ABSTRACT
Myotonic dystrophy type 1 (DM1) involves misregulated alternative splicing for specific genes. We used exon or nucleotide deletion to mimic altered splicing of genes central to muscle excitation-contraction coupling in mice. Mice with forced skipping of exon 29 in the CaV1.1 calcium channel combined with loss of ClC-1 chloride channel function displayed markedly reduced lifespan, whereas other combinations of splicing mimics did not affect survival. The Ca2+/Cl- bi-channelopathy mice exhibited myotonia, weakness, and impairment of mobility and respiration. Chronic administration of the calcium channel blocker verapamil rescued survival and improved force generation, myotonia, and respiratory function. These results suggest that Ca2+/Cl- bi-channelopathy contributes to muscle impairment in DM1 and is potentially mitigated by common clinically available calcium channel blockers.
Subject(s)
Channelopathies , Myotonia , Myotonic Dystrophy , Mice , Animals , Myotonic Dystrophy/drug therapy , Myotonic Dystrophy/genetics , Myotonic Dystrophy/metabolism , Calcium/metabolism , Chlorides/metabolism , Myotonia/metabolism , Verapamil/pharmacology , Verapamil/metabolism , Channelopathies/genetics , Channelopathies/metabolism , Alternative Splicing , Chloride Channels/genetics , Chloride Channels/metabolism , Muscle, Skeletal/metabolismABSTRACT
Myotonic dystrophy type 1 (DM1) involves misregulated alternative splicing for specific genes. We used exon or nucleotide deletion to mimic altered splicing of genes central to muscle excitation-contraction coupling processes in mice. Mice with forced-skipping of exon 29 in CaV1.1 calcium channel combined with loss of ClC-1 chloride channel function showed a markedly reduced lifespan, whereas other combinations of splicing mimics did not affect survival. The Ca2+/Cl- bi-channelopathy mice exhibited myotonia, weakness, and impairment of mobility and respiration. Chronic administration of the calcium channel blocker verapamil rescued survival and improved force generation, myotonia, and respiratory function. These results suggest that Ca2+/Cl- bi-channelopathy contributes to muscle impairment in DM1 and is potentially mitigated by common clinically available calcium channel blockers.
ABSTRACT
Polyriboadenylic [poly(rA)] strands of sufficient length form parallel double helices in acidic and/or ammonium-containing conditions. Poly(rA) duplexes in acidic conditions are held together by A+-A+ base-pairing also involving base interactions with the phosphate backbone. Traditional UV-melting studies of parallel poly(A) duplexes have typically examined homo-duplex formation of a single nucleic acid species in solution. We have adapted a technique utilizing a DNA nanoswitch that detects interaction of two different strands either with similar or differing lengths or modifications. Our method detected parallel duplex formation as a function of length, chemical modifications, and pH, and at a sensitivity that required over 100-fold less concentration of sample than prior UV-melting methods. While parallel polyriboadenylic acid and poly-2'-O-methyl-adenylic acid homo-duplexes formed, we did not detect homo-duplexes of polydeoxyriboadenylic acid strands or poly-locked nucleic acid (LNA)-adenylic strands. Importantly however, a poly-locked nucleic acid (LNA)-adenylic strand, as well as a poly-2'-O-methyl-adenylic strand, formed a hetero-duplex with a polyriboadenylic strand. Overall, our work validates a new tool for studying parallel duplexes and reveals fundamental properties of poly(A) parallel duplex formation. Parallel duplexes may find use in DNA nanotechnology and in molecular biology applications such as a potential poly(rA) tail capture tool as an alternative to traditional oligo(dT) based purification.
