Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 1 of 39
Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle
Alastair Khodabukus and Keith Baar Division of Neurobiology, Physiology and Behavior, University of California Davis, Davis, CA 95616
Corresponding Author: Keith Baar
Email: [email protected]
Keywords: Skeletal muscle, tissue engineering, streptomycin, electrical stimulation
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 2 of 39
2 Abstract Chronic low-frequency electrical stimulation (CLFS) has long been used to induce a fast-to-slow phenotype shift in skeletal muscle. In this study we explore the role of frequency (10 and 20Hz), active time (15-60%) and streptomycin in inducing a fast-toslow shift in engineered muscle. We found that C2C12 engineered muscle could respond to CLFS with an adult-like active time of 60% and found that a constant 10Hz train of 0.6secs followed by 0.4secs rest induced a partial fast-to-slow phenotype shift. Following 2 weeks CLFS, Time-to-peak tension (TPT) (Control (CTL)=40.9 ± 0.2ms; 10Hz=58.5 ± 3.5ms; 20Hz=48.2 ± 2.7ms) and half-relaxation time (1/2RT) (CTL=50.4 ± 0.6ms; 10Hz=76.1 ± 3.3ms; 20Hz=66.6 ± 2.3ms) slowed significantly in a frequency but not active time dependent manner. Streptomycin significantly blunted the slowing of TPT and 1/2RT induced by CLFS by minimizing the fast-to-slow shift in SERCA isoform. Streptomycin (Non-Stim=-42.8 ± 2.5%; Stim=-38.1 ± 3.6%) significantly prevented the improvement in fatigue resistance seen in CTL constructs (Non-Stim=-58.4 ± 3.6%; Stim=-27.8 ± 1.7%). Streptomycin reduced the increase seen in GLUT4 protein following CLFS (CTL=89.4 ± 6.7%; Strep=41.0 ± 4.3%) and prevented increases in the mitochondrial proteins SDH and ATPsynthase. These data demonstrate that streptomycin significantly blunts the fast-to-slow shift induced by CLFS. In the absence of streptomycin, CLFS induced slowing of contractile dynamics and improved fatigue resistance and suggest that this model can be used to study the mechanisms underlying CLFS–induced adaptations in muscle phenotype.
2
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 3 of 39
3 Introduction
Skeletal muscle is a highly plastic tissue capable of changing its molecular, metabolic and functional properties in response to a wide range of factors including developmental maturity [1], electrical stimulation [2], mechanical stimulation [3], and drug treatments [4]. Muscle fibers can be classified as either “slow” or “fast” based on myosin heavy chain isoform and oxidative capacity [5, 6]. Slow fibers have higher mitochondrial content and rely more heavily upon oxidative metabolism, resulting in a more fatigue resistant phenotype compared to fast fibers. Adult muscle phenotype is primarily regulated by neural activity but is highly plastic as demonstrated by cross-innervation [7], denervation [8, 9] and denervation-electrical stimulation [10, 11] studies.
Electromyography (EMG) recordings have shown that slow muscles have a natural tonic firing frequency of 10-20Hz and can fire at least up to 300,000 impulses per day [12, 13]. Chronic low-frequency stimulation (CLFS) protocols that mimic the natural tonic firing pattern of slow phenotype muscles have been shown to induce fast-to-slow fiber type transitions both in vivo [7] and in vitro [11]. Despite CLFS patterns attempting to mimic the natural neural input to slow fibre type muscles (e.g. Soleus), no standardized CLFS protocol has been developed. This is due, in part, to the differential response to CLFS in different muscle groups [10, 14] and animal species [7, 10, 15], showing that no single CLFS pattern exists to transform all muscles into a slow muscle fibre type.
3
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 4 of 39
4 CLFS is typically performed by attaching small programmable stimulator units either on the backs of animals [16] or subcutaneously in animals [17, 18] and humans [19]. External stimulators can be damaged or dislodged by the animals and implantable devices can have limited reuse capability, leading to an increase in experimental cost. Furthermore, studying the mechanisms underlying CLFS induced shifts via inhibitor studies is vastly more expensive and more difficult in vivo compared to in vitro. CLFS studies have been performed in vitro and have been shown to induce changes in myofibrillar and metabolic protein expression [11]. However, performing CLFS in 2D cell culture is hindered by the fact that prolonged continuous stimulation results in rapid myotube detachment and cell cultures typically detach within 7 days without electrical stimulation [20]. Animal studies have shown that changes in myosin isoform can take up to 60 days to appear [21] and can require constant electrical stimulation for 24hrs per day [22] which are technically impossible to perform with 2D cultures due to myotube detachment. Tissue engineered skeletal muscle offers an alternative approach by permitting long-term culture (up to 5 weeks) [23] and allowing stimulation times far greater than 2D cultures permit [14, 20, 24]. In support of this, muscles engineered from primary rat muscle have been shown to adapt to different electrical patterns following 2 weeks of electrical stimulation in vitro [14].
Although engineered muscle is a highly promising model to study muscle physiology [25], several factors including electrical stimulation parameters [20, 24], biomaterial choice [26-28] and nutrient supply [29] need to be optimized to better recreate the in vivo milieu. One such factor is the choice of antibiotics used to minimize bacterial and
4
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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5 fungal contamination. Streptomycin is a widely used antibiotic in cell culture but it decreases protein synthesis and maturation in muscle cultures [30]. Streptomycin also acts as a non-specific calcium blocker including stretch-activated channels (SACs) which can allow conductance of Na+, K+, Mg2+ and Ca2+ ions [31] in response to both mechanical and electrical signals. Streptomycin has been shown to block the increase in force induced by stretch in engineered cardiac tissue in vitro [32] and adult skeletal muscle in vivo [33]. In this paper we first sought to determine a CLFS protocol for muscle engineered from the C2C12 cell line and second we sought to determine whether streptomycin has any effect on the response of engineered skeletal muscle to CLFS.
5
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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6 Materials and Methods 2D Cell Culture The C2C12 myoblast cell line (ATCC) was grown in growth media consisting of high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) until 70% confluent. C2C12 cells were used between passages 6 and 10.
3D Cell Culture Muscles were engineered using fibrin casting as reported previously [23]. Briefly, the muscle constructs were engineered between two 6mm long silk sutures set 12mm apart on Sylgard (PDMS)-coated dishes. 500µl of growth media containing 10U/ml thrombin, 0.2µg/ml genipin, and 0.5µg/ml aprotinin was added to the plate and agitated until it covered the entire surface. 200µl of 20mg/ml fibrinogen was added dropwise and the gels were left to polymerise for 1hr before addition of 100,000 cells. Two days after plating cells, the constructs were switched to differentiation media consisting of highglucose DMEM supplemented with 10% horse serum and penicillin (100U/ml) for 2 days. Following the second day in differentiation media, the constructs were moved to high-glucose DMEM with 7% FBS and penicillin (100U/ml) for the remainder of the experiment as previously found to be optimal [23]. For experiments containing streptomycin, streptomycin was added to the media at a concentration of 100µg/ml.
Electrical Stimulation
6
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 7 of 39
7 Electrical stimulation was performed using a custom made electrical stimulator previously described in detail [24]. Constructs were differentiated for 7 days and then were initially electrically stimulated for 24hrs with an electrical stimulation protocol consisting of a continuous 0.4second 10Hz train followed by a 3.6second rest. The constructs were then electrically stimulated for 14 days with an electrical stimulation protocol consisting of a continuous 0.6second 10Hz train followed by a 0.4second rest, unless stated otherwise. Both the non-stimulated and electrically stimulated constructs were fed daily once electrical stimulation was started.
Contractile Testing Functional testing of the C2C12 constructs was performed as described previously [34]. To determine the contractile properties of the engineered tissue, one of the anchors was freed from the Sylgard substrate and attached to a custom-made force transducer via one of the minutien pins. Rheobase (R50) and chronaxie (C50) were then determined as described previously [34]. Rheobase was calculated as the electric field strength (V/mm) eliciting 50% peak twitch force (Pt) with a 4ms pulse width. Chronaxie was calculated as the pulse width required to elicit 50% peak force at twice rheobase. Once excitability had been determined all impulses were delivered with a 4ms pulse width at 4xR50 as described previously. Fatigue was determined by stimulating for 0.75secs with 0.75secs rest at 50Hz for 3mins at 4 times rheobase with a 4ms pulse width. Cross-sectional area was calculated from the measured width of each construct (at its narrowest point), assuming a rectangular cross section and a depth of 500µm. Specific force was calculated as kilonewtons per square meter: the force generated by the construct (kN) divided by its cross-sectional area (m2).
7
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 8 of 39
8
Western Blot Tissues were washed in ice-cold PBS, and then blot-dried before freezing in liquid nitrogen and storing at −80°C. At the time of processing, samples were powdered in a 1.5mL microcentrifuge tube on dry ice, suspended in 200μL ice-cold sucrose lysis buffer (50mM Tris pH 7.5, 250mM sucrose, 1mM EGTA, 1mM EDTA, 1mM sodium orthovanadate, 50mM sodium fluoride, 5mM Na2(PO4)2, and 0.1% DTT) and shaken at 1,400rpm for 1hr at 4°C in an Eppendorf thermomixer (Hauppauge, NY). The samples were then centrifuged at 4°C for 1min at 10,000g to remove insoluble material. The supernatant was transferred to a new tube, and protein concentration was determined using the DC protein assay (Bio-Rad, CA, USA). Equal aliquots of protein in 1X Laemmli sample buffer were boiled for 5min before separation on a 10% acrylamide gel by SDS–polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Protran, Whatman, Piscataway, NJ) at 100V for 1hr. The membrane was blocked for 1hr in 3% milk in Tris-buffered saline + 0.1% Tween (TBST). Membranes were incubated overnight at 4°C with the appropriate primary antibody in TBST at 1:1,000. The membrane was then washed three times in TBST before incubation for 1hr at room temperature with the appropriate peroxidasecoupled secondary antibody in TBST at 1:10,000 (Pierce, Rockford, IL). Antibody binding was detected using an enhanced chemiluminescence horseradish peroxidase substrate detection kit (Millipore, Billerica, MA). Imaging and band quantification were carried out using a Chemi Genius Bioimaging Gel Doc System (Syngene, Cambridge, UK). The primary antibodies used in this study were MF20, F59, CaF2-5D2 (Hybridoma
8
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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9 Bank, Iowa, USA), total eEF2, SERCA 2a (Cell Signaling, MA, USA), slow MHC (Novacastra, Newcastle, UK), parvalbumin (GeneTex, CA, USA) and total PFK, GLUT4, SDH, ATPsynthase (Santa Cruz Biotechnology, CA, USA).
Statistical Analysis Data is presented as means ± S.E.M. Differences in mean values were compared within groups and significant differences were determined by ANOVA with post hoc TukeyKramer HSD test using Brightstat.com [35]. The significance level was set at (p< 0.05).
9
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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10 Results Effect of frequency, active time and impulse number on contractile properties We first optimised a CLFS protocol by examining the effect of frequency, active time and impulse number on the shift to a slower contractile phenotype following 2 weeks electrical stimulation in the presence of streptomycin (Table 1). Overall we found that time-to-peak tension (TPT) and half-relaxation time (1/2RT) were the contractile properties that displayed the greatest change following CLFS. We found that frequency was the dominant factor over both impulse number and active time in determining the change induced by CLFS. CLFS significantly slowed TPT (CTL=40.9 ± 0.2ms; 10Hz=58.5 ± 3.5ms; 20Hz=48.2 ± 2.7ms) and 1/2RT (CTL=50.4 ± 0.6ms; 10Hz=76.1 ± 3.3ms; 20Hz=66.6 ± 2.3ms) but 10Hz induced greater slowing than 20Hz (Fig 1). Despite the slowing of TPT and 1/2RT we found no change in fatigue resistance, one of the classical changes following CLFS seen in vivo and hypothesised that streptomycin in the media may prevent the metabolic changes required to improve fatigue resistance (Table 1).
Effect of streptomycin on active force production following 2wk electrical stimulation To study the effect of streptomycin on the response of engineered muscle to CLFS in vitro we performed CLFS (continuous 0.6 seconds 10Hz train followed by 0.4 seconds rest) on C2C12 engineered muscles for 2 weeks in the presence or absence of streptomycin. Streptomycin decreased force production in both non-stimulated (CTL=0.014 ± 0.001kN/m2; STREP=0.006 ± 0.0005kN/m2) and stimulated muscles (CTL=0.036 ± 0.002kN/m2; STREP=0.017 ± 0.002kN/m2) (Fig 2A) but did not affect the
10
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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11 fold increase in force seen following CLFS (CTL=165.8 ± 11.2%; STREP=181.8 ± 17.6%)
(Fig 2B). CLFS led to a significant increase (CTL=165.8 ± 11.2%;
STREP=181.8 ± 17.6%) in myosin heavy chain (MHC) protein content compared to non-stimulated muscles (Fig 2C).
Effect of streptomycin on time-to-peak tension following 2wk electrical stimulation We next looked at the change in time-to-peak tension (TPT) and half-relaxation time (1/2RT), classical markers used to help determine muscle phenotype. In non-stimulated muscle streptomycin led to a significant slowing in TPT (CTL=40.9 ± 1.4ms; STREP=50.8 ± 1.2ms). CLFS led to a slowing of TPT (CTL=83.3 ± 2.6ms; STREP=68.5 ± 2.7ms) but this was attenuated in the presence of streptomycin (Fig 3A.). In adult muscle, myosin isoform is the main determinant of TPT so we analysed the relative levels of slow and fast MHC. Relative to total MHC contents we found no change in the relative abundance of the fast MHC isoform following CLFS (Fig 3B). Relative to total MHC, the relative abundance of slow MHC increased following electrical stimulation (CTL=72.2 ± 8.0%; STREP=77.6 ± 8.8ms).
Effect of streptomycin on half-relaxation time following 2wk electrical stimulation Similar to TPT, in non-stimulated muscle, streptomycin led to a significant slowing in 1/2RT (CTL=50.4 ± 1.4ms; STREP=58.3 ± 1.7ms). CLFS resulted in slowing of 1/2RT in both the presence and absence of streptomycin (CTL=94.3 ± 4.2ms; STREP=76.1 ± 3.5ms), though streptomycin significantly attenuated this slowing (Fig 4A).
11
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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12 To determine the cause of the shift in 1/2RT we looked at the levels of the fast specific calcium sequestering protein parvalbumin (Parv) and both the slow and fast isoforms of the calcium uptake protein SERCA (Fig 4B). Following CLFS, the relative increase in the slow SERCA isoform (CTL=58.0 ± 8.6%; STREP=23.6 ± 4.6%) was significantly blunted in the presence of streptomycin (P
Page 1 of 39
Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle
Alastair Khodabukus and Keith Baar Division of Neurobiology, Physiology and Behavior, University of California Davis, Davis, CA 95616
Corresponding Author: Keith Baar
Email: [email protected]
Keywords: Skeletal muscle, tissue engineering, streptomycin, electrical stimulation
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 2 of 39
2 Abstract Chronic low-frequency electrical stimulation (CLFS) has long been used to induce a fast-to-slow phenotype shift in skeletal muscle. In this study we explore the role of frequency (10 and 20Hz), active time (15-60%) and streptomycin in inducing a fast-toslow shift in engineered muscle. We found that C2C12 engineered muscle could respond to CLFS with an adult-like active time of 60% and found that a constant 10Hz train of 0.6secs followed by 0.4secs rest induced a partial fast-to-slow phenotype shift. Following 2 weeks CLFS, Time-to-peak tension (TPT) (Control (CTL)=40.9 ± 0.2ms; 10Hz=58.5 ± 3.5ms; 20Hz=48.2 ± 2.7ms) and half-relaxation time (1/2RT) (CTL=50.4 ± 0.6ms; 10Hz=76.1 ± 3.3ms; 20Hz=66.6 ± 2.3ms) slowed significantly in a frequency but not active time dependent manner. Streptomycin significantly blunted the slowing of TPT and 1/2RT induced by CLFS by minimizing the fast-to-slow shift in SERCA isoform. Streptomycin (Non-Stim=-42.8 ± 2.5%; Stim=-38.1 ± 3.6%) significantly prevented the improvement in fatigue resistance seen in CTL constructs (Non-Stim=-58.4 ± 3.6%; Stim=-27.8 ± 1.7%). Streptomycin reduced the increase seen in GLUT4 protein following CLFS (CTL=89.4 ± 6.7%; Strep=41.0 ± 4.3%) and prevented increases in the mitochondrial proteins SDH and ATPsynthase. These data demonstrate that streptomycin significantly blunts the fast-to-slow shift induced by CLFS. In the absence of streptomycin, CLFS induced slowing of contractile dynamics and improved fatigue resistance and suggest that this model can be used to study the mechanisms underlying CLFS–induced adaptations in muscle phenotype.
2
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 3 of 39
3 Introduction
Skeletal muscle is a highly plastic tissue capable of changing its molecular, metabolic and functional properties in response to a wide range of factors including developmental maturity [1], electrical stimulation [2], mechanical stimulation [3], and drug treatments [4]. Muscle fibers can be classified as either “slow” or “fast” based on myosin heavy chain isoform and oxidative capacity [5, 6]. Slow fibers have higher mitochondrial content and rely more heavily upon oxidative metabolism, resulting in a more fatigue resistant phenotype compared to fast fibers. Adult muscle phenotype is primarily regulated by neural activity but is highly plastic as demonstrated by cross-innervation [7], denervation [8, 9] and denervation-electrical stimulation [10, 11] studies.
Electromyography (EMG) recordings have shown that slow muscles have a natural tonic firing frequency of 10-20Hz and can fire at least up to 300,000 impulses per day [12, 13]. Chronic low-frequency stimulation (CLFS) protocols that mimic the natural tonic firing pattern of slow phenotype muscles have been shown to induce fast-to-slow fiber type transitions both in vivo [7] and in vitro [11]. Despite CLFS patterns attempting to mimic the natural neural input to slow fibre type muscles (e.g. Soleus), no standardized CLFS protocol has been developed. This is due, in part, to the differential response to CLFS in different muscle groups [10, 14] and animal species [7, 10, 15], showing that no single CLFS pattern exists to transform all muscles into a slow muscle fibre type.
3
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 4 of 39
4 CLFS is typically performed by attaching small programmable stimulator units either on the backs of animals [16] or subcutaneously in animals [17, 18] and humans [19]. External stimulators can be damaged or dislodged by the animals and implantable devices can have limited reuse capability, leading to an increase in experimental cost. Furthermore, studying the mechanisms underlying CLFS induced shifts via inhibitor studies is vastly more expensive and more difficult in vivo compared to in vitro. CLFS studies have been performed in vitro and have been shown to induce changes in myofibrillar and metabolic protein expression [11]. However, performing CLFS in 2D cell culture is hindered by the fact that prolonged continuous stimulation results in rapid myotube detachment and cell cultures typically detach within 7 days without electrical stimulation [20]. Animal studies have shown that changes in myosin isoform can take up to 60 days to appear [21] and can require constant electrical stimulation for 24hrs per day [22] which are technically impossible to perform with 2D cultures due to myotube detachment. Tissue engineered skeletal muscle offers an alternative approach by permitting long-term culture (up to 5 weeks) [23] and allowing stimulation times far greater than 2D cultures permit [14, 20, 24]. In support of this, muscles engineered from primary rat muscle have been shown to adapt to different electrical patterns following 2 weeks of electrical stimulation in vitro [14].
Although engineered muscle is a highly promising model to study muscle physiology [25], several factors including electrical stimulation parameters [20, 24], biomaterial choice [26-28] and nutrient supply [29] need to be optimized to better recreate the in vivo milieu. One such factor is the choice of antibiotics used to minimize bacterial and
4
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 5 of 39
5 fungal contamination. Streptomycin is a widely used antibiotic in cell culture but it decreases protein synthesis and maturation in muscle cultures [30]. Streptomycin also acts as a non-specific calcium blocker including stretch-activated channels (SACs) which can allow conductance of Na+, K+, Mg2+ and Ca2+ ions [31] in response to both mechanical and electrical signals. Streptomycin has been shown to block the increase in force induced by stretch in engineered cardiac tissue in vitro [32] and adult skeletal muscle in vivo [33]. In this paper we first sought to determine a CLFS protocol for muscle engineered from the C2C12 cell line and second we sought to determine whether streptomycin has any effect on the response of engineered skeletal muscle to CLFS.
5
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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6 Materials and Methods 2D Cell Culture The C2C12 myoblast cell line (ATCC) was grown in growth media consisting of high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) until 70% confluent. C2C12 cells were used between passages 6 and 10.
3D Cell Culture Muscles were engineered using fibrin casting as reported previously [23]. Briefly, the muscle constructs were engineered between two 6mm long silk sutures set 12mm apart on Sylgard (PDMS)-coated dishes. 500µl of growth media containing 10U/ml thrombin, 0.2µg/ml genipin, and 0.5µg/ml aprotinin was added to the plate and agitated until it covered the entire surface. 200µl of 20mg/ml fibrinogen was added dropwise and the gels were left to polymerise for 1hr before addition of 100,000 cells. Two days after plating cells, the constructs were switched to differentiation media consisting of highglucose DMEM supplemented with 10% horse serum and penicillin (100U/ml) for 2 days. Following the second day in differentiation media, the constructs were moved to high-glucose DMEM with 7% FBS and penicillin (100U/ml) for the remainder of the experiment as previously found to be optimal [23]. For experiments containing streptomycin, streptomycin was added to the media at a concentration of 100µg/ml.
Electrical Stimulation
6
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 7 of 39
7 Electrical stimulation was performed using a custom made electrical stimulator previously described in detail [24]. Constructs were differentiated for 7 days and then were initially electrically stimulated for 24hrs with an electrical stimulation protocol consisting of a continuous 0.4second 10Hz train followed by a 3.6second rest. The constructs were then electrically stimulated for 14 days with an electrical stimulation protocol consisting of a continuous 0.6second 10Hz train followed by a 0.4second rest, unless stated otherwise. Both the non-stimulated and electrically stimulated constructs were fed daily once electrical stimulation was started.
Contractile Testing Functional testing of the C2C12 constructs was performed as described previously [34]. To determine the contractile properties of the engineered tissue, one of the anchors was freed from the Sylgard substrate and attached to a custom-made force transducer via one of the minutien pins. Rheobase (R50) and chronaxie (C50) were then determined as described previously [34]. Rheobase was calculated as the electric field strength (V/mm) eliciting 50% peak twitch force (Pt) with a 4ms pulse width. Chronaxie was calculated as the pulse width required to elicit 50% peak force at twice rheobase. Once excitability had been determined all impulses were delivered with a 4ms pulse width at 4xR50 as described previously. Fatigue was determined by stimulating for 0.75secs with 0.75secs rest at 50Hz for 3mins at 4 times rheobase with a 4ms pulse width. Cross-sectional area was calculated from the measured width of each construct (at its narrowest point), assuming a rectangular cross section and a depth of 500µm. Specific force was calculated as kilonewtons per square meter: the force generated by the construct (kN) divided by its cross-sectional area (m2).
7
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 8 of 39
8
Western Blot Tissues were washed in ice-cold PBS, and then blot-dried before freezing in liquid nitrogen and storing at −80°C. At the time of processing, samples were powdered in a 1.5mL microcentrifuge tube on dry ice, suspended in 200μL ice-cold sucrose lysis buffer (50mM Tris pH 7.5, 250mM sucrose, 1mM EGTA, 1mM EDTA, 1mM sodium orthovanadate, 50mM sodium fluoride, 5mM Na2(PO4)2, and 0.1% DTT) and shaken at 1,400rpm for 1hr at 4°C in an Eppendorf thermomixer (Hauppauge, NY). The samples were then centrifuged at 4°C for 1min at 10,000g to remove insoluble material. The supernatant was transferred to a new tube, and protein concentration was determined using the DC protein assay (Bio-Rad, CA, USA). Equal aliquots of protein in 1X Laemmli sample buffer were boiled for 5min before separation on a 10% acrylamide gel by SDS–polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Protran, Whatman, Piscataway, NJ) at 100V for 1hr. The membrane was blocked for 1hr in 3% milk in Tris-buffered saline + 0.1% Tween (TBST). Membranes were incubated overnight at 4°C with the appropriate primary antibody in TBST at 1:1,000. The membrane was then washed three times in TBST before incubation for 1hr at room temperature with the appropriate peroxidasecoupled secondary antibody in TBST at 1:10,000 (Pierce, Rockford, IL). Antibody binding was detected using an enhanced chemiluminescence horseradish peroxidase substrate detection kit (Millipore, Billerica, MA). Imaging and band quantification were carried out using a Chemi Genius Bioimaging Gel Doc System (Syngene, Cambridge, UK). The primary antibodies used in this study were MF20, F59, CaF2-5D2 (Hybridoma
8
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 9 of 39
9 Bank, Iowa, USA), total eEF2, SERCA 2a (Cell Signaling, MA, USA), slow MHC (Novacastra, Newcastle, UK), parvalbumin (GeneTex, CA, USA) and total PFK, GLUT4, SDH, ATPsynthase (Santa Cruz Biotechnology, CA, USA).
Statistical Analysis Data is presented as means ± S.E.M. Differences in mean values were compared within groups and significant differences were determined by ANOVA with post hoc TukeyKramer HSD test using Brightstat.com [35]. The significance level was set at (p< 0.05).
9
Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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10 Results Effect of frequency, active time and impulse number on contractile properties We first optimised a CLFS protocol by examining the effect of frequency, active time and impulse number on the shift to a slower contractile phenotype following 2 weeks electrical stimulation in the presence of streptomycin (Table 1). Overall we found that time-to-peak tension (TPT) and half-relaxation time (1/2RT) were the contractile properties that displayed the greatest change following CLFS. We found that frequency was the dominant factor over both impulse number and active time in determining the change induced by CLFS. CLFS significantly slowed TPT (CTL=40.9 ± 0.2ms; 10Hz=58.5 ± 3.5ms; 20Hz=48.2 ± 2.7ms) and 1/2RT (CTL=50.4 ± 0.6ms; 10Hz=76.1 ± 3.3ms; 20Hz=66.6 ± 2.3ms) but 10Hz induced greater slowing than 20Hz (Fig 1). Despite the slowing of TPT and 1/2RT we found no change in fatigue resistance, one of the classical changes following CLFS seen in vivo and hypothesised that streptomycin in the media may prevent the metabolic changes required to improve fatigue resistance (Table 1).
Effect of streptomycin on active force production following 2wk electrical stimulation To study the effect of streptomycin on the response of engineered muscle to CLFS in vitro we performed CLFS (continuous 0.6 seconds 10Hz train followed by 0.4 seconds rest) on C2C12 engineered muscles for 2 weeks in the presence or absence of streptomycin. Streptomycin decreased force production in both non-stimulated (CTL=0.014 ± 0.001kN/m2; STREP=0.006 ± 0.0005kN/m2) and stimulated muscles (CTL=0.036 ± 0.002kN/m2; STREP=0.017 ± 0.002kN/m2) (Fig 2A) but did not affect the
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Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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11 fold increase in force seen following CLFS (CTL=165.8 ± 11.2%; STREP=181.8 ± 17.6%)
(Fig 2B). CLFS led to a significant increase (CTL=165.8 ± 11.2%;
STREP=181.8 ± 17.6%) in myosin heavy chain (MHC) protein content compared to non-stimulated muscles (Fig 2C).
Effect of streptomycin on time-to-peak tension following 2wk electrical stimulation We next looked at the change in time-to-peak tension (TPT) and half-relaxation time (1/2RT), classical markers used to help determine muscle phenotype. In non-stimulated muscle streptomycin led to a significant slowing in TPT (CTL=40.9 ± 1.4ms; STREP=50.8 ± 1.2ms). CLFS led to a slowing of TPT (CTL=83.3 ± 2.6ms; STREP=68.5 ± 2.7ms) but this was attenuated in the presence of streptomycin (Fig 3A.). In adult muscle, myosin isoform is the main determinant of TPT so we analysed the relative levels of slow and fast MHC. Relative to total MHC contents we found no change in the relative abundance of the fast MHC isoform following CLFS (Fig 3B). Relative to total MHC, the relative abundance of slow MHC increased following electrical stimulation (CTL=72.2 ± 8.0%; STREP=77.6 ± 8.8ms).
Effect of streptomycin on half-relaxation time following 2wk electrical stimulation Similar to TPT, in non-stimulated muscle, streptomycin led to a significant slowing in 1/2RT (CTL=50.4 ± 1.4ms; STREP=58.3 ± 1.7ms). CLFS resulted in slowing of 1/2RT in both the presence and absence of streptomycin (CTL=94.3 ± 4.2ms; STREP=76.1 ± 3.5ms), though streptomycin significantly attenuated this slowing (Fig 4A).
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Tissue Engineering Part A Streptomycin decreases the functional shift to a slow-phenotype induced by electrical stimulation in engineered muscle (doi: 10.1089/ten.TEA.2014.0462) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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12 To determine the cause of the shift in 1/2RT we looked at the levels of the fast specific calcium sequestering protein parvalbumin (Parv) and both the slow and fast isoforms of the calcium uptake protein SERCA (Fig 4B). Following CLFS, the relative increase in the slow SERCA isoform (CTL=58.0 ± 8.6%; STREP=23.6 ± 4.6%) was significantly blunted in the presence of streptomycin (P
