Skeletal Muscle-Derived Cell Implantation for the Treatment of Fecal Incontinence: A Randomized, Placebo-Controlled Study.

BACKGROUND AND AIMS: Fecal incontinence (FI) improvement following injection of autologous skeletal muscle-derived cells has been previously suggested. This study aimed to test the efficacy and safety of said cells through a multicenter, placebo-controlled study, to determine an appropriate cell dose, and to delineate the target patient population that can most benefit from cell therapy. METHODS: Patients experiencing FI for at least 6 months were randomized to receive a cell-free medium or low or high dose of cells. All patients received pelvic floor electrical stimulation before and after treatment. Incontinence episode frequency (IEF), FI quality of life, FI burden assessed on a visual analog scale, Wexner score, and parameters reflecting anorectal physiological function were all assessed for up to 12 months. RESULTS: Cell therapy improved IEF, FI quality of life, and FI burden, reaching a preset level of statistical significance in IEF change compared with the control treatment. Post hoc exploratory analyses indicated that patients with limited FI duration and high IEF at baseline are most responsive to cells. Effects prevailed or increased in the high cell count group from 6 to 12 months but plateaued or diminished in the low cell count and control groups. Most physiological parameters remained unaltered. No unexpected adverse events were observed. CONCLUSIONS: Injection of a high dose of autologous skeletal muscle-derived cells followed by electrical stimulation significantly improved FI, particularly in patients with limited FI duration and high IEF at baseline, and could become a valuable tool for treatment of FI, subject to confirmatory phase 3 trial(s). (ClinicalTrialRegister.eu; EudraCT Number: 2010-021463-32).

Potency Assay Development: A Keystone for Clinical Use.

Potency can be described as the quantitative measure of biological activity, that is, the ability of an Advanced Therapy Medicinal Product (ATMP) to elicit the intended effect necessary for clinical efficacy. Potency testing is part of the quality control strategy necessary for batch release and is required for market approval application of an ATMP. Thus, it is crucial to develop a reliable and accurate potency assay. As a prerequisite for potency assay development, it is essential to define the mode of action of the product and thereby also the relevant biological activity that should be measured. The establishment of a potency assay should be initiated already during early product development followed by its progressive implementation into an ATMP's manufacturing, quality control and release process. Potency testing is indispensable for clinical use with a wide range of applications. A potency assay is a valuable tool to determine the product's stability, detect the impact of changes in the manufacturing process on the product, demonstrate quality and manufacturing consistency from batch to batch, estimate clinical efficacy and define the effective dose. This chapter describes the requirements and challenges to be considered for potency assay development and the importance of a well-established potency assay for clinical use.

Autologous fibroblast transplantation at the vesico-ureteral junction as potential reconstructive cell replacement in an animal model.

PURPOSE: To evaluate the cellular survival of donor fibroblasts after transplantation at the vesico-ureteral junction (VUJ) and to analyse their potential for reconstructive cell replacement in an animal model as autologous fibroblasts have been used as soft tissue augmentation material for scared and damaged tissue. METHODS: Muscles biopsies were procured from the lower limb muscles of 4 pigs; cytoplasm of fibroblasts was labelled with nano-sized iron oxide particles. Six weeks after taking of the muscle biopsies, fibroblast transplantation was performed, 3 × 10(6) cells suspended in transplantation medium (in 1-ml syringes) were injected at the VUJ using the modified STING technique. Animals were killed 8 weeks later; seeded fibroblasts were identified using prussian blue staining protocol; histological evaluation and morphological analysis were performed by light microscopy (Mayer's haematoxylin-eosin staining); and bladders were scanned by MRI for visualization and localization of the iron-labelled donor cells. RESULTS: Donor fibroblast cell colonization and cellular viability at the VUJ was demonstrated by MRI and histochemically indicating cellular uptake of iron particles at the VUJ. It was also evident that transplanted fibroblasts integrate into the extracellular matrix of the distal ureter augmenting ureteral host tissue. CONCLUSIONS: Labelled implanted autologous fibroblasts were visualized by staining procedure as well as MRI scan demonstrating persistence at the VUJ, suggesting that in vitro expanded fibroblasts survived in vivo after transplantation.

Myogenic progenitor cell transplantation for muscle regeneration following hindlimb ischemia and reperfusion.

BACKGROUND: Muscle is severely affected by ischemia/reperfusion injury (IRI). Quiescent satellite cells differentiating into myogenic progenitor cells (MPC) possess a remarkable regenerative potential. We herein established a model of local application of MPC in murine hindlimb ischemia/reperfusion to study cell engraftment and differentiation required for muscle regeneration. METHODS: A clamping model of murine (C57b/6 J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 h (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1 × 106 cells) obtained from isolated satellite cells were injected intramuscularly. Surface marker expression and differentiation potential of MPC were analyzed in vitro by flow cytometry and differentiation assay. In vivo bioluminescence imaging and histopathologic evaluation of biopsies were performed to quantify cell fate, engraftment and regeneration. RESULTS: 2h WIT induced severe IRI on muscle, and muscle fiber regeneration as per histopathology within 14 days after injury. Bioluminescence in vivo imaging demonstrated reporter protein signals of MPC in 2h WIT animals and controls over the study period (75 days). Bioluminescence signals were detected at the injection site and increased over time. TdTomato expressing MPC and myofibers were visible in host tissue on postoperative days 2 and 14, respectively, suggesting that injected MPC differentiated into muscle fibers. Higher reporter protein signals were found after 2h WIT compared to controls without ischemia, indicative for enhanced growth and/or engraftment of MPC injected into IRI-affected muscle antagonizing muscle damage caused by IRI. CONCLUSION: WIT-induced IRI in muscle requests increased numbers of injected MPC to engraft and persist, suggesting a possible rational for cell therapy to antagonize IRI. Further investigations are needed to evaluate the regenerative capacity and therapeutic advantage of MPC in the setting of ischemic limb injury.

Generation of myogenic progenitor cell-derived smooth muscle cells for sphincter regeneration.

BACKGROUND: Degeneration of smooth muscles in sphincters can cause debilitating diseases such as fecal incontinence. Skeletal muscle-derived cells have been effectively used in clinics for the regeneration of the skeletal muscle sphincters, such as the external anal or urinary sphincter. However, little is known about the in vitro smooth muscle differentiation potential and in vivo regenerative potential of skeletal muscle-derived cells. METHODS: Myogenic progenitor cells (MPC) were isolated from the skeletal muscle and analyzed by flow cytometry and in vitro differentiation assays. The differentiation of MPC to smooth muscle cells (MPC-SMC) was evaluated by immunofluorescence, flow cytometry, patch-clamp, collagen contraction, and microarray gene expression analysis. In vivo engraftment of MPC-SMC was monitored by transplanting reporter protein-expressing cells into the pyloric sphincter of immunodeficient mice. RESULTS: MPC derived from human skeletal muscle expressed mesenchymal surface markers and exhibit skeletal myogenic differentiation potential in vitro. In contrast, they lack hematopoietic surface marker, as well as adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Cultivation of MPC in smooth muscle differentiation medium significantly increases the fraction of alpha smooth muscle actin (aSMA) and smoothelin-positive cells, while leaving the number of desmin-positive cells unchanged. Smooth muscle-differentiated MPC (MPC-SMC) exhibit increased expression of smooth muscle-related genes, significantly enhanced numbers of CD146- and CD49a-positive cells, and in vitro contractility and express functional Cav and Kv channels. MPC to MPC-SMC differentiation was also accompanied by a reduction in their skeletal muscle differentiation potential. Upon removal of the smooth muscle differentiation medium, a major fraction of MPC-SMC remained positive for aSMA, suggesting the definitive acquisition of their phenotype. Transplantation of murine MPC-SMC into the mouse pyloric sphincter revealed engraftment of MPC-SMC based on aSMA protein expression within the host smooth muscle tissue. CONCLUSIONS: Our work confirms the ability of MPC to give rise to smooth muscle cells (MPC-SMC) with a well-defined and stable phenotype. Moreover, the engraftment of in vitro-differentiated murine MPC-SMC into the pyloric sphincter in vivo underscores the potential of this cell population as a novel cell therapeutic treatment for smooth muscle regeneration of sphincters.

Autologous myoblasts and fibroblasts versus collagen for treatment of stress urinary incontinence in women: a randomised controlled trial.

BACKGROUND: Preclinical studies have suggested that transurethral injections of autologous myoblasts can aid in regeneration of the rhabdosphincter, and fibroblasts in reconstruction of the urethral submucosa. We aimed to compare the effectiveness and tolerability of ultrasonography-guided injections of autologous cells with those of endoscopic injections of collagen for stress incontinence. METHODS: Between 2002 and 2004, we recruited 63 eligible women with urinary stress incontinence. 42 of these women were randomly assigned to receive transurethral ultrasonography-guided injections of autologous myoblasts and fibroblasts, and 21 to receive conventional endoscopic injections of collagen. The first primary outcome measure was an incontinence score (range 0-6) based on a 24-hour voiding diary, a 24-hour pad test, and a patient questionnaire. The other primary outcome measures were contractility of the rhabdosphincter and thickness of both the urethra and rhabdosphincter. Analysis was by intention to treat. This trial is registered with Controlled-Trials.com, number CCT-NAPN-16630. FINDINGS: At 12-months' follow-up, 38 of the 42 women injected with autologous cells were completely continent, compared with two of the 21 patients given conventional treatment with collagen. The median incontinence score decreased from a baseline of 6.0 (IQR 6.0-6.0; where 6 represents complete incontinence), to 0 (0-0) for patients treated with autologous cells, and 6.0 (3.5-6.0) for patients treated with collagen (p<0.0001). Ultrasonographic measurements showed that the mean thickness of the rhabdosphincter increased from a baseline of 2.13 mm (SD 0.39) for all patients to 3.38 mm (0.26) for patients treated with autologous cells and 2.32 mm (0.44) for patients treated with collagen (p<0.0001). Contractility of the rhabdosphincter increased from a baseline of 0.58 mm (SD 0.32) to 1.56 mm (0.28) for patients treated with autologous cells and 0.67 mm (0.51) for controls (p<0.0001). The change in the thickness of the urethra after treatment was not significantly different between treatment groups. No adverse effects were recorded in any of the 63 patients. INTERPRETATION: Long-term postoperative results and data from multicentre trials with larger numbers of patients are needed to assess whether injection of autologous cells into the rhabdosphincter and the urethra could become a standard treatment for urinary incontinence.

Myoblast and fibroblast therapy for post-prostatectomy urinary incontinence: 1-year followup of 63 patients.

PURPOSE: We assessed the efficacy and safety of the application of autologous fibroblasts and myoblasts for treatment in post-prostatectomy urinary incontinence after a minimal followup of 1 year. MATERIALS AND METHODS: Sixty-three patients with stress urinary incontinence after radical prostatectomy were treated with transurethral ultrasound guided injections of autologous fibroblasts and myoblasts obtained from skeletal muscle biopsies. All subjects were evaluated preoperatively and 12 months postoperatively in terms of incontinence and Quality of Life Instrument scores, urodynamic parameters, and morphology and function of the urethra and rhabdosphincter. RESULTS: Of the 63 patients 41 were continent 12 months after implantation of cells, 17 showed improvement and 5 did not show any improvement. Incontinence and Quality of Life Instrument scores as well as thickness and contractility of the rhabdosphincter were significantly improved postoperatively. CONCLUSIONS: The use of myoblast and fibroblast therapy represents a minimally invasive, safe and effective treatment for post-prostatectomy incontinence after a followup of 1 year.

Can autologous myoblasts be used as a potential bulking agent?

OBJECTIVE: To investigate the behaviour of donor myoblasts at the vesico-ureteric junction (VUJ) and to evaluate their potential as an autologous bulking agent, as myoblast transplantation has been shown to regenerate damaged or degenerated tissue, and it was postulated that they could be used to treat vesico-ureteric reflux. MATERIALS AND METHODS: Muscle biopsies were obtained from the lower limb muscles of 10 pigs. The quality of the cells was evaluated by electrophysiological and immunohistochemical tests. The cell membranes of myoblasts were labelled with PKH26, a fluorescent dye. Six weeks after taking of the muscle biopsies all pigs underwent cell transplantation; 30 x 10(6) cells suspended in transplantation medium (in 1-mL syringes) were injected at the VUJ, into the proximal urethra and the rhabdosphincter. At the VUJ volumes of 1 mL were injected, whereas in the urethra and rhabdosphincter small cell depots (0.1 mL) were injected. All the pigs were killed 8 weeks later, and the myoblasts and newly formed myofibres were identified using fluorescence microscopy, with a histological evaluation and investigation of potential local inflammatory reaction. RESULTS: Two to three intact layers of autologous myoblasts were found in the outer aspects of the large cell depots in the VUJ. Immunohistochemistry further showed that the myoblasts were only viable at these outermost borders of the large bulking areas, whereas necrosis with red fluorescent cell detritus was visible in the remaining central aspects of the large bulk of cells. By contrast, cells survived and formed myotubes in the wall of the proximal urethra and the rhabdosphincter where the small cell depots had been injected. CONCLUSIONS: In small depots, transplanted autologous myoblasts can survive and differentiate into myofibres, while in a large bulk of cells the vast majority of cells become necrotic. The present results show that myoblasts cannot be used for augmentation of large volumes of tissue or as a bulking agent.

Skeletal muscle-derived cell implantation for the treatment of sphincter-related faecal incontinence.

BACKGROUND: In an earlier pilot study with 10 women, we investigated a new approach for therapy of faecal incontinence (FI) due to obstetric trauma, involving ultrasound-guided injection of autologous skeletal muscle-derived cells (SMDC) into the external anal sphincter (EAS), and observed significant improvement. In the current study, we tested this therapeutic approach in an extended patient group: male and female patients suffering from FI due to EAS damage and/or atrophy. Furthermore, feasibility of lower cell counts and cryo-preserved SMDC was assessed. METHODS: In this single-centre, explorative, baseline-controlled clinical trial, each patient (n = 39; mean age 60.6 ± 13.81 years) received 79.4 ± 22.5 × 106 cryo-preserved autologous SMDC. Changes in FI parameters, Fecal Incontinence Quality of Life (FIQL), anorectal manometry and safety from baseline to 1, 6 and 12 months post implantation were evaluated. RESULTS: SMDC used in this trial contained a high percentage of myogenic-expressing (CD56+) and muscle stem cell marker-expressing (Pax7+, Myf5+) cells. Intervention was well tolerated without any serious adverse events. After 12 months, the number of weekly incontinence episodes (WIE, primary variable), FIQL and patient condition had improved significantly. In 80.6% of males and 78.4% of females, the WIE frequency decreased by at least 50%; Wexner scores and severity of FI complaints decreased significantly, independent of gender and cause of FI. CONCLUSIONS: Injection of SMDCs into the EAS effectively improved sphincter-related FI due to EAS damage and/or atrophy in males and females. When confirmed in a larger, placebo-controlled trial, this minimal invasive procedure has the potential to become first-line therapy for FI. TRIAL REGISTRATION: EU Clinical Trials Register, EudraCT 2010-023826-19 (Date of registration: 08.11.2010).

Development of an in vitro potency assay for human skeletal muscle derived cells.

BACKGROUND: Potency is a quantitative measure of the desired biological function of an advanced therapy medicinal product (ATMP) and is a prerequisite for market approval application (MAA). To assess the potency of human skeletal muscle-derived cells (SMDCs), which are currently investigated in clinical trials for the regeneration of skeletal muscle defects, we evaluated acetylcholinesterase (AChE), which is expressed in skeletal muscle and nervous tissue of all mammals. METHODS: CD56+ SMDCs were separated from CD56- SMDCs by magnetic activated cell sorting (MACS) and both differentiated in skeletal muscle differentiation medium. AChE activity of in vitro differentiated SMDCs was correlated with CD56 expression, fusion index, cell number, cell doubling numbers, differentiation markers and compared to the clinical efficacy in patients treated with SMDCs against fecal incontinence. RESULTS: CD56- SMDCs did not form multinucleated myotubes and remained low in AChE activity during differentiation. CD56+ SMDCs generated myotubes and increased in AChE activity during differentiation. AChE activity was found to accurately reflect the number of CD56+ SMDCs in culture, their fusion competence, and cell doubling number. In patients with fecal incontinence responding to SMDCs treatment, the improvement of clinical symptoms was positively linked with the AChE activity of the SMDCs injected. DISCUSSION: AChE activity was found to truly reflect the in vitro differentiation status of SMDCs and to be superior to the mere use of surface markers as it reflects not only the number of myogenic SMDCs in culture but also their fusion competence and population doubling number, thus combining cell quality and quantification of the expected mode of action (MoA) of SMDCs. Moreover, the successful in vitro validation of the assay proves its suitability for routine use. Most convincingly, our results demonstrate a link between clinical efficacy and the AChE activity of the SMDCs preparations used for the treatment of fecal incontinence. Thus, we recommend using AChE activity of in vitro differentiated SMDCs as a potency measure in end stage (phase III) clinical trials using SMDCs for skeletal muscle regeneration and subsequent market approval application (MAA).

Intramyocardial microdepot injection increases the efficacy of skeletal myoblast transplantation.

OBJECTIVE: Recent progress in the field of cellular cardiomyoplasty has opened new prospects for the treatment of ischemic heart disease and currently moves from bench to bedside. The aim of the present study was to develop a novel cell delivery technique, reducing target tissue damage and improving cell dispersion and engraftment. METHODS: In 30 male Fischer F344 rats an infarction of the left ventricle was generated by ligation of the left anterior descendent artery. Seven days after infarction, either 15 microdepots of 10 microl myoblast cell suspension (microdepot group) or culture medium (control group) were injected into the infarcted region using an automatic pressure injection device, or three depots of 50 microl myoblast cell suspension (macrodepot group) were injected using the standard surgical technique. Echocardiography was performed in all rats before and 6 weeks after cell injection. In all groups the perioperative mortality was below 20%. Six weeks after cell transplantation, a significant improvement of ejection fraction was seen in both myoblast treated groups compared to controls (macrodepot, microdepot, control; 53.7+/-11.9, 70.7+/-2.0, 39.1+/-6.4; P=0.026, P<0.001). The microdepot group showed a more decent improvement than the macrodepot group (70.7+/-2.0 vs. 53.7+/-11.9, P=0.013). In both treated groups, grafted myoblasts differentiated into multinucleated myotubes within host myocardium, however, the engraftment pattern was different and angiogenesis was enhanced in the microdepot group. CONCLUSIONS: Intramyocardial multisite pressure injection allows the safe and reliable transplantation of several myoblast microdepots into an infarcted myocardium and improves the efficacy of myoblast transplantation compared to the standard technique.

Adult stem cell therapy of female stress urinary incontinence.

OBJECTIVES: To investigate the efficacy of transurethral ultrasound (TUUS)-guided injections of autologous myoblasts and fibroblasts in women with incontinence. METHODS: Between January and June 2005, 20 female patients suffering from stress urinary incontinence (SUI) were included. Skeletal muscle biopsies were taken from the left arm to obtain cultures from autologous fibroblasts and myoblasts. By TUUS guidance the fibroblasts were injected into the urethral submucosa and the myoblasts were injected into the rhabdosphincter. A defined incontinence score, quality-of-life score and urodynamic, electromyographic, and laboratory parameters, as well as morphology and function of urethra and rhabdosphincter were evaluated before and up to 2 yr after therapy. RESULTS: Eighteen of 20 patients were cured 1 yr after injection of autologous stem cells and in 2 patients SUI was improved. Two years after therapy 16 of the 18 patients presented as cured, 2 others were improved, and 2 were lost to follow-up. Incontinence and quality-of-life scores were significantly improved postoperatively. The thickness of urethra and rhabdosphincter as well as activity and contractility of the rhabdosphincter were also statistically significantly increased after therapy. CONCLUSIONS: Clinical results demonstrate that SUI can be treated effectively with autologous stem cells. The present data support the conclusion that this therapeutic concept represents an elegant and minimally invasive treatment modality to treat SUI.

Functional and histological changes after myoblast injections in the porcine rhabdosphincter.

OBJECTIVE: Transurethral ultrasound-guided injection of autologous myoblasts has recently been shown to cure urinary stress incontinence. In the present study, the dose-dependent changes in maximal urethral closure pressures after application of myoblasts were investigated in a porcine animal model. METHODS: Myoblast cultures were grown from a porcine muscle biopsy. The biopsy was enzymatically dissociated by using a modified cell dispersion technique. Single myoblasts in suspension were manually collected with a micropipette under microscopic control. Next a clonal myoblast culture was prepared. Before the cells were applied, fluorescence labelling (PKH) was used to assess integration of the injected myoblasts into the rhabdosphincter. With the help of a transurethral ultrasound probe (23 F, 11 MHz) and a special injection system, the myoblasts were injected into the rhabdosphincter of five pigs under direct sonographic control. Into two different areas of the rhabdosphincter, increasing different cell counts were injected (total volume 1.5 ml). At each area, 10 depots of 150 microl volume were injected all along the rhabdosphincter. The following cell counts were used: 1.5 x 10(6), 2.1 x 10(6), 4.2 x 10(6) (low range) 5.69 x 10(6), 8.1 x 10(6), 1.13 x 10(7), 1.6 x 10(7) (mid range) 2.26 x 10(7), 4.4 x 10(7), and 7.8 x 10(7) (high range). To avoid possible cell rejection, we immunosuppressed the pigs with daily cortisone (1g Solu Dacortin) because allogenic myoblasts were used. Urethral pressure profiles (UPPs) were measured before and 3 wk postoperatively before the pigs were put to sleep. The lower urinary tract was removed in all pigs for histological analysis. RESULTS: Histological examination of the specimens revealed that the injected cells had survived at the injection site and had formed new myofibres. Overall the UPP curves revealed dose-dependent changes. Statistically significant increased pressure values of up to more than 300% could be observed in all cases in which higher concentrations of cells had been applied. Increases were also noted in mid range concentrations although not to such a high extent (approximately 150%). Pressure values had even diminished (approximately 50%) after injecting the three lowest concentrations (1.5 x 10(6), 2.1 x 10(6), 4.2 x 10(6)). CONCLUSIONS: The present results show that the effects after application of myoblasts into the rhabdosphincter are dose-dependent.

Autologous myoblasts and fibroblasts for female stress incontinence: a 1-year follow-up in 123 patients.

OBJECTIVE: To assess the efficacy and safety of the application of autologous myoblasts and fibroblasts for treating female stress urinary incontinence (SUI) after a follow-up of >/=1 year. PATIENTS AND METHODS: In all, 123 women with SUI (aged 36-84 years) were treated with transurethral ultrasonography-guided injections with autologous myoblasts and fibroblasts obtained from skeletal muscle biopsies. The fibroblasts were suspended in a small amount of collagen as carrier material and injected into the urethral submucosa, while the myoblasts were implanted into the rhabdosphincter. All patients were evaluated before and 12 months after the injection using the Incontinence and Quality of Life Instrument (I-QOL) scores, urodynamic variables, and morphology and function of the urethra and rhabdosphincter. RESULTS: At 1 year after implanting the cells, 94 of the 119 women (79%) were completely continent, 16 (13%) had a substantial improvement and nine (8%) a slight improvement. Four patients were lost to follow-up. The incontinence and I-QOL scores, and the thickness, contractility and electromyographic activity of the rhabdosphincter were significantly improved after treatment. CONCLUSIONS: These results show the efficacy and safety of transferring autologous myoblasts and fibroblasts in the treatment of female SUI, after a follow-up of 1 year.

Structural determinants of L-type channel activation in segment IIS6 revealed by a retinal disorder.

The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the Ca(V)1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553-7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the Ca(V)1.2 channel. Substitution of Ile-781, the Ca(V)1.2 residue corresponding to Ile-745 in Ca(V)1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779-782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of Ca(V)1.2. Moreover there may be a flexible center of helix bending at positions 779-782 of Ca(V)1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.

A CACNA1F mutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation.

Light stimuli produce graded hyperpolarizations of the photoreceptor plasma membrane and an associated decrease in a voltagegated calcium channel conductance that mediates release of glutamate neurotransmitter. The Ca(v)1.4 channel is thought to be involved in this process. The CACNA1F gene encodes the poreforming subunit of the Ca(v)1.4 channel and various mutations in CACNA1F cause X-linked incomplete congenital stationary night blindness (CSNB2). The molecular mechanism of the pathology underlying the CSNB2 phenotype remains to be established. Recent clinical investigations of a New Zealand family found a severe visual disorder that has some clinical similarities to, but is clearly distinct from, CSNB2. Here, we report investigations into the molecular mechanism of the pathology of this condition. Molecular genetic analyses identified a previously undescribed nucleotide substitution in CACNA1F that is predicted to encode an isoleucine to threonine substitution at CACNA1F residue 745. The I745T CACNA1F allele produced a remarkable approximately -30-mV shift in the voltage dependence of Ca(v)1.4 channel activation and significantly slower inactivation kinetics in an expression system. These findings imply that substitution of this wild-type residue in transmembrane segment IIS6 may have decreased the energy required to open the channel. Collectively, these findings suggest that a gain-of-function mechanism involving increased Ca(v)1.4 channel activity is likely to cause the unusual phenotype.

Membrane properties of single muscle cells of the rhabdosphincter of the male urethra.

BACKGROUND: The electrophysiological properties of myoblast cultures established from the human and porcine rhabdosphincter (RS) and porcine lower limb muscle (LLSKM) were studied to elucidate their potential for tissue engineering applications in the lower urinary tract. METHODS: Muscle biopsies were collected from the prostatic part of the RS, the RS of male pigs, and the porcine LLSKM. Ion channels were studied by means of the patch-clamp technique. RESULTS: Only one subtype each of voltage gated Na+ and Ca2+ channels was observed in porcine RS and LLSKM. Two types of voltage gated Ca2+ channels were identified in human RS cells. The porcine RS and LLSKM myoblasts displayed similar fusion competence. CONCLUSIONS: Porcine RS and LLSKM myoblasts and human RS and human skeletal muscle cells show a high degree of similarity. Injection of autologous skeletal muscle myoboblasts in the lower urinary tract might, therefore, represent a promising approach to treat stress incontinence after radical prostatectomy.