Cystic fibrosis transmembrane conductance regulator in human muscle: Dysfunction causes abnormal metabolic recovery in exercise.
OBJECTIVE
Individuals with cystic fibrosis (CF) have exercise intolerance and skeletal muscle weakness not solely attributable to physical inactivity or pulmonary function abnormalities. CF transmembrane conductance regulator (CFTR) has been demonstrated in human bronchial smooth and cardiac muscle. Using (31)P-magnetic resonance spectroscopy of skeletal muscle, we showed CF patients to have lower resting muscle adenosine triphosphate and delayed phosphocreatine recovery times after high-intensity exercise, suggesting abnormal muscle aerobic metabolism; and higher end-exercise pH values, suggesting altered bicarbonate transport. Our objective was to study CFTR expression in human skeletal muscle.


RESULTS
We studied CFTR expression in human skeletal muscle by Western blot with anti-CFTR antibody (Ab) L12B4 and demonstrated a single band with expected molecular weight of 168kDa. We isolated the cDNA by reverse transcription polymerase chain reaction and directly sequenced a 975bp segment (c. 3,600-4,575) that was identical to the human CFTR sequence. We showed punctate staining of CFTR in sarcoplasm and sarcolemma by immunofluorescence microscopy with L12B4 Ab and secondary Alexa 488-labeled Ab. We confirmed CFTR expression in the sarcotubular network and sarcolemma by electron microscopy, using immunogold-labeled anti-CFTR Ab. We observed activation of CFTR Cl(-) channels with iodide efflux, on addition of forskolin, 3-isobutyl-1-methyl-xanthine, and 8-chlorphenylthio-cyclic adenosine monophosphate, in wild-type C57BL/6J isolated muscle fibers in contrast to no efflux from mutant F508del-CFTR muscle.


CONCLUSIONS
We speculate that a defect in sarcoplasmic reticulum CFTR Cl(-) channels could alter the electrochemical gradient, causing dysregulation of Ca(2+) homeostasis, for example, ryanodine receptor or sarco(endo)plasmic reticulum Ca(2+) adenosine triphosphatases essential to excitation-contraction coupling leading to exercise intolerance and muscle weakness in CF.
Department of Pediatrics, Division of Neurology, Hospital for Sick Children, Toronto, Ontario, Canada.
Anne-Marie
AM
Lamhonwah
Christine E
CE
Bear
Ling Jun
LJ
Huan
Patrick
P
Kim Chiaw
Cameron A
CA
Ackerley
Ingrid
I
Tein
D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000473:pathology* / Q000503:physiopathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D015444:Exercise / Q000502:physiology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016253:Microscopy, Immunoelectron / Q000379:methods; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D009154:Mutation / Q000235:genetics; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D012508:Sarcolemma / Q000378:metabolism / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000378:metabolism
D010726:Phosphodiesterase Inhibitors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin