Patent Application
20140127704
The present invention relates to a kit for diagnosing cardiomyopathy. This invention also relates to a method for predicting a risk of cardiomyopathy for a subject. This invention further relates to a method for screening an agent for treating cardiomyopathy.
A cardiomyopathy is the deterioration of the function of the myocardium. In cardiomyopathy, the heart muscle becomes enlarged, thick, or rigid. A subject with cardiomyopathy is often at risk of dangerous forms of irregular heart beat, heart failure and sudden cardiac death. According to the classification of pathphysiology, the cardiomyopathy encompasses four forms as follows: hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy, restrictive cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy. The common forms of cardiomyopathy are hypertrophic cardiomyopathy and dilated cardiomyopathy.
HCM is an intrinsic myocardial disorder characterized by unexplained left ventricular hypertrophy. Currently, the prevalence of HCM is about 0.2% to 0.5% of the general population. Many mechanisms and determinants cause the occurring of the disease and sarcomere mutation is most frequently reason among them. In HCM, the sarcomeres in the heart increase in size, which results in the thickening of the heart muscle. In addition, the normal alignment of muscle cells is disrupted, a phenomenon known as myocardial disarray. HCM also causes disruptions of the electrical functions of the heart. HCM is most commonly due to a mutation in one of 9 sarcomeric genes that results in a mutated protein in the sarcomere, the primary component of the myocyte.
A nuclear receptor interaction protein (NRIP) is a transcription factor that only expresses in cell nuclei. It is reported that lack of NRIP gene expression in clinical muscular dystrophy and NRIP knockout mice display weaker muscle strength, indicating that NRIP plays a role in muscle function (Zhang Y et al, Differential expression profiling between the relative normal and dystrophic muscle tissues from the same LGMD patient. J Transl Med, 2006, 4: 53).
The relationship of cardiomyopathy and NRIP is not studied in the previous reports.
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The hearts are excised and fixed with 4% paraformaldehyde (PFA) at 4° C. overnight. Some parts of left ventricle posterior wall are used to ultrastructural analysis.
The present invention provides a kit for diagnosing cardiomyopathy, comprising using an antibody or oligonucleotide to measure the expression level of nuclear receptor interaction protein (NRIP).
The present invention also provides a method of predicting a risk of cardiomyopathy for a subject, comprising measuring the expression level of NRIP in a test sample from said subject, wherein a reduction in the expression level of NRIP in the test sample, relative to the expression level of NRIP in a control sample from a non-cardiomyopathy subject, is indicative of a higher risk of cardiomyopathy for said subject.
The present invention further provides a method of screening an agent for treating cardiomyopathy, comprising providing a test agent to a test sample from subject suffering from cardiomyopathy and measuring the expression level of NRIP, wherein an increasing or equal amount in the level of NRIP in the test sample relative to a control sample from a non-cardiomyopathy subject, is indicative of the test agent being the agent for treating cardiomyopathy.
The present invention shows that nuclear receptor interaction protein (NRIP) is a Z-disc protein of sarcomere. The present invention demonstrates that NRIP interacts with α-actinin-2 (ACTN2) and co-localizes with ACTN2 on Z-bands of cardiac muscle by in vitro and in vivo of protein binding assay and immunohistochemistry. Because the previous reports indicate that ACTN2 belongs a Z-disc protein (Sorimachi H et al, J Mol Biol., 1997, Aug. 1; 270(5):688-695), it demonstrates that NRIP is one of Z-disc proteins according to above results. Further, the results of the present invention from NRIP knockout mice model show that left ventricle of NRIP−/− mice is dilated as compared to NRIP+/+ mice and reveal that deficiency of NRIP progressively leads to cardiac hypertrophy.
The present invention shows that NRIP interacts with ACTN2 and is one of Z-disc protein. Moreover, the deficiency of NRIP causes the abnormal cardiac function and leads to hypertrophic cardiomyopathy which is due to left ventricular hypertrophy. According to above results, the present invention can be served as a diagnostic and a therapeutic target for cardiomyopathy or cardiac disease which is caused by deficiency of NRIP. The application of the present invention as follows: to make a pharmaceutical composition of NRIP used to treat cardiomyopathy, to use other agent repairing the abnormal function of NRIP to cure the cardiac disease which was caused by loss of NRIP, and to develop NRIP as a biomarker to identify the occurring risk of cardiomyopathy or progress of cardiac hypertrophy.
As defined herein, the term “cardiomyopathy” refers to a cardiac disease which is induced by loss of NRIP, includes but is not limited to hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy or arrhythmogenic right ventricular cardiomyopathy. The expression level of NRIP, as used herein, is the expression level of protein, RNA or DNA of NRIP. The NRIP, as used herein, is a Z-disc protein and interacts with ACTN2.
The present invention provides a kit for diagnosing cardiomyopathy, comprising an antibody or oligonucleotide for measuring an expression level of NRIP. In a preferred embodiment, the cardiomyopathy is hypertrophic cardiomyopathy.
The present invention also provides a method of predicting a risk of cardiomyopathy for a subject, comprising measuring an expression level of NRIP in a test sample from said subject, wherein a reduction in the expression level of NRIP in the test sample, relative to the expression level of NRIP in a control sample from a non-cardiomyopathy subject, is indicative of a higher risk of cardiomyopathy for said subject. In a preferred embodiment, the said subject and non-cardiomyopathy subject are animal or human. In a preferred embodiment, the test sample and the control sample are blood, serum or plasma.
The present invention further provides a method of screening an agent for treating cardiomyopathy, comprising providing a test agent to a test sample from a subject suffering from cardiomyopathy, and measuring an expression level of NRIP in test sample, wherein an increasing or equal amount in the expression level of NRIP in the test sample, relative to a control sample from a non-cardiomyopathy subject, is indicative of the test agent being the agent for treating cardiomyopathy. In a preferred embodiment, the said subject and non-cardiomyopathy subject are animal or human. In a preferred embodiment, the test sample and the control sample are blood, serum, plasma or cardiac cell.
The examples below are non-limiting and are merely representative of various aspects and features of the present invention.
The present invention used full-length NRIP as bait to perform a yeast two-hybrid screen of a human prostate cDNA library. Under 2×105 of transformants, yeast cells were co-transformed with the bait vector fused to NRIP cDNA and prey vectors fused to the cDNAs from prostate tissues of 25 years old human (Invitrogen). The α-actinin (ACTN) family including ACTN1, ACTN2 and ACTN4 were mostly found from total 145 colonies (Table 1). The result demonstrated that actinins were potentially interacting partners of NRIP.
To further confirm the result of yeast two-hybrid assay, the in vitro binding assay using bacterial expressed NRIP and ACTN was performed (
Since α-Actinin was the major component of the Z-disc and ACTN2 was the cardiac muscle-specific isoform, the localization of NRIP at Z-bands in cardiac muscle was further investigated. By using antibodies against NRIP, ACTN2 and myomesin to perform immunofluorescence staining, NRIP was found to co-localize with ACTN2 on Z-bands of cardiac muscle (
The present invention further examined the cardiac function of wild type (WT, NRIP+/+) and NRIP−/− mice by echocardiography from young to middle aged stage (Table 2). The results showed the measurements of ejection fraction (EF) and fraction shortening (FS) from NRIP−/− mice were lower than WT implying that NRIP−/− mice had cardiac functional defects. Additionally, the dimension of interventricular septum (IVSd), posterior wall (PW) and left ventricle and left ventricle mass were increased with aging showing that NRIP−/− mice were on the process of cardiac hypertrophy. By comparing the hearts excised from WT and NRIP−/− mice at 39 weeks, the sizes of ventricle and atrium of NRIP−/− mice were enlarged (
Many Z-disc proteins have proved that mutations or defects of these proteins disrupt cardiac cytoarchitectural organization and lead to cardiomyopathy. The present invention next investigated the effects of NRIP−/− in sarcommeric structure. Hearts were excised from adult WT (NRIP+/+) and KO (NRIP−/−) mice, and fixed for transmission electron microscopy (TEM) analysis (
One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The animals, and processes and methods for producing them are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.
