Annabelle Z. Caron

A novel hindlimb immobilization procedure for studying skeletal muscle atrophy and recovery in mouse

Skeletal muscle atrophy is a serious concern for patients afflicted by limb restriction due to surgery (e.g., arthrodesis), several articular pathologies (e.g., arthralgia), or simply following cast immobilization. To study the molecular events involved in this immobilization-induced debilitating condition, a convenient mouse model for atrophy is lacking. Here we provide a new immobilization procedure exploiting the normal flexion of the mouse hindlimb using a surgical staple to fix the ventral part of the foot to the distal part of the calf. Histological analysis revealed that our approach induced significant skeletal muscle atrophy by reducing the myofiber size of the tibialis anterior (TA) muscle by 36% compared with the untreated contralateral TA within a few days postimmobilization. Two molecular markers for atrophy, atrogin-1/muscle atrophy F-box (atrogin-1/MAFbx) and muscle ring finger 1 (MuRF-1) mRNAs, were significantly upregulated by 1.9- and 5.9-fold, respectively. Interestingly, our model also revealed the presence of an early inflammatory process during atrophy, characterized by the mRNA upregulation of TNF-alpha, IL-1, and IL-6 (1.9-, 2.4-, and 3.4-fold, respectively) simultaneously with the upregulation of the common leukocyte marker CD45 (6.1-fold). Moreover, muscle rapidly recovered on remobilization, an event associated with significantly increased levels of uncoupling protein-3 and peroxisome proliferator-activated receptor gamma coactivator-1alpha mRNA, key components of prooxidative muscle metabolism. This model offers unexpected new insights into the molecular events involved in immobilization atrophy.

The proteasome inhibitor MG132 reduces immobilization-induced skeletal muscle atrophy in mice

BackgroundSkeletal muscle atrophy is a serious concern for the rehabilitation of patients afflicted by prolonged limb restriction. This debilitating condition is associated with a marked activation of NFκB activity. The ubiquitin-proteasome pathway degrades the NFκB inhibitor IκBα, enabling NFκB to translocate to the nucleus and bind to the target genes that promote muscle atrophy. Although several studies showed that proteasome inhibitors are efficient to reduce atrophy, no studies have demonstrated the ability of these inhibitors to preserve muscle function under catabolic condition.MethodsWe recently developed a new hindlimb immobilization procedure that induces significant skeletal muscle atrophy and used it to show that an inflammatory process characterized by the up-regulation of TNFα, a known activator of the canonical NFκB pathway, is associated with the atrophy. Here, we used this model to investigate the effect of in vivo proteasome inhibition on the muscle integrity by histological approach. TNFα, IL-1, IL-6, MuRF-1 and Atrogin/MAFbx mRNA level were determined by qPCR. Also, a functional measurement of locomotors activity was performed to determine if the treatment can shorten the rehabilitation period following immobilization.ResultsIn the present study, we showed that the proteasome inhibitor MG132 significantly inhibited IκBα degradation thus preventing NFκB activation in vitro. MG132 preserved muscle and myofiber cross-sectional area by downregulating the muscle-specific ubiquitin ligases atrogin-1/MAFbx and MuRF-1 mRNA in vivo. This effect resulted in a diminished rehabilitation period.ConclusionThese finding demonstrate that proteasome inhibitors show potential for the development of pharmacological therapies to prevent muscle atrophy and thus favor muscle rehabilitation.

Transcriptional profiling of skeletal muscle reveals factors that are necessary to maintain satellite cell integrity during ageing.

Skeletal muscle ageing is characterized by faulty degenerative/regenerative processes that promote the decline of its mass, strength, and endurance. In this study, we used a transcriptional profiling method to better understand the molecular pathways and factors that contribute to these processes. To more appropriately contrast the differences in regenerative capacity of old muscle, we compared it with young muscle, where robust growth and efficient myogenic differentiation is ongoing. Notably, in old mice, we found a severe deficit in satellite cells activation. We performed expression analyses on RNA from the gastrocnemius muscle of young (3-week-old) and old (24-month-old) mice. The differential expression highlighted genes that are involved in the efficient functioning of satellite cells. Indeed, the greatest number of up-regulated genes in young mice encoded components of the extracellular matrix required for the maintenance of the satellite cell niche. Moreover, other genes included Wnt inhibitors (Wif1 and Sfrp2) and Notch activator (Dner), which are putatively involved in the interconnected signalling networks that control satellite cell function. The widespread expression differences for inhibitors of TGFbeta signalling further emphasize the shortcomings in satellite cell performance. Therefore, we draw attention to the breakdown of features required to maintain satellite cell integrity during the ageing process.

Protein kinase C phosphorylates the inositol 1,4,5-trisphosphate receptor type 2 and decreases the mobilization of Ca2+in pancreatoma AR4-2J cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor channel, which plays a major (IP(3)R) is an intracellular Ca(2+) role in Ca(2+) signalling. Three isoforms of IP(3)R have been identified (IP(3)R-1, IP(3)R-2 and IP(3)R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP(3)R are poorly understood. AR4-2J cells, which express almost exclusively (~86%) the IP(3)R-2, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) influences IP(3)R-2-mediated Ca(2+) release. Using an immunoprecipitation approach, we confirmed that AR4-2J cells express almost exclusively the IP(3)R-2 isoform. Using an in vitro phosphorylation assay, we showed that the immunopurified IP(3)R-2 was efficiently phosphorylated by exogenous PKC. In intact AR4-2J cells metabolically labelled with (32)Pi, we showed that phorbol-12-myristate-13-acetate (PMA) and Ca(2+) mobilizing agonists cause the phosphorylation of IP(3)R-2. In saponin-permeabilized AR4-2J cells, IP(3)-induced Ca(2+) release was reduced after a pre-treatment with PMA or with exogenous PKC. PMA also reduced the Ca(2+) response of intact AR4-2J cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. These results demonstrate that PKC decreases the Ca(2+)mobilizing activity of IP(3)R-2 and thus exerts a negative feedback on the agonists-induced Ca(2+) response of AR4-2J cells.

Protein kinase A increases the binding affinity and the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

Background information. In endocrine cells, IP3R (inositol 1,4,5‐trisphosphate receptor), a ligand‐gated Ca2+ channel, plays an important role in the control of intracellular Ca2+ concentration. There are three subtypes of IP3R that are distributed differentially among cell types. RINm5F cells express almost exclusively the IP3R‐3 subtype. The purpose of the present study was to investigate the effect of PKA (protein kinase A) on the activity of IP3R‐3 in RINm5F cells.

Angiotensin IV interacts with a juxtamembrane site on AT4/IRAP suggesting an allosteric mechanism of enzyme modulation

Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II, binds to a specific receptor (AT(4)) that has recently been identified as the transmembrane aminopeptidase insulin-regulated aminopeptidase (IRAP) based on the fact that the two proteins share several pharmacological and biochemical properties. Our binding studies indicated that bovine heart expresses relatively large amounts (1.2 pmol/mg protein) of high-affinity binding sites for Ang IV (K(d)=1.8 nM). A photoaffinity-labeling approach combined with mild trypsin digestion revealed that the AT(4) receptor of bovine heart is a single transmembrane domain protein (153 kDa) with a large extracellular fragment (143 kDa). After alkaline denaturation of the AT(4) receptor, trypsin digestion produced two small membrane-associated fragments (16.9 and 6.6 kDa). These results suggest that Ang IV interacts with a juxtamembrane domain of AT(4) receptor. The location of the juxtamembrane site of contact was different from that of the active site of IRAP, suggesting that Ang IV uses an allosteric mechanism to modulate the activity of the AT(4)/IRAP.

Advances in myogenic cell transplantation and skeletal muscle tissue engineering.

Curative treatments are currently not available for people suffering from one of the many prevalent muscle myopathies. One approach to ameliorate these conditions relies on the cell-based transplantation of potential myogenic precursors, or more optimistically, the transfer of engineered skeletal muscle tissue. To date, clinical trials with myogenic stem cell transplantation have met with only modest success while the transplantation of engineered muscle tissue is at the earliest stages of development. Despite the slow progress, these studies have provided insights and avenues that will eventually lead to a powerful therapeutic tool.

The Transcription Factors NFAT and CREB Have Different Susceptibilities to the Reduced Ca2+ Responses Caused by the Knock Down of Inositol Trisphosphate Receptor in HEK 293A Cells

Background/Aims: The inositol 1,4,5-trisphosphate receptor (IP₃R), a ligand-gated Ca²⁺ channel, plays an important role in the control of intracellular Ca²⁺. Three isoforms of IP₃R have been identified and most cell types express different proportions of these isoforms. The purpose of this study was to investigate how IP₃R signalling is involved in the activation of the Ca²⁺-sensitive transcription factors NFAT and CREB. Methods: Each IP₃R isoform expressed in HEK 293A cells was knocked down using selective siRNA. Free intracellular Ca²⁺ was monitored spectrofluometrically. NFAT and CREB activities were measured with luciferase reporter constructs. Results: IP₃R-2-knocked down HEK 293A cells showed a deficient CCh-induced Ca²⁺ response that could be rescued by co-stimulation with VIP, a cAMP increasing agonist. NFAT transcriptional activity, but not CREB transcriptional activity, was significantly reduced in IP₃R-2-knocked down HEK 293A cells. Overexpression of IP₃R-1 could fully compensate for IP₃R-2 knock down to mobilize Ca²⁺ and to activate NFAT. Conclusion: Our results show that the knock down of IP₃R-2 significantly reduced the intracellular Ca²⁺ response of HEK 293 cells. This reduced Ca²⁺ response did not affect the activation of CREB but significantly decreased the activation of NFAT, suggesting that the Ca²⁺ signals required for the activation of NFAT are stronger than those required for the activation of CREB.