Pathogenesis of cardiomyopathy

Abstract

Disclosed within is a mouse, and cells derived therefrom, which are homozygous for a disrupted δ-sarcoglycan gene, the disruption in said gene having been introduced into the mouse or an ancestor of the mouse at an embryonic stage. Said disruption prevents the synthesis of functional δ-sarcoglycan in cells of the mouse and results in the mouse having a reduced amount of β- and ε-sarcoglycan and sarcospan, and a disruption of the sarcoglycan-sarcospan complex in smooth muscle of the mouse. Said disruption also results in a reduced amount of sarcospan, α-, β-, γ-, and ε-sarcoglycan in the sarcolemma of skeletal and cardiac muscles of the mouse, compared to the amounts of said components in a mouse lacking disrupted δ-sarcoglycan genes. Preferred specific disruptions of the δ-sarcoglycan gene are listed. Also disclosed is a mouse, and cells derived therefrom, which are homozygous for a disrupted β-sarcoglycan gene, the disruption in said gene having been introduced into the mouse or an ancestor of the mouse at an embryonic stage. The disruption prevents the synthesis of functional β-sarcoglycan in cells of the mouse and results in the mouse having a reduced amount of δ- and ε-sarcoglycan and sarcospan and α-dystroglycan in smooth muscle of the mouse. The disruption also results in a disruption of the sarcoglycan-sarcospan complex in smooth muscle of the mouse, and a reduced amount of sarcospan, α, γ, δ- and ε-sarcoglycan in the sarcolemma of skeletal and cardiac muscles of the mouse, compared to the amounts of the components in a mouse lacking disrupted β-sarcoglycan genes. Preferred specific disruptions of the β-sarcoglycan gene are listed. A method for treating mammalian autosomal recessive limb-girdle muscular dystrophy type 2F in an individual is also disclosed. The method comprises, providing an expression vector which encodes a wild-type form of δ-sarcoglycan, and introducing the expression vector into skeletal and smooth muscle tissue of the individual under conditions appropriate for expression of the wild-type form of δ-sarcoglycan in said tissues. Examples of expression vectors for use in this method are adenovirus expression vector, a gutted adenovirus expression vector, and an adeno-associated expression vector. Also disclosed are methods for treating mammalian autosomal recessive limb-girdle muscular dystrophy type 2E, and type 2F, in an individual. The methods comprise, providing an expression vector which encodes a wild-type form of β-sarcoglycan, or δ-sarcoglycan, respectively, and introducing the expression vector into skeletal and smooth muscle tissue of the individual under conditions appropriate for expression of the wild-type form of the sarcoglycan gene in said tissues. The δ-sarcoglycan deficient, and β-sarcoglycan deficient mice of the present invention are useful in identifying therapeutic compounds for treatment of an individual diagnosed with δ-sarcoglycan-deficient limb-girdle muscular dystrophy, and β-sarcoglycan-deficient limb-girdle muscular dystrophy, respectively. A therapeutic method for treating ischemic heart disease caused by reduced expression of the sarcoglycan-sarcospan complex in vascular smooth muscle cells of an individual is also provided. The method comprises contacting the vascular smooth muscle cells of the individual with a vascular smooth muscle relaxant, such as Nicorandil. This method is also useful for preventing ischemic injury in skeletal and cardiac muscle of an individual caused by reduced expression of the sarcoglycan-sarcospan complex in the vascular smooth muscle cells of the individual. The method is also useful for treating mammalian autosomal recessive limb-girdle muscular dystrophy type 2F or type 2E in an individual. Other methods provided include a method for identifying a therapeutic compound for the treatment of ischemic heart disease in an individual caused by reduced expression of the sarcoglycan-sarcospan complex in the vascular smooth muscle cells of the individual, and also a method for identifying a therapeutic compound for the prevention of ischemic injury in skeletal and cardiac muscle of an individual which is caused by reduced expression of the sarcoglycan-sarcospan complex in vascular smooth muscle cells of the individual.

1 . A mouse, and cells derived therefrom, homozygous for a disrupted δ-sarcoglycan gene, the disruption in said gene having been introduced into the mouse or an ancestor of the mouse at an embryonic stage, wherein the disruption prevents the synthesis of functional δ-sarcoglycan in cells of the mouse and results in the mouse having a reduced amount of β- and ε-sarcoglycan and sarcospan and a disruption of the sarcoglycan-sarcospan complex in smooth muscle, and a reduced amount of sarcospan, α-, β-, γ-, and ε-sarcoglycan in the sarcolemma of skeletal and cardiac muscles, compared to the amounts of said components in a mouse lacking disrupted δ-sarcoglycan genes.  
     
     
         2 . The mouse, and cells derived therefrom, of    claim 1    wherein the disruption comprises a deletion of a region of 2992 base pairs, including 2277 base pairs of intron  1 , the entire exon  2 , and 576 base pairs of intron  2 , and replacement of the deleted region with a PGK-neomycin cassette as a marker for neomycin resistance.  
     
     
         3 . The mouse, and cells derived therefrom, of    claim 2    wherein the deletion results from the introduction, into embryonic stem cells, of a DNA construct comprising: 
 a) 5 kb of intron  1  and 5 kb of intron  2  of the δ-sarcoglycan gene; and  
 b) a neomycin resistance gene inserted between intron  1  and intron  2  of the δ-sarcoglycan gene, the neomycin resistance gene being in the opposite transcriptional orientation as the δ-sarcoglycan exons replaced, wherein the construct lacks exon  2  of the δ-sarcoglycan gene.  
 
     
     
         4 . A mouse, and cells derived therefrom, homozygous for a disrupted β-sarcoglycan gene, the disruption in said gene having been introduced into the mouse or an ancestor of the mouse at an embryonic stage, wherein the disruption prevents the synthesis of functional β-sarcoglycan in cells of the mouse and results in the mouse having a reduced amount of δ- and ε-sarcoglycan and sarcospan and α-dystroglycan in smooth muscle, and a disruption of the sarcoglycan-sarcospan complex in smooth muscle, and a reduced amount of sarcospan, α-, γ-, δ- and ε-sarcoglycan in the sarcolemma of skeletal and cardiac muscles, compared to the amounts of the components in a mouse lacking disrupted β-sarcoglycan genes.  
     
     
         5 . The mouse, and cells derived therefrom, of    claim 4    wherein the disruption comprises a deletion of a region of about 7.5 kb, including 1606 bp of intron  2 , the entirety of exon  3 , exon  4 , exon  5 , exon  6 , intron  3 , intron  4 , and intron  5 , and 498 bp immediately downstream of exon  6 , and replacement of the deleted region with a PGK-neomycin cassette as a marker for neomycin resistance.  
     
     
         6 . The mouse, and cells derived therefrom, of    claim 5    wherein said disruption results from the introduction, into embryonic stem cells, of a DNA construct comprising: 
 a) 1800 bp of intron  2  and 6500 bp of sequences which begin 498 bp directly downstream of exon  6 , of the β-sarcoglycan gene; and  
 b) a neomycin resistance gene inserted between intron  2  and the sequences downstream of exon  6  of the β-sarcoglycan gene, wherein said construct lacks exon  3 , exon  4 , exon  5 , exon  6 , intron  3 , intron  4 , and intron  5 , of the β-sarcoglycan gene.  
 
     
     
         7 . A method for treating mammalian autosomal recessive limb-girdle muscular dystrophy type 2F in an individual, comprising the steps: 
 a) providing an expression vector which encodes a wild-type form of δ-sarcoglycan; and    b) introducing the expression vector into skeletal and smooth muscle tissue of the individual under conditions appropriate for expression of the wild-type form of δ-sarcoglycan in said tissues.    
     
     
         8 . The method of    claim 7    wherein the expression vector is selected from the group consisting of an adenovirus expression vector, a gutted adenovirus expression vector and an adeno-associated expression vector.  
     
     
         9 . The method of    claim 7    where the expression vector contains a muscle tissue-specific promoter.  
     
     
         10 . The method of    claim 7    wherein the expression vector is introduced into skeletal muscle by intramuscular injection.  
     
     
         11 . A method for treating mammalian autosomal recessive limb-girdle muscular dystrophy type 2E in an individual, comprising the steps: 
 a) providing an expression vector which encodes a wild-type form of β-sarcoglycan; and    b) introducing the expression vector into skeletal and smooth muscle tissue of the individual under conditions appropriate for expression of the wild-type form of β-sarcoglycan in said tissues.    
     
     
         12 . The method of    claim 11    wherein the expression vector is selected from the group consisting of an adenovirus expression vector, a gutted adenovirus expression vector and an adeno-associated expression vector.  
     
     
         13 . The method of    claim 11    where the expression vector contains a muscle tissue-specific promoter.  
     
     
         14 . The method of    claim 11    wherein the expression vector is introduced into skeletal muscle by intramuscular injection.  
     
     
         15 . A method for identifying a therapeutic compound useful for treatment of an individual diagnosed with δ-sarcoglycan-deficient limb-girdle muscular dystrophy, comprising: 
 a) providing a mouse homozygous for a disrupted δ-sarcoglycan gene;  
 b) administering a candidate compound to the mouse of step a); and  
 c) assaying for therapeutic effects on the mouse of step a), the detection of therapeutic effects being an indication that the administered compound is a therapeutic compound for treatment of the individual.  
 
     
     
         16 . The method of    claim 15    wherein the candidate compound is administered to the mouse by means to contact the candidate compound with smooth muscle cells of the mouse.  
     
     
         17 . The method of    claim 15    wherein the candidate compound is administered to the mouse by means to contact the candidate compound with skeletal muscle cells of the mouse.  
     
     
         18 . The method of    claim 15    wherein the candidate therapeutic compound is administered to the mouse by means to contact the candidate compound with cardiac muscle cells of the mouse.  
     
     
         19 . The method of    claim 15    wherein the candidate compound is a gene and the gene is administered under conditions appropriate for expression of the gene in cells of the mouse.  
     
     
         20 . A method for identifying a therapeutic compound useful for treatment of an individual diagnosed with β-sarcoglycan-deficient limb-girdle muscular dystrophy, comprising: 
 a) providing a mouse homozygous for a disrupted β-sarcoglycan gene;  
 b) administering a candidate compound to the mouse of step a); and  
 c) assaying for therapeutic effects on the mouse of step a), the detection of therapeutic effects being an indication that the administered compound is a therapeutic compound for treatment of the individual.  
 
     
     
         21 . The method of    claim 20    wherein the candidate compound is administered to the mouse by means to contact the candidate compound with smooth muscle cells of the mouse.  
     
     
         22 . The method of    claim 20    wherein the candidate compound is administered to the mouse by means to contact the candidate compound with skeletal muscle cells of the mouse.  
     
     
         23 . The method of    claim 20    wherein the candidate therapeutic compound is administered to the mouse by means to contact the candidate compound with cardiac muscle cells of the mouse.  
     
     
         24 . The method of    claim 20    wherein the candidate compound is a gene and the gene is administered under conditions appropriate for expression of the gene in cells of the mouse.  
     
     
         25 . A therapeutic method for treating ischemic heart disease caused by reduced expression of the sarcoglycan-sarcospan complex in vascular smooth muscle cells of an individual, comprising contacting the vascular smooth muscle cells of the individual with a vascular smooth muscle relaxant.  
     
     
         26 . The therapeutic method of    claim 25    wherein the vascular smooth muscle relaxant is Nicorandil.  
     
     
         27 . The method of    claim 25    wherein the reduced expression of the sarcoglycan-sarcospan complex in vascular smooth muscle cells of the individual is due to a defect in the δ-sarcoglycan genes of the individual.  
     
     
         28 . The method of    claim 25    wherein the reduced expression of the sarcoglycan-sarcospan complex in the vascular smooth muscle cells of the individual is due to a defect in the β-sarcoglycan genes of the individual.  
     
     
         29 . A method for preventing ischemic injury in skeletal and cardiac muscle of an individual caused by reduced expression of the sarcoglycan-sarcospan complex in the vascular smooth muscle cells of the individual, the method comprising contacting the vascular smooth muscle cells of the individual with a vascular smooth muscle relaxant.  
     
     
         30 . The method of    claim 29    wherein the vascular smooth muscle relaxant is Nicorandil.  
     
     
         31 . A method for treating mammalian autosomal recessive limb-girdle muscular dystrophy type 2F in an individual, comprising administering a vascular smooth muscle relaxant to the individual.  
     
     
         32 . A method for treating mammalian autosomal recessive limb-girdle muscular dystrophy type 2E in an individual, comprising administering a vascular smooth muscle relaxant to the individual.  
     
     
         33 . A method for identifying a therapeutic compound for the treatment of ischemic heart disease in an individual caused by reduced expression of the sarcoglycan-sarcospan complex in the vascular smooth muscle cells of the individual, comprising: 
 a) providing a mouse which has reduced expression of the sarcoglycan-sarcospan complex in the vascular smooth muscle cells;    b) administering a candidate compound to the mouse of step a) by means to contact the candidate compound with the vascular smooth muscle cells of the mouse; and    c) assaying for therapeutic effects on the mouse of step a), the detection of therapeutic effects being an indication that the administered compound is a therapeutic compound.    
     
     
         34 . The method of    claim 33    wherein the mouse is homozygous for a disrupted δ-sarcoglycan gene.  
     
     
         35 . The method of    claim 33    wherein the mouse is homozygous for a disrupted β-sarcoglycan gene.  
     
     
         36 . A method for identifying a therapeutic compound for the prevention of ischemic injury in skeletal and cardiac muscle of an individual which is caused by reduced expression of the sarcoglycan-sarcospan complex in vascular smooth muscle cells of the individual, the method comprising: 
 a) providing a mouse which has reduced expression of the sarcoglycan-sarcospan complex in vascular smooth muscle cells;    b) administering a candidate compound to the mouse of step a) by means to contact the vascular smooth muscle cells of the mouse; and    c) assaying for protection of the mouse which has received the candidate compound from ischemic injury, the determination of protection being an indication that the administered compound is a therapeutic compound.    
     
     
         37 . The method of    claim 36    wherein the mouse is homozygous for a disrupted δ-sarcoglycan gene.  
     
     
         38 . The method of    claim 36    wherein the mouse is homozygous for a disrupted β-sarcoglycan gene.