Luca Madaro

PKC Theta Ablation Improves Healing in a Mouse Model of Muscular Dystrophy

Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, limiting muscle regeneration and resulting in fibrotic and fatty tissue replacement of muscle, which exacerbates the wasting process in dystrophic muscles. Limiting immune response is thus one of the therapeutic options to improve healing, as well as to improve the efficacy of gene- or cell-mediated strategies to restore dystrophin expression. Protein kinase C θ (PKCθ) is a member of the PKCs family highly expressed in both immune cells and skeletal muscle; given its crucial role in adaptive, but also innate, immunity, it is being proposed as a valuable pharmacological target for immune disorders. In our study we asked whether targeting PKCθ could represent a valuable approach to efficiently prevent inflammatory response and disease progression in a mouse model of muscular dystrophy. We generated the bi-genetic mouse model mdx/θ−/−, where PKCθ expression is lacking in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that muscle wasting in mdx/θ−/− mice was greatly prevented, while muscle regeneration, maintenance and performance was significantly improved, as compared to mdx mice. This phenotype was associated to reduction in inflammatory infiltrate, pro-inflammatory gene expression and pro-fibrotic markers activity, as compared to mdx mice. Moreover, BM transplantation experiments demonstrated that the phenotype observed was primarily dependent on lack of PKCθ expression in hematopoietic cells. These results demonstrate a hitherto unrecognized role of immune-cell intrinsic PKCθ activity in the development of DMD. Although the immune cell population(s) involved remain unidentified, our findings reveal that PKCθ can be proposed as a new pharmacological target to counteract the disease, as well as to improve the efficacy of gene- or cell- therapy approaches.

Intracellular signaling in ER stress-induced autophagy in skeletal muscle cells

Skeletal muscle remodeling in response to muscle disuse and unloading is known to be associated with so‐called ER stress, which, in turn, activates autophagy and contributes to muscle atrophy. Different molecules are involved in ER stress‐induced autophagy, among which PKCθ has recently been described. In this study, we dissected both in vitro and in vivo ER stress‐induced autophagy pathways in muscle. Using C2C12 muscle cells in culture, we demonstrated that PKC activation induced autophagy in the absence of ER stress. We further demonstrated that PKCθ was strongly activated in cultured myoblasts and myotubes during ER stress induced by different stimuli, such as TG or TN treatment, and that it localized into Lc3‐positive autophagic dots upon TG treatment. Neither Akt dephosphorylation nor Foxo or GSK3β activation was observed in these conditions. Moreover, PKCθ inhibition in myoblasts and myotubes prevented ER stress‐induced Lc3 activation and autophagic dot formation, but not ER stress. In vivo, lack of PKCθ prevented both food deprivation‐ and immobilization‐induced autophagy and muscle atrophy, irrespective of Akt pathway inhibition. Taken together, these results demonstrate that PKCθ functions as an ER stress sensor in skeletal muscle, required for ER‐stress‐dependent autophagy activation, and can be proposed as a novel molecular target to maintain muscle homeostasis in response to external stimuli, such as disuse and unloading, still allowing intracellular clearance.—Madaro, L., Marrocco, V., Carnio, S., Sandri, M., Bouché, M. Intracellular signaling in ER stress‐induced autophagy in skeletal muscle cells. FASEB J. 27, 1990–2000 (2013). www.fasebj.org

Thyroid Hormone T3 Counteracts STZ Induced Diabetes in Mouse

This study intended to demonstrate that the thyroid hormone T3 counteracts the onset of a Streptozotocin (STZ) induced diabetes in wild type mice. To test our hypothesis diabetes has been induced in Balb/c male mice by multiple low dose Streptozotocin injection; and a group of mice was contemporaneously injected with T3. After 48 h mice were tested for glucose tolerance test, insulin serum levels and then sacrified. Whole pancreata were utilized for morphological and biochemical analyses, while protein extracts and RNA were utilized for expression analyses of specific molecules. The results showed that islets from T3 treated mice were comparable to age- and sex-matched control, untreated mice in number, shape, dimension, consistency, ultrastructure, insulin and glucagon levels, Tunel positivity and caspases activation, while all the cited parameters and molecules were altered by STZ alone. The T3-induced pro survival effect was associated with a strong increase in phosphorylated Akt. Moreover, T3 administration prevented the STZ-dependent alterations in glucose blood level, both during fasting and after glucose challenge, as well as in insulin serum level. In conclusion we demonstrated that T3 could act as a protective factor against STZ induced diabetes.

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

Using both in vivo and in vitro protein kinase C (PKC) θ mutant models, we found that PKCθ, the PKC isoform predominantly expressed in skeletal muscle, is required for myoblast fusion and myofiber growth, by regulating focal adhesion kinase activity and, in turn, the expression of the pro-fusion genes caveolin-3 and β1D-integrin.

From Innate to Adaptive Immune Response in Muscular Dystrophies and Skeletal Muscle Regeneration: The Role of Lymphocytes

Skeletal muscle is able to restore contractile functionality after injury thanks to its ability to regenerate. Following muscle necrosis, debris is removed by macrophages, and muscle satellite cells (MuSCs), the muscle stem cells, are activated and subsequently proliferate, migrate, and form muscle fibers restoring muscle functionality. In most muscle dystrophies (MDs), MuSCs fail to properly proliferate, differentiate, or replenish the stem cell compartment, leading to fibrotic deposition. However, besides MuSCs, interstitial nonmyogenic cells and inflammatory cells also play a key role in orchestrating muscle repair. A complete understanding of the complexity of these mechanisms should allow the design of interventions to attenuate MDs pathology without disrupting regenerative processes. In this review we will focus on the contribution of immune cells in the onset and progression of MDs, with particular emphasis on Duchenne muscular dystrophy (DMD). We will briefly summarize the current knowledge and recent advances made in our understanding of the involvement of different innate immune cells in MDs and will move on to critically evaluate the possible role of cell populations within the acquired immune response. Revisiting previous observations in the light of recent evidence will likely change our current view of the onset and progression of the disease.

Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ

In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process.

Knock down of caveolin-1 affects morphological and functional hallmarks of human endothelial cells

Caveolin‐1 (CAV1) is the principal structural component of caveolae which functions as scaffolding protein for the integration of a variety of signaling pathways. In this study, we investigated the involvement of CAV1 in endothelial cell (EC) functions and show that siRNA‐induced CAV1 silencing in the human EC line EA.hy926 induces distinctive morphological changes, such as a marked increase in cell size and formation of stress fibers. Design‐based stereology was employed in this work to make unbiased quantification of morphometric properties such as volume, length, and surface of CAV1 silenced versus control cells. In addition, we showed that downregulation of CAV1 affects cell cycle progression at G1/S phase transition most likely by perturbation of AKT signaling. With the aim to assess the contribution of CAV1 to typical biological processes of EC, we report here that CAV1 targeting affects cell migration and matrix metalloproteinases (MMPs) activity, and reduces angiogenesis in response to VEGF, in vitro. Taken together our data suggest that the proper expression of CAV1 is important not only for maintaining the appropriate morphology and size of ECs but it might represent a prospective molecular target for studying key biological mechanisms such as senescence and tumorigenesis. J. Cell. Biochem. 114: 1843–1851, 2013.

Cavin-1 and Caveolin-1 are both required to support cell proliferation, migration and anchorage-independent cell growth in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a childhood soft tissue tumor with broad expression of markers that are typically found in skeletal muscle. Cavin-1 is a recently discovered protein actively cooperating with Caveolin-1 (Cav-1) in the morphogenesis of caveolae and whose role in cancer is drawing increasing attention. Using a combined in silico and in vitro analysis here we show that Cavin-1 is expressed in myogenic RMS tumors as well as in human and primary mouse RMS cultures, exhibiting a broad subcellular localization, ranging from nuclei and cytosol to plasma membrane. In particular, the coexpression and plasma membrane interaction between Cavin-1 and Cav-1 characterized the proliferation of human and mouse RMS cell cultures, while a downregulation of their expression levels was observed during the myogenic differentiation. Knockdown of Cavin-1 or Cav-1 in the human RD and RH30 cells led to impairment of cell proliferation and migration. Moreover, loss of Cavin-1 in RD cells impaired the anchorage-independent cell growth in soft agar. While the loss of Cavin-1 did not affect the Cav-1 protein levels in RMS cells, Cav-1 overexpression and knockdown triggered a rise or depletion of Cavin-1 protein levels in RD cells, respectively, in turn reflecting on increased or decreased cell proliferation, migration and anchorage-independent cell growth. Collectively, these data indicate that the interaction between Cavin-1 and Cav-1 underlies the cell growth and migration in myogenic tumors.

The direct renin inhibitor aliskiren improves vascular remodelling in transgenic rats harbouring human renin and angiotensinogen genes

In the present study, we tested the hypothesis that chronic treatment with the direct rennin inhibitor aliskiren improves the remodelling of resistance arteries in dTGR (double-transgenic rats). dTGR (5 weeks) were treated with aliskiren (3 mg/kg of body mass per day) or ramipril (1 mg/kg of body mass per day) for 14 days and compared with age-matched vehicle-treated dTGR. BP (blood pressure) was similarly reduced in both aliskiren-treated and ramipril-treated rats compared with control dTGR (167±1 and 169±2 mmHg compared with 197±4 mmHg respectively; P<0.05). The M/L (media-to-lumen) ratio assessed on pressurized preparations was equally reduced in aliskiren-treated and ramipril-treated rats compared with controls (6.3±0.5 and 6.4±0.2% compared with 9.8±0.4% respectively; P<0.05). Endothelium-dependent and -independent relaxations were similar among the groups. L-NAME (N(G)-nitro-L-arginine methyl ester) significantly reduced acetylcholine-induced dilation in drug-treated dTGR. This effect was significantly more prominent in aliskiren-treated rats. eNOS (endothelial NO synthase) expression showed a 2-fold increase only in aliskiren-treated dTGR as compared with controls (P<0.01) and ramipril-treated dTGR (P<0.05). Plasma nitrite, as an index of NO production, was significantly increased in dTGR treated with either aliskiren or ramipril compared with controls. Only aliskiren induced a 2-fold increase in plasma nitrite, which was significantly greater than that induced by ramipril (P<0.05). gp91(phox) expression and ROS (reactive oxygen species) production in aorta were significantly and similarly reduced by both drugs. In conclusion, equieffective hypotensive doses of aliskiren or ramipril reduced the M/L ratio of mesenteric arteries and improved oxidative stress in dTGR. However, only aliskiren increased further NO production in the vasculature. Hence, in dTGR, direct renin inhibition induces favourable effects similar to that induced by ACE (angiotensin-converting enzyme) inhibition in improving vascular remodelling through different mechanisms.

Pharmacological Inhibition of PKCθ Counteracts Muscle Disease in a Mouse Model of Duchenne Muscular Dystrophy

Inflammation plays a considerable role in the progression of Duchenne Muscular Dystrophy (DMD), a severe muscle disease caused by a mutation in the dystrophin gene. We previously showed that genetic ablation of Protein Kinase C θ (PKCθ) in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease. Research in context Duchenne muscular dystrophy (DMD) is a severe muscle disease affecting 1:3500 male births. DMD is caused by a mutation in dystrophin gene, coding for a protein required for skeletal and cardiac muscle integrity. Lack of a functional dystrophin is primarily responsible for the muscle eccentric contraction-induced muscle damage, observed in dystrophic muscle. However, inflammation plays a considerable role in the progression of DMD. Glucocorticoids, which have anti-inflammatory properties, are being used to treat DMD with some success; however, long term treatment with these drugs induces muscle atrophy and wasting, outweighing their benefit. The identification of specific targets for anti-inflammatory therapies is one of the ongoing therapeutic options. Although blunting inflammation would not be a “cure” for the disease, the emerging clue is that multiple strategies, addressing different aspects of the pathology, which may eventually converge, may be successful. In this context, we previously showed that genetic ablation of Protein Kinase C θ (PKCθ), an enzyme known to be involved in immune response, in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease.