, D. McGee, B. Karkoska, the Auburn University Division of Laboratory Animal Health, Drs. D. Schwartz, L. Wit, J. Janicki, D. Pascoe, E. Lonergan, C. Bird, R. Paxton, and S. Powers for assistance during this investigation.
| |
FOOTNOTES |
|---|
This work was supported by National Institutes of Health Grant #1R01AR-40342.
Address for reprint requests and other correspondence: W. G. Aschenbach, Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215 (E-mail: Bill.Aschenbach{at}joslin.harvard.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 April 2000; accepted in final form 1 March 2001.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Adams, GR,
Fisher MJ,
and
Meyer RA.
Hypercapnic acidosis and increased H2PO
2.
Armstrong, RB,
and
Laughlin MH.
Metabolic indicators of fibre recruitment in mammalian muscles during locomotion.
J Exp Biol
115:
201-213,
1985
3.
Baker, SK,
McCullaugh KJ,
and
Bonen A.
Training intensity-dependent and tissue-specific increases in lactate uptake and MCT-1 in heart and muscle.
J Appl Physiol
84:
987-994,
1998
4.
Bernocchi, P,
Ceconi C,
Pedersini P,
Pasini E,
Curello S,
and
Ferrari R.
Skeletal muscle metabolism in experimental heart failure.
J Mol Cell Cardiol
28:
2263-2273,
1996[ISI][Medline].
5.
Bigard, AX,
Brunet A,
Guezennec C,
and
Monod H.
Effects of chronic hypoxia and endurance training on muscle capillarity in rats.
Pflügers Arch
419:
225-229,
1991[ISI][Medline].
6.
Bradford, M.
A rapid and sensitive method for quantification of microgram quantities of protein using the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
7.
Brooks, GA.
The lactate shuttle during exercise and recovery.
Med Sci Sports Exerc
18:
360-368,
1986[ISI][Medline].
8.
Brooks, GA,
Dubouchaud H,
Brown MA,
Sicurello JP,
and
Butz EC.
Role of mitochondrial lactate dehydrogenase and lactate oxidation in the intracellular lactate shuttle.
Proc Natl Acad Sci USA
96:
1129-1134,
1999
9.
Brower, GL,
Henegar JR,
and
Janicki JS.
Temporal evaluation of left ventricular remodeling and function in rats with chronic volume overload.
Am J Physiol Heart Circ Physiol
271:
H2071-H2078,
1996
10.
Chati, Z,
Zannad F,
Michel C,
Lherbier B,
Mertes PM,
Escanye J,
Ribuot C,
Robert J,
Villemot JP,
and
Aliot E.
Skeletal muscle phosphate metabolism abnormalities in volume-overload experimental heart failure.
Am J Physiol Heart Circ Physiol
267:
H2186-H2192,
1994
11.
Cooper, RA.
Abnormalities of cell-membrane fluidity in the pathogenesis of disease.
N Engl J Med
297:
371-377,
1977[ISI][Medline].
12.
Delp, MD,
Duan C,
Mattson JP,
and
Musch TI.
Changes in skeletal muscle biochemistry and histology relative to fiber type in rats with heart failure.
J Appl Physiol
83:
1291-1299,
1997
13.
Dubouchaud, H,
Eydoux N,
Granier P,
Prefaut C,
and
Mercier J.
Lactate transport activity in rat skeletal muscle sarcolemmal vesicles after acute exhaustive exercise.
J Appl Physiol
87:
955-961,
1999
14.
Dubouchaud, H,
Grainer P,
Mercier J,
LePeuch C,
and
Prefaut C.
Lactate uptake by skeletal muscle sarcolemmal vesicles decreases after 4 weeks of hindlimb unweighting in rats.
J Appl Physiol
80:
416-421,
1996
15.
Fabiato, A,
and
Fabiato F.
Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles.
J Physiol (Lond)
276:
233-255,
1978
16.
Favero, TG,
Zable AC,
Bowman MB,
Thompson A,
and
Abramson JJ.
Metabolic end products inhibit sarcoplasmic reticulum Ca2+ release and [3H]ryanodine binding.
J Appl Physiol
78:
1665-1672,
1995
17.
Garcia, R,
and
Diebold S.
Simple, rapid, and effective method of producing aortocaval shunts in the rat.
Cardiovasc Res
24:
430-432,
1990
18.
Grimditch, GK,
Barnard RJ,
Kaplan SA,
and
Sternlicht E.
Insulin binding and glucose transport in rat skeletal muscle sarcolemmal vesicles.
Am J Physiol Endocrinol Metab
249:
E398-E408,
1985
19.
Juel, C,
Honig A,
and
Pilegaard H.
Muscle lactate transport studied in sarcolemmal giant vesicles: dependence on fibre type and age.
Acta Physiol Scand
143:
361-365,
1991[ISI][Medline].
20.
Klip, A,
Ramlal T,
Young DA,
and
Holloszy JO.
Insulin induced translocation of glucose transporters in rat hindlimb muscles.
FEBS Lett
224:
224-230,
1987[ISI][Medline].
21.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
22.
Mancini, DM,
Walter G,
Reichek N,
Lenkinski R,
McCully KK,
Mullen JL,
and
Wilson JR.
Contribution of skeletal muscle atrophy to exercise intolerance and altered metabolism in heart failure.
Circulation
85:
1364-1373,
1992
23.
Mancini, DM,
Wilson JR,
Bolinger L,
Li H,
Kendrick K,
Chance B,
and
Leigh JS.
In vivo magnetic resonance spectroscopy measurement of deoxymyoglobin during exercise in patients with heart failure.
Circulation
90:
500-508,
1994
24.
McCullagh, KJ,
and
Bonen A.
Reduced lactate transport in denervated rat skeletal muscle.
Am J Physiol Regulatory Integrative Comp Physiol
268:
R884-R888,
1995
25.
McCullagh, KJ,
Juel C,
O'Brien M,
and
Bonen A.
Chronic muscle stimulation increases lactate transport in rat skeletal muscle.
Mol Cell Biochem
156:
51-57,
1996[ISI][Medline].
26.
McDermott, JC,
and
Bonen A.
Endurance training increases skeletal muscle lactate transport.
Acta Physiol Scand
147:
323-327,
1993[ISI][Medline].
27.
McDermott, JC,
and
Bonen A.
Lactate transport by skeletal muscle sarcolemmal vesicles.
Mol Cell Biochem
122:
113-121,
1993[ISI][Medline].
28.
Pagliassotti, MJ,
and
Donovan CM.
Role of cell type in net lactate removal by skeletal muscle.
Am J Physiol Endocrinol Metab
258:
E635-E642,
1990
29.
Pilegaard, H,
Domino K,
Noland T,
Juel C,
Hellsten Y,
Halestrap AP,
and
Bangsbo J.
Effect of high-intensity exercise training on lactate/H+ transport capacity in human skeletal muscle.
Am J Physiol Endocrinol Metab
276:
E255-E261,
1999
30.
Pilegaard, H,
Juel C,
and
Wibrand F.
Lactate transport studied in sarcolemmal giant vesicles from rats: effect of training.
Am J Physiol Endocrinol Metab
264:
E156-E160,
1993
31.
Pilegaard, H,
Terzis G,
Halestrap A,
and
Juel C.
Distribution of the lactate/H+ transporter isoforms MCT1 and MCT4 in human skeletal muscle.
Am J Physiol Endocrinol Metab
276:
E843-E848,
1999
32.
Roth, DA.
The sarcolemmal lactate transporter: transmembrane determinants of lactate flux.
Med Sci Sports Exerc
23:
925-934,
1991[ISI][Medline].
33.
Roth, DA,
and
Brooks GA.
Lactate and pyruvate transport is dominated by a pH gradient-sensitive carrier in rat skeletal muscle sarcolemmal vesicles.
Arch Biochem Biophys
279:
386-394,
1990[ISI][Medline].
34.
Roth, DA,
and
Brooks GA.
Lactate transport is mediated by a membrane-bound carrier in rat skeletal muscle sarcolemmal vesicles.
Arch Biochem Biophys
279:
377-385,
1990[ISI][Medline].
35.
Roth, DA,
and
Brooks GA.
Training does not affect zero-trans lactate transport across mixed rat skeletal muscle sarcolemmal vesicles.
J Appl Physiol
75:
1559-1565,
1993
36.
Schieffer, B,
Wollert KC,
Berchtold M,
Saal K,
Schieffer E,
Hornig B,
Riede UN,
and
Drexler H.
Development and prevention of skeletal muscle structural alterations after experimental myocardial infarction.
Am J Physiol Heart Circ Physiol
269:
H1507-H1513,
1995
), 5 (
), 10 (
), 20 (
), and
100 mM (
) extravesicular La
