Involvement of Inositol 1,4,5-Trisphosphate in Nicotinic Calcium Responses in Dystrophic Myotubes Assessed by Near-plasma Membrane Calcium Measurement

In skeletal muscle cells, plasma membrane depolarization causes a rapid calcium release from the sarcoplasmic reticulum through ryanodine receptors triggering contraction. In Duchenne muscular dystrophy (DMD), a lethal disease that is caused by the lack of the cytoskeletal protein dystrophin, the cytosolic calcium concentration is known to be increased, and this increase may lead to cell necrosis. Here, we used myotubes derived from control and mdx mice, the murine model of DMD, to study the calcium responses induced by nicotinic acetylcholine receptor stimulation. The photoprotein aequorin was expressed in the cytosol or targeted to the plasma membrane as a fusion protein with the synaptosome-associated protein SNAP-25, thus allowing calcium measurements in a restricted area localized just below the plasma membrane. The carbachol-induced calcium responses were 4.5 times bigger in dystrophic myotubes than in control myotubes. Moreover, in dystrophic myotubes the carbachol-mediated calcium responses measured in the subsarcolemmal area were at least 10 times bigger than in the bulk cytosol. The initial calcium responses were due to calcium influx into the by a from the sarcoplasmic In and the receptor was in calcium in the dystrophic myotubes. of this calcium release in the calcium and muscle in mdx in In skeletal muscle cells, plasma membrane depolarization causes a rapid calcium release from the sarcoplasmic reticulum through ryanodine receptors triggering contraction. In Duchenne muscular dystrophy (DMD), a lethal disease that is caused by the lack of the cytoskeletal protein dystrophin, the cytosolic calcium concentration is known to be increased, and this increase may lead to cell necrosis. Here, we used myotubes derived from control and mdx mice, the murine model of DMD, to study the calcium responses induced by nicotinic acetylcholine receptor stimulation. The photoprotein aequorin was expressed in the cytosol or targeted to the plasma membrane as a fusion protein with the synaptosome-associated protein SNAP-25, thus allowing calcium measurements in a restricted area localized just below the plasma membrane. The carbachol-induced calcium responses were 4.5 times bigger in dystrophic myotubes than in control myotubes. Moreover, in dystrophic myotubes the carbachol-mediated calcium responses measured in the subsarcolemmal area were at least 10 times bigger than in the bulk cytosol. The initial calcium responses were due to calcium influx into the by a from the sarcoplasmic In and the receptor was in calcium in the dystrophic myotubes. of this calcium release in the calcium and muscle in mdx in Duchenne muscular dystrophy used DMD, Duchenne muscular subsarcolemmal SNAP-25, synaptosome-associated protein of used DMD, Duchenne muscular subsarcolemmal SNAP-25, synaptosome-associated protein of is disease that in in muscle that to by or the as the of of is caused by the of dystrophin, a protein localized the plasma membrane In skeletal muscle is with a and a the and the to the plasma membrane that the of in the of the plasma in a and to calcium that of be the muscle that in muscle from or in of the model of DMD, the mdx of cytosolic or membrane in myotubes and skeletal muscle from or mdx this of the concentration of in and this is in the of the disease is that and in mdx is In skeletal muscle and that of nicotinic receptors to plasma membrane a is by the release of from the sarcoplasmic reticulum through the of ryanodine by the of ryanodine receptors and in the skeletal muscle sarcoplasmic reticulum of release receptors In cells, in by the in the The of receptors of to the of and receptors may be in the control of influx through plasma membrane skeletal muscle cells, receptors localized in the as as in the sarcoplasmic reticulum In that the may to that release or the of or skeletal muscle that may a in in skeletal muscle that the plasma membrane depolarization that and receptor in myotubes with a a in responses nicotinic receptor in dystrophic myotubes to Here, we the receptor in dystrophic myotubes plasma aequorin the membrane that aequorin is a to just below the plasma membrane. that nicotinic receptor membrane that receptor in myotubes derived from mdx thus a of were in and at in a of and cell were as with mdx and control were by muscle was of and and into of from were the muscle were in was by of in 10 of with 10 and at the the were with a and the and was The and with The were through a cell and at 10 The cell were in and the were was of a of and with and from to in were and in the were and induced to by the to with 10 and were at in was and with a were at a of of of The was in this was by a were used or aequorin were from were with a a protein or aequorin with the to subsarcolemmal calcium or with cytosolic aequorin to cytosolic calcium The was a from and were with a a the and a a the myotubes were in and with a the a was a a was at and was at a of were the with the used or of subsarcolemmal concentration was in a of myotubes as with the aequorin was in a and 10 and the to the the were at a of in a at was at with a and a as The and is in the and the that to the the the to the measured and the of the aequorin and is the of and was by to 10 with to the was as with myotubes were and at 10 in of the the influx was by with of or with and of 10 at The were times with were with of The of the was measured by expressed in was the and expressed as the was with the or of by a was were the of is the of aequorin were in the the of was the The was and the the of the were with the with the a of a mdx of to be localized at the with the with and was in the to a protein were with control myotubes the of the were 10 with the to cytosolic responses with cytosolic aequorin by and were in with In in with aequorin the responses and to and were by that responses measured with aequorin were due to in a restricted area the plasma Moreover, as in the by were 10 times measured with aequorin than with cytosolic aequorin and that aequorin was localized at the plasma membrane. the to be a membrane the and of the concentration in a of mdx myotubes the of a 10 with myotubes. were with cytosolic aequorin and aequorin were used at or the of with a with with is of The to to a in and the that the receptor be in acetylcholine receptor we measured the of the nicotinic and the of the receptor subsarcolemmal in control and dystrophic myotubes. in with of control and dystrophic cells, a were 10 with the was in control myotubes was in dystrophic myotubes to that the in responses control and dystrophic myotubes and that the may be in the in dystrophic in control and dystrophic is a the of with and in control and dystrophic myotubes. The to to a from the control from the dystrophic by by of with of a of in and of 10 with a receptor the of the the 10 of with the nicotinic receptor responses from in to in the of the and responses in the of were from the responses by that were by the of nicotinic receptors in the of subsarcolemmal in a of mdx myotubes the of with and 10 the of with the to and to a and and a of by a to of the with the and by by may influx through plasma membrane that were by influx through plasma membrane 10 with a the to Moreover, the to a the of with to a that the responses were at least in by influx through plasma membrane influx induced by the of the of a subsarcolemmal induced by in a of mdx myotubes the of a and the of the of subsarcolemmal induced by the of and The to the of influx to a the of a and of the that was in the by that this was due to we used the influx in influx was from with to with Moreover, were with and and the influx was that influx through and from that in dystrophic myotubes responses were at least in due to we the of the reticulum in 10 with the from to the cells, is to the by that the due to influx and release from the sarcoplasmic sarcoplasmic reticulum and of subsarcolemmal in a of mdx myotubes the of the the of with The to of to a of the in in that may be in in dystrophic myotubes. the of this we the of a is through in of the with the from to that the may be in the Moreover, we the of a of at a concentration of the 10 with this the responses from to of the in of subsarcolemmal in a of mdx myotubes the of and with aequorin of with in the of the The to to a as by a of by a to the with the and and may we with a and to as and to responses the was in to and was below that of the due to that the was in in mdx this study we used SNAP-25, a plasma membrane to the photoprotein aequorin to the subsarcolemmal area of control and dystrophic myotubes. of the of and and of skeletal muscle cells, we the of the was by mdx myotubes. that this aequorin was localized at the of mdx myotubes cytosolic that to protein with that and that the protein be to be targeted to the of the plasma membrane In the cytosolic is to be due to of aequorin was with of the responses measured with aequorin with cytosolic The that the to the was by that this is due to the of to in the below the plasma membrane. that aequorin to in a restricted area the plasma membrane that may to the in cell In this restricted area to or that aequorin was localized at the plasma membrane and that is a membrane concentration in myotubes. in to were bigger measured the plasma membrane than in the cytosol. is due to the that aequorin is to and that may in a restricted of in the bulk were 4.5 times bigger in dystrophic myotubes than in control myotubes. is in with in muscle that a subsarcolemmal due to influx in dystrophic as with control Moreover, we that receptor the responses in dystrophic in control myotubes. that the in subsarcolemmal may be control and dystrophic mdx was to was and responses were by a that in a by influx through plasma membrane Moreover, responses were by that in were with the reticulum the responses were and the as the by that responses due to influx through plasma membrane by a of the sarcoplasmic reticulum and a release from the mdx were were with receptor and or were with with that receptors in in dystrophic myotubes. were by the that nicotinic receptor and release in dystrophic a the of that the of by depolarization in myotubes by a a be the in dystrophic as of may be by membrane due to influx through nicotinic receptors that receptor in dystrophic myotubes. the was responses in control that the is in nicotinic responses in this cell that is a of by the of the or were in a aequorin the the of in the with and that release is in we the of in receptor as in of skeletal muscle that to the of the receptor be in the influx in dystrophic that receptors in membrane by the of nicotinic receptors in dystrophic myotubes. The of receptors a and the of dystrophic skeletal muscle control myotubes in muscle that the release from the sarcoplasmic reticulum and muscle is the ryanodine receptor Moreover, in receptor and a to increase in in mdx myotubes that receptors may be in and the muscle that in dystrophic muscle Duchenne muscular dystrophy used DMD, Duchenne muscular subsarcolemmal SNAP-25, synaptosome-associated protein of used DMD, Duchenne muscular subsarcolemmal SNAP-25, synaptosome-associated protein of is disease that in in muscle that to by or the as the of of is caused by the of dystrophin, a protein localized the plasma membrane In skeletal muscle is with a and a the and the to the plasma membrane that the of in the of the plasma in a and to calcium that of be the muscle that in muscle from or in of the model of DMD, the mdx of cytosolic or membrane in myotubes and skeletal muscle from or mdx this of the concentration of in and this is in the of the disease is that and in mdx is In skeletal muscle and that of nicotinic receptors to plasma membrane a is by the release of from the sarcoplasmic reticulum through the of ryanodine by the of ryanodine receptors and in the skeletal muscle sarcoplasmic reticulum of release receptors In cells, in by the in the The of receptors of to the of and receptors may be in the control of influx through plasma membrane In skeletal muscle cells, receptors localized in the as as in the sarcoplasmic reticulum In that the may to that release or the of or skeletal muscle that may a in in skeletal muscle that the plasma membrane depolarization that and receptor in myotubes with a a The in responses nicotinic receptor in dystrophic myotubes to Here, we the receptor in dystrophic myotubes plasma aequorin the membrane that aequorin is a to just below the plasma membrane. that nicotinic receptor membrane that receptor in myotubes derived from mdx thus a of were in and at in a of and cell were as with mdx and control were by muscle was of and and into of from were the muscle were in was by of in 10 of with 10 and at the the were with a and the and was The and with The were through a cell and at 10 The cell were in and the were was of a of and with and from to in were and in the were and induced to by the to with 10 and were at in was and with a were at a of of of The was in this was by a were used or aequorin were from were with a a protein or aequorin with the to subsarcolemmal calcium or with cytosolic aequorin to cytosolic calcium The was a from and were with a a the and a a the myotubes were in and with a the a was a a was at and was at a of were the with the used or of subsarcolemmal concentration was in a of myotubes as with the aequorin was in a and 10 and the to the the were at a of in a at was at with a and a as The and is in the and the that to the the the to the measured and the of the aequorin and is the of and was by to 10 with to the was as with myotubes were and at 10 in of the the influx was by with of or with and of 10 at The were times with were with of The of the was measured by expressed in was the and expressed as the was with the or of by a was were of were in and at in a of and The cell were as with mdx and control were by muscle was of and and into of from were the muscle were in was by of in 10 of with 10 and at the the were with a and the and was The and with The were through a cell and at 10 The cell were in and the were was of a of and with and from to in were and in the were and induced to by the to with 10 and were at in was and with a were at a of of of The was in this was by a were used or aequorin were from were with a a protein or aequorin with the to subsarcolemmal calcium or with cytosolic aequorin to cytosolic calcium The was a from and were with a a the and a a the myotubes were in and with a the a was a a was at and was at a of were the with the used or of subsarcolemmal concentration was in a of myotubes as with the aequorin was in a and 10 and the to the the were at a of in a at was at with a and a as The and is in the and the that to the the the to the measured and the of the aequorin and is the of and was by to 10 with to the was as with myotubes were and at 10 in of the the influx was by with of or with and of 10 at The were times with were with of The of the was measured by expressed in was the and expressed as the was with the or of by a was were the of is the of aequorin were in the the of was the The was and the the of the were with the with the a of a mdx of to be localized at the with the with and was in the to a protein were with control myotubes the of the were 10 with the to cytosolic responses with cytosolic aequorin by and were in with In in with aequorin the responses and to and were by that responses measured with aequorin were due to in a restricted area the plasma Moreover, as in the by were 10 times measured with aequorin than with cytosolic aequorin and that aequorin was localized at the plasma membrane. the to be a membrane in and the that the receptor be in acetylcholine receptor we measured the of the nicotinic and the of the receptor subsarcolemmal in control and dystrophic myotubes. in with of control and dystrophic cells, a were 10 with the was in control myotubes was in dystrophic myotubes to that the in responses control and dystrophic myotubes and that the may be in the in dystrophic in control and dystrophic is a the of with and in control and dystrophic myotubes. The to to a from the control from the dystrophic by by of with of a of in and of 10 with a receptor the of the the 10 of with the nicotinic receptor responses from in to in the of the and responses in the of were from the responses by that were by the of nicotinic receptors in the of subsarcolemmal in a of mdx myotubes the of with and 10 the of with the to and to a and and a of by a to of the with the and by by may influx through plasma membrane that were by influx through plasma membrane 10 with a the to Moreover, the to a the of with to a that the responses were at least in by influx through plasma membrane influx induced by the of the of a subsarcolemmal induced by in a of mdx myotubes the of a and the of the of subsarcolemmal induced by the of and The to the of influx to a the of a and of the that was in the by that this was due to we used the influx in influx was from with to with Moreover, were with and and the influx was that influx through and from that in dystrophic myotubes responses were at least in due to we the of the reticulum in 10 with the from to the cells, is to the by that the due to influx and release from the sarcoplasmic sarcoplasmic reticulum and of subsarcolemmal in a of mdx myotubes the of the the of with The to of to a of the in in that may be in in dystrophic myotubes. the of this we the of a is through in of the with the from to that the may be in the Moreover, we the of a of at a concentration of the 10 with this the responses from to of the in of subsarcolemmal in a of mdx myotubes the of and with aequorin of with in the of the The to to a as by a of by a to the with the and and may we with a and to as and to responses the was in to and was below that of the due to that the was in in mdx myotubes. the of is the of aequorin were in the the of was the The was and the the of the were with the with the a of a mdx of to be localized at the with the with and was in the to a protein were with control myotubes the of the were 10 with the to cytosolic responses with cytosolic aequorin by and were in with In in with aequorin the responses and to and were by that responses measured with aequorin were due to in a restricted area the plasma Moreover, as in the by were 10 times measured with aequorin than with cytosolic aequorin and that aequorin was localized at the plasma membrane. the to be a membrane in and the that the receptor be in acetylcholine receptor we measured the of the nicotinic and the of the receptor subsarcolemmal in control and dystrophic myotubes. in with of control and dystrophic cells, a were 10 with the was in control myotubes was in dystrophic myotubes to that the in responses control and dystrophic myotubes and that the may be in the in dystrophic myotubes. by by of with of a of in and of 10 with a receptor the of the the 10 of with the nicotinic receptor responses from in to in the of the and responses in the of were from the responses by that were by the of nicotinic by by may influx through plasma membrane that were by influx through plasma membrane 10 with a the to Moreover, the to a the of with to a that the responses were at least in by influx through plasma membrane the of a and of the that was in the by that this was due to we used the influx in influx was from with to with Moreover, were with and and the influx was that influx through and from that in dystrophic myotubes responses were at least in due to we the of the reticulum in 10 with the from to the cells, is to the by that the due to influx and release from the sarcoplasmic of the in in that may be in in dystrophic myotubes. the of this we the of a is through in of the with the from to that the may be in the Moreover, we the of a of at a concentration of the 10 with this the responses from to and may we with a and to as and to responses the was in to and was below that of the due to that the was in in mdx myotubes. this study we used SNAP-25, a plasma membrane to the photoprotein aequorin to the subsarcolemmal area of control and dystrophic myotubes. of the of and and of skeletal muscle cells, we the of the was by mdx myotubes. that this aequorin was localized at the of mdx myotubes cytosolic that to protein with that and that the protein be to be targeted to the of the plasma membrane In the cytosolic is to be due to of aequorin was with of the responses measured with aequorin with cytosolic The that the to the was by that this is due to the of to in the below the plasma membrane. that aequorin to in a restricted area the plasma membrane that may to the in cell In this restricted area to or that aequorin was localized at the plasma membrane and that is a membrane concentration in myotubes. in to were bigger measured the plasma membrane than in the cytosol. is due to the that aequorin is to and that may in a restricted of in the bulk were 4.5 times bigger in dystrophic myotubes than in control myotubes. is in with in muscle that a subsarcolemmal due to influx in dystrophic as with control Moreover, we that receptor the responses in dystrophic in control myotubes. that the in subsarcolemmal may be control and dystrophic mdx was to was and responses were by a that in a by influx through plasma membrane Moreover, responses were by that in were with the reticulum the responses were and the as the by that responses due to influx through plasma membrane by a of the sarcoplasmic reticulum and a release from the mdx were were with receptor and or were with with that receptors in in dystrophic myotubes. were by the that nicotinic receptor and release in dystrophic a the of that the of by depolarization in myotubes by a a be the in dystrophic as of may be by membrane due to influx through nicotinic receptors that receptor in dystrophic myotubes. the was responses in control that the is in nicotinic responses in this cell that is a of by the of the or were in a aequorin the the of in the with and that release is in we the of in receptor as in of skeletal muscle that to the of the receptor be in the influx in dystrophic that receptors in membrane by the of nicotinic receptors in dystrophic myotubes. The of receptors a and the of dystrophic skeletal muscle control myotubes in muscle that the release from the sarcoplasmic reticulum and muscle is the ryanodine receptor Moreover, in receptor and a to increase in in mdx myotubes that receptors may be in and the muscle that in dystrophic muscle In this study we used SNAP-25, a plasma membrane to the photoprotein aequorin to the subsarcolemmal area of control and dystrophic myotubes. of the of and and of skeletal muscle cells, we the of the was by mdx myotubes. that this aequorin was localized at the of mdx myotubes cytosolic that to protein with that and that the protein be to be targeted to the of the plasma membrane In the cytosolic is to be due to of aequorin was with of the responses measured with aequorin with cytosolic The that the to the was by that this is due to the of to in the below the plasma membrane. that aequorin to in a restricted area the plasma membrane that may to the in cell In this restricted area to or that aequorin was localized at the plasma membrane and that is a membrane concentration in myotubes. in to were bigger measured the plasma membrane than in the cytosol. is due to the that aequorin is to and that may in a restricted of in the bulk cytosol. were 4.5 times bigger in dystrophic myotubes than in control myotubes. is in with in muscle that a subsarcolemmal due to influx in dystrophic as with control Moreover, we that receptor the responses in dystrophic in control myotubes. that the in subsarcolemmal may be control and dystrophic myotubes. In mdx was to was and responses were by a that in a by influx through plasma membrane Moreover, responses were by that in were with the reticulum the responses were and the as the by that responses due to influx through plasma membrane by a of the sarcoplasmic reticulum and a release from the In mdx were were with receptor and or were with with that receptors in in dystrophic myotubes. were by the that nicotinic receptor and release in dystrophic a the of that the of by depolarization in myotubes by a a be the in dystrophic as of may be by membrane due to influx through nicotinic receptors that receptor in dystrophic myotubes. the was responses in control that the is in nicotinic responses in this cell that is a of by the of the or were in a aequorin the the of in the with and that release is in we the of in receptor as in of skeletal muscle that to the of the receptor be in the influx in dystrophic In that receptors in membrane by the of nicotinic receptors in dystrophic myotubes. The of receptors a and the of dystrophic skeletal muscle control myotubes in muscle that the release from the sarcoplasmic reticulum and muscle is the ryanodine receptor Moreover, in receptor and a to increase in in mdx myotubes that receptors may be in and the muscle that in dystrophic muscle with the cell and and of The and of of

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