Biological Bypass Bridge with Sodium Channels, Calcium Channels and/or Potassium Channels to Compensate for Conduction Block in the Heart

Abstract

This invention provides a bypass bridge comprising a tract of gap junction-coupled cells having a first end and a second end, both ends capable of being attached to two selected sites in a heart so as to allow the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites, wherein the cells functionally express a sodium channel. The invention also provides related methods of making the bypass bridge, methods of implanting same in a heart, and methods of treating a disorder associated with an impaired conduction in a subject's heart.

1 . An atrioventricular bypass bridge comprising a tract of gap junction-coupled adult human mesenchymal stem cells obtained from blood, wherein the tract
 i. is formed in vitro,   ii. is at least about 0.5 mm in length,   iii. has a first end that is capable of being attached to a first site in the atrium of the heart and a second end that is capable of being attached to a second site in the ventricle of the heart so as to allow the conduction of at least one of a pacemaker signal, pacemaker current, electrical signal, and electrical current across the tract between the two sites, and   iv. wherein the adult mesenchymal stem cells functionally express both a sodium channel and a potassium channel which channels are each encoded by a respective nucleic acid that has been introduced into the cells.   
     
     
         2 . The bypass bridge of  claim 1 , wherein the mesenchymal stem cells further functionally express a pacemaker ion channel encoded by a nucleic acid that has been introduced into the cells which channel induces a pacemaker in the cells. 
     
     
         3 - 6 . (canceled) 
     
     
         7 . The bypass bridge of  claim 1  or  2 , wherein the sodium channel is a SKM-1 channel. 
     
     
         8 . The bypass bridge of  claim 7 , wherein the SKM-1 channel comprises an alpha subunit. 
     
     
         9 . The bypass bridge of  claim 8 , wherein the SKM-1 channel further comprises an accessory subunit. 
     
     
         10 . The bypass bridge of  claim 1 , wherein the cells in the tract further functionally express a calcium channel encoded by a nucleic acid that has been introduced into the cells. 
     
     
         11 . The bypass bridge of  claim 1 , wherein the potassium channel comprises the potassium inwardly-rectifying channel 12 (Kir2.1) alpha subunit or potassium inwardly-rectifying channel, subfamily 1, member 12 (Kir2.2) alpha subunit. 
     
     
         12 . The bypass bridge of  claim 11 , wherein the potassium channel further comprises an accessory subunit. 
     
     
         13 . The bypass bridge of  claim 10 , wherein the calcium channel is an L-type calcium channel. 
     
     
         14 . The bypass bridge of  claim 13 , wherein the L-type calcium channel comprises an alpha subunit and accessory subunits. 
     
     
         15 . The bypass bridge of  claim 1 , wherein cells in the tract further functionally express one or more of at least one cardiac connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel. 
     
     
         16 . The bypass bridge of  claim 15 , wherein the at least one connexin is Cx43, Cx40, or Cx45. 
     
     
         17 . The bypass bridge of  claim 2 , wherein the pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or chimera thereof, and (b) a MiRP1 beta subunit. 
     
     
         18 . The bypass bridge of  claim 17 , wherein the pacemaker ion channel is expressed in cells in the first end of the tract. 
     
     
         19 . The bypass bridge of  claim 18 , wherein the cells expressing the pacemaker ion channel are located in a region extending 0.5 mm from the first end. 
     
     
         20 . The bypass bridge of  claim 17 , wherein the chimeric HCN channel provides an improved characteristic, as compared to a wild-type HCN channel, selected from the group consisting of faster kinetics, more positive activation, increased expression, increased stability, preserved or enhanced cAMP responsiveness, and preserved or enhanced neurohumoral response. 
     
     
         21 . The bypass bridge of  claim 17 , wherein the HCN chimera comprises portions derived from more than one HCN channel isoform. 
     
     
         22 . The bypass bridge of  claim 21 , wherein the portions are an amino terminal portion, an intramembranous portion, and a carboxy terminal portion. 
     
     
         23 . The bypass bridge of  claim 21 , wherein at least one portion of the HCN chimera is derived
 from an animal species which is different from the animal species from which at least one of the other two portions is derived.   
     
     
         24 . The bypass bridge of  claim 21 , wherein the HCN chimera is mHCN112, mHCN212, mHCN312, mHCN412, mHCN114, mHCN214, mHCN314, mHCN414, hHCN112, hHCN212, hHCN312, hHCN412, hHCN114, hHCN214, hHCN314, or hHCN414. 
     
     
         25 . The bypass bridge of  claim 24 , wherein the HCN chimera is hHCN212 having the sequence set forth in SEQ ID NO: 2. 
     
     
         26 . The bypass bridge of  claim 24 , wherein the HCN chimera is mHCN212 having the sequence set forth in SEQ ID NO: 6. 
     
     
         27 . The bypass bridge of  claim 21 , wherein at least one portion of the chimera is derived from a HCN channel containing a mutation which provides an improved characteristic, as compared to a wild-type HCN channel, selected from the group consisting of faster kinetics, more positive activation, increased expression, increased stability, preserved or enhanced cAMP responsiveness, and preserved or enhanced neurohumoral response. 
     
     
         28 . The bypass bridge of  claim 17 , wherein the HCN channel is a mutant channel derived from mHCN2 having the sequence set forth in SEQ ID NO: 14 and comprises E324A-mHCN2, Y331A-mHCN2, R339A-mHCN2, or Y331A, E324A-mHCN2. 
     
     
         29 . The bypass bridge of  claim 28 , wherein the mutant HCN channel is E324A-HCN2. 
     
     
         30 . The bypass bridge of  claim 19 , wherein the pacemaker current is conducted by electrotonic conduction. 
     
     
         31 . The bypass bridge of  claim 17 , wherein the pacemaker current is actively propagated by an action potential. 
     
     
         32 . The bypass bridge of  claim 31 , wherein the action potential is a sodium-dependent action potential. 
     
     
         33 . The bypass bridge of  claim 31 , wherein cells in the tract further functionally express an L-type calcium channel and the action potential is a calcium-dependent action potential. 
     
     
         34 . A method of making a bypass bridge for implantation in a heart comprising:
 (a) transfecting a cell with, and functionally expressing therein, a nucleic acid encoding a sodium channel; and   (b) growing the transfected cell into a tract of cells having a first and a second end capable of being attached to two selected sites in the heart, wherein the cells are physically interconnected via electrically conductive gap junctions.   
     
     
         35 . The method of  claim 34  for making a bypass bridge, further comprising transfecting cells in the tract with a nucleic acid encoding a pacemaker ion channel, wherein the nucleic acid is functionally expressed so as to induce a pacemaker current in the cells. 
     
     
         36 . The method of  claim 34  or  35 , wherein the cells are human adult mesenchymal stem cells. 
     
     
         37 . The method of  claim 34  or  35 , further comprising transfecting the cell with, and expressing therein, at least one nucleic acid encoding one or more of at least one cardiac connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel, such that implantation of a bypass bridge in a heart changes the voltage-time course of repolarization and/or refractoriness of the heart. 
     
     
         38 . The method of  claim 41 , wherein the pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or a mutant or chimera thereof, and (b) a MiRP1 beta subunit. 
     
     
         39 . A method of implanting a bypass bridge in a heart comprising:
 (a) making a bypass bridge by the method of  claim 34 ;   (b) selecting a first and a second site in the heart; and   (c) attaching the first end of the tract to the first site and the second end of the tract to the second site; so as to thereby implant a bypass bridge in the heart that allows the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites.   
     
     
         40 . The method of  claim 39 , wherein the a pacemaker and/or electrical signal/current is generated in the atrium by the sinus node or an electronic pacemaker. 
     
     
         41 . The method of  claim 39 , further comprising transfecting cells in the tract with a nucleic acid encoding a pacemaker ion channel, wherein the nucleic acid is functionally expressed so as to induce a pacemaker current in the cells. 
     
     
         42 . The method of  claim 41 , wherein the pacemaker ion channel is expressed in cells in the first end of the tract. 
     
     
         43 . The method of  claim 42 , wherein the cells expressing the pacemaker ion channel are located in a region extending 0.5 mm from the first end. 
     
     
         44 . The method of  claim 39  or  41 , wherein the first site is in an atrium and the second site is in a ventricle, so as to allow propagation of a pacemaker and/or electrical signal/current across the tract from the atrium to the ventricle. 
     
     
         45 . The method of  claim 39  or  41 , wherein the cells are stem cells, cardiomyocytes, fibroblasts or skeletal muscle cells engineered to express connexins, or endothelial cells. 
     
     
         46 . The method of  claim 44 , wherein the stem cells are adult mesenchymal stem cells or embryonic stem cells, wherein said cells are substantially incapable of differentiation. 
     
     
         47 . The method of  claim 46 , wherein the stem cells are human adult mesenchymal stem cells or human embryonic stem cells. 
     
     
         48 . The method of  claim 39  or  41 , further comprising transfecting the cells in the tract with, and expressing therein, at least one nucleic acid encoding one or more of at least one cardiac connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel, so as to change the voltage-time course of repolarization and/or refractoriness of the heart. 
     
     
         49 . The method of  claim 48 , wherein the at least one cardiac connexin is Cx43, Cx40, or Cx45. 
     
     
         50 . The method of  claim 41 , wherein the pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or a mutant or chimera thereof, and (b) a MiRP1 beta subunit. 
     
     
         51 . A method of treating a disorder associated with an impaired conduction in a subject's heart comprising: (a) transfecting a cell with a nucleic acid encoding a sodium channel, wherein the cell functionally expresses the sodium channel; (b) growing the transfected cell into a tract of cells having a first end and a second end, wherein the cells are physically interconnected via electrically conductive gap junctions; (c) selecting a first site and a second site in the heart between which sites conduction is impaired; and (d) so as to allow the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites and thereby treat the subject. 
     
     
         52 . A method of treating a disorder associated with an impaired conduction and impaired sinus node activity in a subject's heart comprising:
 (a) transfecting a cell with at least one nucleic acid encoding a sodium channel and a pacemaker ion channel, wherein the cell functionally expresses the sodium channel and the pacemaker ion channel;   (b) growing the transfected cell into a tract of cells having a first end and a second end, wherein the cells are physically interconnected via electrically conductive gap junctions;   (c) selecting a first site in the left atrium of the heart and a second site, between which sites conduction is impaired; and   (d) attaching the first end of the tract to the first site and the second end of the tract to the second site; so as to allow the propagation of a pacemaker and/or electrical signal/current generated by the sinus node and/or tract of cells between the two sites and thereby treat the subject.   
     
     
         53 . The method of  claim 51  or  52 , wherein the cells are human adult mesenchymal stem cells or human embryonic stem cells, wherein said cells are substantially incapable of differentiation. 
     
     
         54 . The method of  claim 51  or  52 , further comprising transfecting the cells with, and expressing therein, at least one nucleic acid encoding one or more of at least one connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel, so as to change the voltage-time course of repolarization and/or refractoriness of the heart. 
     
     
         55 . The method of  claim 52 , wherein the pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or a mutant or chimera thereof, and (b) a MiRP1 beta subunit. 
     
     
         56 . An atrioventricular bypass tract made by growing mesenchymal stem cells into a strip with two ends and transfecting the mesenchymal stems cells with a nucleic acid encoding as a sodium channel so that the mesenchymal stem cells functionally express the sodium channel, wherein the strip can be attached to a heart so as to create a tract between an atrium of the heart and a ventricle of the heart, which tract is capable of propagating electrical signals.