Patent Application
20130274838
The invention features an optically-controlled biological device comprising a non-excitable cell expressing a light-gated ion channel protein and capable of forming gap junction channels with cardiomyocytes, and an optical stimulation unit.
The heart's natural pacemaker is a small mass of specialized cells called the sinoatrial (SA) node, which initiates and maintains the heart's normal rhythm (referred to as normal sinus rhythm). The sinoatrial node consists of only a few thousand electrically active pacemaker cells that generate spontaneous rhythmic action potentials that subsequently propagate to induce coordinated muscle contractions of the atria and ventricles. The rhythm is modulated, but not initiated, by the autonomic nervous system.
Thus, normal cardiac depolarization is initiated by the SA node. From the sinus node the wave of depolarization spreads across the atria to the atrioventricular (AV) node. At the AV node the impulse is delayed briefly and atrial contraction is completed. The depolarization wave then proceeds to the bundle of His where it follows two pathways, travelling along the right and left bundle branches. The impulse travels the length of the bundles along the interventricular septum to the base of the heart. The bundles divide into the Purkinje system, which distributes the wave of depolarization to the ventricle walls, initiating ventricular contraction.
Cardiac disease can result, for example, when an event disrupts or alters the generation of the impulse or disrupts or alters the conduction of the impulse to the atria or ventricles. For example, when an event interrupts the heart's normal beat, either intermittent or sustained arrhythmias can occur. Cardiac arrhythmia is a group of conditions in which the muscle contraction of the heart is irregular and/or is faster (tachycardia) or slower (bradycardia) than normal. The most common causes of bradycardias are reduced SA node pacemaker activity and depressed conduction. Slowing of the SA node pacemaker, sinus bradycardia, can be caused by excessive parasympathetic tone, hyperthyroidism, and administration of drugs including β-adrenergic blockers and calcium channel blockers. In addition, sinus bradycardia occurs in sinus node dysfunction (tachycardia-bradycardia syndrome) commonly seen in the elderly. Depressed conduction causing bradycardia can occur, for example, when conduction of the sinus impulse to the ventricles is impaired (referred to as a block). Three regions of the heart are particularly vulnerable to block, SA node, the AV node, and the His-Purkinje system.
Thus, SA block occurs when impulses arising in the SA node fail to depolarize the atria. AV block is clinically sorted into three categories: first-degree AV block is an abnormal delay in the AV conduction (prolonging the PR interval); second-degree AV block is more severe because some, but not all, P waves fail to activate the ventricles; and third-degree AV block no impulses are conducted from the atria to the ventricles. Second-degree AV block may in turn be categorized as type 1, in which the blocked P waves are proceeded by progressive prolongation of the PR interval; and type 2, in which there is no such pattern. Type 2 second-degree AV block is often a result of lesions of the His-Purkinje system.
A tachyarrythmia is a tachycardia associated with an irregularity in the normal heart rhythm. Tachyarrythmias include tachycardia (a series of premature systoles), fibrillation (disorganized activation where there is no effective beating), and flutter (a rapid regular activation of the atria or ventricle). Mechanisms that account for tachyarrhythmias include accelerated pacemaker activity, reentry, and triggered depolarizations. Accelerated firing of a pacemaker cell of the SA node causes sinus tachycardia. Reentry occurs when a single impulse traveling through the heart gives rise to two or more propagated responses. Reentrant arrhythmias may be caused by abnormal conduction (decremental conduction and unidirectional block), inhomogeneties of the action potential, and abnormal conducting structures.
Malfunction or loss of pacemaker cells can occur due to disease or aging. For example, acute myocardial infarction 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.
A variety of implantable devices have been developed to address cardiac pacing disorders. Electronic pacemakers are lifesaving devices that provide a regular heartbeat in settings where the sinoatrial node, atrioventricular conduction, or both, have failed. They also have been adapted to the therapy of congestive heart failure. One of the major indications for electronic pacemaker therapy is high degree of heart block, such that a normally functioning sinus node impulse cannot propagate to the ventricle, resulting in ventricular arrest and/or fibrillation, and death. Another major indication for electronic pacemaker therapy is sinoatrial node dysfunction, in which the sinus node fails to initiate a normal heartbeat, thereby compromising cardiac output.
Implantable cardioverter defibrillators (ICDs) are used to treat patients at risk for recurrent, sustained ventricular tachycardia or fibrillation. These devices deliver electrical pulses or shocks to help correct the irregular heartbeats.
In a first aspect, the invention features an optically-controlled biological device comprising: (a) a non-excitable cell expressing a light-gated ion channel protein and capable of forming a gap junction channel with a cardiomyocyte when implanted in a subject, and (b) an optical stimulation unit. In one embodiment the device is a cardiac pacemaker. In one embodiment, the device is an implantable cardioverter defibrillator. In one embodiment, the device is an optical-mechanical actuator.
In another aspect, the invention features a method of treating a subject with a cardiac pacing disorder, comprising: (a) delivering in proximity to the heart of the subject a non-excitable cell expressing a light-gated ion channel protein, wherein the non-excitable cell forms a gap junction channel with a cardiomyocyte of the subject; and (b) providing an optical stimulation unit.
In one embodiment, the light-gated ion channel protein is one or more proteins selected from the group consisting of channelrhodopsin-1 (ChR1), channelrhodopsin-2 (ChR2), Volvox channelrhodopsin (VChR1), halorhodopsin (Halo/NpHR), archaerhodopsin-3 (Arch), and Leptosphaeria maculans rhodopsin (Mac).
In one embodiment, the non-excitable cell is engineered to express one or more connexin proteins. In one embodiment, the non-excitable cell is engineered to express one or more connexins selected from the group consisting of connexin 40, connexin 43, and connexin 45. In one embodiment, the non-excitable cell endogenously expresses one or more connexin proteins.
In one embodiment, the non-excitable cell is a stem cell, an endothelial cell, a fibroblast or an adipocyte. In one embodiment, the non-excitable cell is a stem cell. In other embodiments the non-excitable cell is pluripotent or totipotent. In other embodiments the non-excitable cell is an embryonic stem cell, an induced pluripotent stem cell, or a mesenchymal stem cell. In one embodiment, the non-excitable cell is a human mesenchymal stem cell. In one embodiment the non-excitable cell is substantially incapable of differentiating after it is implanted in the subject.
In one embodiment, the optically-controlled biological device comprises between about 5,000 to about 1.5 million cells. In other embodiments the biological device comprises between about 700,000 to about 1.0 million cells. In one embodiment, the biological device comprises at least about 5,000 cells; at least about 10,000 cells; at least about 50,000 cells; at least about 100,000 cells; at least about 200,000 cells; at least about 500,000 cells; at least about 700,000 cells; or at least about 1 million cells.
In one embodiment, the optically-controlled biological device comprises a number of non-excitable cells such that the ratio of non-excitable cells to target cells is from about 1:20 to about 1:100; from about 1:20 to about 1:500; from about 1:30 to about 1:70; from about 1:50 to about 1:100; from about 1:100 to about 1:200; from about 1:200 to about 1:300; from about 1:300 to about 1:400; from about 1:400 to about 1:500; and from about 1:500 to about 1:1000.
In one embodiment, the optical stimulation unit comprises a light source wherein the light source a laser, a laser diode, a light emitting diode, an organic light emitting diode, an incandescent light source, or an organic light source. In one embodiment, the optical stimulation unit comprises one or more sensors for detecting cardiac pacing in the atrium, ventricles, or both.
In one embodiment, the cardiac pacing disorder is selected from the group consisting of cardiac arrhythmia, reentrant arrhythmia, bradycardia, tachycardia, sinus bradycardia, sinus tachycardia, ventricular tachycardia, supraventricular tachycardia, ventricular fibrillation, atrial flutter, atrial fibrillation, pro-arrhythmic cardiac alternans, sinus node dysfunction, first degree heart block, type 1 second degree heart block, type 2 second degree heart block, third degree heart block, SA block, and SV block.
In one embodiment, the non-excitable cell is delivered to one or more of the sinoatrial node, the atrioventricular node, Bachmann's bundle, the atrioventricular junction region, His branch, left bundle branch, right bundle branch, Purkinje fibers, right atrial muscle, left atrial muscle, or ventricular muscle.
The invention features an optically-controlled biological device comprising (a) a biological component comprising a non-excitable cell expressing a light-gated ion channel protein and capable of forming a gap junction channel with a target cell (e.g., a cardiomyocyte), and (b) an optical stimulation unit.
In one aspect the invention features a donor cell engineered to express a light-gated ion channel protein, which forms a tandem cell unit with a target cell via gap junction channels between the donor and target cell. Stimulation of the donor cell by light causes depolarization or repolarization of the membrane potential of the donor cell, which in turn triggers depolarization or repolarization of the target cell. In one embodiment the donor cell is a non-excitable cell.
In one embodiment, a non-excitable cell that is capable of forming a gap junction with the target cell is engineered to express a light-gated ion channel. A non-excitable cell is a cell that does not normally generate an action potential and possesses only passive conduction properties. However, the non-excitable cell may be manipulated such that it will contribute to the depolarization and/or repolarization of an excitable cell to which it is coupled in response to certain stimuli, as in the present invention, when such a cell is engineered to express a light-gated ion channel. Cell types include, but are not limited to, stem cells, fibroblasts, endothelial cells, adipocytes, or other cell that can be implanted in a subject.
An implantable cell means a cell that can be implanted or administered into a subject. Preferably, the biological component of the present invention comprises an implantable cell capable of gap junction-mediated communication with cardiomyocytes or other target cells. In one embodiment, the cell is substantially incapable of differentiation when implanted into a subject. In other embodiments the cell is pluripotent or totipotent. In one embodiment, the cell is an embryonic or adult stem cell. In one embodiment, the cell is an induced pluripotent stem cell. In one embodiment, the cell is an adult mesenchymal stem cell. In one embodiment, the cell is an adult human mesenchymal stem cell.
The absence of rejection, if nonautologous cells are employed, will facilitate the long-term use of the cell-based biological component. In this regard, cells could be obtained from an autologous source. Thus, in one embodiment, the non-excitable cell is obtained from the subject and manipulated to express a light-activated ion channel. In a further embodiment the cell is engineered to express one or more connexins. In other embodiments, the non-excitable cell is immunoprivileged. For example, evidence suggests that hMSCs cells are immunoprivileged [1]. In addition, no cellular or humoral rejection was evident six weeks following injection of hMSCs into canine hearts [2]. Thus, allogeneic solutions based on the immunoprivileged status of hMSCs provides off-the-shelf cells, that could be ready for implantation.
Light-gated ion channels are transmembrane proteins that form an ion channel that opens and/or closes in response to light.
Light-gated ion channels useful in the present invention include, but are not limited to those listed in Table 1, as well as mutations and other variations and alterations of the light-gated ion channels such as chimeras and light-gated ion channels comprising one or more deletions or insertions. Such mutations or alterations may, for example, alter the wavelength of light at which the light-gated ion channel is activated, alter the specificity of the channel for certain ions, and/or alter the kinetics of the light-gated ion channel. For example, C128 mutations of ChR2 provide a light-gated ion channel that can be opened by a blue light pulse and closed by a green or yellow light pulse. Mutating the E123 residue, on the other hand, can accelerate channel kinetics. A C-terminal truncation of ChR2 (ChR21-315) is substantially as active as the full-length protein. Light-gated ion channels also include variations that have been mammalian codon-optimized.
A chimera of a light-gated ion channel comprises portions of more than one type of light-gated ion channels. For example, a chimera may comprise portions of ChR1 and ChR2 or VChR1, and so forth. In addition, a light-gated ion channel chimera includes an ion channel comprising portions of an light-gated ion channel derived from different species. For example, one portion of the channel may be derived from a human and another portion may be derived from a non-human.
For the tandem cell unit to be functional (to fire an action potential upon light excitation), proper gap junctional coupling between the donor and target cell is needed to close the ion current loop. The major connexins expressed in human cardiac muscle are Cx43, Cx40, and Cx45. Gap junction channels can be formed when the target and donor cells express one or more connexins.
The cDNA sequence and deduced amino acid sequence of connexin 40 (Cx40, also known as GJA5) has been characterized from several species including mouse, rat, and human. Sequences of the Cx40 proteins are available from public databases. The GenBank accession number for human Cx40 is NM—181703.
The cDNA sequence and deduced amino acid sequence of connexin 43 (Cx43, also known as GJA1) has been characterized from several species including mouse, rat, and human. Sequences of the Cx43 proteins are available from public databases. The GenBank accession number for human Cx43 is BC026329.
The cDNA sequence and deduced amino acid sequence of connexin 45 (Cx45, also known as GJC1) has been characterized from species including mouse and human. Sequences of the Cx45 proteins are available from public databases. The GenBank accession number for human Cx45 is NM—005497.
The use of a particular light-gated ion channel isoform as described herein (e.g., the expression of the light-gated ion channel in a non-excitable cell) encompasses the use of a light-gated ion channel exhibiting at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity with that isoform. In embodiments of the invention comprising portions of a particular light-gated ion channel isoform, for example, the use of a N-terminal portion of a particular isoform encompasses the use of a N-terminal portion of the channel exhibiting at least about 60%, at least about 70%, at least about 80%, or at least about 90% identity with the N-terminus of that isoform. In addition, the use of a C-terminal portion of a particular light-gated ion channel isoform encompasses the use of a C-terminal portion of a light-gated ion channel exhibiting at least about 60%, at least about 70%, at least about 80%, or at least about 90% identity with the C-terminus of that isoform.
The use of a particular connexin isoform as described herein (e.g., the expression of the connexin in a non-excitable cell) encompasses the use of a connexin exhibiting at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity with that isoform. In embodiments of the invention comprising portions of a particular connexin isoform, the use of a N-terminal portion of a particular connexin isoform encompasses the use of a N-terminal portion of a connexin exhibiting at least about 60%, at least about 70%, at least about 80%, or at least about 90% identity with the N-terminus of that connexin isoform. In addition, the use of a C-terminal portion of a particular connexin isoform encompasses the use of a C-terminal portion of a connexin exhibiting at least about 60%, at least about 70%, at least about 80%, or at least about 90% identity with the C-terminus of that connexin isoform.
To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, 80%, or 90% or more of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The percent identity between two light-gated ion channel amino acid sequences or between two connexins amino acid sequences can be determined using the Needleman et al. algorithm [28]. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989).
The invention also encompasses polypeptides having sufficient similarity so as to perform one or more of the same functions performed by the light-gated ion channel or the connexin molecule. Thus, the light-gated ion channel or connexin includes isoforms exhibiting at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% similarity with the light-gated ion channel or connexin protein. Similarity is determined by considering conserved amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in the art (see e.g., [21]). The comparison of sequences and determination of percent identity and similarity between two polypeptides can be accomplished using mathematical algorithms known in the art. (see e.g., [22-26]). A non-limiting example of such a mathematical algorithm is described in Karlin et al. [27].
A light-gated ion channel or connexin according to the present invention, may also be a polypeptide encoded by a nucleic acid sequence capable of hybridizing to the nucleic acid sequence of a light-gated ion channel or connexin set forth above under stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. [29] and encodes a functionally equivalent gene product; or under less stringent conditions, such as moderately stringent conditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C., yet which still encodes a functionally equivalent light-gated ion channel or connexin protein.
In another aspect of the invention, variant light-gated ion channel or connexin polypeptides that differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these can be used in the methods of the present invention. Variant polypeptides can be fully functional or can lack function in one or more activities.
To create certain biological components of the present invention a nucleic acid encoding a light-gated ion channel is delivered to an implantable cell. In other embodiments, nucleic acids encoding two or more light-gated ion channels are delivered to the implantable cell. In certain embodiments a nucleic acid encoding a connexin is delivered to the implantable cell. In other embodiments, nucleic acids encoding two or more connexins are delivered to the implantable cell. In one embodiment, the nucleic acid is transfected using electroporation. Electroporation is a technique in which exposure of cells to a brief pulse of high voltage transiently opens pores in the cell membranes that allow macromolecules, such as DNA and proteins, to enter the cell.
Other methods of introducing genes into a cell include viral infection using, for example, adenovirus, adeno-associated virus (AAV), and lentivirus; liposome-mediated transfection (lipofection); transfection using a chemical transfection reagent; heat shock transfection; or microinjection. AAV, a small parvovirus associated with adenovirus, cannot replicate on its own and requires co-infection with adenovirus or herpesvirus in order to replicate. In the absence of helper virus, AAV enters a latent phase during which it stably integrates into the host cell genome. This latent phase makes AAV attractive for applications involving transfer of genes of up to about 4.4 kb, as the gene inserted into AAV can persist in the host cell genome for a long period [30]. Lentivirus, a member of the retroviral family, provides a potentially interesting alternative [31-32]. Unlike adenoviruses, electroporation and the use of lentiviral vectors allow persistent transgene expression without eliciting host immune responses.
The cell-based biological component of the present invention functions as a biological actuator converting the light from the optical stimulation unit to an electrical signal that can depolarize and/or repolarize cardiac tissue via gap junction connections with cardiomyocytes. Furthermore, for muscle cells (cardiac, skeletal or smooth muscle cells), the optical signal leads to electromechanical response, ending in mechanical contraction. Thus, the biological actuator converts the optical signal into mechanical contraction.
In one embodiment, the cell-based biological component is administered to one or more selected sites of the heart of a subject. Several methods to achieve focal delivery are feasible; for example, the use of catheters and needles, and/or growth on a matrix and a “glue.” Whatever approach is selected, the delivered cells should not disperse too far from the target site. Such dispersion could introduce unwanted electrical effects within the heart or in other organs. It is noteworthy that in a preliminary study involving injection of up to about 106 hMSCs into the LV subepicardium of six adult dogs, nests of hMSCs were consistently found adjacent to the injection site but not at a distance [2].
In various embodiments, implantable cells are administered onto or into the heart by injection, catheterization, surgical insertion, or surgical attachment. The delivery site is determined at the time of administration, based on the subject's pathology, to give the optimal activation and hemodynamic response. Thus, the chosen site could be the sinoatrial (SA) node, Bachmann's bundle, the atrioventricular junctional region, His branch, left or right bundle branch, Purkinje fibers, right or left atrial muscle, or right or left ventricular muscle, the appropriate site being known to one of ordinary skill in the art.
In another embodiment, implantable cells are locally administered by injection or catheterization directly onto or into the heart. In further embodiments, the cell is systemically administered by injection or catheterization into a coronary blood vessel or a blood vessel proximate to the heart. In still further embodiments, the cell is injected onto or into an area of an atrium or ventricle of the heart. In other embodiments, the cell is injected onto or into the left atrium, a wall of a ventricle, a bundle branch of a ventricle, or the proximal LV conducting system of the heart.
The isoform or type of light-gated ion channel expressed in the non-excitable cells may also be changed depending on the delivery site. In addition, different levels of expression of the ion channel gene may be desirable in different delivery sites. Such different levels of expression may be obtained by using different promoters to drive expression at a desired level.
In certain embodiments, the biological component comprises between 5,000 to 1.5 million cells. In other embodiments the biological component comprises between about 700,000 to about 1.0 million cells. In one embodiment, the biological component comprises at least about 5,000 cells; at least about 10,000 cells; at least about 50,000 cells; at least about 100,000 cells; at least about 200,000 cells; at least about 500,000 cells; at least about 700,000 cells; or at least about 1 million cells.
In certain embodiments the number of cells administered to form the biological component is determined in terms of a ratio of donor cells delivered to cells in the target tissue (e.g., the ratio of non-excitable cells to cardiomyocytes). In one embodiment, the ratio is from about 1:10 to about 1:1000. In other embodiments the ratio of donor cells to target cells is from about 1:10 to about 1:20; from about 1:20 to about 1:30; from about 1:30 to about 1:40; from about 1:40 to about 1:50; from about 1:50 to about 1:60; from about 1:60 to about 1:70; from about 1:70 to about 1:80; from about 1:80 to about 1:90; from about 1:90 to about 1:100; from about 1:20 to about 1:100; from about 1:20 to about 1:500; from about 1:30 to about 1:70; from about 1:50 to about 1:100; from about 1:100 to about 1:200; from about 1:200 to about 1:300; from about 1:300 to about 1:400; from about 1:400 to about 1:500; and from about 1:500 to about 1:1000.
In one aspect, the invention provides a functional “tandem cell unit,” formed by a subject's target cell (e.g., a cardiomyocyte) and a non-excitable cell, acting as a donor for exogenous ion channels (e.g., channelrhodopsin2). For this unit to be functional (to fire an action potential upon light excitation), a proper gap junctional coupling is needed for closing the ion current loops.
Connexin 40 (Cx40), Connexin 43 (Cx43), and Connexin 45 (Cx45) are the major connexins expressed in the human heart. The gap junction channels formed by these connexins help maintain normal conduction velocity in the heart as the currents associated with action potential propagation move from myocyte to myocyte via gap junctions.
Thus, in one aspect the biological component of the optically-controlled biological device comprises a non-excitable cell expressing a light-gated ion channel protein and is capable of forming gap a junction channel with a cardiomyocyte when implanted in a subject. In one embodiment, the non-excitable cell endogenously expresses one or more connexin proteins. In one embodiment, the non-excitable cell has been engineered to express one or more connexins. In one embodiment, the non-excitable cell has been engineered to express one or more of Cx40, Cx43, and/or Cx45.
The light-gated ion channels when expressed in a cell affect the flow of ions across the cell membrane in response to light. The change in the ion flow corresponds to a change in the electrical properties of the cell such as the membrane potential. When the cell is coupled to an adjacent cell via a gap junction, the electrical properties of the adjacent cell are affected as well.
Cardiomyocytes have a negative membrane potential at rest. Stimulation above a threshold value induces the opening of voltage-gated ion channels allowing a flood of cations into the cell. The positively charged ions entering the cell cause the depolarization characteristic of an action potential. Following depolarization, a brief repolarization takes place with the efflux of potassium through fast acting potassium channels. Depolarization causes the opening of voltage-gated calcium channels and closing of the potassium channels and is followed by a titrated release of Ca2+ from t-tubules. This influx of calcium causes calcium-induced calcium release from the sarcoplasmic reticulum, and free Ca2+ causes muscle contraction. After a delay, slow acting potassium channels reopen and the resulting flow of K+ out of the cell causes repolarization to the resting state. Thus, repolarization is the return of the ions to their previous resting state, which corresponds with relaxation of the myocardial muscle.
In one embodiment, the cell expressing the light-gated ion channel (the donor cell) is stimulated by light causing the depolarization of one or more adjacent target cells (such as cardiomyocytes) via gap junctions connecting the donor cells to the target cells. In another embodiment, light is used to suppress depolarization in the target cells. In one embodiment, the cell expressing the light-gated ion channel (the donor cell) is stimulated by light causing the repolarization of one or more adjacent target cells (such as cardiomyocytes) via gap junctions connecting the donor cells to the target cells.
Manipulation of repolarization by light (when repolarization-related light-sesnitive ion channels are used, e.g. Halo, Arch, Mac) can alter the shape of the action potential (AP). In many pro-arrhythmic states, a disease-related AP prolongation takes place. Longer AP can be pro-arrhythmic by increasing the chances for early after-depolarization events (EAD)—abnormal secondary, undesired firings of APs. For example, class Ib and IV anti-arrhythmic drugs currently aim at shortening repolarization by manipulation of Na+ or Ca2+ ion channels. Optical suppression of such events (EADs) by light-forced or facilitated repolarization can be anti-arrhythmic without affecting contractile function (as in class IV Ca2+ channel blockers).
One aspect of the invention features a donor cell engineered to express a light-gated ion channel protein, which forms a tandem cell unit with a target cell via gap junction channels between the donor and target cell. In one embodiment the target cell is a syncytial cell (i.e., a cell from a syncytial structure, such as the heart, bladder, liver, or gastrointestinal tract). In one embodiment the target cell is a cardiomyocyte. In other embodiments the target cell is a skeletal muscle cell or a smooth muscle cell.
In one embodiment, the optical stimulation unit comprises a power supply, control circuitry, and one of more light sources. In further embodiments the optical stimulation unit also comprises one or more sensors.
The optical stimulating unit comprises one or more light sources. The light source can be any appropriate source of light capable of stimulating the light-gated ion channel expressed in the cells of the biological component. Such light sources include, but are not limited to, a laser, a laser diode, a light-emitting diode (LED), a organic light-emitting diode (OLED), an incandescent light source, an organic light source, or any other switchable light source with the ability to modulate the output at suitable rates. In one embodiment, the optical simulation unit comprises two or more light sources. In one embodiment, the optical stimulation unit comprises one or more optical fibers to deliver light to the biological component.
In one embodiment the optically-controlled biological device functions as a cardiac pacemaker. In one embodiment the optically-controlled biological device functions as a cardioverter/defibrillator. In one embodiment the optically-controlled biological device functions as both a cardiac pacemaker and a cardioverter/defibrillator.
In one embodiment the optical stimulating unit comprises one or more sensors that detect the status of one or more cardiac chambers.
The control circuitry (including programming) for cardiac pacing is known in the art and depends upon the type of cardiac pacing required. Pacemaker type is described by three (or four) letter code as described in Table 2.
The appropriate application to treat cardiac disease of the particular types of pacemakers is known to one of skill in the art.
The control circuitry (including programming) for ICDs is also known in the art. Such programming includes protocols for termination of ventricular tachycardia such as burst pacing (short bursts of paced beats delivered up to about 90% of the rate of the VT) and ramp pacing (short bursts of paced beats at a rate increasing up to about 90% of the rate of the VT).
The optical stimulation unit may use one or a combination of various methods known in the art to determine if an arrhythmia is normal or if it is ventricular tachycardia or ventricular fibrillation. For example, rate discrimination evaluates the rate of the ventricles and compares it to the rate in the atria. Rhythm discrimination determines the regularity of the ventricular tachycardia. Generally, ventricular tachycardia is regular. If the rhythm is irregular, it is usually due to conduction of an irregular rhythm that originates in the atria, such as atrial fibrillation. Morphology discrimination checks the morphology of each ventricular beat and compares it to a normally conducted ventricular impulse for the subject, which is often an average of multiple beats of the subject taken in the recent past.
In one embodiment the optically-controlled biological devices described herein are used in the treatment of a subject with a cardiac pacing disorder. Cardiac pacing disorders include, but are not limited to, cardiac arrhythmia, reentrant arrhythmia, bradycardia, tachycardia, sinus bradycardia, sinus tachycardia, ventricular tachycardia, supraventricular tachycardia, ventricular fibrillation, atrial flutter, atrial fibrillation, and pro-arrhythmic cardiac alternans.
In one embodiment the optically-controlled biological devices described herein functions as an optical-mechanical actuator modulating the function of target tissues including skeletal muscle and smooth muscle.
The use of cells expressing ChR2 as described herein demonstrates the feasibility of preparing light-activated biological pacemakers, but also demonstrates the formation of a tandem cell unit to influence the function of syncytial tissues. Thus, the device of the invention can also be used to deliver, for example, a signal to hyperpolarize smooth muscle (including vascular smooth muscle and bladder smooth muscle) inducing relaxation.
The term “subject” as used herein refers to any organism in need of treatment, or requiring preventative therapy, for cardiac disease with the methods and devices of the invention. In one embodiment the subject is a human.
It is to be understood and expected that variations in the principles of the invention disclosed herein may be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application. The following examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way.
Plasmid Preparation:
The plasmid, pcDNA3.1/hChR2(H134R)-EYFP, was obtained from Addgene. It was expanded in bacteria (DH5a) on a LB+ampicillin agar plate overnight at 37° C. Selected colonies were further grown in LB-ampicillin medium with agitation, and the plasmid DNA was extracted into TE buffer using the Qiagen High Speed kit (Qiagen, Valencia, Calif.). Plasmid DNA (dsDNA) was quantified using the absorption ratio at 260 nm vs. 280 nm. After confirming the identity of the plasmid (by gel and spectrophotometry), it was stored at −20° C. in TE buffer at the obtained concentration (typically 2-4 μg/ml), and later diluted to 1 μg/ml for transfection.
Transfection:
HEK293 cells (ATCC, Manassas, Va.) were used as a model non-excitable cell and were transfected with the plasmid using Lipofectamine 2000 (Invitrogen) as directed: 4 μg of DNA and 10 μg of Lipofectamine in 250 μl medium for a 35 mm dish with cells. Gene expression was examined by EYFP signal the next day. 48 hours after transfection, cells were switched to selection medium, containing 500 μg/ml Geneticin (GIBCO Invitrogen). The selected cells with high fluorescence signal were maintained in Geneticin (500 μg/ml) containing culture medium at 37° C. in a humidified atmosphere incubator with 5% CO2 and 95% air. Expanded HEK cell cultures showing near 100% expression were frozen at −80 C for later use. Immediately prior to use, the HEK-ChR2 cells were grown in DMEM (Dulbecco's Modified Eagle's Medium, GIBCO Invitrogen) supplemented with 10% FBS (fetal bovine serum, Sigma-Aldrich, St Louis, Mo.) and 1% penicillin-streptomycin (Sigma) at 37° C., 5% CO2. Expression and functional properties were confirmed in passages 2 to 20 and used in co-culture experiments with cardiomyocytes.
Confirmation and Analysis of Light-Triggered ChR2-Current in the Cell Delivery System:
ChR2 cell membrane expression was confirmed in virtually 100% of the transfected HEK cells (
The kinetics of light-triggered ChR2-current was examined as the cells were clamped at −80 mV after obtaining whole-cell configuration, and light pulses of variable duration and spacing were applied sequentially. For the analysis of the current kinetics—activation and deactivation time-constants—nonlinear sigmoidal curve fit was applied to the rising and the falling portion upon light on/off pulse (
To validate the tandem cell unit (TCU) strategy for cardiac optogenetics, as a proof of principle, we developed a stable HEK cell line expressing a variant of ChR2.
Confirmation of ChR2 functionality was done by whole-cell voltage clamp. Quantification of the steady-state light-sensitive ion current in single HEK-ChR2 cells (
Recent comprehensive characterization of ChR2 current kinetics indicates fast activation (<5 ms), deactivation (<10 ms) and inactivation (<50 ms) [33], thus making it suitable as excitatory (action potential—generating) current for cardiomyocytes during external optical pacing at relevant frequencies (5-12 Hz for rodents, 1-3 Hz for humans). Indeed, our kinetics characterization (
In contrast to the robust expression of ChR2 in HEK cells, much lower yield was seen when directly transfecting cardiomyocytes with ChR2 using nucleofector electroporation. This prevented direct synthesis of a large-scale ventricular syncytium from ChR2-expressing myocytes. Nevertheless, individual ChR2-expressing neonatal rat ventricular myocytes were excitable and contracting when optically stimulated and quiescent otherwise.
Primary Cardiomyocyte Cell Culture:
Neonatal Sprague-Dawley rats were sacrificed and cardiomyocytes were isolated by an approved Stony Brook University IACUC protocol as previously described [37]. Briefly, the ventricular portion of the hearts was excised and washed free of blood. The tissue was cut into small pieces and enzymatically digested with trypsin at 4° C. (1 mg/ml, USB, Cleveland, Ohio), then with collagenase at 37° C. (1 mg/ml, Worthington, Lakewood, N.J.) the next morning. Cardiac fibroblasts were removed by a two-stage pre-plating process. In some transfection experiments, these cardiac fibroblasts were used in conjunction with electroporation.
Co-Culture of Cardiomyocytes with HEK-ChR2 Cells:
Cardiomyocytes were plated onto fibronectin-coated glass coverslips at high density: 4×105 cells/cm2 for the control myocyte group and 3.5×105 cells/cm2 for the co-culture groups, mixed with approximately 7,700 or 3,500 HEK cells (for 45:1 and 100:1 initial plating ratios) onto glass bottom dishes in M199 medium (GIBCO Invitrogen) supplemented with 10% fetal bovine serum (GIBCO Invitrogen) for the first 2 days and then reduced to 2%. Cultures were maintained in an incubator at 37° C. with 5% CO2 for 4 to 5 days before functional measurements.
Direct Expression of ChR2-EYFP in Neonatal Rat Cardiomyocytes and Cardiac Fibroblasts:
Freshly isolated neonatal rat cardiomyocytes were transfected by electroporation using a Nucleofector device (Amaxa Lonza, Gaithersburg, Md.) as follows: 4 μg of plasmid DNA was mixed with 100 μl of nucleofector solution for transfecting 4×106 cells. Transfected cells were incubated in normal culture conditions. Similar conditions were used to transfect cardiac fibroblasts via electroporation. Expression of fluorescence was detected 24 to 48 hours after transfection using confocal fluorescence imaging.
Direct Expression of ChR2-EYFP in Mesenchymal Stem Cells:
Human mesenchymal stem cells (hMSC) were purchased from Clonetics/BioWhittaker, Walkersville, Md., USA, and cultured in mesenchymal stem cell growth medium—Poietics-MSCGM (BioWhittaker). Canine mesenchymal stem cells (cMSC) were isolated from the bone marrow of adult dogs and cultured in Poietics-MSCGM. Flow cytometry revealed 93.9% CD44+ and 6.1% cells were CD34+. Cells with spindle-like morphology were selected after flow cytometry characterization and replated for use. Transfected cells were incubated in normal culture conditions. Transfect via electroporation as described above. Expression of fluorescence was detected 24 to 48 hours after transfection using confocal fluorescence imaging.
Image Processing:
Background fluorescence was first subtracted for images of control (non-transfected) and transfected cells of the same type. Then the remaining integral fluorescence over identical areas was used to form a ratio (transfected/average control) in order to quantify and compare different cell types (
Immunocytochemistry:
After 6 days of co-culture, samples were fixed for 10 min in freshly made 3.7% formaldehyde solution, followed by extensive washing in phosphate buffer solution (PBS). Cells were then permeabilized in a solution of PBS containing 0.2% Triton X-100 and 5% FBS for 5 min. The permeabilization solution was removed with 3×10 min washes in 1% FBS solution. Cells were then labeled simultaneously with mouse anti-connexin 43 (Invitrogen) for gap junctions and with rabbit anti-α-actinin (Sigma) primary antibodies diluted in 0.5% BSA solution, overnight at 4° C. The next day the cells were washed and then incubated in 0.5% BSA solution containing the appropriate secondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG and Alexa 647 goat anti-mouse IgG) for 1 h, rinsed with 1% FBS solution, and then mounted in VectaShield (with DAPI) (Vector Laboratories) for imaging using the Olympus FluoView™ FV1000 imaging system with 60× oil lens (NA=1.42).
Here, we exploit this particular aspect (high coupling) characteristic for muscle tissues to develop a non-viral cell delivery system for expression of exogenous light-sensitive ion channels.
We used voltage clamp under blue light excitation (470/40 nm) to confirm functionality of ChR2—a large inward current was obtained. Co-cultures of neonatal rat cardiomyocytes and HEK cells (in ratio 50:1) were prepared to implement the “tandem cell unit” concept on a large scale (samples having >2 cm×2 cm area). Furthermore, immunocytochemistry confirmed the structural prerequisites for “tandem cell unit” formation, i.e., proximity and gap junctional coupling between the donor and host cells (
Adult mongrel dogs were euthanized as per IACUC protocol at Stony Brook University by intravenous injection of sodium pentobarbitone (80 mg/kg body weight) and the heart was removed. Canine ventricular cells were isolated using a modified Langendorff procedure [38] perfusing a wedge of the left ventricle through a coronary artery with 0.5 mg ml-1 collagenase (Worthington) and 0.08 mg ml−1 protease (Sigma) for 10 min before tissue digestion. Prior to plating, isolated cardiomyocytes were stored in Kraft-Brühe (KB) solution (in mM: KCl, 83; K2HPO4, 30; MgSO4, 5; Na-Pyruvic Acid, 5; β-OH-Butyric Acid, 5; Creatine, 5; Taurine, 20; Glucose, 10; EGTA, 0.5; KOH, 2; and Nae-ATP, 5; pH was adjusted to 7.2 with KOH) at room temperature. The canine ventricular myocytes were plated onto laminin-coated glass coverslips (10 μg/ml, Invitrogen) and incubated at 37° C. to ensure attachment. HEK-ChR2 cells were added within 24 h at low density to stimulate formation of individual cell pairs and the co-culture was maintained in Medium 199 (Gibco) supplemented with 15% FBS, 2 mM 1-glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 50 μg ml−1 gentamicin.
Dual patch clamp experiments were performed within 48 hours after plating. Briefly, experiments were carried out on heterologous (HEK-ChR2— canine myocyte) cell pairs within 48 hours after plating, as described previously [39]. A dual whole-cell voltage-clamp method was used to control and record the membrane potential of both cells and to measure associated membrane and junctional currents [39-40]. Each cell of a pair was voltage clamped at the same potential by two separate patch clamp amplifiers (Axopatch 200, Axon Instruments). To record junctional conductance, brief voltage steps (±10 mV, 400 ms) were applied to one cell of a pair, whereas the other cell was held at constant voltage and the junctional currents were recorded from the unstepped cell. Membrane and action potentials were recorded in current-clamp mode.
For electrical recordings, glass coverslips with adherent cells were transferred to an experimental chamber mounted on the stage of an inverted microscope (Olympus-IX71) equipped with a fluorescence imaging system. The chamber was perfused at room temperature (˜22° C.) with bath solution containing (in mM): NaCl, 140; MgCl2, 1; KCl, 5; CaCl2, 2; HEPES, 5 (pH 7.4); glucose, 10. Perfusion with 200 μM of carbenoxolone (Sigma) was used to block cell-cell communication. The patch pipettes were filled with solution containing (in mM): K+ aspartate−, 120; NaCl, 10; MgATP, 3; HEPES, 5 (pH 7.2); EGTA, 10 (pCa ˜8). Patch pipettes were pulled from glass capillaries (code GC150F10; Harvard Apparatus) with a horizontal puller (DMZ-Universal, Zeitz-Instrumente). When filled, the resistance of the pipettes measured 1-4MΩ.
Immunostaining of Co-Culture:
For immunocytochemistry, the co-cultures were fixed and permeabilized with 3.7% formaldehyde and 0.02% Triton-X 100 before being stained with a monoclonal mouse antibody against sarcomeric α-actinin (Sigma). Samples were visualized using goat anti-mouse antibody conjugated with fluorophore Alexa 546 (Invitrogen) and imaged on the Olympus FluoView confocal system.
Western Blots of Cx43 and α-Tubulin:
Cells from three groups (HEK293, HEK+ChR2 and stably transfected HeLa+Cx43 cells) were collected, lysed and centrifuged to obtain a pellet. The pellets were re-suspended in cold RIPA buffer (R0278, Sigma), protease Inhibitor cocktail (P2714, Sigma), sodium orthovanadate (S-6508, Sigma) and PMSF (P-7626, Sigma); after centrifugation, the supernatants were transferred to pre chilled microtubes. Protein concentration for each sample was determined by the Bradford assay. Total protein of 30 micrograms from each lysate was mixed with equal volume of laemmli sample buffer (161-0737, Bio-Rad, Hercules, Calif.) containing β-mercaptoethanol and boiled for 5 minutes at 95° C. After centrifugation, samples were loaded on a SDS-polacrylamide gel. MagicMark XP Protein Standard (LC5602, Invitrogen) was loaded along with the samples. After separation by electrophoresis at 115 V for 90 minutes in tris-glycine/SDS buffer, proteins were transferred to immobilon-P membrane (Millipore, Billerica, Mass.) by electrophoresis at 100 V for 60 minutes in tris-glycine/methanol buffer. Nonspecific antibody binding was blocked for 1 hour by 5% blotting grade blocker non-fat dry milk (Bio-Rad) dissolved in 1×TBST. The following antibodies were used: primary anti-Cx43 antibody raised in rabbit (C 6219, Sigma), secondary goat-anti-rabbit antibody (sc-2004, Santa Cruz); a primary antibody for α-tubulin at 55 kD (sc-8035, Santa Cruz), and a secondary goat-anti-mouse antibody for tubulin from Pierce, Rockford, Ill. The secondary antibodies were detected using SuperSignal West Femto Maximum Sensitivity Substrate (34095, Pierce) and images obtained by exposing the membrane to HyBlot CL autoradiography film. Quantification of the Cx43 bands relative to the α-tubulin bands was done using a buil-in routine in ImageG.
Carbenoxolone Treatment to Test Effects of Cell Coupling on Tandem Cell Units:
Carbenoxolone, CBX, (Sigma), a gap junctional uncoupler [39], was used at a concentration 200 μM in the dual-patch experiments with canine cardiomyocytes and HEK-ChR2 or in the cardiac syncytium of neonatal rat cardiomyocytes and HEK-ChR2. In the latter case, CBX was applied for 20 min (without perfusion) in the co-cultures of HEK-ChR2 cells and cardiomyocytes. Contractility movies were recorded in response to optical pacing before and during administration of carbenoxolone, and upon washout to assess the role of gap junctional coupling in the functionality of the tandem cell unit.
The TCU approach for cardiac optogenetics, i.e., inscribing light-sensitivity into cardiomyocytes and cardiac tissue without their direct genetic modifications, was validated in cell pairs of CM and HEK-ChR2 cells, as well as in a synthesized large-scale cardiac syncytium. ChR2-expressing donor cells were most often found to aggregate in small clusters among the neonatal CMs rather than disperse as single cells (
Functional response of TCUs to optical stimulation was confirmed in cell pairs of adult canine CM and HEK-ChR2 cells using dual-clamp to estimate coupling (
In a functional TCU pair, the cardiomyocytes generated normal action potentials upon stimulation by blue light (470 nm, 0.13 mW/mm2, 10 ms pulses) indistinguishable from electrically-triggered ones (
Two-dimensional optical mapping over a large field of view (about 2.2 cm) was done with a custom-developed macroscopic system [37, 38] allowing for ultra-high spatio-temporal resolution. The system (
Movies of propagation were acquired using Cam Ware (pco, Germany) data acquisition software. Raw data were binned (2×2) and analyzed in custom-developed Matlab software to extract quantitative information about calcium transient morphology, conduction velocity etc. Activation maps (based on time of maximum rise in Ca2+) and phase movies (using the Hilbert transform) were generated after filtering spatially (Bartlett filter, 5-pixel kernel) and temporally (Savitsky-Golay, order 2, width 7) [37, 41].
Electrical and Optical Pacing:
For records of electrically-triggered activity, cells were paced by Pt electrodes, connected to a computer-driven Myopacer stimulator (IonOptix, Milton, Mass.). Excitation pulses for light-triggered activity were delivered through the bottom of the dish from an optically focused light from an LED (470 nm, 1.35 cd, 20 mA, 5 mm, 30 deg angle) from Optek Technology (Carrollton, Tex.) or from a fiber-optics coupled high-power blue LED (470 nm, 1.6 A), Thor Labs (Newton, N.J.), connected to the TTL output of a second computer-driven Myopacer stimulator. Irradiance (in mW/mm2) was measured at the cell monolayer site using a Newport digital optical power meter Model 815 (Newport, Irvine, Calif.), with a sensor area of 0.4 cm×0.4 cm, with wavelength set at 470 nm.
Recording Light-Triggered Contractions:
Microscopic imaging for confirmation of gene expression or for documenting contractions by optical excitation was done with the Olympus FluoView™ FV1000 confocal system at room temperature. An EMCCD camera (ImagEM EMCCD camera from Hamamatsu, Bridgewater, N.J.) attached to the Olympus FluoView™ FV1000 microscope was used to record contractility movies at 20 fps with 60× oil lens (NA=1.42) using SlideBook 5 software (Intelligent Imaging Innovations, Denver, Colo.). In addition, contractility response was also documented by automatic optical tracking of cell length [42] at 250 Hz using an IonOptix videosystem (IonOptix) attached to a Nikon TE2000 inverted microscope.
Synchronized wave propagation is essential for normal functionality of the heart and efficient mechanical contraction. Lethal arrhythmias occur when the generation or propagation of these excitation waves is adversely altered (failure to initiate, abnormal propagation velocity and/or path). Hence, arrhythmias are complex spatio-temporal phenomena and require matching imaging tools to dissect. We have developed ultrahigh resolution optical mapping system, allowing for measurements of propagating voltage and calcium waves using fast fluorescence indicator dyes over centimeter-scale with subcellular resolution [41]. Excitation waves were triggered by a computer-controlled light source (Xenon lamp with a liquid light guide, filtered at 470/40 nm and focused under the sample at a circular spot with diameter of 4 mm). Interestingly, irradiance (0.9 mW/cm2) for supra-threshold stimulation was 2-3 orders of magnitude lower than previously reported for use in neurons with ChR2. This very low intensity of light needed, is an important factor in confirming a lack of heat-related effects, no phototoxicity, and in considering future implantable devices. The discrepancy with the reported light levels for neurons and cardiomyocyte monolayers here, may stem from different cellular properties, single vs. multicellular preparations and different focusing of the light. Optical mapping of calcium waves triggered by localized electrical and optical stimulation in the same sample, revealed similar conduction velocities and calcium transient morphologies (away from the stimulus site) for both types of stimulation, thus confirming equivalent triggering abilities for both (
Synthesized optically-excitable cardiac tissue was subjected to functional testing. Synchronized wave propagation is essential for the heart's normal functionality and efficient mechanical contraction; lethal arrhythmias occur when the generation or propagation of these excitation waves is altered (failure to initiate, abnormal propagation velocity and/or path). Accordingly, we have developed an ultra-high resolution optical mapping system [41, 37] to dissect cardiac wave propagation during external pacing or arrhythmic activity over a centimeter-scale (>2 cm) with subcellular resolution (22 μm/pix) at 200 fps using fast voltage and calcium-sensitive dyes [41]. This optical mapping system was made compatible with simultaneous optical excitation (
Optical mapping of propagating waves triggered by localized electrical and optical stimulation in the same sample, revealed similar conduction velocities and calcium transient morphologies, thus confirming equivalent triggering abilities for both modes of stimulation (
The response of the syncytium to optical excitation was captured by constructing a strength-duration curve, describing minimum irradiance over a range of pulse duration values for a point excitation of the 2D syncytium (2 mm fiber-optic coupled controllable LED) (
Considering the electromechanical nature of cardiomyocytes, we also show direct light-triggered muscle contraction, confirming intact excitation-contraction coupling in single myocytes or hybrid cardiac tissue (
Energy Needs in Cardiac Optogenetics:
Previous studies in neuroscience have reported optical energies used to excite single neurons or brain tissue [8, 44, 45] in a wide range of high values (approximately 8 to 75 mW/mm2). The well-coupled spatially-extended cardiac tissue was expected to present higher load for optical stimulation, thus possibly requiring even higher irradiance values. Yet, surprisingly, in Bruegmann et al.'s study [35], significantly lower light levels (0.5 to 7 mW/mm2) were sufficient to optically stimulate cardiac tissue in vitro or in vivo for a wide range of pulse durations.
In our TCU approach, during optical pacing in the cell pairs or in the two-dimensional cardiac syncytium, we measured irradiance at 470 nm as low as 0.006 mW/mm2. The strength-duration curve (
When comparing the TCU strategy to direct expression of ChR2 in the native myocytes (mostly by viral methods) there are at least several important differences (
For a spatially-extended tissue, when the cell pair (TCU) is connected to some “load” of excitable cells (CMs), the system can be abstracted to a Source-Neighbor-Load (S-N-L) triad for easier analysis. A simplified equivalent circuit of such a triad is presented in
For the simplified circuit in
In determining how the elements of the circuit determine the “ease of excitation” of the system, i.e what fraction of ID does Im constitute, our simplified analysis only considers steady-state passive response and ignores the kinetics of the ion channels and/or transient processes, which are admittedly very important.
The following observations can be made:
1) Higher Zm for the source, i.e., Zm(D)>Zm(CM), makes the source closer to an “ideal current source” (with infinite internal resistance/impedance) and therefore increases the “ease of excitation”, i.e., for a given light-induced ID, more current Im will be available for excitation of the neighbors in the TCU case, which follows directly from Eqn (1).
2) Higher load, i.e., low ZL, such that Zm>>ZL, and ZT≈ZL→0, that may occur in very well-coupled, spatially-extended tissue (higher dimensionality) will indeed make it harder to excite by making Im a smaller fraction of ID—see simplified combination of Eqns (1) and (2) for this case:
3) Very low load, i.e., high ZL, such that ZL>Zm, and ZT≈Zm) applies for small cell clusters and/or highly uncoupled tissue, and highlights the importance of the coupling to the immediate neighbors, Zi and the membrane impedance of the immediate neighbors. The ease of excitation will improve by higher coupling to the neighbors for CM-CM (low Zi) in this case compared to D-CM and lower Zm of the neighbors (possibly determined by the equivalent K+ conductance in the neighbor CMs), as seen from the simplified Eqn. (4).
4) Overall, better coupling for S-N, but reduced coupling (and equivalent conductivity) for N-L works towards ease of excitation. It is important to note, however, that for spatially distributed D-cells, the S-N coupling in the S-N-L triad plays a dual role, i.e., it affects both the S-N charge transfer and the equivalent “load” presented by the tissue. All parameters indicated in d act in parallel and actual simulations, quantification is needed to fully uncover the extent of their individual contribution.
This application claims priority to U.S. Application No. 61/394,256 filed Oct. 18, 2010, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/56716 | 10/18/2011 | WO | 00 | 7/1/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61394256 | Oct 2010 | US |
