Caveolin-3 and Caveolin-1 Interaction Decreases Channel Dysfunction Due to Caveolin-3 Mutations.

Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in CAV3 lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.

Aberrant Adenosine Triphosphate Release and Impairment of P2Y2-Mediated Signaling in Sarcoglycanopathies.

Sarcoglycanopathies, limb-girdle muscular dystrophies (LGMD) caused by genetic loss-of-function of the membrane proteins sarcoglycans (SGs), are characterized by progressive degeneration of skeletal muscle. In these disorders, muscle necrosis is associated with immune-mediated damage, whose triggering and perpetuating molecular mechanisms are not fully elucidated yet. Extracellular adenosine triphosphate (eATP) seems to represent a crucial factor, with eATP activating purinergic receptors. Indeed, in vivo blockade of the eATP/P2X7 purinergic pathway ameliorated muscle disease progression. P2X7 inhibition improved the dystrophic process by restraining the activity of P2X7 receptors on immune cells. Whether P2X7 blockade can display a direct action on muscle cells is not known yet. In this study, we investigated eATP effects in primary cultures of myoblasts isolated from patients with LGMDR3 (α-sarcoglycanopathy) and in immortalized cells isolated from a patient with LGMDR5 (γ-sarcoglycanopathy). Our results demonstrated that, owing to a reduced ecto-ATPase activity and/or an enhanced release of ATP, patient cells are exposed to increased juxtamembrane concentrations of eATP and display a higher susceptivity to eATP signals. The purinoceptor P2Y2, which proved to be overexpressed in patient cells, was identified as a pivotal receptor responsible for the enhanced ATP-induced or UTP-induced Ca2+ increase in affected myoblasts. Moreover, P2Y2 stimulation in LDMDR3 muscle cells induced chemotaxis of immune cells and release of interleukin-8. In conclusion, a higher eATP concentration and sensitivity in primary human muscle cells carrying different α-SG or γ-SG loss-of-function mutations indicate that eATP/P2Y2 is an enhanced signaling axis in cells from patients with α-/γ-sarcoglycanopathy. Understanding the basis of the innate immune-mediated damage associated with the dystrophic process may be critical in overcoming the immunologic hurdles associated with emerging gene therapies for these disorders.

[Diagnostics and treatment of statin-associated muscle symptoms]. / Diagnostik und Therapie von statinassoziierten Muskelsymptomen.

Statins are among the most frequently prescribed drugs in Germany. Their benefits in lowering cardiovascular risk are beyond dispute. Nevertheless, many patients complain of side effects from statin therapy, including statin-associated muscle symptoms (SAMS) in particular. Despite their relative frequency, it is difficult to objectively diagnose them, as the time until appearance of first symptoms, the nature of the complaints and the severity of muscle problems vary widely. This narrative review summarizes the causes of SAMS as well as new possibilities regarding their diagnosis and therapy.

The Danger Signal Extracellular ATP Is Involved in the Immunomediated Damage of α-Sarcoglycan-Deficient Muscular Dystrophy.

In muscular dystrophies, muscle membrane fragility results in a tissue-specific increase of danger-associated molecular pattern molecules (DAMPs) and infiltration of inflammatory cells. The DAMP extracellular ATP (eATP) released by dying myofibers steadily activates muscle and immune purinergic receptors exerting dual negative effects: a direct damage linked to altered intracellular calcium homeostasis in muscle cells and an indirect toxicity through the triggering of the immune response and inhibition of regulatory T cells. Accordingly, pharmacologic and genetic inhibition of eATP signaling improves the phenotype in models of chronic inflammatory diseases. In α-sarcoglycanopathy, eATP effects may be further amplified because α-sarcoglycan extracellular domain binds eATP and displays an ecto-ATPase activity, thus controlling eATP concentration at the cell surface and attenuating the magnitude and/or the duration of eATP-induced signals. Herein, we show that in vivo blockade of the eATP/P2X purinergic pathway by a broad-spectrum P2X receptor-antagonist delayed the progression of the dystrophic phenotype in α-sarcoglycan-null mice. eATP blockade dampened the muscular inflammatory response and enhanced the recruitment of forkhead box protein P3-positive immunosuppressive regulatory CD4+ T cells. The improvement of the inflammatory features was associated with increased strength, reduced necrosis, and limited expression of profibrotic factors, suggesting that pharmacologic purinergic antagonism, altering the innate and adaptive immune component in muscle infiltrates, might provide a therapeutic approach to slow disease progression in α-sarcoglycanopathy.

eATP/P2X7R Axis: An Orchestrated Pathway Triggering Inflammasome Activation in Muscle Diseases.

In muscle ATP is primarily known for its function as an energy source and as a mediator of the "excitation-transcription" process, which guarantees muscle plasticity in response to environmental stimuli. When quickly released in massive concentrations in the extracellular space as in presence of muscle membrane damage, ATP acts as a damage-associated molecular pattern molecule (DAMP). In experimental murine models of muscular dystrophies characterized by membrane instability, blockade of eATP/P2X7 receptor (R) purinergic signaling delayed the progression of the dystrophic phenotype dampening the local inflammatory response and inducing Foxp3+ T Regulatory lymphocytes. These discoveries highlighted the relevance of ATP as a harbinger of immune-tissue damage in muscular genetic diseases. Given the interactions between the immune system and muscle regeneration, the comprehension of ATP/purinerigic pathway articulated organization in muscle cells has become of extreme interest. This review explores ATP release, metabolism, feedback control and cross-talk with members of muscle inflammasome in the context of muscular dystrophies.

Clinical and molecular consequences of exon 78 deletion in DMD gene.

We present a 13-year-old patient with persistent increase of serum Creatine Kinase (CK) and myalgia after exertion. Skeletal muscle biopsy showed marked reduction of dystrophin expression leading to genetic analysis of DMD gene by MLPA, which detected a single deletion of exon 78. To the best of our knowledge, DMD exon 78 deletion has never been described in literature and, according to prediction, it should lead to loss of reading frame in the dystrophin gene. To further assess the actual effect of exon 78 deletion, we analysed cDNA from muscle mRNA. This analysis confirmed the absence of 32 bp of exon 78. Exclusion of exon 78 changes the open reading frame of exon 79 and generate a downstream stop codon, producing a dystrophin protein of 3703 amino acids instead of 3685 amino acids. Albeit loss of reading frame usually leads to protein degradation and severe phenotype, in this case, we demonstrated that deletion of DMD exon 78 can be associated with a functional protein able to bind DGC complex and a very mild phenotype. This study adds a novel deletion in DMD gene in human and helps to define the compliance between maintaining/disrupting the reading frame and clinical form of the disease.

The ubiquitin ligase tripartite-motif-protein 32 is induced in Duchenne muscular dystrophy.

Activation of the proteasome pathway is one of the secondary processes of cell damage, which ultimately lead to muscle degeneration and necrosis in Duchenne muscular dystrophy (DMD). In mdx mice, the proteasome inhibitor bortezomib up-regulates the membrane expression of members of the dystrophin complex and reduces the inflammatory reaction. However, chronic inhibition of the 26S proteasome may be toxic, as indicated by the systemic side-effects caused by this drug. Therefore, we sought to determine the components of the ubiquitin-proteasome pathway that are specifically activated in human dystrophin-deficient muscles. The analysis of a cohort of patients with genetically determined DMD or Becker muscular dystrophy (BMD) unveiled a selective up-regulation of the ubiquitin ligase tripartite motif-containing protein 32 (TRIM32). The induction of TRIM32 was due to a transcriptional effect and it correlated with disease severity in BMD patients. In contrast, atrogin1 and muscle RING-finger protein-1 (MuRF-1), which are strongly increased in distinct types of muscular atrophy, were not affected by the DMD dystrophic process. Knock-out models showed that TRIM32 is involved in ubiquitination of muscle cytoskeletal proteins as well as of protein inhibitor of activated STAT protein gamma (Piasγ) and N-myc downstream-regulated gene, two inhibitors of satellite cell proliferation and differentiation. Accordingly, we showed that in DMD/BMD muscle tissue, TRIM32 induction was more pronounced in regenerating myofibers rather than in necrotic muscle cells, thus pointing out a role of this protein in the regulation of human myoblast cell fate. This finding highlights TRIM32 as a possible therapeutic target to favor skeletal muscle regeneration in DMD patients.

Enhancement of Muscle T Regulatory Cells and Improvement of Muscular Dystrophic Process in mdx Mice by Blockade of Extracellular ATP/P2X Axis.

Infiltration of immune cells and chronic inflammation substantially affect skeletal and cardiac muscle degeneration in Duchenne muscular dystrophy. In the immune system, extracellular adenosine triphosphate (ATP) released by dying cells is sensed as a danger associated molecular pattern through P2 purinergic receptors. Specifically, the P2X7 subtype has a prominent role in regulating immune system physiology and contributes to inflammasome activation also in muscle cells. Here, we show that in vivo blockade of the extracellular ATP/P2X purinergic signaling pathway by periodate-oxidized ATP delayed the progression of the dystrophic phenotype and dampened the local inflammatory response in mdx mice, a spontaneous mouse model of dystrophin deficiency. Reduced infiltration of leukocytes and macrophages and decreased expression of IL-6 were revealed in the muscles of periodate-oxidized ATP-treated mdx mice. Concomitantly, an increase in Foxp3(+) immunosuppressive regulatory T cells was observed and correlated with enhanced myofiber regeneration. Moreover, we detected reduced concentrations of profibrotic cytokines, including transforming growth factor-ß and connective tissue growth factor, in muscles of periodate-oxidized ATP-treated mdx mice. The improvement of inflammatory features was associated with increased strength and reduced necrosis, thus suggesting that pharmacologic purinergic antagonism altering the adaptive immune component in the muscle infiltrates might represent a promising therapeutic approach in Duchenne muscular dystrophy.

Impairment of ceramide synthesis causes a novel progressive myoclonus epilepsy.

OBJECTIVE: Alterations of sphingolipid metabolism are implicated in the pathogenesis of many neurodegenerative disorders. METHODS: We identified a homozygous nonsynonymous mutation in CERS1, the gene encoding ceramide synthase 1, in 4 siblings affected by a progressive disorder with myoclonic epilepsy and dementia. CerS1, a transmembrane protein of the endoplasmic reticulum (ER), catalyzes the biosynthesis of C18-ceramides. RESULTS: We demonstrated that the mutation decreases C18-ceramide levels. In addition, we showed that downregulation of CerS1 in a neuroblastoma cell line triggers ER stress response and induces proapoptotic pathways. INTERPRETATION: This study demonstrates that impairment of ceramide biosynthesis underlies neurodegeneration in humans.

Deficiency of hyccin, a newly identified membrane protein, causes hypomyelination and congenital cataract.

We describe a new autosomal recessive white matter disorder ('hypomyelination and congenital cataract') characterized by hypomyelination of the central and peripheral nervous system, progressive neurological impairment and congenital cataract. We identified mutations in five affected families, resulting in a deficiency of hyccin, a newly identified 521-amino acid membrane protein. Our study highlights the essential role of hyccin in central and peripheral myelination.

Subclinical myopathy in a child with neutral lipid storage disease and mutations in the PNPLA2 gene.

We report a 14-year-old-boy with markedly elevated serum creatine kinase (CK) levels, in whom massive triglyceride storage was found in peripheral blood leukocytes and in muscle biopsy. Sequencing PNPLA2, the gene encoding the adipose triglyceride lipase (ATGL) and responsible for the neutral lipid storage disease with myopathy (NLSDM), we identified two heterozygous mutations, including a previously reported nonsense and a novel missense mutation in the patatin domain of the gene. Lipid storage myopathy can be clinically silent in childhood and presenting only with hyperCKemia.

Neuromuscular disorders of glycogen metabolism.

Disorders of glycogen metabolism are inborn errors of energy homeostasis affecting primarily skeletal muscle, heart, liver, and, less frequently, the central nervous system. These rare diseases are quite variable in age of onset, symptoms, morbidity, and mortality. This review provides an update on disorders of glycogen metabolism affecting skeletal muscle exclusively or predominantly. From a pathogenetic perspective, we classify these diseases as primary, if the defective enzyme is directly involved in glycogen/glucose metabolism, or secondary, if the genetic mutation affects proteins which indirectly regulate glycogen or glucose processing. In addition to summarizing the most recent clinical reports in this field, we briefly describe animal models of human glycogen disorders. These experimental models are greatly improving the understanding of the pathogenetic mechanisms underlying the muscle degenerative process associated to these diseases and provide in vivo platforms to test new therapeutic strategies.

"suMus," a novel digital system for arm movement metrics and muscle energy expenditure.

Objective: In the field of non-treatable muscular dystrophies, promising new gene and cell therapies are being developed and are entering clinical trials. Objective assessment of therapeutic effects on motor function is mandatory for economical and ethical reasons. Main shortcomings of existing measurements are discontinuous data collection in artificial settings as well as a major focus on walking, neglecting the importance of hand and arm movements for patients' independence. We aimed to create a digital tool to measure muscle function with an emphasis on upper limb motility. Methods: suMus provides a custom-made App running on smartwatches. Movement data are sent to the backend of a suMus web-based platform, from which they can be extracted as CSV data. Fifty patients with neuromuscular diseases assessed the pool of suMus activities in a first orientation phase. suMus performance was hence validated in four upper extremity exercises based on the feedback of the orientation phase. We monitored the arm metrics in a cohort of healthy volunteers using the suMus application, while completing each exercise at low frequency in a metabolic chamber. Collected movement data encompassed average acceleration, rotation rate as well as activity counts. Spearman rank tests correlated movement data with energy expenditure from the metabolic chamber. Results: Our novel application "suMus," sum of muscle activity, collects muscle movement data plus Patient-Related-Outcome-Measures, sends real-time feedback to patients and caregivers and provides, while ensuring data protection, a long-term follow-up of disease course. The application was well received from the patients during the orientation phase. In our pilot study, energy expenditure did not differ between overnight fasted and non-fasted participants. Acceleration ranged from 1.7 ± 0.7 to 3.2 ± 0.5 m/sec2 with rotation rates between 0.9 ± 0.5 and 2.0 ± 3.4 rad/sec. Acceleration and rotation rate as well as derived activity counts correlated with energy expenditure values measured in the metabolic chamber for one exercise (r = 0.58, p < 0.03). Conclusion: In the analysis of slow frequency movements of upper extremities, the integration of the suMus application with smartwatch sensors characterized motion parameters, thus supporting a use in clinical trial outcome measures. Alternative methodologies need to complement indirect calorimetry in validating accelerometer-derived energy expenditure data.

Targeting gut dysbiosis against inflammation and impaired autophagy in Duchenne muscular dystrophy.

Nothing is known about the potential implication of gut microbiota in skeletal muscle disorders. Here, we provide evidence that fecal microbiota composition along with circulating levels of short-chain fatty acids (SCFAs) and related metabolites are altered in the mdx mouse model of Duchenne muscular dystrophy (DMD) compared with healthy controls. Supplementation with sodium butyrate (NaB) in mdx mice rescued muscle strength and autophagy, and prevented inflammation associated with excessive endocannabinoid signaling at CB1 receptors to the same extent as deflazacort (DFZ), the standard palliative care for DMD. In LPS-stimulated C2C12 myoblasts, NaB reduces inflammation, promotes autophagy, and prevents dysregulation of microRNAs targeting the endocannabinoid CB1 receptor gene, in a manner depending on the activation of GPR109A and PPARγ receptors. In sum, we propose a novel disease-modifying approach in DMD that may have benefits also in other muscular dystrophies.

Telemedicine in Neuromuscular Diseases During Covid-19 Pandemic: ERN-NMD European Survey.

BACKGROUND: Telemedicine (TM) contributes to bridge the gap between healthcare facilities and patients' homes with neuromuscular disease (NMD) because of mobility issues. However, its deployment is limited due to difficulties evaluating subtle neurological signs such as mild weakness or sensory deficits. The COVID-19 pandemic has disrupted healthcare delivery worldwide, necessitating rapid measures implementation by health care providers (HCPs) to protect patients from acquiring SARS-CoV-2 while maintaining the best care and treatment. OBJECTIVES: Given the challenges faced by remote healthcare assistance of NMD patients, we aim to evaluate the use of TM in NMD during the COVID-19 pandemic. METHODS: Based on the Model for Assessment-of-Telemedicine-Applications (MAST), we conducted a survey amongst clinicians of the ERN EURO NMD (European-Reference-Network-for-Rare-Neuromuscular-Diseases). RESULTS: Based on 42 responses over 76 expected ones, our results show that the COVID-19 pandemic significantly increased the number of HCPs using TM (from 60% to 100%). The TM types most used during the COVID-19 period are teleconsultation and consultation by phone, particularly in the context of symptoms worsening in NMD patients with COVID-19 infection. Most European HCPs were satisfied when using TM but as a complementary option to physical consultations. Many responses addressed the issue of technical aspects needing improvement, particularly for elderly patients who need caregivers' assistance for accessing the TM platform. CONCLUSIONS: TM has been essential during COVID-19, but its use still presents some limitations for NMD patients with cognitive deficits or for first-time diagnosis. Thus, TM should be used as complement to, rather than substitute, for face-to-face consultations.

P2X7 Receptor Antagonist Reduces Fibrosis and Inflammation in a Mouse Model of Alpha-Sarcoglycan Muscular Dystrophy.

Limb-girdle muscular dystrophy R3, a rare genetic disorder affecting the limb proximal muscles, is caused by mutations in the α-sarcoglycan gene (Sgca) and aggravated by an immune-mediated damage, finely modulated by the extracellular (e)ATP/purinoceptors axis. Currently, no specific drugs are available. The aim of this study was to evaluate the therapeutic effectiveness of a selective P2X7 purinoreceptor antagonist, A438079. Sgca knockout mice were treated with A438079 every two days at 3 mg/Kg for 24 weeks. The P2X7 antagonist improved clinical parameters by ameliorating mice motor function and decreasing serum creatine kinase levels. Histological analysis of muscle morphology indicated a significant reduction of the percentage of central nuclei, of fiber size variability and of the extent of local fibrosis and inflammation. A cytometric characterization of the muscle inflammatory infiltrates showed that A438079 significantly decreased innate immune cells and upregulated the immunosuppressive regulatory T cell subpopulation. In α-sarcoglycan null mice, the selective P2X7 antagonist A438079 has been shown to be effective to counteract the progression of the dystrophic phenotype and to reduce the inflammatory response. P2X7 antagonism via selective inhibitors could be included in the immunosuppressant strategies aimed to dampen the basal immune-mediated damage and to favor a better engraftment of gene-cell therapies.

Therapeutic potential of proteasome inhibition in Duchenne and Becker muscular dystrophies.

Duchenne muscular dystrophy (DMD) and its milder allelic variant, Becker muscular dystrophy (BMD), result from mutations of the dystrophin gene and lead to progressive muscle deterioration. Enhanced activation of proteasomal degradation underlies critical steps in the pathogenesis of the DMD/BMD dystrophic process. Previously, we demonstrated that treatment with the proteasome inhibitor MG-132 rescues the cell membrane localization of dystrophin and the dystrophin glycoprotein complex in mdx mice, a natural genetic mouse model of DMD. The current work aims to thoroughly define the therapeutic potential in dystrophinopathies of Velcade, a drug that selectively blocks the ubiquitin-proteasome pathway. Velcade is particularly intriguing since it has been approved for the treatment of multiple myeloma. Therefore, its side effects in humans have been explored. Velcade effects were analyzed through two independent methodological approaches. First, we administered the drug systemically in mdx mice over a 2-week period. In this system, Velcade restores the membrane expression of dystrophin and dystrophin glycoprotein complex members and improves the dystrophic phenotype. In a second approach, we treated with the compound explants from muscle biopsies of DMD or BMD patients. We show that the inhibition of the proteasome pathway up-regulates dystrophin, alpha-sarcoglycan, and beta-dystroglycan protein levels in explants from BMD patients, whereas it increases the proteins of the dystrophin glycoprotein complex in DMD cases.

The spectrum of GNE mutations: allelic heterogeneity for a common phenotype.

Hereditary inclusion body myopathy (IBM2) was mainly reported in Middle Eastern Jewish patients. Distal myopathy with rimmed vacuoles has been described as a worldwide distributed distal myopathy. Both diseases are caused by mutations of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. Herein we report two patients: an Egyptian Muslim patient with the "common" Middle Eastern mutation (M712T), rarely described in non-Jewish patients; and an Italian patient carrying a novel GNE mutation (L179F) in the epimerase domain. Our patients share common clinical and histopathological features, with some interesting aspects. The first patient presented a clinical deterioration during her first pregnancy confirming that an increased requirement of sialic acid during pregnancy may trigger a clinical worsening. The second patient showed a slowly progressive deterioration, different from other patients carrying mutations in the epimerase domain, who had a severe and rapid progression.

Effects of non-euphoric plant cannabinoids on muscle quality and performance of dystrophic mdx mice.

BACKGROUND AND PURPOSE: Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, results in chronic inflammation and irreversible skeletal muscle degeneration. Moreover, the associated impairment of autophagy greatly contributes to the aggravation of muscle damage. We explored the possibility of using non-euphoric compounds present in Cannabis sativa, cannabidiol (CBD), cannabidivarin (CBDV) and tetrahydrocannabidivarin (THCV), to reduce inflammation, restore functional autophagy and positively enhance muscle function in vivo. EXPERIMENTAL APPROACH: Using quantitative PCR, western blots and [Ca2+ ]i measurements, we explored the effects of CBD and CBDV on the differentiation of both murine and human skeletal muscle cells as well as their potential interaction with TRP channels. Male dystrophic mdx mice were injected i.p. with CBD or CBDV at different stages of the disease. After treatment, locomotor tests and biochemical analyses were used to evaluate their effects on inflammation and autophagy. KEY RESULTS: CBD and CBDV promoted the differentiation of murine C2C12 myoblast cells into myotubes by increasing [Ca2+ ]i mostly via TRPV1 activation, an effect that undergoes rapid desensitization. In primary satellite cells and myoblasts isolated from healthy and/or DMD donors, not only CBD and CBDV but also THCV promoted myotube formation, in this case, mostly via TRPA1 activation. In mdx mice, CBD (60 mg·kg-1 ) and CBDV (60 mg·kg-1 ) prevented the loss of locomotor activity, reduced inflammation and restored autophagy. CONCLUSION AND IMPLICATIONS: We provide new insights into plant cannabinoid interactions with TRP channels in skeletal muscle, highlighting a potential opportunity for novel co-adjuvant therapies to prevent muscle degeneration in DMD patients. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Caveolin-3 T78M and T78K missense mutations lead to different phenotypes in vivo and in vitro.

Caveolins are the principal protein components of caveolae, invaginations of the plasma membrane involved in cell signaling and trafficking. Caveolin-3 (Cav-3) is the muscle-specific isoform of the caveolin family and mutations in the CAV3 gene lead to a large group of neuromuscular disorders. In unrelated patients, we identified two distinct CAV3 mutations involving the same codon 78. Patient 1, affected by dilated cardiomyopathy and limb girdle muscular dystrophy (LGMD)-1C, shows an autosomal recessive mutation converting threonine to methionine (T78M). Patient 2, affected by isolated familiar hyperCKemia, shows an autosomal dominant mutation converting threonine to lysine (T78K). Cav-3 wild type (WT) and Cav-3 mutations were transiently transfected into Cos-7 cells. Cav-3 WT and Cav-3 T78M mutant localized at the plasma membrane, whereas Cav-3 T78K was retained in a perinuclear compartment. Cav-3 T78K expression was decreased by 87% when compared with Cav-3 WT, whereas Cav-3 T78M protein levels were unchanged. To evaluate whether Cav-3 T78K and Cav-3 T78M mutants behaved with a dominant negative pattern, Cos-7 cells were cotransfected with green fluorescent protein (GFP)-Cav-3 WT in combination with either mutant or WT Cav-3. When cotransfected with Cav-3 WT or Cav-3 T78M, GFP-Cav-3 WT was localized at the plasma membrane, as expected. However, when cotransfected with Cav-3 T78K, GFP-Cav-3 WT was retained in a perinuclear compartment, and its protein levels were reduced by 60%, suggesting a dominant negative action. Accordingly, Cav-3 protein levels in muscles from a biopsy of patient 2 (T78K mutation) were reduced by 80%. In conclusion, CAV3 T78M and T78K mutations lead to distinct disorders showing different clinical features and inheritance, and displaying distinct phenotypes in vitro.