muscle biology therapies - the fast skeletal ......for fast skeletal muscle fibers. ck-2017357 does...
TRANSCRIPT
REFERENCES
INTRODUCTION
Direct modulation of muscle contractility at the level of the contractile apparatus is atherapeutic approach with applicability to several diseases. Previous discovery effortsdirected at cardiac muscle resulted in the identification of CK-1827452, a small moleculedirect activator of cardiac myosin that increases cardiac contractility and is currentlybeing studied in Phase II clinical trials in patients with systolic heart failure. Similarly, asmall molecule activator of the skeletal sarcomere may have equal utility in increasingmuscle function in patient groups where skeletal muscle weakness is a feature.
CK-2017357 is a fast skeletal troponin activator that was discovered as part of ascreening and chemical optimization process using detergent treated skeletal musclemyofibrils from rabbit muscle. In biochemical assays, it sensitizes the fast skeletalmyofibril ATPase activity to calcium, shifting the pCa relationship to the left withoutaffecting enzymatic activity at low and high calcium concentrations.
The objective of this study was to evaluate whether CK-2017357 changes forcedevelopment in native skeletal muscle preparations in vitro, using skinned and livingskeletal muscle fibers, and in situ, where nerve and blood supply are left intact.
An additional objective was to test whether the increase in calcium sensitivity of thesarcomere effected by CK-2017357 would alter the rate of fatigue of flexor digitorumfibers in vitro since one of the causes for skeletal muscle fatigue is decreased myoplasmicCa2+ due to impaired sarcoplasmic reticulum (SR) Ca2+ function (Allen et al., 2007).
Figure 1. Effect of CK-2017357 on skinned skeletal fibers from (A) rabbit psoas muscle (fast skeletal muscle, n=8),(B) rat soleus muscle (slow skeletal muscle), (C) rat cardiac muscle. Single skinned fibers were attached to a model400A force transducer (Aurora Scientific) at 10°C and force measured after incubation with increasing concentrationof buffered calcium and the indicated concentration of CK-2017357 (force is plotted as a percent of maximalcontraction measured at pCa 4).
Isometric skinned fiber analysis: Muscle fibers for skinned fiber studies were prepared usinga protocol based on Lynch and Faulker, 1998. Single muscle fibers were dissected in rigorbuffer at 4°C (20 mM MOPS, 5 mM MgCl2, 120 mM potassium acetate, 1 mM EGTA, pH 7.0)and attached to a 400A force transducer (Aurora Scientific, Ontario, Canada) with 2-4 µl ofa 5% solution of methylcellulose in acetone. Fibers were incubated at 10°C in relax buffer(20 mM MOPS, 5.5 mM MgCl2, 132 mM potassium acetate, 4.4 mM ATP, 22 mM creatinephosphate, 1 mg/mL creatine kinase, 1 mM DTT, 44 ppm antifoam, pH 7.0) and baselinetension adjusted. Tension was generated by incubating fibers in relax buffer supplementedwith 1 mM EGTA and 10 nM to 100 µM free calcium ions (labeled as pCa 8 to pCa 4, addedas different volumes of a 15 mM solution of CaCl2 and calculated using the web resourcehttp://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm). Compound was added tothese buffers from a DMSO stock (final DMSO concentration 1%).
In vitro muscle analysis: Adult male Sprague-Dawley rats were euthanized with isofluraneand a small branch of the flexor digitorum brevis (FDB) was dissected from the foot inoxygenated Krebs solution at 4°C (1 mM NaH2PO4, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 137mM NaCl, 11 mM glucose and 1 mM NaHCO3). Muscles were attached with silk thread to thefixed lever arm and force transducer of an 801A in vitro analysis system (Aurora Scientific,Ontario, Canada) and incubated in Krebs solution at 20°C. After length adjustment, muscleswere stimulated via field electrodes with 350 ms trains (5, 10, 20, 30, 50, 80, 100 Hz, 1 msstimuli) over a 2 minute period. This was repeated every 10 minutes. For compoundtreatment, muscles were perfused with DMSO (0.1%) or CK-2017357 (1-10 µM).
Fatigue assays were performed by pre-incubating FDB muscles at resting tension with DMSO(0.1%) or CK-2017357 (5 µM) for 60 minutes at 4°C. Incubating temperature was then raisedto 30°C and a force frequency relationship established (350 ms trains at 5, 10, 20, 30, 50, 80,100 Hz, 1 ms stimuli). Stimulation frequency was adjusted to achieve a force of 50% ofmaximal (FMax50) and muscles were stimulated at this frequency every 6 seconds for 15minutes.
At the end of each assay, the length and weight of each muscle were recorded, andmeasured force was normalized to the cross sectional area of the muscle (N/cm2, describedin Segal and Faulkner, 1985).
In situ muscle analysis: In situ studies were based on experimental procedures describedby Brooks et al., 1990. Rats were placed under anesthesia using isoflurane and the distalend of the extensor digitorum longus (EDL) muscle and its associated tendon were isolated.The knee was immobilized with a clamp and the tendon cut and tied to the arm of a forcetransducer (806C, Aurora Scientific) using silk suture. The muscle was stimulated directly viathe peroneal nerve at the upper thigh with a pair of stainless steel hook electrodes. Musclelength was adjusted to produce maximum isometric force (Lo) and then stimulated every 2minutes with a 30 Hz train (1 ms stimuli, 350 ms duration) for the course of the experiment.CK-2017357 was administered as a 2 minute bolus in increments up to a total of 10 mg/kgvia the femoral artery as a solution (50% PEG300/10% EtOH/40% cavitron). For analysis ofthe force/frequency relationship, muscles were stimulated at 10 Hz to 200 Hz before andafter treatment with 10 mg/kg CK-2017357 over a 2 minute period. At the end of eachassay, the length and weight of the muscle was recorded, and measured force normalizedto the cross sectional area of the muscle (N/cm2).
METHODS
RESULTS
1 Allen D.G., Lamb G.D. and Westerblad, H. J. Appl. Physiol. 2007; 104:296-305.
2 Lynch G., Faulkner J. American Journal of Physiology. 1998; 275: C1548-54.
3 Brooks, S.V., Faulkner, J.A. and McCubbrey, D.A. Journal of Applied Physiology 68:1282-1285, 1990.
4 Segal, S.S. and Faulkner, J.A. American Journal of Physiology, 248:C265-270, 1985.
CONCLUSIONS
1 The skeletal troponin activator, CK-2017357,increases sub-maximal force development infast skeletal rabbit muscle in vitro.
2 In living rat FDB preparations, increases inforce are coupled to frequency-independentincreases in relaxation time.
3 Skinned slow skeletal muscle fibers areapproximately ten fold less responsive to CK-2017357 than skinned fast skeletal musclefibers, confirming the compound specificityfor fast skeletal muscle fibers. CK-2017357does not activate cardiac muscle.
4 CK-2017357 increases sub-maximal forcedevelopment in the EDL muscle of rats afterarterial administration of compound. Incontrast to in vitro results, the increases inrelaxation time are attenuated and directlyproportional to increases in force.
5 CK-2017357 reduces isometric fatigue in FDBmuscle in vitro. This finding is possibly linkedto the reduced stimulation frequency requiredto elicit the same starting force in treatedmuscle as compared to untreated muscle.
These data are consistent with the mechanismof action of the fast skeletal troponinactivator, CK-2017357. In skinned musclefibers, CK-2017357 increases the sensitivity ofskeletal muscle to calcium and in living muscleto the frequency of stimulation, each of which results in an increase in muscle force development at sub-maximal muscleactivation. In addition, CK-2017357 reducesisometric muscle fatigue in vitro.
These findings may translate into functionalimprovements in skeletal muscle performanceand efficiency in conditions marked by muscleweakness by improving the extent of musclefiber recruitment during physical activity.
THE FAST SKELETAL TROPONIN ACTIVATOR, CK-2017357, INCREASES SKELETAL MUSCLE FORCEAND REDUCES MUSCLE FATIGUE IN VITRO AND IN SITU
Alan J Russell, Ken Lee, Jim J Hartman, David Marquez, Richard Hansen, Alex Muci, Bradley Morgan, Zhiheng Jia, David J. Morgans Jr., Fady MalikCytokinetics, Inc., South San Francisco, CA, USA
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10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
eM
etri
cat
30H
z Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Forc
e
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n(N
/cm
2)
Spec
ific
Ten
sio
n(N
/cm
2)
**
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n,%
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n,%
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n,%
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n(N
/cm
2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
eFo
rce
at10
Hz
*
*
* p<0.05
1200
Tim
e
5 Hz
DMSO 1µM 2µM 5µM 10µM
900
1000
tio
n
*
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n(N
/cm
2 )at3
0Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
eM
etri
cat
30H
z Force
RT1/2
0
10
20
30
40
50
60
0
Perc
ent
of
Max
imal
Forc
e
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n(N
/cm
2)
Spec
ific
Ten
sio
n(N
/cm
2)
**
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n,%
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n,%
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n,%
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n(N
/cm
2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
eFo
rce
at10
Hz
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
eH
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
eH
alf
Rel
axat
ion
Tim
e(1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n(N
/cm
2 )at3
0Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
eM
etri
cat
30H
z Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Forc
e
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n(N
/cm
2)
Spec
ific
Ten
sio
n(N
/cm
2)
**
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n,%
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n,%
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n,%
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n(N
/cm
2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCaTe
nsi
on
, % o
f M
axim
al C
on
tro
l
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)Pe
rcen
t o
f M
axim
al F
orc
e
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z *
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000Pe
rcen
t o
f B
asel
ine
Hal
f R
elax
atio
nTi
me
(10H
z)*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCaTe
nsi
on
, % o
f M
axim
al C
on
tro
l
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z *
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCaTe
nsi
on
, % o
f M
axim
al C
on
tro
l
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
45678
0
20
40
60
80
100
120
DMSO 1%0.1µM CK-2017357
10µM CK-20173571µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rabbit Psoas (Fast Skeletal Muscle)Rat Soleus (Slow Skeletal Muscle)
456780
20
40
60
80
100
120
140
DMSO 1%10uM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
45678
0
20
40
60
80
100
120
DMSO 1%60µM CK-2017357
pCa
Ten
sio
n, %
of
Max
imal
Co
ntr
ol
Rat Cardiac Muscle
0
10
20
30
40
50
60
70
5 Hz 10 Hz 20 Hz 30 Hz 50 Hz 80 Hz 100 Hz
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
0.1% DMSO10uM CK2017357
* p<0.05
80
100
120
140
160
180
0 2 4 6 8 10 12
Concentration CK-2017357 (µM)
Concentration CK-2017357 (µM)
Perc
ent
of
Bas
elin
e Fo
rce
at
10H
z
*
*
* p<0.05
0
200
400
600
800
1000
1200
0 0 2 4 6 8 10 1250 100 150 200 250
Time (mins)
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e
5 Hz
10 Hz
20 Hz
30 Hz
50 Hz
80 Hz
100 Hz
DMSO 1µM 2µM 5µM 10µM
0
100
200
300
400
500
600
700
800
900
1000
Perc
ent
of
Bas
elin
e H
alf
Rel
axat
ion
Tim
e (1
0Hz)
*
*
*** p<0.05
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80
Time (minutes)
Spec
ific
Ten
sio
n (
N/c
m2 ) a
t 30
Hz
(A) (B) (C) (D)
2 mg/Kg 4 mg/Kg 6 mg/Kg 10 mg/Kgaccumulating dose
0
10
20
30
40
50
60
10 Hz 30 Hz 40 Hz 50 Hz 60 Hz 70 Hz 80 Hz 100 Hz 200 Hz
Pre-dose
10 mg/Kg CK2017357
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12
Dose CK-2017357 (mg/Kg)
Perc
ent
of
Bas
elin
e M
etri
c at
30
Hz Force
RT1/2
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700 800 900 1000
Time (seconds) Time (seconds)
Perc
ent
of
Max
imal
Fo
rce
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0.1% DMSO (n=5)
5 uM CK-2017357 (n=6)
0
5
10
15
20
25
30
35
40
45
0 200 400 600 800 1000
Stimulation Frequency (Hz)
Spec
ific
Ten
sio
n (
N/c
m2)
Spec
ific
Ten
sio
n (
N/c
m2)
* *
*
CK-2017357 Selectively Activates Fast Skeletal Muscle
Figure 3. CK-2017357 Increases Force in Rat Extensor Digitorum Longus (EDL) Muscle in situ. (A) The EDL muscle(approx 90% fast fiber composition) was stimulated every 2 minutes at 30 Hz via the peroneal nerve. CK-2017357was then administered as a 2 minute intra-arterial bolus in four cumulative doses up to 10 mg/kg (n=5, error bars+/– S.E.M.). Force is plotted as N/cm2, normalized to the weight and cross-sectional area of the muscle. (B)Force/Frequency plot pre- and post-treatment with 10 mg/kg CK-2017357 (n=5, error +/– sd). (C) CK-2017357increases relaxation time in proportion to force in situ. Plotted is the percent increase in force and half relaxationtime (RT1/2) at 30 Hz following escalating doses of CK-2017357 (n=7, error +/– S.E.M.).
Figure 4. CK-2017357 Decreases Isometric Fatigue Development in Rat Flexor Digitorum Brevis (FDB) Muscle in vitro.FDB muscles were pre-incubated for 30 minutes at 4°C in Krebs buffer at resting tension with DMSO (0.1%) or CK-2017357 (5 µM). Incubation temperature was then raised to 30°C and a force frequency relationship established(350 ms trains at 5, 10, 20, 30, 50, 80, 100 Hz). Stimulation frequency was adjusted to achieve a force of 50% ofmaximal (FMax50) and muscles were stimulated at this frequency every 6 seconds for 15 minutes. StimulationFrequencies required to achieve FMax50 were 34+/– 5.8 Hz, DMSO; 20.5 +/– 4.9 Hz, 5 mM CK-2017357 (p<0.01 byunpaired students T-Test). (A) Graph showing the percent of maximal force over time (+/– S.E.M.). (B) Graph showingthe effect of CK-2017357 upon absolute tension generation expressed as Specific Tension (N/cm2), normalized to thecross-sectional area of the muscle.
Figure 2. CK-2017357 Increases Force Development in Rat Flexor Digitorum Brevis (FDB) Muscle in vitro. FDB muscles(approx 85% fast fiber composition) were incubated at 20°C in Krebs buffer. (A) Effect of 10 ĶM CK-2017357 on theforce/frequency relationship in FDB muscles (specific tension +/– S.D.; * p<0.05 vs pretreatment; n=6). (B) Graphshowing the average change in force (+/– S.D.) at 10 Hz vs concentration of CK-2017357 (n=7, * p<0.05 vs DMSO bypaired students T-Test). (C) Result from a single experiment showing the change in half relaxation time of themuscle with time at seven different stimulation frequencies with increasing concentrations of CK-2017357. (D) Graphshowing the average change in relaxation time (error +/– S.D.) at 10 Hz vs concentration of CK-2017357 (n=7, *p<0.05 vs DMSO by paired students T-Test).
CK-2017357 Increases Force Development in Living Muscle