Fiber Type Properties of the Forelimb Muscles of Sloths (Xenarthra: Pilosa)

Abstract

Sloths exhibit a range of arboreal locomotion that involves suspension by one or more limbs for extended periods of time. Despite these abilities that require great strength, their skeletal muscle mass is quite low; however, their overall limb form has been extremely modified for suspensory behaviors. One modification may be the expression of only slow-twitch isoforms of myosin and muscle fibers with high fatigue resistance. This hypothesis was tested using a combination of histochemical (aerobic vs. anaerobic metabolism) and immunohistochemical analyses to determine muscle fiber types in the major forelimb flexors of both two-toed (Choloepus hoffmanni, N=2) and three-toed (Bradypus variegatus, N=4) sloth species. The results indicate the primary expression of only two myosin fiber types, slow MHC-1 and fast MHC-2A. Both fiber types are strongly reactive for oxidative potential, and all muscles analyzed in both species show a relatively broad distribution of slow, oxidative fibers. It is also observed that sloth forelimb muscle have a large amount of extracellular matrix in between segregated regions of muscle contractile tissue. Collectively, the findings suggest that sloths have evolved slow-contracting, highly fatigue resistant muscles for suspensory behaviors. The physiological properties observed are further consistent with economical force production. However, while slow fiber type specialization provides a means to sustain muscle force, large amounts of force that may be required for suspension are more dependent on muscle architecture and the passive and active force properties of their muscles.

Full text

0!
20!
40!
60!
80!
100!
120!
Percent MHC Fiber Type (%)
fast MHC-2A!
slow MHC-1!
Results
Team: Tyler Tsangaris-Braatz, Paije Kiraly, Amanda Svenson, Abdel Ruhman Yusuf
Mentor: Michael T. Butcher, Ph.D.
Fiber Type Properties of the Forelimb Muscles of Sloths (Xenarthra: Pilosa)
Purpose
Tree sloths have low muscle mass
1
, but they are capable of
suspending themselves underneath tree branches for
extended periods of time
2
, a behavior that requires great
strength. This paradox of having both high strength and
endurance is difficult to reconcile by our current
understanding of the physiological properties of mammalian
skeletal muscle. This study aims to identify physiological
modifications to the forelimb muscles of two-toed
(
Choloepus
) and three-toed (
Bradypus
) sloths that allow for
sustained high force contractions. This will be achieved by
determining the myosin fiber type and enzymatic properties
of sloth muscles
3,4
.
Myosin Heavy Chain (MHC) fiber properties
5
• MHC-1:
slow-twitch
, oxidative; Small size and force
• MHC-2A:
fast-twitch
, highly oxidative; Intermediate size and force
• MHC-2X:
fast-twitch
, moderately oxidative; Large size and force
Hypothesis
Tree sloths will have a heterogeneous distribution of
large and fast, highly oxidative myosin fiber types.
Methodology
• A
total of
N=
6 animals (4,
Bradypus
; 2,
Choloepus
)
• Muscle tissue blocks to cork and flash frozen in isopentane
cooled in liquid nitrogen
4
• Serial sections (10 µm) cut on a cryostat at -22ºC and
mounted to frosted glass microscope slides
• Reacted sloth muscle tissue in NADH incubations for
oxidative (aerobic) potential
3
• Reacted sloth muscle tissue against monoclonal antibodies
(mAbs) for its slow vs. fast MHC isoform expression
4
• Calculated percent distribution of myosin fiber types for
suites of
n=
5 limb muscles/individual
• Quantified fiber cross-sectional area in a subset of fibers
identified as MHC-1 or MHC-2A
3,4
References
1. Grand TI. 1978. Smithsonian Institution Press, Washington
DC, p 231-241.
2. Hayssen V. 2010.
Mammal Spec
42: 19-32.
3. Hazimihalis PJ et al. 2013.
Anat Rec
296: 96-107.
4. Rupert JE et al. 2014.
Anat Rec
297: 1364−1376.
5. Schiaffino S, Reggiani C. 2011.
Physiol Rev
91: 1447-1531.
Conclusions/Future Research*
• The hypothesis is partially supported
• High fatigue resistant fibers sustain muscle force
• MHC-1 fibers have large CSA to enhance force production
• Grip force is dependent on other properties (e.g. passive)
• Minimizing contractile tissue economizes force production
• Quantify hybrid fibers and their properties*
Figure 1. Reactions from m. latissimus dorsi. Panels are labeled with the mAbs reacted (or dark
NADH reaction for aerobic potential, panel B). Individual fibers labeled (1) is a positive reaction
against slow MHC-1. Fibers labeled (2A) is a positive reaction against fast MHC-2A. The arrow
points to extracelluar matrix demonstrating a heavy investment of collagen fibers. The (*) is
the same fiber in each serial section.
Figure 2. Reactions from m. subscapularis. Panels are labeled with the mAbs reacted (or
control, panel B). Individual fibers labeled (1) is a positive reaction against slow MHC-1.
Individual fibers labeled (2A) is a positive reaction against fast MHC-2A. The arrow points to a
MHC-1-2A hybrid fiber. The (*) is the same fiber in each serial section.
*
*
1
1
2A
2A
1
2A
A B
C D
Choloepus
hoffmanni
*
*
1
1-2A
A
2A
*
B
C D
1
2A
100 µm
Bradypus
variegatus
2A
100 µm
Figure 3. A. Mean % myosin fiber type distribution in the forelimb muscles of both species. B. Mean (± standard
deviation) of myosin fiber CSA in the forelimb muscles of both species.
Ch
are data for
Choloepus hoffmanni
;
Bv
are
data for
Bradypus variegatus
. All data are grouped proximodistal by major anatomical region of the forelimb in the
order: shoulder (left, proximal), arm, and forearm (right, distal).
A B
*
Shoulder
Arm
Forearm
Ch Bv
Ch Bv Ch Bv
0!
500!
1000!
1500!
2000!
2500!
3000!
3500!
MHC Fiber Type CSA (µm
2
)
slow MHC-1!
fast MHC-2A!
Shoulder
Arm
Forearm
Ch Bv
Ch Bv Ch Bv