T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases

In skeletal muscle, the excitation-contraction (EC) coupling machinery mediates the translation of the action potential transmitted by the nerve into intracellular calcium release and muscle contraction. EC coupling requires a highly specialized membranous structure, the triad, composed of a central T-tubule surrounded by two terminal cisternae from the sarcoplasmic reticulum. While several proteins located on these structures have been identified, mechanisms governing T-tubule biogenesis and triad formation remain largely unknown. Here, we provide a description of triad structure and plasticity and review the role of proteins that have been linked to T-tubule biogenesis and triad formation and/or maintenance specifically in skeletal muscle: caveolin 3, amphiphysin 2, dysferlin, mitsugumins, junctophilins, myotubularin, ryanodine receptor, and dihydhropyridine Receptor. The importance of these proteins in triad biogenesis and subsequently in muscle contraction is sustained by studies on animal models and by the direct implication of most of these proteins in human myopathies

A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing

The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in ∼12days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 ± 0.8mV/cm2) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease

Physiopathological rnechanisms underlying myotubular myopathy in mice lacking myotubularin

Le but de celle étude est d'identifier les mécanismes moléculaircs liés à la pathogenèse de XLMTM. Pour cela. nous avons analysé le transcriptome de muscles squelettiques déficients en MTM 1 et trouvé plusieurs voies cellulaires dérégulées. Parmi lesquellThe aim of our study is 10 identify the molecular mechanisms leading 10 XLMTM pathogenesis. For this, we have analyzed the transcriplome of MTM1 KO skclctal muscle and found several deregulatcd pathways. among them. the pathway of Cal+ homeostasie. Sinc

Mécanismes physiopathologiques de la myopathie myotubulaire chez les souris déficientes en myotubularin

Le but de celle étude est d'identifier les mécanismes moléculaircs liés à la pathogenèse de XLMTM. Pour cela. nous avons analysé le transcriptome de muscles squelettiques déficients en MTM 1 et trouvé plusieurs voies cellulaires dérégulées. Parmi lesquelles. la voie de l'homéostasie calcique. En prenant en compte que la faiblesse musculaire est très sévère dans les souris KO-MTM 1 et que la protéine MTM 1 surexprimée dans les muscles squclettiques est associée au triad, J'ai focalisé mes études sur les gènes impliqués dans l'homéostasie calcique. En utilisant de Q-PCR et WB, j'ai identifié plusieurs dérégulations dans cette voie. Nous avons donc, mesuré le transit de calcium intracellulaire dans les fibres musculaires de control et de souris KO-MTM 1. Le pic de transit calcique était largement réduit dans les fibres musculaires déficientes en MTM 1. Ce qui suggère un défaut du couplage E-C dû à une altération de relâchement calcique de réticulum sarcoplasmic qui pourrait être à l'origine de faiblesse musculaire de souris KO-MTM l. J'ai également analysé le triad du muscle squelettique et trouvé des altérations ultrastructurales qui peuvent contribuer au couplage E-C. Ces résultats ensemble améliorent notre compréhension du mécanisme pathologiques de XLMTM.The aim of our study is 10 identify the molecular mechanisms leading 10 XLMTM pathogenesis. For this, we have analyzed the transcriplome of MTM1 KO skclctal muscle and found several deregulatcd pathways. among them. the pathway of Cal+ homeostasie. Since muscle wcakncss is very severe in MTM1 KO mice and Ihe overcxpressed myolubularin is associated to triads in skeletal muscle. we focuscd on genes involvcd in calcium homcostasis. Using Q-PCR and WB, we identified several deregulation in this pathway. We thus measurcd intracel1ular Ca2+ transients in muscle fibres from control and MTM1 KO mice. The peak Ca2+ transicnt was strongly reduced in the MTM1 dcficient fibres. Thcse results strongly suggcst that failure of excitation-contraction coupling duc to alteration of Ca2+ release from the SR accounts for muscle weakness in MTM1 KO mice. We also analyscd the triads in skeletal muscle and found ultrastructural alterations Ithat may contribute to the E-C uncoupling. These results provide novel insights into the pathomechanism of XLMTM.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Impaired Sarcoplasmic Reticulum Calcium Release In Skeletal Muscle Fibers From Myotubularin-Deficient Mice

International audienc

Calcification Propensity (T50) Predicts a Rapid Decline of Renal Function in Kidney Transplant Recipients

Background: Serum creatinine level, proteinuria, and interstitial fibrosis are predictive of renal prognosis. Fractional excretion of phosphate (FEP)/FGF23 ratio, tubular reabsorption of phosphate (TRP), serum calcification propensity (T50), and Klotho’s serum level are emerging as determinants of poor kidney outcomes in CKD patients. We aimed at analysing the use of FGF23, FEP/FGF23, TRP, T50, and Klotho in predicting the rapid decline of renal function in kidney allograft recipients. Methods: We included 103 kidney allograft recipients in a retrospective study with a prospective follow-up of 4 years. We analysed the predictive values of FGF23, FEP/FGF23, TRP, T50, and Klotho for a rapid decline of renal function defined as a drop of eGFR > 30%. Results: During a follow-up of 4 years, 23 patients displayed a rapid decline of renal function. Tertile of FGF23 (p value = 0.17), FEP/FGF23 (p value = 0.78), TRP (p value = 0.62) and Klotho (p value = 0.31) were not associated with an increased risk of rapid decline of renal function in kidney transplant recipients. The lower tertile of T50 was significantly associated with eGFR decline >30% with a hazard ratio of 3.86 (p = 0.048) and remained significant in multivariable analysis. Conclusion: T50 showed a strong association with a rapid decline of renal function in kidney allograft patients. This study underlines its role as an independent biomarker of loss of kidney function. We found no association between other phosphocalcic markers, such as FGF23, FEP/FGF23, TRP and Klotho, with a rapid decline of renal function in kidney allograft recipients

Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action.

The thiazide-sensitive NaCl cotransporter (NCC) is important for renal salt handling and blood-pressure homeostasis. The canonical NCC-activating pathway consists of With-No-Lysine (WNK) kinases and their downstream effector kinases SPAK and OSR1, which phosphorylate NCC directly. The upstream mechanisms that connect physiological stimuli to this system remain obscure. Here, we have shown that aldosterone activates SPAK/OSR1 via WNK1. We identified 2 alternatively spliced exons embedded within a proline-rich region of WNK1 that contain PY motifs, which bind the E3 ubiquitin ligase NEDD4-2. PY motif-containing WNK1 isoforms were expressed in human kidney, and these isoforms were efficiently degraded by the ubiquitin proteasome system, an effect reversed by the aldosterone-induced kinase SGK1. In gene-edited cells, WNK1 deficiency negated regulatory effects of NEDD4-2 and SGK1 on NCC, suggesting that WNK1 mediates aldosterone-dependent activity of the WNK/SPAK/OSR1 pathway. Aldosterone infusion increased proline-rich WNK1 isoform abundance in WT mice but did not alter WNK1 abundance in hypertensive Nedd4-2 KO mice, which exhibit high baseline WNK1 and SPAK/OSR1 activity toward NCC. Conversely, hypotensive Sgk1 KO mice exhibited low WNK1 expression and activity. Together, our findings indicate that the proline-rich exons are modular cassettes that convert WNK1 into a NEDD4-2 substrate, thereby linking aldosterone and other NEDD4-2-suppressing antinatriuretic hormones to NCC phosphorylation status

A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing

The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in ∼12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 ± 0.8 mV/cm(2)) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease