Controlling pathways that regulate muscle contraction in the heart

Abstract

This invention provides methods of regulating contraction of a subject's heart and of treating heart failure and cardiac arrhythmia. This invention also provides methods of obtaining compounds that bind to, and activate or inhibit the activation of a type 2 ryanodine (RyR2) receptor, and methods for screening for compounds that alleviate heart disease.

What is claimed is:  
     
         1 . A method of regulating contraction of a subject's heart by administering to the subject a compound which regulates protein kinase A (PKA) phosphorylation of a type 2 ryanodine (RyR2) receptor of the subject's heart.  
     
     
         2 . The method of  claim 1 , wherein PKA phosphorylation of the RyR2 receptor causes dissociation of a FKBP12.6 binding protein from the RyR2 receptor.  
     
     
         3 . A method of treating a subject's heart failure by administering to the subject a compound which decreases protein kinase A (PKA) phosphorylation of a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's heart failure.  
     
     
         4 . The method of  claim 3 , wherein PKA phosphorylation of the RyR2 receptor causes dissociation of a FKBP12.6 binding protein from the RyR2 receptor.  
     
     
         5 . A method of treating a subject's heart failure by administering to the subject a compound which decreases dissociation of a FKBP12.6 binding protein from a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's heart failure.  
     
     
         6 . A method of treating a subject's heart failure by administering to the subject a compound which mimics binding of a FKBP12.6 binding protein to a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's heart failure.  
     
     
         7 . A method of treating a subject's cardiac arrhythmia by administering to the subject a compound which decreases protein kinase A (PKA) phosphorylation of a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's cardiac arrhythmia.  
     
     
         8 . The method of  claim 7 , wherein PKA phosphorylation of the RyR2 receptor causes dissociation of a FKBP12.6 binding protein from the RyR2 receptor.  
     
     
         9 . The method of  claim 7 , wherein the cardiac arrhythmia is a ventricular fibrillation or a ventricular tachycardia.  
     
     
         10 . A method of treating a subject's cardiac arrhythmia by administering to the subject a compound which decreases dissociation of a FKBP12.6 binding protein from a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's cardiac arrhythmia.  
     
     
         11 . The method of  claim 10 , wherein the cardiac arrhythmia is a ventricular fibrillation or a ventricular tachycardia.  
     
     
         12 . A method of treating a subject's cardiac arrhythmia by administering to the subject a compound which mimics binding of a FKBP12.6 binding protein to a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's cardiac arrhythmia.  
     
     
         13 . The method of  claim 12 , wherein the cardiac arrhythmia is a ventricular fibrillation or a ventricular tachycardia.  
     
     
         14 . A method for identifying a chemical compound which specifically binds to a type 2 ryanodine (RyR2) receptor, which comprises contacting cells expressing the RyR2 receptor, or contacting a fraction containing sacroplasmic reticulum or endoplasmic reticulum from a cell extract of said cells, with the chemical compound under conditions suitable for binding and detecting specific binding of the chemical compound to the RyR2 receptor.  
     
     
         15 . A method involving competitive binding for identifying a chemical compound which specifically binds to a type 2 ryanodine (RyR2) receptor, which comprises separately contacting cells expressing the RyR2 receptor, or separately contacting a fraction containing sacroplasmic reticulum or endoplasmic reticulum from a cell extract of said cells, with both the chemical compound and a second chemical compound known to bind to the RyR2 receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the RyR2 receptor, a decrease in binding of the second chemical compound to the RyR2 receptor in the presence of the chemical compound indicating that the chemical compound binds to the RyR2 receptor.  
     
     
         16 . The method of  claim 14  or  15 , wherein the chemical compound is not previously known to bind to a type 2 ryanodine (RyR2) receptor.  
     
     
         17 . A chemical compound identified by the method of  claim 16 .  
     
     
         18 . A method of screening a plurality of chemical compounds not known to bind to a type 2 ryanodine (RyR2) receptor to identify a compound which specifically binds to the RyR2 receptor, which comprises: 
 (a) contacting cells expressing the RyR2 receptor, or contacting a fraction containing sacroplasmic reticulum or endoplasmic reticulum from a cell extract of said cells, with a compound known to bind specifically to the RyR2 receptor;    (b) contacting the cells or fraction of step (a) with the plurality of compounds not known to bind specifically to the RyR2 receptor, under conditions permitting binding of compounds known to bind to the RyR2 receptor;    (c) determining whether the binding of the compound known to bind to the RyR2 receptor is reduced in the presence of the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so    (d) separately determining the binding to the RyR2 receptor of each compound included in the plurality of compounds, so as to thereby identify any compound included therein which specifically binds to the RyR2 receptor.    
     
     
         19 . A compound identified by the method of  claim 18 .  
     
     
         20 . A method for determining whether a chemical compound activates a type 2 ryanodine (RyR2) receptor, which comprises contacting cells expressing the RyR2 receptor, or contacting a fraction containing sacroplasmic reticulum or endoplasmic reticulum from a cell extract of said cells, with the chemical compound under conditions suitable for activation of the RyR2 receptor and measuring RyR2 receptor activation in the presence and in the absence of the chemical compound, an increase in RyR2 receptor activation in the presence of the chemical compound indicating that the chemical compound activates the RyR2 receptor.  
     
     
         21 . The method of  claim 20 , wherein the chemical compound is not previously known to activate a type 2 ryanodine (RyR2) receptor.  
     
     
         22 . A chemical compound identified by the method of  claim 21 .  
     
     
         23 . A method of screening a plurality of chemical compounds not known to activate a type 2 ryanodine (RyR2) receptor to identify a compound which activates the RyR2 receptor which comprises: 
 (a) contacting cells expressing the RyR2 receptor, or contacting a fraction containing sacroplasmic reticulum or endoplasmic reticulum from a cell extract of said cells, with the plurality of compounds not known to activate the RyR2 receptor, under conditions permitting activation of the RyR2 receptor;    (b) determining whether the activity of the RyR2 receptor is increased in the presence of one or more of the compounds; and if so    (c) separately determining whether the activation of the RyR2 receptor is increased by any compound included in the plurality of compounds, so as to thereby identify each compound which activates the RyR2 receptor.    
     
     
         24 . A chemical compound identified by the method of  claim 23 .  
     
     
         25 . A method for determining whether a chemical compound inhibits activation of a type 2 ryanodine (RyR2) receptor, which comprises separately contacting cells expressing the RyR2 receptor, or separately contacting a fraction containing sacroplasmic reticulum or endoplasmic reticulum from a cell extract of cells, with both the chemical compound and a second chemical compound known to activate the RyR2 receptor, and with only the second chemical compound, under conditions suitable for activation of the RyR2 receptor, and measuring RyR2 receptor activation in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller RyR2 receptor activation in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the RyR2 receptor.  
     
     
         26 . The method of  claim 25 , wherein the chemical compound is not previously known to inhibit activation of a type 2 ryanodine (RyR2) receptor.  
     
     
         27 . A chemical compound identified by the method of  claim 26 .  
     
     
         28 . A method of screening a plurality of chemical compounds not known to inhibit the activation of a type 2 ryanodine (RyR2) receptor to identify a compound which inhibits the activation of the RyR2 receptor, which comprises: 
 (a) contacting cells expressing the RyR2 receptor, or contacting a fraction containing sacroplasmic reticulum or endoplasmic reticulum from a cell extract of said cells, with the plurality of compounds in the presence of a known RyR2 receptor activator, under conditions permitting activation of the RyR2 receptor;    (b) determining whether the amount of activation of the RyR2 receptor is reduced in the presence of one or more of the compounds, relative to the amount of activation of the RyR2 receptor in the absence of such one or more compounds; and if so    (c) separately determining whether each such compound inhibits activation of the RyR2 receptor for each compound included in the plurality of compounds, so as to thereby identify any compound included in such plurality of compounds which inhibits the activation of the RyR2 receptor.    
     
     
         29 . A chemical compound identified by the method of  claim 28 .  
     
     
         30 . The method of any of  claim 14 ,  15 ,  18 ,  20 ,  23 ,  25 , or  28 , wherein the RyR2 receptor is a human RyR2 receptor.  
     
     
         31 . The method of any of  claim 14 ,  15 ,  18 ,  20 ,  23 ,  25 , or  28 , wherein the nucleic acid encoding the RyR2 receptor is endogenous to the cell.  
     
     
         32 . The method of any of  claim 14 ,  15 ,  18 ,  20 ,  23 ,  25 , or  28 , wherein the nucleic acid encoding the RyR2 receptor is transfected into the cell.  
     
     
         33 . The method of  claim 32 , wherein a beta adrenergic receptor is co-expressed with the RyR2 receptor.  
     
     
         34 . The method of  claim 32 , wherein the cell is a bacterial cell, a yeast cell, an insect cell, an amphibian cell, a plant cell or a mammalian cell.  
     
     
         35 . The method of  claim 34 , wherein the mammalian cell is a HEK293 cell, a Chinese hamster ovary (CHO) cell, a COS-7 cell, a LM(tk-) cell, a mouse embroyonic fibroblast NIH-3T3 cell, a mouse Y1 cell, a 293 human embryonic kidney cell, or a HeLa cell.  
     
     
         36 . The method of  claim 34 , wherein the insect cell is an Sf9 cell, an Sf21 cell or a Trichoplusia ni 5B-4 cell.  
     
     
         37 . The method of  claim 34 , wherein the amphibian cell is a Xenopus oocyte cell or a Xenopus melanophore cell.  
     
     
         38 . The method of any of  claim 20 ,  23 ,  25 , or  28 , wherein activation of the RyR2 receptor is measured by measuring protein kinase A phosphorylation of the RyR2 receptor.  
     
     
         39 . The method of  claim 25  or  28 , wherein the cells are cardiac cells from a subject with a failing heart.  
     
     
         40 . The method of  claim 39 , wherein the subject is an animal in which heart failure has been induced by rapid cardiac pacing or a human.  
     
     
         41 . A method for making a composition of matter which specifically binds to a RyR2 receptor which comprises identifying a chemical compound using the method of  claim 14 ,  15 , or  18 , and then synthesizing the chemical compound or a novel structural and functional analog or homolog thereof.  
     
     
         42 . A method for making a composition of matter which activates a RyR2 receptor which comprises identifying a chemical compound using the method of  claim 20  or  23 , and then synthesizing the chemical compound or a novel structural and functional analog or homolog thereof.  
     
     
         43 . A method for making a composition of matter which inhibits the activation of a RyR2 receptor which comprises identifying a chemical compound using the method of  claim 25  or  28 , and then synthesizing the chemical compound or a novel structural and functional analog or homolog thereof.  
     
     
         44 . A pharmaceutical composition comprising (a) an amount of a chemical compound identified using the method of  claim 20  or  23 , or a novel structural and functional homolog or analog thereof, capable of passing through a cell membrane and effective to activate a RyR2 receptor and (b) a pharmaceutically acceptable carrier capable of passing through the cell membrane.  
     
     
         45 . A pharmaceutical composition comprising (a) an amount of a chemical compound identified using the method of  claim 25  or  28 , or a novel structural and functional homolog or analog thereof, capable of passing through a cell membrane and effective to inhibit the activation of a RyR2 receptor and (b) a pharmaceutically acceptable carrier capable of passing through the cell membrane.  
     
     
         46 . A method for preparing a composition which comprises admixing a carrier and a pharmaceutically effective amount of a chemical compound identified by the method of any of  claim 14 ,  15 ,  18 ,  20 ,  23 ,  25 , or  28 , or a novel structural and functional anaolog or homolog thereof.  
     
     
         47 . A method of treating a subject with a heart disease which comprises administering to the subject a therapeutically effective amount of a chemical compound identified by the method of any of  claim 14 ,  15 ,  18 ,  20 ,  23 ,  25 , or  28 , or a novel structural and functional anaolog or homolog thereof.  
     
     
         48 . A method of  claim 47 , wherein the heart disease is a cardiac failure or a cardiac arrhythmia.  
     
     
         49 . A method of  claim 48 , wherein the cardiac arrhythmia is a ventricular fibrillation or a ventricular tachycardia.  
     
     
         50 . Use of a chemical compound identified by the method of  claim 20  or  23  for the preparation of a pharmaceutical composition for treating an abnormality, wherein the abnormality is alleviated by activating a RyR2 receptor.  
     
     
         51 . A use of  claim 50 , wherein the abnormality is a heart disease.  
     
     
         52 . Use of a chemical compound identified by the method of  claim 25  or  28  for the preparation of a pharmaceutical composition for treating an abnormality, wherein the abnormality is alleviated by inhibiting the activation of a RyR2 receptor.  
     
     
         53 . A use of  claim 52 , wherein the abnormality is a heart disease.  
     
     
         54 . A use of a  claim 53 , wherein the heart disease is a cardiac failure or a cardiac arrhythmia.  
     
     
         55 . A use of  claim 54 , wherein the cardiac arrhythmia is a ventricular fibrillation or a ventricular tachycardia.