Methods for treating cardiac disease by modifying an N-terminal domain of troponin I

Abstract

The invention provides methods for identifying modulators of cardiac troponin I activity as well as methods for using N-terminally truncated forms of cTnI to monitor the function of the circulatory system in general, and the heart in particular, of an organism such as man. Additionally, the invention provides methods of treating circulatory diseases, disorders or conditions, such as cardiac diseases and conditions, by administering a therapeutically effective amount of a modulator of the N-terminal structure of cTnI, such as a molecule capable of binding to the N-terminus of full-length cTnI, a molecule that modulates cTnI phosphorylation and/or N-terminal proteolytic cleavage, or by administering N-terminally truncated cTnI or a polynucleotide encoding that protein or polypeptide.

1 . A method for monitoring cardiac function comprising measuring the level of a cardiac troponin I polypeptide selected from the group consisting of N-terminally truncated cardiac troponin I, an N-terminal fragment of cardiac troponin I and a modified cardiac troponin I in a patient.  
     
     
         2 . The method according to  claim 1  wherein the measuring comprises contacting a sample from the patient with an antibody specifically recognizing the peptide consisting of the sequence set forth in SEQ ID NO:5 and determining the level of binding of the antibody to the sample.  
     
     
         3 . The method according to  claim 1  wherein said measurement determines the relative proportion of cardiac troponin I that is truncated.  
     
     
         4 . The method according to  claim 1  wherein said cardiac troponin I polypeptide is measured in a blood sample from said patient.  
     
     
         5 . The method according to  claim 1  wherein said N-terminally truncated cardiac troponin I lacks amino acid residues selected from the group consisting of residues 1-26 of SEQ ID NO:2, residues 1-27 of SEQ ID NO:2 and residues 1-30 of SEQ ID NO:2.  
     
     
         6 . The method according to  claim 1  wherein the truncated cardiac troponin I is truncated human cardiac troponin I.  
     
     
         7 . A method for treating a cardiac disorder by increasing ventricular relaxation comprising administering a therapeutically effective amount of a protein selected from the group consisting of an N-terminally truncated cardiac troponin I and a modified cardiac troponin I.  
     
     
         8 . The method according to  claim 7  wherein said administering comprises producing the N-terminally truncated cardiac troponin I by expressing a nucleic acid encoding the truncated cardiac troponin I in a host cell of an organism being treated.  
     
     
         9 . A method of screening for a modulator of the N-terminal structure of cardiac troponin I (cTnI) comprising 
 (a) incubating a full-length cardiac troponin I in the presence and absence of a candidate modulator;    (b) assessing the N-terminal structure of the cardiac troponin I; and    (c) identifying a candidate modulator as a modulator of N-terminal structure of cardiac troponin I when the N-terminal structure of cTnI in the absence of said modulator differs from the N-terminal structure of cTnI in the presence of said modulator.    
     
     
         10 . The method according to  claim 9  further comprising protein kinase A in said incubating step, wherein said modulator is identified as a modulator of cardiac troponin I phosphorylation.  
     
     
         11 . The method according to  claim 10  wherein said measuring comprises determining the binding between said cardiac troponin I and a specific anti-cardiac troponin I antibody that exhibits differential binding to a non-phosphorylated cTnI and a phosphorylated cTnI.  
     
     
         12 . The method according to  claim 9  wherein said measuring comprises determining the binding between said cardiac troponin I and a specific anti-cardiac troponin I antibody that exhibits differential binding to a non-modified cTnI and a modified cTnI.  
     
     
         13 . A method of screening for a modulator of cardiac troponin I (cTnI) activity comprising 
 (a) incubating a full-length cardiac troponin I in the presence and absence of a candidate modulator;    (b) assessing cardiac troponin I activity; and    (c) identifying a candidate modulator as a modulator of cardiac troponin I activity when the activity of cTnI in the absence of said candidate modulator differs from the activity of cTnI in the presence of said candidate modulator.    
     
     
         14 . The method according to  claim 13  wherein the assessing comprises determining the conformation of cTnI in the presence and absence of the candidate modulator.  
     
     
         15 . The method according to  claim 14  wherein the conformation of a region of cTnI other than the N-terminal region is assessed.  
     
     
         16 . A method of screening for a modulator of cardiac troponin calcium (Ca 2+ ) binding affinity comprising 
 (a) incubating a cardiomyocyte preparation comprising a cardiac troponin I comprising a serine residue selected from the group consisting of Ser23 of SEQ ID NO:2 and Ser24 of SEQ ID NO:2 in the presence and absence of a candidate modulator;    (b) measuring the level of contraction of the preparation; and    (c) identifying a candidate modulator as a modulator of cardiac troponin I when the level of contraction of the preparation in the absence of said modulator differs from the level in the presence of said modulator.    
     
     
         17 . The method according to  claim 16  wherein the level of contraction of the cardiomyocyte preparation is measured by determining the level of Ca 2+ -induced actomyosin ATPase activity.  
     
     
         18 . A method of treating a cardiac disorder by increasing ventricular relaxation comprising administering a therapeutically effective amount of a modulator according to  claim 9  to a patient in need.  
     
     
         19 . The method according to  claim 18  wherein the modulator is an inhibitor of cardiac troponin I.  
     
     
         20 . A method of screening for a modulator of cardiac troponin I proteolytic cleavage comprising 
 (a) contacting cytoplasmic components of a cardiomyocyte and a cardiac troponin I comprising a serine residue selected from the group consisting of Ser23 of SEQ ID NO:2 and Ser24 of SEQ ID NO:2 in the presence and absence of a candidate modulator;    (b) measuring the level of N-terminal truncation of said cardiac troponin I; and    (c) identifying a candidate modulator as a modulator of cardiac troponin I proteolytic cleavage when the cleavage level of cTnI in the absence of said modulator differs from the cleavage level of cTnI in the presence of said modulator.    
     
     
         21 . The method according to  claim 20  wherein said modulator increases said proteolytic cleavage.  
     
     
         22 . A method for treating a cardiac disorder by increasing ventricular relaxation comprising administering a therapeutically effective amount of the modulator according to  claim 21  to a patient in need.  
     
     
         23 . A pharmaceutical composition for monitoring or treating a cardiac disorder comprising an agent selected from the group consisting of a modified cardiac troponin I, an N-terminally truncated cardiac troponin I and a modulator thereof, in a pharmaceutically acceptable carrier.  
     
     
         24 . The pharmaceutical composition according to  claim 23  wherein the cardiac disorder is cardiac failure.  
     
     
         25 . A kit for monitoring or treating a cardiac disorder comprising an agent selected from the group consisting of a modified cardiac troponin I, an N-terminally truncated cardiac troponin I, and a modulator thereof, and a protocol for the use thereof to monitor or treat said cardiac disorder.  
     
     
         26 . The kit according to  claim 25  wherein the cardiac disorder is cardiac failure.  
     
     
         27 . The kit according to  claim 25  wherein the modulator is a polypeptide that specifically binds to a peptide consisting of the sequence set forth in SEQ ID NO:5.  
     
     
         28 . A kit for screening for a modulator of a cardiac troponin I activity selected from the group consisting of cTnI phosphorylation, cTnI N-terminal structural modification, cardiac troponin I-mediated troponin Ca 2+  binding affinity and N-terminal cleavage of cardiac troponin I, comprising a protocol for assaying for said modulator and a cardiac troponin I holoprotein or an N-terminally truncated cardiac troponin I.