The loss and recovery of muscle mass and function following injury and during rehabilitation varies among individuals.While recent expression profiling studies have illustrated transcriptomic responses to muscle disuse and remodeling, how these changes contribute to the physiological responses are not clear.In this study, we quantified the effects of immobilization and subsequent rehabilitation training on muscle size and identified molecular pathways associated with muscle responsiveness in an orthopaedic patient cohort study.The injured leg of 16 individuals with ankle injury was immobilized for a minimum of 4 weeks, followed by a 6-week rehabilitation program.The maximal cross-sectional area (CSA) of the medial gastrocnemius muscle of the immobilized and control legs were determined by T1-weighted axial MRI images.Genome-wide mRNA profiling data were used to identify molecular signatures that distinguish the patients who responded to immobilization and rehabilitation and those who were considered minimal responders.RESULTS: Using 6% change as the threshold to define responsiveness, a greater degree of changes in muscle size was noted in high responders (-14.9 ± 3.6%) compared to low responders (0.1 ± 0.0%) during immobilization.In addition, a greater degree of changes in muscle size was observed in high responders (20.5 ± 3.2%) compared to low responders (2.5 ± 0.9%) at 6-week rehabilitation.Microarray analysis showed a higher number of genes differentially expressed in the responders compared to low responders in general; with more expression changes observed at the acute stage of rehabilitation in both groups.Pathways analysis revealed top molecular pathways differentially affected in the groups, including genes involved in mitochondrial function, protein turn over, integrin signaling and inflammation.This study confirmed the extent of muscle atrophy due to immobilization and recovery by exercise training is associated with distinct remodeling signature, which can potentially be used for evaluating and predicting clinical outcomes.
ABSTRACT Autosomal recessive polycystic kidney disease (ARPKD) is caused primarily by mutations in PKHD1 , encoding fibrocystin (FPC), but Pkhd1 mutant mice fail to express renal cystic disease. In contrast, the renal lesion in Cys1 cpk/cpk ( cpk ) mice with loss of the cystin protein, closely phenocopy ARPKD. Recent identification of patients with CYS1 -related ARPKD prompted the investigations described herein. We analyzed cystin and FPC expression in mouse models ( cpk , rescued- cpk ( r - cpk ), Pkhd1 mutants) and cortical collecting duct (CCD) cell lines (wild type ( wt), cpk) . We found that cystin deficiency led to diminished FPC in both cpk kidneys and CCD cells. In r-cpk kidneys, FPC increased and siRNA of Cys1 in wt CCD cells reduced FPC. Conversely, FPC deficiency in Pkhd1 mutants did not affect cystin levels. Cystin deficiency and the associated reduction in FPC levels impacted the architecture of the primary cilium, but not ciliogenesis. Similar Pkhd1 mRNA levels in wt, cpk kidneys and CCD cells suggested posttranslational mechanisms directed FPC loss and studies of cellular protein degradation systems revealed selective autophagy as a possible mechanism. Loss of FPC from the NEDD4 E3 ubiquitin ligase complexes caused reduced polyubiquitination and elevated levels of functional epithelial sodium channel (NEDD4 target) in cpk cells. We propose that cystin is necessary to stabilize FPC and loss of cystin leads to rapid FPC degradation. FPC removal from E3-ligase complexes alters the cellular proteome and may contribute to cystogenesis through multiple mechanisms, that include MYC transcriptional regulation.
Abstract Autosomal recessive polycystic kidney disease (ARPKD) is a hereditary hepato-renal fibrocystic disorder and a significant genetic cause of childhood morbidity and mortality. Mutations in the Polycystic Kidney and Hepatic Disease 1 ( PKHD1 ) gene cause all typical forms of ARPKD. Several mouse strains carrying diverse genetically engineered disruptions in the orthologous Pkhd1 gene have been generated. The current study describes a novel spontaneous mouse recessive mutation causing a cystic liver phenotype resembling the hepato-biliary disease characteristic of human ARPKD. Here we describe mapping of the cystic liver mutation to the Pkhd1 interval on Chromosome 1 and identification of a frameshift mutation within Pkhd1 exon 48 predicted to result in premature translation termination. Mice homozygous for the new mutation, symbollzed Pkhd1 cyli , lack renal pathology, consistent with previously generated Pkhd1 mouse mutants that fail to recapitulate human kidney disease. We have identified a profile of alternatively spliced Pkhd1 renal transcripts that are distinct in normal versus mutant mice. The Pkhd1 transcript profile in mutant kidneys is consistent with predicted outcomes of nonsense-associated alternative splicing (NAS) and nonsense mediated decay (NMD). Overall levels of Pkhd1 transcripts in mutant mouse kidneys were reduced compared to kidneys of normal mice, and Pkhd1 encoded protein in mutant kidneys was undetectable by immunoblotting. We suggest that in Pkhd1 cyli /Pkhd1 cyli (cyli) mice, mutation-promoted Pkhd1 alternative splicing in the kidney yields transcripts encoding low-abundance protein isoforms lacking exon 48 encoded amino acid sequences that are sufficiently functional so as to attenuate expression of a renal cystic disease phenotype.
