Myosin heavy chain profiles and body composition are different in old versus young Standardbred mares.

Abstract

There are limited data on age-related changes in body composition or skeletal muscle in the horse. Therefore, the purpose of this study was to investigate any differences in muscle myosin heavy chain (MHC) and body composition associated with aging. Twenty-three young (4-8 years) and eight old (20+ years) unfit Standardbred mares were evaluated. Rump fat thickness was measured using B-mode ultrasound and per cent body fat (% fat) was calculated. Needle muscle biopsies were obtained from right gluteus medius muscle. MHC composition was determined via sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three MHC isoforms were subsequently identified as type I, type IIA, and type IIX and quantified using a scanning and densometric system. There were no significant differences (p>0.05) between old and young mares in fat (%) (19.0+/-6.4 vs 20.5+/-5.4), fat mass (kg) (102.3+/-39.9 vs 106.9+/-37.1), or body weight (kg) (529.4+/-34.9 vs 512.7+/-57.7). However, the old mares had significantly (p<0.05) greater lean body mass than the young mares (427.1+/-24.5 vs 405.7+/-37.9). Aged mares had significantly (p<0.05) less type I (7.8+/-2.9% vs 12.1+/-4.4%) and IIA (27.8+/-7.1% vs 36.1+/-9.5%) fibres than the young group but more type IIX (64.6+/-4.7% vs 51.8+/-11.1%). The MHC data are consistent with the age-related changes seen in other species.

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Myosin heavy chain profiles and body composition are different
in old versus young Standardbred mares
Robert A. Lehnharda, Kenneth H. McKeeverb,*,
Charles F. Kearnsb, Matthew D. Beekleyc
aDepartment of Exercise Science, University of Maine, Orono, ME, USA
bEquine Science Center, Department of Animal Science, Rutgers the State University of New Jersey, 84 Lipman Drive, New Brunswick, NJ 08901, USA
cDepartment of Physical Education, United States Military Academy, West Point, NY 10996, USA
Accepted 24 December 2002
Abstract
There are limited data on age-related changes in body composition or skeletal muscle in the horse. Therefore, the purpose of this
study was to investigate any differences in muscle myosin heavy chain (MHC) and body composition associated with aging. Twenty-
three young (4–8 years) and eight old (20+ years) unfit Standardbred mares were evaluated. Rump fat thickness was measured using
B-mode ultrasound and per cent body fat (% fat) was calculated. Needle muscle biopsies were obtained from right gluteus medius
muscle. MHC composition was determined via sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Three MHC isoforms
were subsequently identified as type I, type IIA, and type IIX and quantified using a scanning and densometric system. There were
no significant differences (p > 0:05) between old and young mares in fat (%) (19:0 ? 6:4 vs 20:5 ? 5:4), fat mass (kg) (102:3 ? 39:9 vs
106:9 ? 37:1), or body weight (kg) (529:4 ? 34:9 vs 512:7 ? 57:7). However, the old mares had significantly (p < 0:05) greater lean
body mass than the young mares (427:1 ? 24:5 vs 405:7 ? 37:9). Aged mares had significantly (p < 0:05) less type I (7:8 ? 2:9% vs
12:1 ? 4:4%) and IIA (27:8 ? 7:1% vs 36:1 ? 9:5%) fibres than the young group but more type IIX (64:6 ? 4:7% vs 51:8 ? 11:1%).
The MHC data are consistent with the age-related changes seen in other species.
? 2003 Elsevier Ltd. All rights reserved.
Keywords: Horse; Aging; Muscle fibre; Fat mass; Fat-free mass
1. Introduction
Recent surveys have indicated that up to 15% of the
equine population is over 20 years of age with many of
these horses continuing to perform in athletic activities
(McKeever and Malinowski, 1997). More horses are in
their late teens and are in the prime of their performance
careers, competing in trail rides, dressage, driving, three-
day eventing, and other athletic competitions. Age ap-
pears to affect cardiopulmonary function in the horse
(McKeever and Malinowski, 1997; McKeever et al.,
1998). It has also been shown that older horses may be
at increased risk of hyperthermia due to changes in fluid
and electrolyte status leading to the impairment of car-
diovascular function and heat dissipation mechanisms
(McKeever et al., 2000). While those data have docu-
mented age-related changes in central factors affecting
exercise capacity in the horse, few studies have examined
peripheral changes associated with aging.
Many workers have examined muscle fibre type in the
horse (Essen-Gustavsson and Lindholm, 1985; Lovell
and Rose, 1991; Rivero, 1996a,b; Rivero and Serrano,
1999; Rivero et al., 1993a,b; Rivero et al., 1997; Snow
et al., 1981; Snow and Guy, 1980); however, only two
papers to date have attempted to present limited data
grouped by age (Essen et al., 1980; Rivero et al.,
1993a,b). Unfortunately, the mean age of the oldest age
group in one study was 15 years (Rivero et al., 1993a,b)
while the other study grouped together the horses from
10 to 28 years. This intermediate age group (10–16
The Veterinary Journal 167 (2004) 59–66
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*Corresponding author. Tel.: +1-732-932-9390; fax: +1-732-932-
6996.
E-mail address: mckeever@aesop.rutgers.edu (K.H. McKeever).
1090-0233/$ - see front matter ? 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S1090-0233(03)00045-5

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years) is an age where many horses are still in their
prime athletically and are considered to be physiologi-
cally analogous to 40+ year old humans (Masoro, 1995).
Fibre type distribution is one set of measures of
muscle function that may be altered by aging. Other
measures that may have a more important bearing on the
ability to perform exercise are body composition and,
more importantly, total fat-free mass (FFM). The pre-
dominant component of a horse?s FFM is muscle mass
and a recent study has documented the strong correla-
tion between FFM and performance in elite Standard-
bred horses (Kearns et al., 2002a). We are unaware of
any experiments that have examined these parameters in
older horses, analogous to geriatric humans. As men-
tioned, many horses continue to perform athletic activ-
ities beyond 20 years of age, a period when changes in
body composition and changes in fibre type profile may
limit the ability to perform exercise. Therefore, the object
of this study was to test the hypothesis that aging induces
differences in body composition (fat mass and muscle
mass) and muscle fibre profile in the horse.
2. Materials and methods
2.1. Animals
Thirty-one Standardbred mares were used in this
study. Twenty-three were four to eight years old and
classified as ‘‘young’’. Eight were 20 years or older and
classified as ‘‘old’’. All the horses were housed as a
group on pasture. They were fed 6kg/day alfalfa and
grass hay, and 3kg/day of a commercially available
grain twice per day, morning and evening. All of the
horses were unfit and had been housed at Rutgers under
similar conditions and had not received any exercise
training for several years. None of the younger horses
had made it to the track prior to arriving at Rutgers. In
the case of the old mares, they were brood mares prior
to donation to the study and any training they may have
received occurred almost 20 years prior to the experi-
ment. Water was provided ad libitum. All of the meth-
ods and procedures used in this study were reviewed and
approved for use by the Rutgers University Institutional
Animal Care Review Board.
2.2. Body composition
Per cent body fat was estimated using the equation
from Kane et al. (1987) where:
% fat ¼ 2:47 þ 5:47 ? ðrump fat thickness in cmÞ:
Rump fat thickness was measured using B-mode ultra-
sonography. The measurement site was determined by
placing the probe over the rump at approximately 5cm
lateral from the midline, at the centre of the pelvic bone.
The region was scanned and the position of maximal fat
thickness was used as the measured site. The calculated
average coefficient of variation (CV) based on six ani-
mals for this rump fat thickness determination was
3:6 ? 0:7%. Fat weight (kg) was calculated by multi-
plying total body weight (kg) by the percentage of body
fat. FFM (kg) was then calculated by subtracting the fat
weight from the total body weight.
2.3. Muscle biopsies
Muscle biopsies were obtained from each horse at a
depth of 2cm into the gluteus medialis. A Bergstrom
biopsy needle was used to obtain each tissue sample and
the muscle sample was immediately frozen to )80?C for
later analysis.
2.4. Myofibillar isolation
Myosin fibrils were isolated from each tissue sample
using methods previously described by LaFramboise
et al. (1992). Briefly, the samples were removed from the
)80?C freezer and powdered under liquid nitrogen. The
powder was then placed in glass-on-glass homogeniza-
tion tubes and homogenized in a buffer solution of
300mM NaCl, 100mM NaH2PO4, 50mM Na2HPO4,
1mM MgCl2, 10mM Na4P2O2, and 10mM EDTA.
Extracts were then centrifuged at 13,000g for 30min at
4?C. The supernatant was recovered and diluted in a 9?
volume precipitation buffer of 1mM EDTA and 0.1% b-
mercaptoethanol. These diluted extracts were then
stored overnight at 4?C to allow precipitation of the
myosin filaments. The solution was subsequently cen-
trifuged at 13,000g for 30min at 4?C and the resulting
pellet was re-suspended (1:1) in a 0.5M NaCl and
10mM NaH2PO2buffer. This suspension was then di-
luted 1:100 in a SDS buffer consisting of 62.5mM Tris–
HCl, 2% SDS, 10% glycerol, 5% b-mercaptoethanol,
and 0.001% bromophenol blue at a pH of 6.8. The
samples were then boiled for 3min and stored at )80?C.
2.5. Myosin heavy chains
Myosin heavy chain (MHC) composition was deter-
mined using one-dimensional SDS–polyacrylamide gel
electrophoresis as described by Talmadge and Roy
(1993).Briefly,1–3lgofproteinwereloadedontoa20cm
longverticalgelandelectrophoresedfor24hat4?Cusing
a 14% bis-acrylamide stacking and 8% bis-acrylamide
separating gel. This gel was stained with Coomassie blue
R-250anddestainedwitha10%/10%methanolandacetic
acid solution. The relative concentration of MHC was
determined by scanning the gel using a NIH com-
puterized image analysis system (Scion Image for Win-
dows,ScionCorporation).Thecoefficientofvariationfor
this measure in our laboratory was less than 5%.
60
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2.6. Statistical analysis
An ANOVA was used to determine any significant
difference (p60:05) between group means. Post-hoc
differences were determined using the Tukey test and
correlation coefficients were derived using the Pearson
product moment (Sigma Stat 2.0).
3. Results
There were no differences (p > 0:05) between old and
young mares for total body weight, rump fat thickness,
per cent body fat, or fat weight. The old mares, however,
had significantly (p < 0:05) greater lean body weight
than the young mares (Table 1).
The old group could be clearly divided in two by
appearance. The animals were either very lean (n ¼ 5)
(‘‘skinny’’), or very fat (n ¼ 3) (‘‘fat’’). The skinny old
mares had significantly smaller rump fat thickness,
lower per cent body fat, and less fat weight than both the
fat old mares and the young mares. The skinny old
mares had significantly less body weight than the fat old
mares but not compared to the young mares. The skinny
old mares also had greater (p < 0:05) lean body weight
than the young mares but there was no significant
Table 1
Body compositional characteristics of all mares
Group
n
Rump fat (cm)% FatBody mass (kg) Fat mass (kg)Fat-free mass (kg)
Old
‘‘Skinny’’
‘‘Fat’’
Young
8
5
3
3:0 ? 1:2
2:1 ? 0:2a
4:4 ? 0:4b
3:4 ? 1:0c
19:0 ? 6:4
14:2 ? 1:0a
26:6 ? 2:4b
20:5 ? 5:4c
529:4 ? 34:9
507:8 ? 27:0a
563:1 ? 9:2b
512:7 ? 57:7a
102:3 ? 39:9
71:8 ? 3:2a
150:0 ? 16:0b
107:0 ? 37:1c
427:1 ? 24:5?
436:0 ? 27:5
413:1 ? 7:0a;b
405:8 ? 37:9b
23
Means (Young, ‘‘Skinny’’ and ‘‘Fat’’ mares) with different lettersa;b;care different (p < 0:05) from each other.
*Old mares (all) different (p < 0:05) from Young mares.
Fig. 1. Scatter diagrams and linear regression lines relating (A) fat mass to body mass; (B) fat-free mass to body mass and (C) per cent body fat to
body mass.
R.A. Lehnhard et al. / The Veterinary Journal 167 (2004) 59–66
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difference in this parameter when compare to the old fat
mares. In turn, the old fat group had larger (p < 0:05)
rump fat thickness, per cent body fat, and fat mass when
compared to the young mares. These comparisons are
shown in Table 1.
Plots of individual values for body mass against fat
mass and FFM are illustrated in Fig. 1. Significant re-
lationships between body mass and both fat mass
(r ¼ 0:735; p < 0:001; Fig. 1A) and FFM (r ¼ 0:706;
p < 0:001; Fig. 1B) were found. Also, there was a sig-
nificant relationship between per cent fat (r ¼ 0:570;
P < 0:001; Fig. 1C) and body mass.
The results of the gel electrophoresis demonstrated
that the old mares had a smaller proportion (p < 0:05)
of type I MHC (7:8 ? 2:9% vs 12:1 ? 4:4%) and IIA
MHC (27:8 ? 7:1% vs 36:1 ? 9:5%) than the young
mares, respectively. Conversely, the old mares had a
larger proportion(p < 0:05)
(64:6 ? 4:7% vs 51:8 ? 11:1%) than young mares. These
mean values?SE are presented in Fig. 2.
oftype IIXMHC
4. Discussion
The significant finding of this study was that older
horses had a change in myosin heavy chain (MHC)
distribution when compared to young horses. On a
functional level it appears that the older horse has a shift
in MHC population away from that which would be
conducive to endurance exercise. These data may, in
part, explain the decrease in maximal aerobic capacity
documented in other studies (McKeever et al., 1998).
One also could argue that the differences in muscle fibre
profile and body composition seen in the present study
may have been influenced by prior training status;
however, as noted in Section 2 the mares used in the
present study were broodmares and had not been
trained for the several years they were housed at the
university research facility. However, it has been
suggested that an age-related decrease in the type II to
type I MHC isoform ratio and muscle atrophy in hu-
mans is associated with a reduction in the maximal
aerobic capacity regardless of training status (Proctor
and Joiner, 1997). Such may be the case in the horse.
The prevalent belief of age-related atrophy seen in
human skeletal muscle is primarily due to a reduction in
the number and size of muscle fibres (Kirkendall and
Garrett, 1998; Lexell, 1995; Lexell et al., 1988). How-
ever, data from rats (Eddinger et al., 1985) have raised
questions as to whether this shift in fibre profile is due to
aging or other factors. Due to the large variation in
activity and/or hormonal levels common in humans,
interpretation of data gathered from muscle biopsy is
difficult (Staron, 1997). Data from rats have demon-
strated that type IID/X, the presumed ‘‘fast-twitch’’ fi-
bres, made up a significant portion of rat muscle mass
(Delp and Duan, 1996) and the percentage of type IID
and IIC did not change with aging (Eddinger et al.,
1985). These findings are similar to data seen in senes-
cent beagles (Haidet and Parsons, 1991). Per cent fibre
type distribution was not different in the triceps, semi-
tendinosus, and gastrocnemius of young or old dogs,
although there was a reduction in type II fibre area in
the old (Haidet and Parsons, 1991). In the vastus medi-
alis muscle of swine, there was a reduction of slow fibres
in older animals (Cappello et al., 2000). These data,
along with findings from the present study, suggest that
the reduction of type II fibres seen in older humans may
not be associated with aging alone. Aging itself may not
cause a loss of type II fibres. In fact, data from a 12-year
longitudinal study in humans (Frontera et al., 2000)
reported a reduction in the percentage of type I fibres
with a concomitant increase in the percentage of type
IIB fibres. The authors of that study speculated that
since 20–53% of muscle from older subjects co-express
two or three MHC isoforms (Andersen et al., 1999),
histochemical techniques used in previous studies could
misclassify fibres that co-express ‘‘slow’’ and ‘‘fast’’
isoforms (Frontera et al., 2000).
4.1. Muscle fibre typing
Fibre typing in the horse has been widely performed
using the muscle biopsy technique. The traditional
staining method has been used to determine the per-
centage of type I, type IIA, and type IIB fibres in dif-
ferent muscle groups (Snow and Guy, 1980; van den
Hoven et al., 1985), as well as at different sampling
depths (Kline et al., 1987). Fibre types have also been
profiled for different breeds of horse (Snow and Guy,
1980; Strull and Albert, 1981). There are a number of
ways to differentiate muscle fibre type, including meta-
bolic enzymes, MHC composition, and myosin ATPase
(mATPase) activity. It is now clear that metabolic
enzymes do not correlate with MHC or mATPase
Fig. 2. Percentage distribution of the myosin heavy chain (MHC)
isoforms measured in old and young mares. An asterisk (*) denotes a
significant difference between the young and old groups.
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activities (Pette and Staron, 2000). MHC composition
and mATPase activities correlate reasonably well, but it
has been recently shown that a single muscle fibre can
contain several MHCs at once (termed hybrid fibres)
which are not identified by mATPase staining or even
the gel electrophoresis technique used in this paper
(Pette and Staron, 2000). These hybrid fibres are im-
portant in that they likely happen during fibre type
transitions (Pette and Staron, 2000).
A limited number of studies have been performed in
the horse utilizing the myosin content to describe
muscle fibre composition (Barrey et al., 1999; Linnane
et al., 1999; Rivero and Serrano, 1999; Snow et al.,
1981). To date, there have been no studies to determine
MHC composition as it relates to age. Therefore, the
observed shift in the fibre profile with aging seen in the
horses of the present study is new information that may
shed additional light on the special needs of the older
equine athlete. On a functional level it appears that the
older horse has a shift in fibre type population away
from that which would be conducive to endurance ex-
ercise. This shift in fibre profile is similar to that seen in
disuse atrophy (Serrano et al., 2000) and aging models
and may explain some of the decrease in maximal
aerobic capacity seen in the older horse (McKeever
et al., 1998).
Age has been studied as a factor of fibre type distri-
bution in various breeds including Thoroughbreds,
Standardbreds, Quarter Horses, Andalusians and Ara-
bians (Bechtel and Kline, 1987; Gunn, 1991; Gunn,
1995; Rivero et al., 1993a,b; Roneus and Lindholm,
1991). These studies have employed staining techniques
to determine fibre composition and have spanned ages
pre-natal to beyond 20 years. While some of these
studies have included horses older than 20 years in their
analysis (Essen et al., 1980; Rivero et al., 1993a,b), the
aged horse was not the focus of the study by Rivero and
co-workers (1993a,b). Essen et al. (1980) grouped horses
as either Foals (two months) or Adults (1–28 years). In
that study, horses 10–28 years of age had a larger ratio
of type I/II than foals. While this is different from the
results presented here, the aged horses of the present
study had an average age of 27 years. Horses of less than
20 years of age may be more similar to younger horses in
terms of body composition and metabolism than they
are of horses older than 20 years (Malinowski et al.,
2002). It is possible that addition adaptations occur
beyond 20 years of age in horses. On the other hand,
when the issue of aging is discussed, it has been in ref-
erence to younger horses between the ages of one to six
years of age (Bechtel and Kline, 1987; Roneus and
Lindholm, 1991). These studies have been reported
documenting an increase in type I percentage and a
decrease in type IIB percentage with aging in young
horses that occur in both mares and stallions (Roneus
and Lindholm, 1991; Rivero et al., 1993a,b).
Typically, muscle biopsies have been used to study
the effects of training in horses. Pre-and post-test designs
have been utilized in both endurance and high intensity
interval training regimes to determine effects on fibre
composition. Results of these studies have shown no
changes in respective fibre type numbers, but increases
in area percentage do occur in specific fibre types with
specific training regimes (endurance vs high intensity
interval) (Beekley et al., 2003; Essen-Gustavsson and
Lindholm, 1985; Hodgson and Rose, 1987; Hodgson
et al., 1986; Lopez-Rivero et al., 1992; Rivero, 1996a,b;
Rivero et al., 1995; Roneus and Lindholm, 1991). Fibre
adaptations to detraining have also been studied in
different breeds, again with no changes seen in fibre
composition (Guy and Snow, 1977; Sinha et al., 1993).
Recently it has been demonstrated that long-term en-
durance training induces a reversible transition of MHC
from MHC IIX to MHC I (Serrano et al., 2000). Race
performance is yet another parameter that has been in-
vestigated as a function of muscle fibre type. It has been
shown that a high percentage of type I and type IIA
fibres exists in high performing racehorses (Rivero et al.,
1993a,b; Rivero, 1996a,b). To date, there have been no
published data regarding the potential effect of exercise
on the MHC profile of older horses. Therefore, it is
unknown whether older horses would demonstrate
similar plasticity as do younger horses in either their
MHC profile or per cent area of these fibres.
4.2. Body composition
A larger body mass was associated with a larger fat
mass and FFM in both young and old mares. However,
per cent body fat significantly increased with body mass
signifying that these mares are accumulating excess fat.
Excess body fat accumulation, or obesity, is linked to
insulin resistance in humans (Arita et al., 1999). These
findings are in agreement with recent data in older
horses (Malinowski et al., 2002). Older horses appear to
be insulin resistant because they required more insulin
than younger horses to successfully manage an acute
glucose load (Malinowski et al., 2002). This insulin re-
sistance/hyperinsulinaemia can increase the risk of
laminitis (Field and Jeffcott, 1989; Pass et al., 1998),
exertional rhabdomyolysis (Valentine et al., 1998), os-
teochondritis dissecans (Pagan et al., 2001; Ralston,
1995), colic and other serious metabolic problems
(Cohen et al., 1999).
Another significant finding of the present study was
that older horses had a change in body composition
when compared to young horses. Morphometrically, the
old mares had two aged phenotypes, the ‘‘skinny mares’’
and the ‘‘fat mares’’. These data are similar to another
study in older horses that also saw two distinct pheno-
types (Ralston and Breuer, 1996). Using a body condi-
tion score (BCS) system (Henneke et al., 1983), horses
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