# Patent

## Title
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.

## Background
This application claims the benefit of each of U.S. Provisional Application Nos. 60/704,210, filed Jul. 29, 2005; 60/701,312, filed Jul. 21, 2005; and 60/781,723, filed Mar. 14, 2006, the entire contents of each of which is incorporated herein by reference.


This invention was made with government support under NIH Grant No. HL-28958 awarded by the National Institutes of Health. The government has certain rights in the invention.


Throughout this application, various publications are referenced in parentheses by author name and date, patent number, patent application or publication number. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to those skilled therein as of the date of the invention described and claimed herein. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention.
FIELD OF THE INVENTION
The present invention relates to a bypass bridge comprising a tract of gap junction-coupled cells that functionally express a sodium channel, the two ends of the tract being attachable to two selected sites in a heart so as to allow the conduction of a pacemaker and/or pacemaker and/or electrical signal/current across the tract between the two sites.
BACKGROUND OF THE INVENTION
Electronic pacemakers are lifesaving devices that provide a regular heartbeat in settings where the sinoatrial node, atrioventricular conduction, or both, have failed. Thus, one of the major indications for electronic pacemaker therapy is high degree heart block, such that a normally functioning sinus node impulse cannot propagate to the ventricle. The result is ventricular arrest and/or fibrillation, and death.
Malfunction or loss of pacemaker cells can occur due to disease or aging. For example, acute myocardial infarction (MI) kills millions of people each year and generally induces in survivors marked reductions in myocyte number and cardiac pump function. Adult cardiac myocytes divide only rarely, and the usual responses to myocyte cell loss include compensatory hypertrophy and/or congestive heart failure, a disease with a significant annual mortality. There have been recent reports of the delivery of bone marrow-derived and/or circulating human mesenchymal stem cells (hMSCs) to the hearts of post-myocardial infarct patients resulting in some improvement of mechanical performance (Strauer et al., 2002; Perin et al., 2003) in the absence of overt toxicity. The presumption in these and other animal studies (Orlic et al., 2001) is that the hMSCs integrate into the cardiac syncytium and then differentiate into new heart cells restoring mechanical function.
An alternative application of cell therapy, described herein, involves growing cells such as hMSCs into a bypass bridge comprising a tract of gap junction-coupled cells that can be used to conduct pacemaker and/or electrical current/signals across a region of the heart exhibiting impaired electrical conduction.
SUMMARY OF THE INVENTION
The invention disclosed herein 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. In certain embodiments of the bypass bridge, cells in the tract further functionally express a pacemaker ion channel which induces a pacemaker current so as to induce a pacemaker current in the cells. In preferred embodiments, the first end of the tract of cells is capable of being attached to the atrium and the second end is capable of being attached to the ventricle, so as to form an atrioventricular (AV) bridge that allows conduction of a pacemaker and/or electrical signal/current across the tract from the atrium to the ventricle.
The invention also provides 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 optionally a nucleic acid encoding a pacemaker ion 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.
The invention further provides a method of implanting a bypass bridge in a heart comprising (a) making a bypass bridge by any of the methods disclosed herein, (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.
The present invention also provides 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) 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 conduction of a pacemaker and/or electrical signal/current across the tract between the two sites and thereby treat the subject.
This invention further provides 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.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Alignment of mouse and human HCN2 polypeptide sequences. The mouse and human HCN2 polypeptide sequences are aligned for maximum correspondence. The amino acid sequences of mHCN2 and hHCN2 are set forth in SEQ ID NO: 14 and SEQ ID NO: 16, respectively.
FIG. 2. Amino acid sequence of the human HCN212 chimeric channel. The shaded N-terminal portion of the sequence is derived from hHCN2; the underlined intramembranous portion from hHCN1; and the C-terminal portion (without shading or underline) from hHCN2. The amino acid sequence of the hHCN212 chimeric channel is set forth in SEQ ID NO: 2. This 889-amino acid long chimeric hHCN212 sequence shows 91.2% identity with the 863-amino acid long mHCN212 sequence in 893 residues overlap when aligned for maximum correspondence.
FIG. 3. Amino acid sequence of the mouse HCN212 chimeric channel. The shaded N-terminal portion of the sequence is derived from mouse HCN2; the underlined intramembranous portion from mouse HCN1; and the C-terminal portion (without shading or underline) from mouse HCN2. The amino acid sequence of the mouse HCN212 chimeric channel is set forth in SEQ ID NO: 6. This 863-amino acid long chimeric mHCN212 sequence shows 91.2% identity with the 889-amino acid long hHCN212 sequence in 893 residues overlap when aligned for maximum correspondence.
FIG. 4. Identification of connexins in gap junctions of human mesenchymal stem cells (hMSCs). Immunostaining of Cx43 (A), Cx40 (B) and Cx45 (C). D, Immunoblot analysis of Cx43 in canine ventricle myocytes and hMSCs. Whole cell lysates (120 μg) from ventricle cells or hMSCs were resolved by SDS, transferred to membranes, and blotted with Cx43 antibodies. Molecular weight markers are indicated.
FIG. 5. Macroscopic and single channel properties of gap junctions between hMSC pairs. Gap junction currents (Ij) elicited from hMSCs using a symmetrical bipolar pulse protocol (10 s, from ±10 mV to ±110 mV, Vh=0 mV) showed two types of voltage-dependent current deactivation: symmetrical (A) and asymmetrical (B). C, summary plots of normalized instantaneous (∘) and steady-state (●) gj versus Vj. Left panel, quasi-symmetrical relationship from 5 pairs; continuous line, Boltzmann fit: Vj,0=−70/65 mV, gj,min=0.29/0.34, gj,max=0.99/1.00, z=2.2/2.3 for negative/positive Vj. Right panel, asymmetrical relationship from 6 pairs; Boltzmann fit for negative Vj: Vj,0=−72 mV, gj,min=0.25, gj,max=0.99, z=1.5. D and E, single channel recordings from pairs of hMSCs. Pulse protocol (V1 and V2) and associated multichannel currents (I2) recorded from a cell pair during maintained Vj of ±80 mV. The discrete current steps indicate the opening and closing of single channels. Dashed line: zero current level. The all points current histograms on the right-hand side reveal a conductance of  ̃50 pS.
FIG. 6. Macroscopic properties of functions in cell pairs between a hMSC and HeLa cell expressing only Cx40, Cx43 or Cx45. In all cases hMSC to Hela cell coupling was tested 6 to 12 after hours initiating co-culture. A, Ij elicited in response to a series of 5-s voltage steps (Vj) in hMSC-HeLaCx43 pairs. Top, symmetrical current deactivation; bottom, asymmetrical current voltage dependence. B, Macroscopic Ij recordings from hMSC-HelaCx40 pairs exhibit symmetrical (top panel) and asymmetrical (bottom panel) voltage dependent deactivation. C, Asymmetric Ij from hMSC-HeLaCx43 pair exhibits voltage dependent gating when Cx45 side is relatively negative. Ij recorded from hMSC. D, gj,ss plots versus Vj from pairs between hMSC and transfected HeLa cells. Left panel, hMSC-HeLaCx43 pairs, quasi-symmetrical relationship (●) and asymmetrical relationship (∘); continuous and dashed lines are Boltzmann fits (see text for details). Middle panel, symmetrical (●) and asymmetrical (∘) relationships from hMSC-HeLaCx40 pairs; the continuous and dashed lines correspond to Boltzmann fits (see text for details). Right panel, asymmetrical relationship from hMSC-HeLaCx45 cell pairs; continuous line, Boltzmann fit for positive Vj (see text for details). E, Cell-to-cell Lucifer Yellow (LY) spread in cell pairs: from an hMSC to an hMSC (upper panel), from a HeLaCx43 to an hMSC (middle panel), and from an hMSC to a HeLaCx43 (bottom panel). In all cases a pipette containing 2 mM LY was attached to the left-hand cell in the whole-cell configuration. Epifluorescent micrographs taken at 12 min after dye injection show LY spread to the adjacent (right-hand) cell. The simultaneously measured junctional conductance revealed gj of  ̃13 nS,  ̃16 nS, and  ̃18 nS of the pairs, respectively. Cell Tracker green was used to distinguish hMSCs from HeLa cells or vice versa in all experiments.
FIG. 7. Macroscopic and single channel properties of gap junctions between hMSC-canine ventricle cell pairs. Myocytes were plated between 12 and 72 h and co-cultured with hMSCs for 6 to 12 h before measuring coupling. A, Localization of Cx43 for hMSC-canine ventricle cell pairs. Most of Cx43 was localized to the ventricular cell ends and a small amount of Cx43 was present along the lateral borders. The intensive Cx43 staining was detected between the end of the rod-shaped ventricular cell (middle cell) and the hMSC (right cell). There is no detectable Cx43 staining between the ventricular cell and the hMSC on the left side. B, Top, phase-contrast micrograph of a hMSC-canine ventricular myocyte pair. Bottom, monopolar pulse protocol (V1 and V2) and associated macroscopic junctional currents (I2) exhibiting asymmetrical voltage dependence. C, Top, multichannel current elicited by symmetrical biphasic 60 mV pulse. Dashed line, zero current level; dotted lines, represent discrete current steps indicative of opening and closing of channels. The current histograms yielded a conductance of 40-50 pS. Bottom, multichannel recording during maintained Vj of 60 mV. The current histograms revealed several conductances of 48-64 pS with several events with conductance of 84 pS to 99 pS (arrows) which resemble operation of Cx43, heterotypic Cx40-Cx43 and/or homotypic Cx40 channels.
FIG. 8. Inactivation properties of a sodium channel suitable for incorporation into a cardiac bypass bridge. The inactivation properties of the SKM-1 sodium channel measured in Xenopus oocytes are shown. Left, The holding potential ranged from −100 mV to −55 mV and the test potential was +30 mV. Right, averaged inactivation curves for 11 oocytes.
FIG. 9. I-V relationship of a sodium channel suitable for incorporation into a cardiac bypass bridge. Shown here is I-V relationship for the SKM-1 channel recorded in Xenopus oocytes. Left, Raw data recorded from a holding potential of −80 mV to potentials between −70 mV and +50 mV in 10 mV increments. Right, Peak inward I-V relationship.
FIG. 10. Biophysical properties of a calcium channel suitable for incorporation into a cardiac bypass bridge. CaV1.2, alpha2 and gamma, P1.b and red fluorescent reporter were co-expressed in HEK293 cells. Ca2+ current was recorded in the red fluorescent cells. The cells were held at −50 mV. Test pulses (60 ms) were from −40 mV to +50 mV with a 10 mV interval.



DETAILED DESCRIPTION OF THE INVENTION
The present 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. In certain embodiments of the bypass bridge, cells in the tract further functionally express a pacemaker ion channel which induces a pacemaker current so as to induce a pacemaker current in the cells. In preferred embodiments, the first end of the tract of cells is capable of being attached to the atrium and the second end is capable of being attached to the ventricle, so as to form an atrioventricular (“AV”) bridge that allows conduction of a pacemaker and/or electrical signal/current across the tract from the atrium to the ventricle and thereby stimulate contraction of the ventricle.
The invention also 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 potassium channel or calcium channel instead of a sodium channel. The embodiments disclosed herein for the bypass bridge expressing a sodium channel are equally applicable to the bypass bridge expressing a potassium or calcium channel.
Exemplary cells that may be used for growing the tract include, but are not limited to, stem cells, cardiomyocytes, fibroblasts or skeletal muscle cells engineered to express at least one cardiac connexin, or endothelial cells. In preferred embodiments, the stem cells are adult mesenchymal stem cells (MSCs) or embryonic stem cells (ESCs), wherein said stem cells are substantially incapable of differentiation. In various embodiments, the MSCs are autologous, allogeneic or heterogenic relative to the subject into whose heart the bypass bridge is to be introduced, and the subject is a human being. As used herein, a “subject” shall mean any animal or artificially modified animal. Animals include, but are not limited to, humans, non-human primates, dogs, cats, cows, horses, sheep, pigs, rabbits, ferrets, rodents such as mice, rats and guinea pigs, and birds such as chickens and turkeys. Artificially modified animals include, but are not limited to, SCID mice with human immune systems. In a preferred embodiment, the subject is a human.
In more preferred embodiments of the instant bypass bridge, the stem cells are human adult mesenchymal stem cells (hMSCs) or human embryonic stem cells (hESCs), wherein the stem cells are substantially incapable of differentiation. In other preferred embodiments, the hMSCs (a) express CD29, CD44, CD54, and HLA class I surface markers; and (b) do not express CD14, CD34, CD45, and HLA class II surface markers. In additional embodiments, the hMSCs have been passaged at least 9 times, preferably 9-12 times. In further embodiments, the cells further express at least one cardiac connexin. In still further embodiments, the at least one cardiac connexin is Cx43, Cx40, or Cx45.
Thus, the present invention is based on the use of cell therapy to construct in the heart a bypass tract incorporating functional sodium ion channels, and optionally pacemaker channels, and optionally or alternatively potassium or calcium channels. The bypass bridge can be used to conduct a pacemaker and/or electrical signal/current between any two sites in the heart between which there is impaired pacemaker and/or electrical conduction. For example, the bypass may be used as an AV bridge to take over or supplement the function of a diseased AV node. Human MSCs may be grown in culture on a non-bioreactive matrix into a strip or tract of cells containing two ends. Once growth is complete, one end of the tract may be attached, e.g., by sutures, to a first selected site in the heart such as an atrium, and the other end may attached to a second selected site such as a ventricle. In a bypass bridge used as an AV bridge, for example, pacemaker and/or electrical signals/current generated by the sinus node to activate the atria will propagate across the artificially constructed tract to excite the ventricle. In this way the normal sequence of atrioventricular activation will be maintained.
Human MSCs may be prepared in several ways including, but not limited to, the following:
1: In culture without incorporation of additional molecular determinants of conduction. Here the cells' own ability to form gap junctions that communicate pacemaker and/or electrical signals are used as a means to conduct an electrotonic wave from one site in the heart to another.
2: In culture following transfection of the cells, such as by electroporation or viral infection, to introduce a gene for at least one of the cardiac connexins Cx43, Cx40 or Cx45, to enhance formation of gap junctions and thereby facilitate cell-to-cell propagation of pacemaker/electrical signals.
3: In culture following transfection of the cells to introduce a nucleic acid encoding the alpha and the accessory subunits of an L-type calcium channel, thereby increasing the likelihood of not just electrotonic propagation of a wavefront, but its active propagation by an action potential.
4: In culture following transfection of the cells to introduce a nucleic acid encoding the alpha subunit, with or without the accessory subunits, of a sodium channel, thereby increasing the likelihood of not just electrotonic propagation or calcium-dependent propagation of a wavefront, but its active propagation by a sodium-dependent action potential.
5: In culture following transfection of the cells to introduce a nucleic acid encoding the calcium and/or sodium channel subunits as in 3 and/or 4, and a nucleic acid encoding an alpha subunit, with or without the accessory subunits of a potassium channel, thereby increasing the likelihood of not just active propagation by an action potential, but additional control of the initial resting potential and its voltage-time course of repolarization and refractoriness.
6: Combinations of steps 2 through 5.
The preparation of a bypass bridge from hMSCs thus prepared allows conduction of pacemaker and/or electrical signals/current between any two selected sites in the heart. In the case of an AV bridge, it will not only will facilitate propagation of signals from atrium to ventricle, but provides sufficient delay from atrial to ventricular contraction to maximize ventricular filling and emptying, thus mimicking the normal activation and contractile sequence of the heart. Moreover, the cells in the bypass bridge, preferably cells at one end of the tract, may also be further transfected with a nucleic acid encoding a pacemaker current channel, wherein said transfected cells express a pacemaker current. Increases or decreases in calcium current, sodium current, potassium current or pacemaker current (If) may be obtained by increasing or reducing in the cells the expression of the ion channels carrying these currents. These approaches, when used with gene therapy and stem cell technology to improve atrial impulse initiation in the setting of sinus node disease offer a completely physiologic system rather than its electronic replacement. Thus, in embodiments of this invention, the bypass bridge expresses one or more genes encoding a pacemaker ion channel and thereby induces a pacemaker current.
In different embodiments of this invention, the nucleic acid encoding the ion channel or connexin gene is introduced into the cell by infection with a viral vector, plasmid transformation, cosmid transformation, electroporation, lipofection, transfection using a chemical transfection reagent, heat shock transfection, or microinjection. In further embodiments, the viral vector is an adenoviral, an adeno-associated viral (AAV), or a retroviral vector.
In embodiments of the instant bypass bridge, the sodium channel is a SKM-1 channel. In certain embodiments, the SKM-1 channel comprises an alpha subunit. In other embodiments, the SKM-1 channel further comprises an accessory subunit. In additional embodiments, the tract further functionally expresses a potassium channel. The potassium channel may comprise a Kir2.1 or Kir2.2 alpha subunit, and may further comprise an accessory subunit. In different embodiments of the bypass bridge, cells in the tract further functionally express an L-type calcium channel, which may comprise an alpha subunit and accessory subunits. In further embodiments, cells in the tract forming the bypass bridge 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, so as to change the voltage-time course of repolarization and/or refractoriness of the heart. In various embodiments, the at least one cardiac connexin is Cx43, Cx40, or Cx45.
In various embodiments of the bypass bridge that expresses a pacemaker ion channel, said 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. The HCN channel may be any of HCN1, HCN2, HCN3 or HCN4. In embodiments of this invention, the pacemaker channels are engineered to operate at progressively slower rates, so as to equate to primary and subsidiary pacemakers similar to the sinus node and Purkinje system. In preferred embodiments, the pacemaker ion channel is expressed in cells in the first end of the tract. In more preferred embodiments, the cells expressing the pacemaker ion channel are located in a region extending 0.5 mm from the first end.
Hyperpolarization-activated cation currents, termed If, Ih, or Iq, were initially discovered in heart and nerve cells over 20 years ago (for review, see DiFrancesco, 1993; Pape, 1996). These currents, carried by Na+ and K+ ions, contribute to a wide range of physiological functions, including cardiac and neuronal pacemaker activity, the setting of resting potentials, input conductance and length constants, and dendritic integration (see Robinson and Siegelbaum, 2003; Biel et al., 2002). The hyperpolarization-activated, cyclic nucleotide-gated (HCN) family of ion channel subunits has been identified by molecular cloning (for review, see Clapham, 1998; Santoro and Tibbs, 1999; Biel et al., 2002), and when heterologously expressed, each of the four different HCN isoforms generates channels with the principal properties of native If, confirming that HCN channels are the molecular correlate of this current.
As used herein, a “HCN channel” shall mean a hyperpolarization-activated, cyclic nucleotide-gated ion channel responsible for the hyperpolarization-activated cation currents that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. There are four HCN isoforms: HCN1, HCN2, HCN3 and HCN4. All four isoforms are expressed in brain; HCN1, HCN2 and HCN4 are also prominently expressed in heart, with HCN4 and HCN1 predominating in sinoatrial node and HCN2 in the ventricular specialized conducting system. “mHCN” designates murine or mouse HCN; “hHCN” designates human HCN.
HCN channels, similar to voltage-gated K+ (Kv) channels, have four subunits, each of which has six transmembrane segments, S1-S6: the positively charged S4 domain forms the major voltage sensor, whereas S5 and S6, together with the S5-S6 linker connecting the two, form the pore domain containing the ion permeation pathway and the gates that control the flow of ions (Larsson, 2002). Mutational studies on HCN channels indicate that mutations in the S4 voltage sensor, the S4-S5 linker implicated in the coupling of voltage sensing to pore opening and closing, the S5, S6 and S5-S6 linker which form the pore, the C-linker, and the C-terminal cyclic nucleotide binding domain (CNBD), may be particularly important in affecting HCN channel activity. In embodiments of the bypass bridge expressing a mutant HCN, said mutant 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. Mutant HCN channels for inducing pacemaker activity in cells is also described in U.S. Provisional Application Nos. 60/781,723 (filed Mar. 14, 2006) and 60/832,515, entitled “Chimeric HCN Channels,” which is being filed concurrently with the subject application (Jul. 21, 2006). Mutant HCN channels are also discussed in U.S. application Ser. No. 10/342,506. The preceding applications are herein incorporated by reference in their entirety.
In certain embodiments of the present invention, the mutant HCN channel carries at least one mutation in S4 voltage sensor, the S4-S5 linker, S5, S6, the S5-S6 linker, and/or the C-linker, and the CNBD which mutations result in one or more of the above discussed improved characteristics. In other embodiments, the HCN mutant is E324A-HCN2, Y331A-HCN2, R339A-HCN2, or Y331A, E324A-HCN2. In preferred embodiments, the mutant HCN channel is E324A-HCN2.
In addition to the mutations noted above, many mutations in different HCN isoforms have been reported. These include R318Q, W323A, E324A, E324D, E324K, E324Q, F327A, T330A and Y331A, Y331D, Y331F, Y331K, D332A, M338A, R339A, R339C, R339D, R339E and R339Q in HCN2 made by Chen et al. (2001a) to investigate in greater detail the role of the E324, Y331 and R339 residues in voltage sensing and activation. Chen et al. (2001b) have also reported the R538E and R591E mutations in mHCN1; Tsang et al. (2004) have reported G231A and M232A in mHCN1; Vemana et al (2004) have reported R247C, T249C, K250C, I251C, L252C, S253C, L254C, L258C, R259C, L260C, S261C, C318S, S338C in mHCN2; Macri and Accili (2004) have reported S306Q, Y331D AND G404S in mHCN2; and Decher et al. (2004) have reported Y331A, Y331D, Y331S, R331FD, R339E, R339Q, I439A, S441A, S441T, D443A, D443C, D443E, D443K, D443N, D443R, R447A, R447D, R447E, R447Y, Y449A, Y449D, Y449F, Y449G, Y449W, Y453A, Y453D, Y453F, Y453L, Y453W, P466Q, P466V, Y476A, Y477A and Y481A in mHCN2. The entire contents of all of the above publications are incorporated herein by reference. Certain of the reported mutations listed above may confer, singly or in combination, beneficial characteristics on the HCN channel with regard to creating a biological pacemaker. The invention disclosed herein encompasses all mutations in HCN channels, singly or in combinations, which improve pacemaker activity of the channel such as by providing faster kinetics, more positive activation, increased expression and/or stability, preserved cyclic interval responsiveness, and/or preserved or enhanced neurohumoral response.
Mutations are identified herein by a designation with provides the single letter abbreviation of the amino acid residue that underwent mutation, the position of that residue within a polypeptide, and the single letter abbreviation of the amino acid residue to which the residue was mutated. Thus, for example, E324A identifies a mutant polypeptide in which the glutamate residue (E) at position 324 was mutated to alanine (A). Y331A, E324A-HCN2 indicates a mouse HCN2 having a double mutation, one in which tyrosine (Y) at position 331 was mutated to alanine (A), and the other in which the glutamate residue at position 324 was mutated to alanine.
In general terms, HCN polypeptides can be divided into three major domains: (1) a cytoplasmic amino terminal domain; (2) the membrane spanning domains and their linking regions; and (3) a cytoplasmic carboxy-terminal domain. The N-terminal domain does not appear to play a major role in channel activation (Biel et al., 2002). However, the membrane spanning domains with their linking regions play an important role in determining the kinetics of gating, whereas the CNBD is largely responsible for the ability of the channel to respond to the sympathetic and parasympathetic nervous systems that respectively raise and lower cellular cAMP levels.
In embodiments of the bypass bridge expressing a HCN chimera, the chimeric HCN channel preferably 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. HCN chimeras for inducing pacemaker activity in cells is described in detail in U.S. Provisional Application No. 60/715,934 (filed Sep. 9, 2005) and 60/832,515, entitled “Chimeric HCN Channels,” which is being filed concurrently with the subject application (Jul. 21, 2006), both of which are herein incorporated by reference in their entirety.
As used herein, a “HCN chimera” or “chimeric HCN channel” shall mean a HCN channel comprising portions of more than one HCN channel isoform. Thus, a chimera may comprise portions of HCN1 and HCN2 or HCN3 or HCN4, and so forth. In preferred embodiments, the portions are an amino terminal portion, an intramembranous portion, and a carboxy terminal portion. In other preferred embodiments, the portions are derived from human HCN isoforms. In addition, an HCN chimera encompasses an ion channel comprising portions of HCN channels derived from different animal species. Accordingly, in various embodiments of the bypass bridge, 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. For example, one portion of the channel may be derived from a human and another portion may be derived from a non-human.
In additional embodiments of the instant bypass bridge, the HCN chimera is mHCN112, mHCN212, mHCN312, mHCN412, mHCN114, mHCN214, mHCN314, mHCN414, hHCN112, hHCN212, hHCN312, hHCN412, hHCN114, hHCN214, hHCN314, or hHCN414. In different embodiments, the HCN chimera is mHCN112, mHCN212, mHCN312, mHCN412, mHCN114, mHCN214, mHCN314, mHCN414, hHCN112, hHCN212, hHCN312, hHCN412, hHCN114, hHCN214, hHCN314, or hHCN414. In a preferred embodiment, the HCN chimera is hHCN212 having the sequence set forth in SEQ ID NO: 2 (see FIG. 2). In yet another preferred embodiment, the HCN chimera is mHCN212 having the sequence set forth in SEQ ID NO: 6 (see FIG. 3).
As used herein, the term “HCNXYZ” (wherein X, Y and Z are any one of the integers 1, 2, 3 or 4, with the proviso that at least one of X, Y and Z is a different number from at least one of the other numbers) shall mean a chimeric HCN channel polypeptide comprising three contiguous portions in the order XYZ, wherein X is an N-terminal portion, Y is an intramembrane portion, and Z is a C-terminal portion, and wherein the number X, Y or Z designates the HCN channel from which that portion is derived. For example, HCN112 is an HCN chimera with a N-terminal portion and intramembrane portion from HCN1 and a C-terminal portion from HCN2.
In certain embodiments of the bypass bridge, the HCN chimera comprises an amino terminal portion contiguous with an intramembrane portion contiguous with a carboxy terminal portion, wherein each portion is a portion of an HCN channel or a portion of a mutant thereof, and wherein one portion derives from an HCN channel or a mutant thereof which is different from the HCN channel or mutant thereof from which at least one of the other two portions derive. Thus, in various embodiments, 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 portion from 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. In certain embodiments, the mutant HCN channel contains a mutation in a region of the channel selected from the group consisting of the S4 voltage sensor, the S4-S5 linker, S5, S6 and S5-S6 linker, the C-linker, and the CNBD. In other embodiments, the mutant portion is derived from mHCN2 having the sequence set forth in SEQ ID NO: 14 (see FIG. 1) and comprises E324A-mHCN2, Y331A-mHCN2, R339A-mHCN2, or Y331A,E324A-mHCN2. In preferred embodiments, the mutant portion comprises E324A-mHCN2.
The pacemaker activity of a HCN channel may be enhanced by co-expressing the HCN channel with its beta subunit, MiRP1, which increases the magnitude of the current expressed and/or speeds its kinetics of activation. See U.S. Pat. No. 6,783,979 and Qu et al. (2004), the entire contents of which are incorporated herein by reference.
In certain embodiments of the bypass bridge disclosed herein, the pacemaker current is conducted by electrotonic conduction. In other embodiments, the pacemaker current is actively propagated by an action potential. In further embodiments, the action potential is a sodium-dependent action potential. In additional embodiments, cells in the tract further functionally express an L-type calcium channel and the action potential is a calcium-dependent action potential.
The present invention also provides a kit comprising a packaging material containing therein (a) any of the bypass bridges disclosed herein. The kit may also contain labeling and instructions for implanting the bypass bridge in a heart, so as to thereby treat a disorder associated with impaired conduction, or a disorder associated with both impaired conduction and impaired sinus node activity, in a subject's heart in a subject's heart.
This invention also provides a tandem pacemaker system comprising (1) any of the bypass bridges disclosed herein, and (2) an electronic pacemaker and/or a biological pacemaker, wherein the bypass bridge operates in tandem with the electronic and/or biological pacemaker to more effectively treat a cardiac rhythm condition compared to the use of the biological or electronic pacemaker alone. In various embodiments, the bypass bridge is an AV bridge. The biological pacemaker or electronic pacemaker may have a pacing level at, around, above or below the normal resting sinus node pacemaker level. In embodiments where a biological pacemaker or electronic pacemaker is used in tandem with an AV bridge described herein, the natural AV node may be ablated. Further details of tandem pacemaker systems comprising a bypass bridge may be found in U.S. Provisional Application Nos. 60/701,312 (filed Jul. 21, 2005) and 60/781,723 (filed Mar. 14, 2006), and U.S. Ser. No. 11/490,997, filed concurrently herewith on Jul. 21, 2006, entitled “Tandem Cardiac Pacemaker System.” The proceeding applications are herein incorporated by reference in their entirety.
The invention also provides 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. An embodiment of this method further comprises 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. In preferred embodiments, the pacemaker ion channel is expressed in cells in the first end of the tract. In more preferred embodiments, the cells expressing the pacemaker ion channel are located in a region extending 0.5 mm from the first end. In these embodiments, the transfection method used to introduce the pacemaker channel gene(s) to the first end may be locally applied, or separated from the distal portions of the tract using physical barriers, in order to restrict transfection to first end portion.
In various embodiments, 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. In preferred embodiments of the instant methods, the cells are hMSCs or hESCs, wherein said cells are substantially incapable of differentiation. In further embodiments, the human adult mesenchymal stem cells (a) express CD29, CD44, CD54, and HLA class I surface markers; and (b) do not express CD14, CD34, CD45, and HLA class II surface markers. In still further embodiments, the human adult mesenchymal stem cells have been passaged at least 9 times.
Additional embodiments of the instant methods for making a bypass bridge further comprise 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.
Variations of the instant methods for making a bypass bridge comprise (a) growing a 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, before (b) transfecting the cells in the tract with, and functionally expressing therein, a nucleic acid encoding a sodium channel, and optionally other ion channel genes and/or a pacemaker channel gene.
This invention further provides a method of implanting a bypass bridge in a heart comprising (a) making a bypass bridge comprising a tract of cells by any of the methods disclosed herein, (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. Other embodiments further comprise 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. In preferred embodiments, the pacemaker ion channel is expressed in cells in the first end of the tract. In more preferred embodiments, the cells expressing the pacemaker ion channel are located in a region extending 0.5 mm from the first end. In other preferred embodiments, 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. In different embodiments of the instant methods, the a pacemaker and/or electrical signal/current is generated in the atrium by the sinus node, an electronic pacemaker, a biological pacemaker, or cells within the bypass bridge expressing a pacemaker current. In further embodiments, 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.
In additional embodiments of the instant methods, the cells in the tract of the bypass bridge are stem cells, cardiomyocytes, fibroblasts or skeletal muscle cells engineered to express at least one cardiac connexins, or endothelial cells. In various embodiments, the stem cells are adult MSCs or ESCs, wherein said cells are substantially incapable of differentiation. In preferred embodiments, the stem cells are hMSCs or hESCs, wherein said stem cells are substantially incapable of differentiation. Other embodiments further comprise transfecting the cells 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. In further embodiments, the at least one connexin is Cx43, Cx40, or Cx45.
The present invention also provides 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 subject's heart 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 conduction of a pacemaker and/or electrical signal/current across the tract between the two sites and thereby treat the subject.
As used herein, “treating” a disorder shall mean causing the subject afflicted with the disorder to experience a reduction, remission or regression of the disorder and/or its symptoms. In various embodiments, recurrence of the disorder and/or its symptoms is prevented. In preferred embodiments, the subject is cured of the disorder and/or its symptoms. To “functionally express” a nucleic acid shall mean that a cell or other biological system into which the nucleic acid has been introduced produces a functional polypeptide encoded by the nucleic acid. The encoded polypeptide itself may also be said to be functionally expressed.
This invention further provides 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.
In embodiments of the instant methods, the cells are hMSCs or hESCs, wherein said stem cells are substantially incapable of differentiation. Various embodiments further comprise 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. In further embodiments, 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.
The present invention encompasses a variety of kits comprising the bypass bridge of the instant invention, as well as methods for making a bypass bridge, methods of implanting a bypass bridge in a heart, and methods of treating a disorder, corresponding to each of the different embodiments of the bypass bridge disclosed herein. These variant kits and methods are not all individually described in detail herein but will be readily evident and understood to one of ordinary skill in the art based on the disclosure of the different embodiments of the bypass bridge. Accordingly, the various embodiments described herein may be equally applied as appropriate to the bypass bridges, kits and methods of this invention.
The following Examples are presented to aid in understanding the invention, and are not intended, and should not be construed, to limit in any way the invention set forth in the claims which follow thereafter. These Examples do not include detailed descriptions of experimental methods that are well known to those of ordinary skill in the art, such as methods used in the construction of recombinant nucleic acid vectors, transfection of host cells with such recombinant vectors, and the functional expression of genes in transfected cells. Detailed descriptions of such conventional methods are provided in numerous publications, including Sambrook et al. (1989), the contents of which are hereby incorporated herein in their entirety.
EXAMPLE 1
Culture of Gap Junction-Coupled Human Mesenchymal Stem Cells
Cell Cultures
Human mesenchymal stem cells (hMSCs; mesenchymal stem cells, human bone marrow; PoieticsTM) were purchased from Clonetics/BioWhittaker (Walkersville, Md., USA), cultured in mesenchymal stem cell (MCS) growth medium and used from passages 2-4. Isolated and purified hMSCs can be cultured for many passages (12) without losing their unique properties, i.e., normal karyotype and telomerase activity (van den Bos et al., 1997; Pittenger et al., 1999).
HeLa cells transfected with rat Cx40, rat Cx43 or mouse Cx45 were cocultured with hMSCs. Production, characterization and culture conditions of transfected HeLa cells have been previously described (Elfgang et al., 1995; Valiunas et al., 2000; 2002).
Anti-Connexin Antibodies, Immunofluorescent Labeling, and Immunoblot Analysis
Commercially available mouse anticonnexin monoclonal and polyclonal antibodies (Chemicon International, Temecula, Calif.) of Cx40, Cx43 and Cx45 were used for immunostaining and immunoblots as described earlier (Laing and Beyer, 1995). Fluorescein-conjugated goat antimouse or antirabbit IgG (ICN Biomedicals, Inc., Costa Mesa, Calif.) was used as secondary antibody.
Electrophysiological Measurements Across Gap Junctions
Glass coverslips with adherent cells were transferred to an experimental chamber perfused at room temperature ( ̃22° C.) with bath solution containing (mM): NaCl, 150; KCl, 10; CaCl2, 2; Hepes, 5 (pH 7.4); glucose, 5. The patch pipettes were filled with solution containing (mM): potassium aspartate, 120; NaCl, 10; MgATP, 3; Hepes, 5 (pH 7.2); EGTA, 10 (pCa  ̃8); filtered through 0.22 μm pores. When filled, the resistance of the pipettes measured 1-2 MΩ. Experiments were carried out on cell pairs using a double voltage-clamp. This method permitted control of the membrane potential (Vm) and measurement of the associated junctional currents (Ij).
Dye Flux Studies
Dye transfer through gap junction channels was investigated using cell pairs. Lucifer Yellow (LY; Molecular Probes) was dissolved in the pipette solution to reach a concentration of 2 mM. Fluorescent dye cell-to-cell spread was imaged using a 16 bit 64 000 pixel grey scale digital CCD-camera (LYNXX 2000T, SpectraSource Instruments, Westlake Village, Calif.) (Valiunas et al., 2002). In experiments with heterologous pairs, LY was always injected into the cells which were tagged with Cell Tracker Green. The injected cell fluorescence intensity derived from LY is 10-15 times higher than the initial fluorescence from Cell Tracker Green.
Human MSCs Express Connexins
The connexins, Cx43 and Cx40, were immunolocalized, as evidenced by typical punctate staining, along regions of intimate cell-to-cell contact and within regions of the cytoplasm of the hMSCs grown in culture as monolayers (FIGS. 4A, B). Cx45 staining was also detected, but unlike that of Cx43 or Cx40, was not typical of connexin distribution in cells. Rather, it was characterized by fine granular cytoplasmic and reticular-like staining with no readily observed membrane-associated plaques (FIG. 4C). This does not exclude the possibility that Cx45 channels exist but does imply that their number relative to Cx43 and Cx40 homotypic, heterotypic and heteromeric channels is low. FIG. 4D illustrates Western blot analysis for canine ventricle myocytes and hMSCs with a Cx43 polyclonal antibody which adds further proof of Cx43 presence in hMSCs.
Gap Junctional Coupling Between hMSCs and Various Cell Lines
Gap junctional coupling among hMSCs is demonstrated in FIG. 5. Junctional currents recorded between hMSC pairs show quasi-symmetrical (FIG. 5A) and asymmetrical (FIG. 5B) voltage dependency arising in response to symmetrical 10-s transjunctional voltage steps (Vj) of equal amplitude but opposite sign starting from ±10 mV to ±110 mV using increments of 20 mV. These behaviors are typically observed in cells which co-express Cx43 and Cx40 (Valiunas et al., 2001).
FIG. 5C summarizes the data obtained from hMSC pairs. The values of normalized instantaneous (gj,inst, ∘) and steady state conductances (gj,ss, ●) (determined at the beginning and at the end of each Vj step, respectively) were plotted versus Vj. The left panel shows a quasi-symmetrical relationship from five hMSC pairs. The continuous curves represent the best fit of data to the Boltzmann equation with the following parameters: half-deactivation voltage, Vj,0=−70/65 mV; minimum gj, gj,min=0.29/0.34; maximum gj, gj,max=0.99/1.00; gating charge, z=2.2/2.3 for negative/positive Vj, respectively. Summarized plots from six asymmetrical cases are shown in the right panel. The gj,ss declined in sigmoidal fashion at negative Vj and showed a reduced voltage sensitivity to positive Vj. Boltzman fitting for negative Vj revealed the following values: Vj,0=−72 mV, gj,min=0.25, gj,max=0.99, z=1.5.
FIGS. 5D and E illustrate typical multichannel recordings from a hMSC pair. Using 120 mM K aspartate as a pipette solution, channels were observed with unitary conductances of 28-80 pS range. Operation of channels with  ̃50 pS conductance (see FIG. 5D) is consistent with previously published values (Valiunas et al., 1997; 2002) for Cx43 homotypic channels. This does not preclude the presence of other channel types, it merely suggests that Cx43 forms functional channels in hMSCs.
To further define the nature of the coupling, hMSCs were co-cultured with human HeLa cells stably transfected with Cx43, Cx40, and Cx45 (Elfgang et al., 1995) and it was found that hMSCs were able to couple to all these transfectants. FIG. 6A shows an example of junctional currents recorded between an hMSC and HeLaCx43 cell pairs that manifested symmetrically and asymmetrically voltage dependent currents in response to a series (from +10 mV to +110 mV) of symmetrical transjunctional voltage steps (Vj). The quasi-symmetric record suggests that the dominant functional channel is homotypic Cx43 while the asymmetric record suggests the activity of another connexin in the hMSC (presumably Cx40 as shown by immunohistochemistry, see FIG. 4) that could be either a heterotypic or heteromeric form or both. These records are similar to those published for transfected cells: heterotypic and mixed (heteromeric) forms of Cx40 and Cx43 (Valiunas et al., 2000; 2001). Co-culture of hMSCs with HeLa cells transfected with Cx40 (FIG. 6B) also revealed symmetric and asymmetric voltage dependent junctional currents consistent with the co-expression of Cx43 and Cx40 in the hMSCs similar to the data for Cx43 HeLa-hMSC pairs. HeLa cells transfected with Cx45 coupled to hMSCs always produced asymmetric junctional currents with pronounced voltage gating when Cx45 (HeLa) side was negative (FIG. 6C). This is consistent with the dominant channel forms in the hMSC being Cx43 and Cx40 as both produce asymmetric currents when they form heterotypic channels with Cx45 (Valiunas et al., 2000; 2001). This does not exclude Cx45 as a functioning channel in hMSCs but it does indicate that Cx45 is a minor contributor to cell to cell coupling in hMSCs. The lack of visualized plaques in the immunostaining for Cx45 (FIG. 7) further supports this interpretation.
The summarized plots of gj,ss versus Vj from pairs between hMSC and transfected HeLa cells are shown in FIG. 6D. The left panel shows the results from hMSC-HeLaCx43 pairs. For symmetrical data (●, four preparations), Boltzmann fits (continuous lines) yielded the following parameters: Vj,0=−61/65 mV, gj,min=0.24/0.33, gj,max=0.99/0.99, z=2.4/3.8 for negative/positive Vj. For asymmetrical data (∘, three preparations), the Boltzmann fit (dashed line) at negative Vj values revealed the following parameter values: Vj,0=−70 mV, gj,min=0.31, gj,max=1.00, z=2.2. The middle panel shows data from hMSC-HeLaCx40 pairs including three symmetrical (●) and two asymmetrical (∘) gj,ss-Vj relationships. The continuous lines correspond to a Boltzmann fit to symmetrical data (Vj,0=−57/76 mV, gj,min=0.22/0.29, gj,max=1.1/1.0, z=1.4/2.3; negative/positive Vj) and the dashed line is a fit to the asymmetrical data (Vj,0=−57/85 mV, gj,min=0.22/0.65, gj,max=1.1/1.0, z=1.3/2.2; negative/positive Vj). The data from the six complete experiments from hMSC-HeLaCx45 cell pairs are shown on the right panel. The gj,ss plot versus Vj was strongly asymmetrical and the best fit of the data to the Boltzmann equation at positive Vj values revealed following parameter values: Vj,0=31 mV, gj,min=0.07, gj,max=1.2, z=1.8.
FIG. 6E shows Lucifer Yellow transfer from an hMSC to an hMSC (upper panel), from a HeLaCx43 to an hMSC (middle panel), and from an hMSC to a HeLaCx43 (bottom panel). The junctional conductance of the cell pairs was simultaneously measured by methods described earlier (Valiunas et al., 2002) and revealed conductances of  ̃13,  ̃16 and  ̃18 nS, respectively. The transfer of Lucifer Yellow was similar to that previously reported for homotypic Cx43 or co-expressed Cx43 and Cx40 in HeLa cells (Valiunas et al., 2002). Cell Tracker Green (Molecular Probes) was always used in one of the two populations of cells to allow heterologous pairs to be identified (Valiunas et al., 2000). Lucifer Yellow was always delivered to the cell containing cell tracker. The fluorescence intensity generated by the Cell Tracker Green was 10-15 times less than fluorescence intensity produced by the concentration of Lucifer Yellow delivered to the source cell.
Human MSCs were also co-cultured with adult canine ventricular myocytes as shown in FIG. 7. Immunostaining for Cx43 was detected between the rod-shaped ventricular myocytes and hMSCs as shown in FIG. 7A. The hMSCs couple electrically with cardiac myocytes. Both macroscopic (FIG. 7B) and multichannel (FIG. 7C) records were obtained. Junctional currents in FIG. 7B are asymmetrical while those in FIG. 7C show unitary events of the size range typically resulting from the operation of homotypic Cx43 or heterotypic Cx43-Cx40 or homotypic Cx40 channels (Valiunas et al., 2000; 2001). Heteromeric forms are also possible whose conductances are the same or similar to homotypic or heterotypic forms.
The studies of cell pairs have demonstrated effective coupling of hMSC to other hMSC (13.8±2.4 nS, n=14), to HeLaCx43 (7.9±2.1 nS, n=7), to HeLaCx40 (4.6±2.6 nS, n=5), to HeLaCx45 (11±2.6 nS, n=5), and to ventricular myocytes (1.5±1.3 nS, n=4).
These data suggest that MSCs should readily integrate into electrical syncytia of many tissues, promoting repair or serving as the substrate for a therapeutic delivery system. In particular, the data support the possibility of using hMSCs as a therapeutic substrate for repair of cardiac tissue. Other syncytia such as vascular smooth muscle or endothelial cells should also be able to couple to the hMSCs because of the ubiquity of Cx43 and Cx40 (Wang et al., 2001; Beyer, 1993). Thus, they may also be amenable to hMSCs-based therapeutics. For example, hMSCs can be transfected to express ion channels which then can influence the surrounding synctial tissue. Alternatively, the hMSCs can be transfected to express genes that produce small therapeutic molecules capable of permeating gap junctions and influencing recipient cells. Further, for short term therapy, small molecules can be directly loaded into hMSCs for delivery to recipient cells. The success of such approaches is dependent on gap junction channels as the final conduit for delivery of the therapeutic agent to the recipient cells. The feasibility of the first approach has been demonstrated herein by delivering HCN2-transfected hMSCs to the canine heart where they generate a spontaneous rhythm.
EXAMPLE 2
Ion Channels Suitable for Incorporation into a Cardiac Bypass Bridge
As previously described herein, hMSCs form gap junctions that permit a tract of physically connected cells to conduct electrical signals by electrotonic conduction. Cell-to-cell propagation of electrical signals may be facilitated by functionally expressing in the cells one or more nucleic acids encoding at least one of the cardiac connexins Cx43, Cx40 or Cx45 in order to enhance formation of gap junctions. The expression in the cells of a nucleic acid(s) encoding the alpha subunit, with or without the accessory subunits, of a sodium channel, or the alpha and accessory subunits of an L-type calcium channel, also increases the likelihood of not just electrotonic propagation of a wavefront, but its active propagation by a sodium-dependent or calcium-dependent action potential. In addition, expression of a potassium channel in the cells both increases the likelihood of active propagation by an action potential and provides a means of controlling the initial resting potential and its voltage-time course of repolarization and refractoriness.
The biophysical properties of a SKM-1 sodium channel and a L-type calcium channel were assayed to investigate their suitability for providing an action potential in a cardiac bypass bridge. The inactivation properties of the SKM-1 sodium channel measured in Xenopus oocytes are shown in FIG. 8, and the I-V relationship for the SKM-1 channel in Xenopus oocytes is shown in FIG. 9. The CaV1.2, alpha2&gamma, P1.b and red fluorescent reporter were co-expressed in HEK293 cells. FIG. 10 shows the Ca2+ current recorded in the red fluorescent cells.
The SKM-1 sodium channel and L-type calcium channel are used to “fine-tune” the conductivity of a bypass bridge to the extent that this is needed. One embodiment of the bypass bridge described herein is an AV bridge. Incorporation of sodium channels in an AV bridge can be viewed as counterintuitive in that the sodium channel is usually associated with very rapid conduction, whereas an AV bypass should conduct more slowly to operate most efficiently. However, because the degree of coupling among the cells can be manipulated it is possible to use a channel that routinely propagates rapidly and expect this to conduct more slowly. Incorporation of calcium channels, for inward calcium current, will elevate the plateau of the cardiac action potential and prolong repolarization and refractoriness. In addition, functionally expressing potassium channels would accelerate repolarization and can thus shorten refractoriness. Therefore, by manipulating the sodium, calcium and potassium currents and cell coupling, the characteristics of the cardiac electrical activity can be correspondingly manipulated.
REFERENCES


    U.S. Provisional Application No. 60/704,210, filed Jul. 29, 2005 by Brink P R et al.
    U.S. Pat. No. 6,783,979, issued Aug. 31, 2004 to Rosen M R et al.
    U.S. Provisional Application No. 60/832,515, entitled “Chimeric HCN Channels,” filed Jul. 21, 2006.
    U.S. Provisional application Ser. No. 11/490,997, entitled “Tandem Cardiac Pacemaker System,” filed Jul. 21, 2006.
    Biel M, Schneider A, Wahl C (2002) Cardiac HCN channels: Structure, function, and modulation. Trends Cardiovasc Med 12: 202-216.
    Beyer E C (1993) Gap junctions. Int Rev Cytol 137C: 1-37 1993.
    Clapham D E (1998) Not so funny anymore: pacing channels are cloned. Neuron 21: 5-7.
    DiFrancesco D (1993) Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol 55: 455-472.
    Elfgang C, Eckert R, Lichtenberg-Frate H, Butterweck A, Traub O, Klein R A, Hulser D F, Willecke K. (1995) Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Biol 129: 805-817.
    Larsson H P (2002) The Search Is on for the Voltage Sensor-to-gate Coupling. J Gen Physiol 120: 475-481.
    Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson S M, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine D M, Leri A, Anversa P (2001) Bone marrow cells regenerate infarcted myocardium. Nature 410: 701-705.
    Pape H C (1996) Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. Annu Rev Physiol 58: 299-327.
    Perin E C, Geng Y J, Willerson J T (2003) Adult stem cell therapy in perspective. Circulation 107: 935-938.
    Qu J, Kryukova Y, Potapova I A, Doronin S V, Larsen M, Krishnamurthy G, Cohen I S, Robinson R B (2004) MiRP1 modulates HCN2 channel expression and gating in cardiac myocytes. J Biol Chem 279: 43497-43502.
    Robinson R B, Siegelbaum S A (2003) Hyperpolarization-activated cation currents: from molecules to physiological function. Annu Rev Physiol 65: 453-480.
    Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.
    Santoro B, Tibbs G R (1999) The HCN Gene Family: Molecular Basis of the Hyperpolarization-Activated Pacemaker Channels. Ann NY Acad Sci 868: 741-764.
    Strauer B E, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg R V, Kogler G, Wernet P (2002) Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 106; 1913-1918.
    Valiunas V, Beyer E C, Brink P R (2002) Cardiac gap junction channels show quantitative differences in selectivity. Circ Res 91: 104-111.
    Valiunas V, Bukauskas F F, Weingart R (1997) Conductances and selective permeability of connexin43 gap junction channels examined in neonatal rat heart cells. Circ Res 80: 708-719.
    Valiunas V, Gemel J, Brink P R, Beyer E C (2001) Gap junction channels formed by coexpressed connexin40 and connexin43. Am J Physiol Heart Circ Physiol 281: H1675-H1689.
    Valiunas V, Weingart R, Brink P R (2000) Formation of heterotypic gap junction channels by connexins 40 and 43. Circ Res 86: E42-E49.
    Wang H Z, Day N, Valcic M, Hsieh K, Serels S, Brink P R, Christ G J (2001a) Intercellular communication in cultured human vascular smooth muscle cells. Am J Physiol Cell Physiol 281: C75-C88.

## Claims

1. An atrioventricular bypass bridge comprising a tract of gap junction-coupled adult human mesenchymal stem cells obtained from bone marrow, 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. The bypass bridge of claim 2, wherein the pacemaker ion channel comprises either a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or chimera thereof.

4. The bypass bridge of claim 3, wherein the pacemaker ion channel is an HCN chimera that comprises portions of more than one HCN channel isoform.

5. The bypass bridge of claim 4, wherein the portions are selected from the group consisting of an amino terminal portion, an intramembranous portion, and a carboxy terminal portion.

6. The bypass bridge of claim 4, wherein at least one portion of the HCN chimera is encoded by a nucleic acid from a first animal species and at least one of the other two portions is encoded by a nucleic acid from a second animal species.

7. The bypass bridge of claim 4, wherein the HCN chimera is mHCNI12, mHCN212, mHCN312, mHCN412, mHCN114, mHCN214, mHCN314, mHCN414, hHCN112, hHCN212, hHCN312, hHCN412, hHCNI14, hHCN214, hHCN314, or hHCN414.

8. The bypass bridge of claim 7, wherein the HCN chimera is hHCN212 having the sequence set forth in SEQ ID NO:2.

9. The bypass bridge of claim 7, wherein the HCN chimera is mHCN212 having the sequence set forth in SEQ ID NO:6.

10. The bypass bridge of claim 3, wherein the HCN channel is a mutant mHCN2 channel having the sequence set forth in SEQ ID NO: 14 modified such that it comprises E324A-mHCN2, Y331A-mHCN2, R339A-mHCN2, or Y331A,E324A-mHCN2.

11. The bypass bridge of claim 10, wherein the mutant mHCN2 channel comprises E324A-mHCN2.

12. The bypass bridge of claim 3, wherein the pacemaker current is actively propagated by an action potential.

13. The bypass bridge of claim 12, wherein the action potential is a sodium-dependent action potential.

14. The bypass bridge of claim 12, wherein cells in the tract further functionally express a calcium channel and the action potential is a calcium-dependent action potential.

15. The bypass bridge of claim 2, wherein the pacemaker ion channel is expressed in mesenchymal stem cells located in the first end of the tract.

16. The bypass bridge of claim 15, wherein the pacemaker ion channel is expressed in mesenchymal stem cells located in a region extending 0.5 mm from the first end.

17. The bypass bridge of claim 16, wherein the pacemaker current is conducted by electrotonic conduction.

18. The bypass bridge of claim 1, wherein the sodium channel is a skeletal muscle sodium channel (SKM-1).

19. The bypass bridge of claim 18, wherein the skeletal muscle sodium channel (SKM-1) comprises an alpha subunit.

20. The bypass bridge of claim 19, wherein the skeletal muscle sodium channel (SKM-1) further comprises an accessory subunit.

21. 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.

22. The bypass bridge of claim 21, wherein the calcium channel is an L-type calcium channel, wherein the alpha subunit is optionally-CaV1.2.

23. 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.

24. The bypass bridge of claim 23, wherein the potassium channels further comprises an accessory subunit.

25. The bypass bridge of claim 1, wherein the mesenchymal stem cells further functionally express a connexin that is a member of the group consisting of Cx43, Cx40, and Cx45.