Identifying dynamic membrane structures with atomic-force microscopy and confocal imaging

Combining the biological specificity of fluorescence microscopy with topographical features revealed by atomic force microscopy (AFM) provides new insights into cell biology. However, the lack of systematic alignment capabilities especially in scanning-tip AFM has limited the combined application approach as AFM drift leads to increasing image mismatch over time. We present an alignment correction method using the cantilever tip as a reference landmark. Since the precise tip position is known in both the fluorescence and AFM images, exact re-alignment becomes possible. We used beads to demonstrate the validity of the method in a complex artificial sample. We then extended this method to biological samples to depict membrane structures in fixed and living human fibroblasts. We were able to map nanoscale membrane structures, such as clathrin-coated pits, to their respective fluorescent spots. Reliable alignment between fluorescence signals and topographic structures opens possibilities to assess key biological processes at the cell surface such as endocytosis and exocytosis

A molecular signature of myalgia in myotonic dystrophy 2

Background: Chronic muscle pain affects close to 20% of the population and is a major health burden. The underlying mechanisms of muscle pain are difficult to investigate as pain presents in patients with very diverse histories. Treatment options are therefore limited and not tailored to underlying mechanisms. To gain insight into the pathophysiology of myalgia we investigated a homogeneous group of patients suffering from myotonic dystrophy type 2 (DM2), a monogenic disorder presenting with myalgia in at least 50% of affected patients. Methods: After IRB approval we performed an observational cross-sectional cohort study and recruited 42 patients with genetically confirmed DM2 plus 20 healthy age and gender matched control subjects. All participants were subjected to an extensive sensory-testing protocol. In addition, RNA sequencing was performed from 12 muscle biopsy specimens obtained from DM2 patients. Findings: Clinical sensory testing as well as RNA sequencing clearly separated DM2 myalgic from non-myalgia patients and also from healthy controls. In particular pressure pain thresholds were significantly lowered for all muscles tested in myalgic DM2 patients but were not significantly different between non-myalgic patients and healthy controls. The expression of fourteen muscle expressed genes in myalgic patients was significantly up or down-regulated in myalgic compared to non-myalgic DM2 patients. Interpretation: Our data support the idea that molecular changes in the muscles of DM2 patients are associated with muscle pain. Further studies should address whether muscle-specific molecular pathways play a significant role in myalgia in order to facilitate the development of mechanism-based therapeutic strategies to treat musculoskeletal pain

Full-length dysferlin transfer by the hyperactive Sleeping Beauty transposase restores dysferlin-deficient muscle

Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysf(prmd) Prkdc(scid)/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy

Altered expression of cyclin A 1 in muscle of patients with facioscapulohumeral muscle dystrophy (FSHD-1)

Objectives Cyclin A1 regulates cell cycle activity and proliferation in somatic and germ-line cells. Its expression increases in G1/S phase and reaches a maximum in G2 and M phases. Altered cyclin A1 expression might contribute to clinical symptoms in facioscapulohumeral muscular dystrophy (FSHD). Methods Muscle biopsies were taken from the Vastus lateralis muscle for cDNA microarray, RT-PCR, immunohistochemistry and Western blot analyses to assess RNA and protein expression of cyclin A1 in human muscle cell lines and muscle tissue. Muscle fibers diameter was calculated on cryosections to test for hypertrophy. Results cDNA microarray data showed specifically elevated cyclin A1 levels in FSHD vs. other muscular disorders such as caveolinopathy, dysferlinopathy, four and a half LIM domains protein 1 deficiency and healthy controls. Data could be confirmed with RT-PCR and Western blot analysis showing up-regulated cyclin A1 levels also at protein level. We found also clear signs of hypertrophy within the Vastus lateralis muscle in FSHD-1 patients. Conclusions In most somatic human cell lines, cyclin A1 levels are low. Overexpression of cyclin A1 in FSHD indicates cell cycle dysregulation in FSHD and might contribute to clinical symptoms of this disease

Human satellite cells have regenerative capacity and are genetically manipulable

Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon-mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies

Linking cytoarchitecture to metabolism: sarcolemma-associated plectin affects glucose uptake by destabilizing microtubule networks in mdx myofibers

BACKGROUND: Duchenne muscular dystrophy (DMD) is one of the most frequent forms of muscular disorders. It is caused by the absence of dystrophin, a core component of the sarcolemma-associated junctional complex that links the cytoskeleton to the extracellular matrix. We showed previously that plectin 1f (P1f), one of the major muscle-expressed isoforms of the cytoskeletal linker protein plectin, accumulates at the sarcolemma of DMD patients as well as of mdx mice, a widely studied animal model for DMD.Based on plectin's dual role as structural protein and scaffolding platform for signaling molecules, we speculated that the dystrophic phenotype observed after loss of dystrophin was caused, at least to some extent, by excess plectin. Thus, we hypothesized that elimination of plectin expression in mdx skeletal muscle, while probably resulting in an overall more severe phenotype, may lead to a partial phenotype rescue. In particular, we wanted to assess whether excess sarcolemmal plectin contributes to the dysregulation of sugar metabolism in mdx myofibers. METHODS: We generated plectin/dystrophin double deficient (dKO) mice by breeding mdx with conditional striated muscle-restricted plectin knockout (cKO) mice. The phenotype of these mice was comparatively analyzed with that of mdx, cKO, and wild-type mice, focusing on structural integrity and dysregulation of glucose metabolism. RESULTS: We show that the accumulation of plectin at the sarcolemma of mdx muscle fibers hardly compensated for their loss of structural integrity. Instead, it led to an additional metabolic deficit by impairing glucose uptake. While dKO mice suffered from an overall more severe form of muscular dystrophy compared to mdx or plectin-deficient mice, sarcolemmal integrity as well as glucose uptake of their myofibers were restored to normal levels upon ablation of plectin. Furthermore, microtubule (MT) networks in intact dKO myofibers, including subsarcolemmal areas, were found to be more robust than those in mdx mice. Finally, myotubes differentiated from P1f-overexpressing myoblasts showed an impairment of glucose transporter 4 translocation and a destabilization of MT networks. CONCLUSIONS: Based on these results we propose that sarcolemma-associated plectin acts as an antagonist of MT network formation in myofibers, thereby hindering vesicle-mediated (MT-dependent) transport of glucose transporter 4. This novel role of plectin throws a bridge between extra-sarcomeric cytoarchitecture and metabolism of muscle fibers. Our study thus provides new insights into pathomechanisms of plectinopathies and muscular dystrophies in general

Generation of three age and gender matched pairs of human induced pluripotent stem cells derived from myoblasts (MDCi011-A, MDCi012-A, MDCi013-A) and from peripheral blood mononuclear cells (MDCi011-B, MDCi012-B, MDCi013-B) from the same donor

We describe the generation and characterization of three pairs of human induced pluripotent stem cell (hiPSC) lines reprogrammed from myoblasts and from peripheral blood mononuclear cells (PBMCs) of the same donor. All donors were free of neuromuscular disorders, female and between 47 and 50 years of age. For reprogramming we used Sendai-virus delivery of the four Yamanaka factors. The pluripotent identity of the hiPSC lines was confirmed by the expression of pluripotency markers and their capacity to differentiate into all three germ layers. These hiPSCs constitute a tool to study tissue of origin specific differences in the identity of hiPSCs

Generation of two human induced Pluripotent Stem Cell lines derived from myoblasts (MDCi014-A) and from peripheral blood mononuclear cells (MDCi014-B) from the same donor

We describe the generation and characterization of two human induced pluripotent stem cell (hiPSCs) lines reprogrammed from myoblasts and from peripheral blood mononuclear cells (PBMCs) from the same donor. The donor was free of neuromuscular disorders, male and 18 years of age. For reprogramming we used Sendai-virus delivery of the four Yamanaka factors. The pluripotent identity of the hiPSC lines was confirmed by the expression of pluripotency markers and their capacity to differentiate into all three germ layers. These hiPSCs constitute a tool to study tissue of origin specific differences in the identity of hiPSCs

Subclinical myocardial injury in patients with Facioscapulohumeral muscular dystrophy 1 and preserved ejection fraction - assessment by cardiovascular magnetic resonance

Background: Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is an autosomal dominant and the third most common inherited muscle disease. Cardiac involvement is currently described in several muscular dystrophies (MD), but there are conflicting reports in FSHD1. Mostly, FSHD1 is recognized as MD with infrequent cardiac involvement, but sudden cardiac deaths are reported in single cases. The aim of this study is to investigate whether subclinical cardiac involvement in FSHD1 patients is detectable in preserved left ventricular systolic function applying cardiovascular magnetic resonance (CMR). Methods: We prospectively included patients with genetically confirmed FSHD1 (n = 52, 48 ± 15 years) and compared them with 29 healthy age-matched controls using a 1.5 T CMR scanner. Myocardial tissue differentiation was performed qualitatively using focal fibrosis imaging (late gadolinium enhancement (LGE)), fat imaging (multi-echo sequence for fat/water-separation) and parametric T2- and T1-mapping for quantifying inflammation and diffuse fibrosis. Extracellular volume fraction was calculated. A 12-lead electrocardiogram and 24-h Holter were performed for the assessment of MD-specific Groh-criteria and arrhythmia. Results: Focal fibrosis by LGE was present in 13 patients (25%,10 men), fat infiltration in 7 patients (13%,5 men). T2 values did not differ between FSHD1 and healthy controls. Native T1 mapping revealed significantly higher values in patients (global native myocardial T1 values basal: FSHD1: 1012 ± 26 ms vs. controls: 985 ± 28 ms, p < 0.01, medial FSHD1: 994 ± 37 ms vs. controls: 982 ± 28 ms, p = 0.028). This was also evident in regions adjacent to focal fibrosis, indicating diffuse fibrosis. Groh-criteria were positive in 1 patient. In Holter, arrhythmic events were recorded in 10/43 subjects (23%). Conclusions: Patients with FSHD1 and preserved left ventricular ejection fraction present focal and diffuse myocardial injury. Longitudinal multi-center trials are needed to define the impact of myocardial changes as well as a relation between myocardial injury and arrhythmias on long-term prognosis and therapeutic decision-making. Trial Registration: ISRCTN registry with study ID ISRCTN13744381