Kwok-Kuen Cheung

Abundant and dynamic expression of G protein‐coupled P2Y receptors in mammalian development

Extracellular ATP mediates diverse biological effects by activating two families of receptors, the P2X and P2Y receptors. There is growing evidence to show that activation of G protein‐coupled P2Y receptors can produce trophic effects in many cell types. Yet the expression and function of the P2Y receptors in development has rarely been studied and has never been investigated in mammalian development. This study used the reverse transcription‐polymerase chain reaction and immunohistochemistry to demonstrate the abundant and dynamic expression of P2Y receptors in rat development. These receptors were expressed in a wide range of embryonic structures, notably somites, skeletal muscle, the central and peripheral nervous system, the heart, lung, and liver. All the P2Y receptors studied were expressed as early as embryonic day 11, when most embryonic organs were far from being functional and still in the process of being formed. P2Y receptor proteins were strongly expressed in temporary, developmental structures that do not have a correlate in the adult animal, including the somites (P2Y1, P2Y2, and P2Y4) and the floor plate of the neural tube (P2Y1). P2Y receptors were also dynamically expressed, with receptor mRNA and protein being both up‐ and down‐regulated at different developmental stages. The down‐regulation of the P2Y1, 2, and 4 receptor proteins in skeletal muscle and heart, and the disappearance of the P2Y4 receptor from the brainstem and ventral white matter of the spinal cord postnatally, demonstrated that many P2Y receptors were likely to be involved in functions specific to embryonic life. Thus, these findings strongly suggest that P2Y receptors play an important role in the development of many tissues, and pioneer further studies into the role of purinergic signalling in development. Developmental Dynamics 228:254–266, 2003.

Expression of P2X purinoceptors during rat brain development and their inhibitory role on motor axon outgrowth in neural tube explant cultures

Extracellular ATP is well known as a neurotransmitter and neuromodulator in the CNS of adults. However, little is known about the involvement of ATP during the development of mammalian brain. In the present study, we have examined the expression pattern of P2X receptor subtype mRNA and protein during perinatal rat brain development (from embryonic day (E) 10 to postnatal day (P) 16 brain). While P2X3 receptors appeared early at E11, they declined in the stages that follow. P2X2 and P2X7 receptors were expressed from E14 onwards, while P2X4, P2X5 and P2X6 receptors were expressed from P1 onwards. P2X1 receptor expression was not observed in any of the developmental ages examined. We investigated the effect of 100 microM ATP and alpha,beta-methylene ATP (alpha,beta-meATP; selective agonist for P2X1, P2X2/3 and P2X3 receptors) on motor axon outgrowth in collagen-embedded neural tube explant cultures. Both ATP- and alpha,beta-meATP-treated neural tubes showed a significant reduction in neurite outgrowth compared with the control explants. This inhibitory effect could not be reproduced by uridine triphosphate. In conclusion, all P2X receptor subtypes, except for P2X1, were strongly represented in the developing rat brain. ATP was shown to inhibit motor axon outgrowth during early embryonic neurogenesis, most likely via the P2X3 receptor. It is speculated that P2X7 receptors may be involved in programmed cell death during embryogenesis and that P2X4, P2X(5) and P2X6 receptors might be involved in postnatal neurogenesis.

Localization of P2X3 receptors and coexpression with P2X2 receptors during rat embryonic neurogenesis.

It is well known that extracellular ATP mediates rapid excitatory signaling by means of the ionotropic P2X receptors. One of its subunits, the P2X3 receptor, is well documented to be associated with sensory innervation in adult animals. It is speculated that the P2X3 receptor may have already been present in the early sensory system. The aim of this study was to investigate the distribution of the P2X3 receptor during neurogenesis by using immunohistochemistry on rat embryos from embryonic day (E)9.5–18.5. The P2X3 receptor was first identified in the hindbrain neural tube and the sensory ganglia in E11–11.5 embryos. At E14.5, the optic tract and retina, nucleus tractus solitarius, mesencephalic trigeminal nucleus, and sensory nerves in both respiratory and digestive tract showed positive staining. The facial nucleus, the prepositus hypoglossal nucleus, and the sympathetic ganglia also showed P2X3 immunoreactivity, even though these are not sensory associated. P2X3 immunoreactivity was detected in the vestibular nucleus, the nerves in mesentery, bladder, and kidney in E16.5 and in nerves in vibrissae in E18.5. P2X3 immunoreactivity in the facial nucleus, spinal trigeminal tract, the mesencephalic trigeminal nucleus, and the vestibular nucleus were undetectable in postnatal day 16 rat brainstem. The P2X3 receptor was coexpressed with the P2X2 receptor in nucleus tractus solitarius, dorsal root ganglion, nodose ganglion, and the taste bud in E16.5 embryo, which was 5 days later than the first appearance of the native P2X3 receptor. In summary, we present a detailed expression pattern of the P2X3 receptor during neurogenesis and report that P2X3 immunoreactivity is down‐regulated in early postnatal brainstems. J. Comp. Neurol. 443:368–382, 2002.

Electrical Stimulation Influences Satellite Cell Proliferation and Apoptosis in Unloading-Induced Muscle Atrophy in Mice

Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU+/Pax7+ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention.

Pulsed electromagnetic fields (PEMF) promote early wound healing and myofibroblast proliferation in diabetic rats.

Reduced collagen deposition possibly leads to slow recovery of tensile strength in the healing process of diabetic cutaneous wounds. Myofibroblasts are transiently present during wound healing and play a key role in wound closure and collagen synthesis. Pulsed electromagnetic fields (PEMF) have been shown to enhance the tensile strength of diabetic wounds. In this study, we examined the effect of PEMF on wound closure and the presence of myofibroblasts in Sprague-Dawley rats after diabetic induction using streptozotocin. A full-thickness square-shaped dermal wound (2 cm × 2 cm) was excised aseptically on the shaved dorsum. The rats were randomly divided into PEMF-treated (5 mT, 25 Hz, 1 h daily) and control groups. The results indicated that there were no significant differences between the groups in blood glucose level and body weight. However, PEMF treatment significantly enhanced wound closure (days 10 and 14 post-wounding) and re-epithelialization (day 10 post-wounding), although these improvements were no longer observed at later stages of the wound healing process. Using immunohistochemistry against α-smooth muscle actin (α-SMA), we demonstrated that significantly more myofibroblasts were detected on days 7 and 10 post-wounding in the PEMF group when compared to the control group. We hypothesized that PEMF would increase the myofibroblast population, contributing to wound closure during diabetic wound healing.

Expression and association of TRPC1 with TRPC3 during skeletal myogenesis

Introduction: TRPC1 and TRPC3 proteins are widely expressed in skeletal muscles in forming calcium‐permeable channels. Herein we characterize the expression pattern of TRPC transcripts during skeletal myogenesis in C2C12 myoblasts. Methods: We used polymerase chain reaction and Western blotting to detect expression levels, immunohistochemistry for subcellular localization, and co‐immunoprecipitation techniques to assess interaction. Results: TRPC1 localizes to the cytoplasm and is enriched in the perinuclear region in undifferentiated myoblasts. Expression of TRPC1 increases significantly during myogenesis and resides mainly in differentiated myocytes and myotubes. TRPC3 is absent in undifferentiated myoblasts, is dramatically upregulated in differentiated culture, and is preferentially expressed in myotubes. Physical interaction of TRPC1–TRPC3 was observed, suggesting the possible existence of heteromers. Conclusions: Expression of TRPC1 and TRPC3 is tightly regulated during myogensis. Evidence of TRPC1–TRPC3 interaction was first demonstrated in a muscle cell line. The functional consequences of this interaction remain to be established. Muscle Nerve 44: 358–365, 2011

Adaptive responses of TRPC1 and TRPC3 during skeletal muscle atrophy and regrowth

Introduction: We assessed the time‐dependent changes of transient receptor potential canonical type 1 (TRPC1) and TRPC3 expression and localization associated with muscle atrophy and regrowth in vivo. Methods: Mice were subjected to hindlimb unloading for 7 or 14 days (7U, 14U) followed by 3, 7, or 14 days of reloading (3R, 7R, 14R). Results: Soleus muscle mass and tetanic force were reduced significantly at 7U and 14U and recovered by 14R. Recovery of muscle fiber cross‐sectional area was observed by 28R. TRPC1 mRNA was unaltered during the unloading‐reloading period. However, protein expression remained depressed through 14R. Decreased localization of TRPC1 to the sarcolemma was observed. TRPC3 mRNA and protein expression levels were decreased significantly during the early phase of reloading. Conclusions: Given the known role of these channels in muscle development, changes observed in TRPC1 and TRPC3 may relate closely to muscle atrophy and remodeling processes. Muscle Nerve 49: 691–699, 2014

In Vivo and ex Vivo Approaches to Studying the Biomechanical Properties of Healing Wounds in Rat Skin

An evaluation of wound mechanics is crucial in reflecting the wound healing status. The present study examined the biomechanical properties of healing rat skin wounds in vivo and ex vivo. Thirty male Sprague-Dawley rats, each with a 6 mm full-thickness circular punch biopsied wound at both posterior hind limbs were used. The mechanical stiffness at both the central and margins of the wound was measured repeatedly in five rats over the same wound sites to monitor the longitudinal changes over time of before wounding, and on days 0, 3, 7, 10, 14, and 21 after wounding in vivo by using an optical coherence tomography-based air-jet indentation system. Five rats were euthanized at each time point, and the biomechanical properties of the wound tissues were assessed ex vivo using a tensiometer. At the central wound bed region, the stiffness measured by the air-jet system increased significantly from day 0 (17.2%), peaked at day 7 (208.3%), and then decreased progressively until day 21 (40.2%) as compared with baseline prewounding status. The biomechanical parameters of the skin wound samples measured by the tensiometer showed a marked reduction upon wounding, then increased with time (all p < 0.05). On day 21, the ultimate tensile strength of the skin wound tissue approached 50% of the normal skin; while the stiffness of tissue recovered at a faster rate, reaching 97% of its prewounded state. Our results suggested that it took less time for healing wound tissues to recover their stiffness than their maximal strength in rat skin. The stiffness of wound tissues measured by air-jet could be an indicator for monitoring wound healing and contraction.

The involvement of TRPC1 in skeletal muscle regrowth after unloading‐induced atrophy

Decreased mechanical loading results in skeletal muscle atrophy. The transient receptor potential canonical type 1 (TRPC1) protein is implicated in this process. Investigation of the regulation of TRPC1 in vivo has rarely been reported. In the present study, we employ the mouse hindlimb unloading and reloading model to examine the involvement of TRPC1 in the regulation of muscle atrophy and regrowth, respectively. We establish the physiological relevance of the concept that manipulation of TRPC1 could interfere with muscle regrowth processes following an atrophy‐inducing event. Specifically, we show that suppressing TRPC1 expression during reloading impairs the recovery of the muscle mass and slow myosin heavy chain profile. Calcineurin appears to be part of the signalling pathway involved in the regulation of TRPC1 expression during muscle regrowth. These results provide new insights concerning the function of TRPC1. Interventions targeting TRPC1 or its downstream or upstream pathways could be useful for promoting muscle regeneration.

The involvement of transient receptor potential canonical type 1 in skeletal muscle regrowth after unloading-induced atrophy

Decreased mechanical loading results in skeletal muscle atrophy. The transient receptor potential canonical type 1 (TRPC1) protein is implicated in this process. Investigation of the regulation of TRPC1 in vivo has rarely been reported. In the present study, we employ the mouse hindlimb unloading and reloading model to examine the involvement of TRPC1 in the regulation of muscle atrophy and regrowth, respectively. We establish the physiological relevance of the concept that manipulation of TRPC1 could interfere with muscle regrowth processes following an atrophy‐inducing event. Specifically, we show that suppressing TRPC1 expression during reloading impairs the recovery of the muscle mass and slow myosin heavy chain profile. Calcineurin appears to be part of the signalling pathway involved in the regulation of TRPC1 expression during muscle regrowth. These results provide new insights concerning the function of TRPC1. Interventions targeting TRPC1 or its downstream or upstream pathways could be useful for promoting muscle regeneration.