Belt electrode tetanus muscle stimulation reduces denervation-induced atrophy of rat multiple skeletal muscle groups.

Belt electrode-skeletal muscle electrical stimulation (B-SES) involves the use of belt-shaped electrodes to contract multiple muscle groups simultaneously. Twitch contractions have been demonstrated to protect against denervation-induced muscle atrophy in rats, possibly through mitochondrial biosynthesis. This study examined whether inducing tetanus contractions with B-SES suppresses muscle atrophy and identified the underlying molecular mechanisms. We evaluated the effects of acute (60 Hz, 5 min) and chronic (60 Hz, 5 min, every alternate day for one week) B-SES on the tibialis anterior (TA) and gastrocnemius (GAS) muscles in Sprague-Dawley rats using belt electrodes attached to both ankle joints. After acute stimulation, a significant decrease in the glycogen content was observed in the left and right TA and GAS, suggesting that B-SES causes simultaneous contractions in multiple muscle groups. B-SES enhanced p70S6K phosphorylation, an indicator of the mechanistic target of rapamycin complex 1 activity. During chronic stimulations, rats were divided into control (CONT), denervation-induced atrophy (DEN), and DEN + electrically stimulated with B-SES (DEN + ES) groups. After seven days of treatment, the wet weight (n = 8-11 for each group) and muscle fiber cross-sectional area (CSA, n = 6 for each group) of the TA and GAS muscles were reduced in the DEN and DEN + ES groups compared with that in the CON group. The DEN + ES group showed significantly higher muscle weight and CSA than those in the DEN group. Although RNA-seq and pathway analysis suggested that mitochondrial biogenesis is a critical event in this phenomenon, mitochondrial content showed no difference. In contrast, ribosomal RNA 28S and 18S (n = 6) levels in the DEN + ES group were higher than those in the DEN group, even though RNA-seq showed that the ribosome biogenesis pathway was reduced by electrical stimulation. The mRNA levels of the muscle proteolytic molecules atrogin-1 and MuRF1 were significantly higher in DEN than those in CONT. However, they were more suppressed in DEN + ES than those in DEN. In conclusion, tetanic electrical stimulation of both ankles using belt electrodes effectively reduced denervation-induced atrophy in multiple muscle groups. Furthermore, ribosomal biosynthesis plays a vital role in this phenomenon.

Pulsed electrical stimulation and amino acid derivatives promote collagen gene expression in human dermal fibroblasts.

Several collagen types are important for maintaining skin structure and function. Previous reports show that l-hydroxyproline (Hyp), N-acetyl-l-hydroxyproline (AHyp), and l-alanyl-l-glutamine (Aln-Gln) are biological active substances with collagen synthesis-promoting effects. In this study, we combined the promotive effects of pulsed electrical stimulation (PES) with three amino acid derivatives in human dermal fibroblasts. Fibroblasts were exposed to PES with a 4,800 Hz pulse frequency and a voltage at 1 or 5 V for 15 min. The gene expression of type I and III collagen (fibrillar collagen), type IV and VII collagen (basement membrane collagen and anchoring fibril collagen) were measured by RT-PCR 48 h after PES. PES alone promoted the expression of COL1A1 and COL3A1 at 5 V but did not alter that of COL4A1 and COL7A1. Each AAD and the AAD mixture promoted the expression of COL4A1 and COL7A1 but either repressed, or did not alter, that of COL1A1 and COL3A1. Compared to treatment with each AAD, PES at 5 V with Hyp promoted the expression of COL1A1 and COL3A1, enhanced COL3A1 expression with AHyp, and stimulated COL3A1 expression with Aln-Gln, while COL4A1 and COL7A1 expressions were not affected. PES and the AAD mixture significantly promoted COL4A1 expression in a voltage-dependent manner, and COL1A1 and COL3A1 demonstrated a similar but nonsignificant trend, whereas COL7A1 expression was not affected. The combination of PES with each AAD or the AAD mixture may improve skin structure and function by increasing the expression of basement membrane collagen and dermal fibrillar collagen.

Effects of pulsed electrical stimulation on α-smooth muscle actin and type I collagen expression in human dermal fibroblasts.

Pulsed electrical stimulation (PES) is known to affect cellular activities. We previously found PES to human dermal fibroblasts (HFs) promoted platelet-derived growth factor subunit A (PDGFA) gene expression, which enhanced proliferation. In this study, we investigated PES effects on fibroblast collagen production and differentiation into myofibroblasts. HFs were electrically stimulated at 4800 Hz and 5 V for 60 min. Imatinib, a specific inhibitor of PDGF receptors, was treated before PES. After 6 h of PES, PDGFA, α-smooth muscle actin (α-SMA), and collagen type I α1 chain gene expressions were upregulated in PES group. Imatinib suppressed the promoted expression except for PDGFA. Immunofluorescence staining and enzyme-linked immunosorbent assay showed the production of α-SMA and collagen I was enhanced in PES group but suppressed in PES + imatinib group at 48 h after PES. Therefore, PES promotes the production of α-SMA and collagen I in fibroblasts, which is triggered by PDGFA that is upregulated early after PES.

Staining of stratum corneum with fluorescent ε-poly-L-lysine and its application to evaluation of skin conditions.

BACKGROUND: ε-Poly-L-lysine (PLL) is a cationic polymer consisting of 25 to 35 L-lysine residues that adheres to the surface of skin as well as hair. However, the properties of PLL regarding its adhesion to the skin remain to be elucidated. In this study, we examined the staining of stratum corneum (SC) with fluorescence-labeled PLL and explored its relationship with skin condition. MATERIALS AND METHODS: Alexa Fluor 488-labeled PLL (AF-PLL) was reacted with tape-stripped stratum corneum (SC), and the staining properties were monitored by fluorescence microscopy. Clinical study was performed by measuring the water content of the cheek SC and transepidermal water loss (TEWL), and the tape-stripped SC was subjected to staining with AF-PLL. RESULTS: AF-PLL staining of the SC was inhibited at acidic pH or by the addition of high concentration of salt solution, suggesting the involvement of ionic interaction between PLL and the SC, at least in part. The AF-PLL staining was inhibited by unlabeled PLL or various alkyl amines, but not by L-lysine monomer. AF-PLL staining was observed inside the corneocytes as well as surrounding cornified envelope. Clinical study revealed that AF-PLL staining intensity of the SC was negatively correlated with its water content and positively correlated with its TEWL. CONCLUSION: PLL can efficiently adhere to SC and AF-PLL staining of SC can be applied to evaluate skin conditions.

Low-frequency electrical stimulation of bilateral hind legs by belt electrodes is effective for preventing denervation-induced atrophies in multiple skeletal muscle groups in rats.

Belt electrode skeletal muscle electrical stimulation (B-SES) can simultaneously contract multiple muscle groups. Although the beneficial effects of B-SES in clinical situations have been elucidated, its molecular mechanism remains unknown. In this study, we developed a novel rodent B-SES ankle stimulation system to test whether low-frequency stimulation prevents denervation-induced muscle atrophy. Electrical stimulations (7‒8 Hz, 30 min) with ankle belt electrodes were applied to Sprague-Dawley rats daily for one week. All animals were assigned to the control (CONT), denervation-induced atrophy (DEN), and DEN + electrical stimulation (ES) groups. The tibialis anterior (TA) and gastrocnemius (GAS) muscles were used to examine the effect of ES treatment. After seven daily sessions of continuous stimulation, muscle wet weight (n = 8-11), and muscle fiber cross-sectional area (CSA, n = 4-6) of TA and GAS muscles were lower in DEN and DEN + ES than in CON. However, it was significantly higher in DEN than DEN + ES, showing that ES partially prevented muscle atrophy. PGC-1α, COX-IV, and citrate synthase activities (n = 6) were significantly higher in DEN + ES than in DEN. The mRNA levels of muscle proteolytic molecules, Atrogin-1 and Murf1, were significantly higher in DEN than in CONT, while B-SES significantly suppressed their expression (p < 0.05). In conclusion, low-frequency electrical stimulation of the bilateral ankles using belt electrodes (but not the pad electrodes) is effective in preventing denervation-induced atrophy in multiple muscles, which has not been observed with pad electrodes. Maintaining the mitochondrial quantity and enzyme activity by low-frequency electrical stimulation is key to suppressing muscle protein degradation.

Effect of belt electrode-skeletal muscle electrical stimulation on immobilization-induced muscle fibrosis.

PURPOSE: Macrophage accumulation in response to decreasing myonuclei may be the major mechanism underlying immobilization-induced muscle fibrosis in muscle contracture, an intervention strategy suppressing these lesions is necessary. Therefore, this research investigated the effect of belt electrode-skeletal muscle electrical stimulation (B-SES), a new electrical stimulation device, to the macrophage accumulation via myonuclei decrease in immobilization-induced muscle fibrosis. MATERIALS AND METHODS: 18 Wistar male rats were divided into the control group, immobilization group (with plaster cast fixation to immobilize the soleus muscles in a shortened position for 2 weeks), and B-SES group (with muscle contractile exercise through B-SES during the immobilization period). B-SES stimulation was performed at a frequency of 50 Hz and an intensity of 4.7 mA, muscle contractile exercise by B-SES was applied to the lower limb muscles for 20 minutes/session (twice a day) for 2 weeks (6 times/week). The bilateral soleus muscles were used for histological, immunohistochemical, biochemical, and molecular biological analyses. RESULTS: The number of myonuclei was significantly higher in the B-SES group than in the immobilization group, and there was no significant difference between the B-SES and control groups. The cross-sectional area of type I and II myofibers in the immobilization and B-SES groups was significantly lower than that in the control group, and the cross-sectional area of type I myofibers in the B-SES group was higher than that in the immobilization group. However, Atrogin-1 and MuRF-1 mRNA expression in the immobilization and B-SES groups was significantly higher than those in the control group. Additionally, the number of macrophages, IL-1ß, TGF-ß1, and α-SMA mRNA expression, and hydroxyproline expression was significantly lower in the control and B-SES groups than those in the immobilization group. CONCLUSION: This research surmised that muscle contractile exercise through B-SES prevented immobilization-induced muscle fibrosis, and this alteration suppressed the development of muscle contracture.

Effects of pulsed electrical stimulation on growth factor gene expression and proliferation in human dermal fibroblasts.

Human dermal fibroblast proliferation plays an important role in skin wound healing, and electrical stimulation (ES) promotes skin wound healing. Although the use of ES for skin wound healing has been investigated, the mechanism underlying the effects of ES on cells is still unclear. This study examined the effects of pulsed electrical stimulation (PES) on human dermal fibroblasts. Normal adult human dermal fibroblasts were exposed to a frequency of 4800 Hz, voltage of 1-5 V, and PES exposure time of 15, 30, and 60 min. Dermal fibroblast proliferation and growth factor gene expression were investigated for 6-48 h post PES. Dermal fibroblast proliferation significantly increased from 24 to 48 h post PES at a voltage of 5 V and PES exposure time of 60 min. Under the same conditions, post PES, platelet-derived growth factor subunit A (PDGFA), fibroblast growth factor 2 (FGF2), and transforming growth factor beta 1 (TGF-ß1) expression significantly increased from 6 to 24 h, 12 to 48 h, and 24 to 48 h, respectively. Imatinib, a specific inhibitor of platelet-derived growth factor receptor, significantly inhibited the proliferation of dermal fibroblasts promoted by PES, suggesting that PDGFA expression, an early response of PES, was involved in promoting the cell proliferation. Therefore, PES at 4800 Hz may initially promote PDGFA expression and subsequently stimulate the expression of two other growth factors, resulting in dermal fibroblast proliferation after 24 h or later. In conclusion, PES may activate the cell growth phase of wound healing.

Cardiac cycle-synchronized electrical muscle stimulator for lower limb training with the potential to reduce the heart's pumping workload.

BACKGROUND: The lower limb muscle may play an important role in decreasing the heart's pumping workload. Aging and inactivity cause atrophy and weakness of the muscle, leading to a loss of the heart-assisting role. An electrical lower limb muscle stimulator can prevent atrophy and weakness more effectively than conventional resistance training; however, it has been reported to increase the heart's pumping workload in some situations. Therefore, more effective tools should be developed. METHODS: We newly developed a cardiac cycle-synchronized electrical lower limb muscle stimulator by combining a commercially available electrocardiogram monitor and belt electrode skeletal muscle electrical stimulator, making it possible to achieve strong and wide but not painful muscle contractions. Then, we tested the stimulator in 11 healthy volunteers to determine whether the special equipment enabled lower limb muscle training without harming the hemodynamics using plethysmography and a percutaneous cardiac output analyzer. RESULTS: In 9 of 11 subjects, the stimulator generated diastolic augmentation waves on the dicrotic notches and end-diastolic pressure reduction waves on the plethysmogram waveforms of the brachial artery, showing analogous waveforms in the intra-aortic balloon pumping heart-assisting therapy. The heart rate, stroke volume, and cardiac output significantly increased during the stimulation. There was no change in the systolic or diastolic blood pressure during the stimulation. CONCLUSION: Cardiac cycle-synchronized electrical muscle stimulation for the lower limbs may enable muscle training without harmfully influencing the hemodynamics and with a potential to reduce the heart's pumping workload, suggesting a promising tool for effectively treating both locomotor and cardiovascular disorders.

Pulsed electric current induces the differentiation of human keratinocytes.

Although normal human keratinocytes are known to migrate toward the cathode in a direct current (DC) electric field, other effects of the electric stimulation on keratinocyte activities are still unclear. We have investigated the keratinocyte differentiation under monodirectional pulsed electric stimulation which reduces the electrothermal and electrochemical hazards of a DC application. When cultured keratinocytes were exposed to the electric field of 3 V (ca. 100 mV/mm) or 5 V (ca. 166 mV/mm) at a frequency of 4,800 Hz for 5 min a day for 5 days, cell growth under the 5-V stimulation was significantly suppressed as compared with the control culture. Expression of mRNAs encoding keratinocyte differentiation markers such as keratin 10, involucrin, transglutaminase 1, and filaggrin was significantly increased in response to the 5-V stimulation, while the 3-V stimulation induced no significant change. After the 5-V stimulation, enhanced immunofluorescent stainings of involucrin and filaggrin were observed. These results indicate that monodirectional pulsed electric stimulation induces the keratinocyte differentiation with growth arrest.

Skin collagen reproduction increased by ascorbic acid derivative iontophoresis by frequent-reversal bipolar electric stimulation.

The effect of the iontophoresis of ascorbic acid (Vitamin C; VC) derivative with frequent-reversal bipolar electric stimulation on the production of collagen in rat skin was evaluated in terms of hydroxyproline content through high-performance liquid chromatography. First, a control group was not given electrical stimulation and four groups were stimulated with a unipolar pulse for 0.5-10 min every day for one week. The hydroxyproline level in the skin was increased depending on the length of the stimulation. Second, a control group was not given any electrical stimulation, and three groups were treated with (a) VC solution without any stimulation, (b) a bipolar pulse for 10 min with saline, or (c) a bipolar pulse for 5 min with the VC solution. Significant increases were found in all the stimulation groups, although these treated with the VC solution without any stimulation did not have any effects compared to the control. Thus, in order to increase the hydroxyproline levels in skin, a VC must be delivered with bipolar stimulation as a method of iontophoresis. These results suggest that our newly developed electric stimulation is effective at increasing skin collagen content, and that bipolar stimulation is more effective on the iontophoresis of not only VC but also some medicines such as low- and high-molecular drugs directed to the target organ (7).