Defined Electrical Stimulation Emphasizing Excitability for the Development and Testing of Engineered Skeletal Muscle

Abstract

Electrical stimulation is required for the maturation of skeletal muscle and as a way to nondestructively monitor muscle development. However, the wrong stimulation parameters can result in electrochemical damage that impairs muscle development/regeneration. The goal of the current study was to determine what aspect of an electrical impulse, specifically the pulse amplitude or pulse width, was detrimental to engineered muscle function and subsequently how engineered muscle responded to continuous electrical stimulation for 24 h. Acute stimulation at a pulse amplitude greater than six-times rheobase resulted in a 2.4-fold increase in the half-relaxation time (32.3±0.49 ms vs. 77.4±4.35 ms; p<0.05) and a 1.59-fold increase in fatigability (38.2%±3.61% vs. 60.6%±4.52%; p<0.05). No negative effects were observed when the pulse energy was increased by lengthening the pulse width, indicating electrochemical damage was due to electric fields at or above six-times rheobase. Continuous stimulation for 24 h at electric fields greater than 0.5 V/mm consistently resulted in ∼2.5-fold increase in force (0.30±0.04 kN/m² vs. 0.67±0.06 kN/m²; p<0.05). Forty per cent of this increase in force was dependent on the mammalian target of rapamycin (RAP) complex 1 (mTORC1), as RAP prevented this portion of the increase in force (CON=0.30±0.04 kN/m² to 0.67±0.06 kN/m² compared with RAP=0.21±0.01 kN/m² to 0.37±0.04 kN/m²; p<0.05). Since there was no increase in myosin heavy chain, the remaining increase in force over the 24 h of stimulation is likely due to cytoskeletal rearrangement. These data indicate that electrochemical damage occurs in muscle at a voltage field greater than six-times rheobase and therefore optimal muscle stimulation should be performed using lower electric fields (two- to four-times rheobase).

Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro

“…Electrical stimulation was delivered to the constructs using LABview software (National Instruments) connected to a custom built amplifier and in turn attached to the stainless steel electrodes. Based on a previously published protocol (Khodabukus & Baar, 2012) with some slight modifications, stimulation consisted of 5 bipolar 1 ms pulses delivered at 1 V/mm and 10 Hz with 3.5 s rest periods. After 24 hr, stimulation was terminated and constructs were tested as described above in section 2.6.…”

Modulation of cell function by electric field: a high-resolution analysis

“…The regulatory effect of EFs has been demonstrated on different cell types such as neurons, osteoblasts, myoblasts, fibroblasts, endothelial cells, muscle cell and epithelial cells [8][9][10][11][12][13][14]. These effects result from manipulation of the native EF in the ionic extracellular environment and across the cell membrane [15 -17].…”

Mechanistic investigation of adult myotube response to exercise and drug treatment in vitro using a multiplexed functional assay system

“…The collected data demonstrate that this multiplexed cantilever array can be used to effectively monitor rates of muscle fatigue in vitro. While previous work from a number of groups (17,24,30,32) has used long-term stimulation and creatine treatment to evaluate molecular changes during cellular development in vitro, this study is the first to assess the effect of such treatment applications on long-term functional performance. Such data are important for confirming that the molecular changes observed in vitro correlate to alterations in functional output, thereby validating such studies for producing predictions of whole tissue responses in vivo.…”