CN1840186A - Use of calmodulin kinase ii inhibitors to treat myocardial dysfunction in structural heart disease - Google Patents

Abstract

The present invention provides a method for treating structural heart disease in a subject, comprising administering an effective amount of an inhibitor of CaMKII to the subject, whereby the administration of the inhibitor treats the structural heart disease in the subject. Also provided are transgenic animal models for treating structural heart disease. Further provided is a means of screening for a compound that can treat structural heart disease.

Description

With the myocardial dysfunction in the cam kinase II inhibitor for treating structural heart disease

The application is to be the dividing an application of Chinese patent application 02824101.0 " with the myocardial dysfunction in the cam kinase II inhibitor for treating structural heart disease " on October 1st, 2002 applying date.

The present invention requires to submit to October 1 calendar year 2001, serial number is 60/326,576 and submit to October 8 calendar year 2001, serial number is 60/328,010 U.S. Provisional Application No..60/326,576 and 60/328,010 provisional application is incorporated herein by reference in full herein.

The present invention is under the HL03727 of national sanitary portion and HL62494 financial aid, obtains that the support of government finishes.Government enjoys some right among the present invention.

Technical field

The present invention relates to the inhibition of cam kinase II (CaMKII).More specifically, structural heart disease be treated or be prevented to the inhibition of CaMKII can, for example, and the contractile dysfunction that occurs after the myocardial infarction, perhaps DCM (dilated cardiomyopathy).

Background technology

Myocardial infarction is to have the deformity and the main causes of death of significance in the U.S. and other many countries of the whole world, and also is the reason of about 2/3 heart failure case. ⁷

Some causes that the incident (as the congenital sudden change of myocardial infarction, untreated hypertension, contractile protein) of disease can cause comprising the common heart disease phenotype of chambers of the heart chamber expansion, and weaken contractile function (promptly reducing the ejection fraction of heart shrinkage period) by each ventricle thus cause the clinical syndrome of heart failure. ⁷DCM (dilated cardiomyopathy) comprises the disease entity of two kinds of uniquenesses.DCM (dilated cardiomyopathy) used herein comprises ischemic cardiomyopathy, and this disease is that to weaken with left ventricle dilatation and contractile function be the disease entity of feature.When the normal compensatory of the not infarcted myocardium of surviving after the myocardial infarction is plump when not enough, just this disease may appear. ⁷" DCM (dilated cardiomyopathy) " also can be included under the situation that does not have myocardial infarction, and the myocardium weight that causes owing to the genetic abnormality of myocardium protein increases, and contractile function weakens. ⁷DCM (dilated cardiomyopathy) experimenter is the experimenter who causes cardiac contractility to reduce owing to ventricular dilatation.Therefore, compare with the experimenter who compensates infarcted myocardium with the not infarcted myocardium plumpness of survival, DCM (dilated cardiomyopathy) and contractile dysfunction experimenter's dysfunction is different and even more serious.In addition, DCM (dilated cardiomyopathy) and contractile dysfunction disease that the experimenter takes a disease are different from and comprise unusual lax other heart disease (being cardiac diastolic function obstacle and arrhythmia).

The therapy deficiency that can be used for heart failure, thereby need new Therapeutic Method.Heart is that plumpness by remaining cardiac muscle is to keep normal contraction as far as possible to the reaction of infraction.Yet when plumpness was not enough to compensate, the result was that DCM (dilated cardiomyopathy) will cause heart failure and death with the contractile function that weakens. ¹⁹Although medical therapy after preventing myocardial infarction heart dysfunction and heart failure aspect make substantial progress, ¹⁵These problems but remain an important and unsolved public health difficult problem.

It is effective or gratifying not having a kind of pharmacological treatments at DCM (dilated cardiomyopathy), and many experimenters are dead or need to carry out heart transplantation in case-specific.At present, the pharmacological treatments utilized that is used to alleviate heart failure experimenter's heart dysfunction and reduce its mortality rate can be divided into three main kinds: angiotensin converting enzyme (ACE) inhibitor, Beta-3 adrenergic receptor (β AR) antagonist and aldosterone antagonists.Although mortality rate has reduced, with comparing with age contrast experimenter of no heart failure, the mortality risk that the experimenter bore of these Drug therapys of process remains remarkable have been increased.ACE inhibitor, ¹¹β AR antagonist ⁴(at least a type) aldosterone receptor antagonist ¹²All can significantly reduce the sickness rate of heart dysfunction and heart failure after the myocardial infarction and alleviate occurring degree.Other available pharmacological treatments comprises nitroglycerin, diuretic, positivity inotropic agent (cardiac tonic), and brain natriuretic peptide (BNP).Several medicaments in back can relief of symptoms, but irrelevant with the reduction of heart failure experimenter mortality rate.

The cough phenomenon that ACE inhibitor and 10% experimenter occur is relevant, and can be narrow in bilateral renal arteries or the stable phase of other serious nephropathy between cause renal failure. ⁷β AR antagonist is then relevant with sexual impotence and depression, and avoids and be used for asthmatic subjects; In addition, under the initiation of β AR antagonist, the heart failure, hypotension, bradycardia, the heart-block that worsen may further appear in the experimenter, and tired. ⁷Aldosterone receptor antagonist then can cause significant hyperkalemia of 10% male subject and poignant gynecomastia. ⁷, ¹²It is also relevant with many difficult problems not to be proved the medicament that helps reducing mortality rate; Improve symptom though it should be noted that the many cardiac tonic of continuous discovery most, improved mortality rate by causing fatal arrhythmia in fact probably. ⁷By comparison, be known that at present CaMKII suppresses to reduce the arrhythmia phenomenon of animal model, ²⁰, ²¹Thereby represented and a kind ofly can but not increase the weight of ARR new method by cardiac function enhancing.Present available pharmacological treatments is invalid and be subject to its significant harmful side effect, and the progress that therefore has the new therapy of improvement effect and less serious side effects just becomes an important public health target.

Cam kinase II is present in the heart, as the intracellular Ca of heart ²⁺Concentration just was activated when improving, and with combine Ca ²⁺The bonded a kind of enzyme of protein calmodulin, CaM. ³The CaMKII activity may improve in severe cardiac myopathy subject, but CaMKII never causes the DCM (dilated cardiomyopathy) in the heart failure situation or shrinks and degenerate.

The invention provides by suppressing CaMKII and improve (raising) myocardium shrinkage function, with the method for treatment DCM (dilated cardiomyopathy) and heart failure.The present invention further provides by the transgenic of selectivity CaMKII peptide for inhibiting AC3-I and crossed expression, realized mouse model at the CaMKII inhibition of heart.Therefore, this AC3-I transgenic mice is a kind of new important tool that can check chronic CaMKII inhibition effect in the heart disease.

Summary of the invention

The method of the myocardial dysfunction after the invention provides a kind of treatment or preventing experimenter's myocardial infarction, this method comprises cam kinase II (CaMKII) inhibitor that gives experimenter's effective dose, gives the myocardium shrinkage function after this inhibitor can improve experimenter's myocardial infarction.

The invention provides a kind of the diagnosis at quilt is suffering among the experimenter of DCM (dilated cardiomyopathy) or other structural heart disease, treatment or prevention appear at the method for the myocardial dysfunction in DCM (dilated cardiomyopathy) or other structural heart disease (as the lular heart disease in latter stage), this method comprises the CaMKII inhibitor that gives experimenter's effective dose, gives the experimenter could be treated or prevent to this inhibitor DCM (dilated cardiomyopathy) or other structural heart disease.

The invention provides a kind of the diagnosis at quilt is suffering among the experimenter of DCM (dilated cardiomyopathy), treatment or prevention appear at the method for the myocardial dysfunction in the DCM (dilated cardiomyopathy), this method comprises the CaMKII inhibitor that gives experimenter's effective dose, gives the experimenter could be treated or prevent to this inhibitor DCM (dilated cardiomyopathy) or other structural heart disease.

The invention provides a kind of the diagnosis at quilt and suffer among the experimenter of DCM (dilated cardiomyopathy), improve the method for its myocardial contractility, this method comprises the CaMKII inhibitor that gives experimenter's effective dose, gives the myocardial contractility that this inhibitor can improve the experimenter.

The present invention further provides a kind of in by the cardiac dysfunction and/or the experimenter that reduces of contractility of diagnosis after suffering from myocardial infarction, improve the method for its myocardial contractility, this method comprises the CaMKII inhibitor that gives experimenter's effective dose, gives the myocardial contractility that this inhibitor can improve the experimenter.

The present invention further provides among a kind of experimenter of the myocardial contractility reduction after being suffered from myocardial infarction by diagnosis, improve the method for its myocardial contractility, this method comprises the CaMKII inhibitor that gives experimenter's effective dose, gives the myocardial contractility that this inhibitor can improve the experimenter.

The invention provides evaluation and can treat the method for the chemical compound of structural heart disease, this method comprises: the cardiac contractility of a) measuring the animal that suffers from structural heart disease; B) this chemical compound is given animal in the step a); C) cardiac contractility of animal determination step b); And detection compares with the cardiac contractility of animal in the step a), the increase of the cardiac contractility of animal in the step b), and the detection that cardiac contractility is increased identifies the chemical compound that can treat structural heart disease.

The invention provides a kind of method of the experimenter's of treatment structural heart disease, this method comprise give experimenter's effective dose pass through the inventive method compounds identified.

The invention provides a kind of transgenic animal of expressing the nucleic acid of coding CaMKII inhibitor.

The present invention also provides a kind of transgenic animal of expressing the nucleic acid of a kind of peptide of coding, and this peptide comprises the peptide of the SEQ ID NO:8 that is called as AC3-C.

The present invention further provides a kind of dual transgenic animal, this animal can be expressed the nucleic acid of coding CaMKII inhibitor, and expresses the nucleic acid of coding calcineurin.

Description of drawings

Fig. 1 .CaMKI, II and IV, and at AC3-I and the CaMKII peptide for inhibiting of AC3-C transgenic mice expression in vivo and the domain structural diagrams of control peptide of this research engineeringization.CaMKII and IV all have one by the CaM-land with suppress the adjusting territory (shaded rectangle) that (AI) district constitutes certainly.CaM land (the 296-309 of CaMKII, number according to CaMKII, and by the italics labelling) with the CaM land among the CaMKIV closely similar (under the identical aminoacid line being arranged), and similarly suppress the activation of CaMKIV at the peptide for inhibiting of the CaM binding sequence among the CaMKII ¹⁷AC3-I is according to the AI district foundation that be the CaMKII at center with Thr286 (indicating arrow), and CaMKII is different with AI district among the CaMKIV.The CaM land of CaMKI or AI district all not with CaMKII or IV homology, but, form with the AI district and correlatedly to be, still can suppress CaMKI at the peptide for inhibiting of the CaM land of CaMKII or IV, because CaM has a helical structure usually in conjunction with the territory, but often lack the primary sequence homology.

Fig. 2 .A-E.AC3-I mice has normal heart size and contractile function.Among A and the B figure, between AC3-I mice and the brood birth mice contrast of wild type (WT), the ultrasoundcardiogram left ventricle size no significant difference of diastole (A) or systole (B).Among C and the D figure, between AC3-C mice and the contrast of the brood birth of WT mice, the interventricular septal thickness zero difference of diastole (C) or systole (D).Among the E figure, the WT of AC3-I and baseline is brood between the contrast of birth mice, and the ejection fraction of left ventricle shortens zero difference.In all diagrams, blank post is WT, and the shade post is AC3-I transgenic (TG).In all diagrams, each kind is that the research mice quantity (n) of correspondence in the numbering (marking in figure E) is all identical.

Fig. 3 A-B. compares with the brood birth mice contrast of wild type (WT), and the AC3-I mice has significantly reduced heart CaMKII activity (A), and left ventricular ejection fraction shortening (B) significantly preferably behind the myocardial infarction surgical operation.The CaMKII determination of activity obtains from complete heart tissue homogenate thing.

Fig. 4. determine AC3-I and the intravital transgene expression of AC3-C mice by the GFP western blotting.Kind system numbering expression Western blotting that indicates in the bracket and the characteristic (is standardized data according to AC3-I5) in the corresponding quantitatively phosphor imaging results.The inherited character of all transgenic mices is all confirmed by the southern blotting technique analysis, but the AC3-I kind is 3 not express transgenic.

DCM (dilated cardiomyopathy) has appearred in Fig. 5 A-C.AC3-C transgenic mice (is 1 from kind).During the diastole, the left ventricular internal diameter (LVID) of AC3-C (B) has shown the phenotype of expansion obviously greater than AC3-I mice (B, * * P＜0.01 is 4 from kind).Heart shrinkage period, the intravital LVID shortening of AC3-C (B) obviously is less than AC3-I (A, * * * P＜0.001) mice, has shown the ventricular function that reduces.Compare with LVID, during diastole or the contraction, interventricular septum (IVS) and the left ventricular posterior wall (LVPW) of AC3-I and AC3-C mice all do not have significant difference.C. compare with AC3-I mice (P＜0.001), left ventricular ejection fraction shortens remarkable reduction in the AC3-C body.D. between AC3-I and AC3-C mice, there is not the difference on the heart rate.

Fig. 6 A-B. compares with the brood birth mice of wild type (WT) with the AC3-C mice, and total CaMKII activity of the ventricle homogenate in AC3-I source has remarkable reduction.A. total CaMKII activity.Do not rely on the part of Ca2+ in the B.CaMKII activity.

Fig. 7 A-D.CaMKII inhibitory action has been improved the cardiac systolic function behind the myocardial infarction surgical operation.To 3 weeks behind the myocardial infarction surgical operation not the result that measures of the ultrasoundcardiogram of anesthetized mice show that the CaMKII inhibitory action significantly protected the left ventricular function of mice.A. compare with the brood birth of wild type mice contrast (WT) or via the WT mice that non-activity KN-93 congener KN-92 (30 μ mol/Kg body weight) handled every day, the mice of handling every day via CaMKII inhibitor KN-93 (with 1 and 10 μ mol/Kg body weight dosage) and have left ventricle (LV) ejection fraction that the transgenic target decides the inhibiting AC3-I mice of CaMKII and shorten remarkable increase is all arranged.P＜0.001 by variance analysis (ANOVA) represents to adopt Bonferroni-to proofread and correct the significant difference that the t check is compared with WT with asterisk.B. the LV internal diameter (LVID) during the diastole is the index of LV ventricular dilatation.The LVID that respectively organizes during the diastole does not have significant difference.C. LV rear wall (LVPW) wall thickness during the diastole is an index of weighing the LV plumpness of not blocking wall.The LVPW that respectively organizes during the diastole does not have significant difference.D. have the transgenic target and decide the mice that the heart rate of the inhibiting AC3-I mice of CaMKII is slower than other all groups significantly.

Fig. 8. in the DCM (dilated cardiomyopathy) situation, the CaMKII inhibitory action has been alleviated left ventricle dilatation, has also improved the left ventricle contractile function.Compare with the CAN+ transgenic mice, left ventricle (LV) expansion of dual transgenic mice (AC3-I+/CAN+) is eased, and the LV ejection fraction shortens and also improved. ¹⁰Calcineurin (CAN+) transgenic mice of not anesthesia and the dual transgenic AC3-I+/CAN+ mice of interbreeding are carried out result's demonstration that ultrasoundcardiogram is measured: compare with the CAN+ mice, during the diastole of AC3-I+/CAN mice, LV internal diameter (LVID) significantly reduces, interventricular septum (IVS) and LV rear wall (LVPW) then thicken, and this shows the phenotype that can partly alleviate DCM (dilated cardiomyopathy) by the fixed CaMKII inhibitory action of transgenic target.Measurement parameter unanimity among preceding 4 width of cloth figure: left ventricular internal diameter (LVID), interventricular septum (IVS), left ventricular posterior wall (LVPW).Compare with the CAN+ mice, the left ventricle contractile function of dual transgenic AC3-I+/CAN+ mice has significantly improved, and shows the contractile function that can improve heart by the dirty CaMKII inhibitory action of transgenic target centering.By comparison, there is not significant difference between the heart rate of AC3-I+/CAN+ and CAN+ transgenic animal.All mices were for 4～8 ages in week.

The method of Fig. 9 A-B. interbreeding AC3-I or AC3-C mice and calcineurin (CAN) transgenic mice.A. the box indicating breeding is right.Main contrast is to carry out between the positive mice of CAN, and optional contrast (data that provide as shown in Figure 8) also has been provided.B.PCR result shows that the interbreeding between AC3-I and the CAN mice is successful, and dual transgenic mice can be identified by standard pcr.

The specific embodiment

Should be pointed out that singulative " a kind of ", " one " and " being somebody's turn to do " used in this description and the claims comprise plural object if context does not indicate clearly in addition.Therefore, for example, " a kind of inhibitor " mentioned comprises the multiple copy of this inhibitor, also can comprise the inhibitor that surpasses a particular types.

The invention provides a kind of after being suffered from myocardial infarction among the experimenter of myocardium shrinkage function obstacle by diagnosis, the method of myocardium shrinkage function obstacle after treatment or the prevention myocardial infarction, this method comprises cam kinase II (CaMKII) inhibitor that gives experimenter's effective dose, gives the myocardium shrinkage function obstacle after experimenter's myocardial infarction can be treated or prevent to this inhibitor.Usually, " effective dose " of inhibitor refers to obtain the needed amount of one or more desired result of expection." myocardial infarction " means the ischemic injury of heart, (cardiac muscle) necrosis of part cardiac muscle or apoptosis, the i.e. apoptosis of its cardiac." ischemic injury " means owing to flow to the blood of organ or tissue and interrupt, i.e. ischemia incident and cause infringement or potential damage to organ or tissue.Running through in full, used " experimenter " means individuality.Therefore, " experimenter " can comprise domestic animal, as cat, Canis familiaris L. etc., and domestic animal (as cattle, horse, pig, sheep, goat etc.), laboratory animal (as mice, rabbit, rat, Cavia porcellus etc.) and birds.Preferred experimenter is a mammal, and is as primate, more preferably human.

This experimenter is diagnosed as the patient who suffers from myocardial infarction.This experimenter is diagnosed as the patient who suffers from infraction back heart dysfunction.This experimenter is diagnosed as the patient who suffers from myocardial infarction, this patient thereby have the high risk that infraction back heart dysfunction occurs.In addition, this experimenter is diagnosed as the patient who suffers from DCM (dilated cardiomyopathy) or have heart failure symptoms, and wherein this heart failure symptoms reduces relevant causing by any with ventricular chamber expansion phenotype and myocardium shrinkage function.This experimenter is diagnosed as the patient that myocardial contractility reduces.The method of diagnosing cardiac infarction, infraction back heart dysfunction, myocardial contractility reduction and DCM (dilated cardiomyopathy) is well-known to those skilled in the art.Differentiation suffers from infraction after-contraction dysfunction or the DCM (dilated cardiomyopathy) experimenter also is well-known to those skilled in the art with the method for suffering from the experimenter of myocardial infarction or myocardial hypertrophy.Will be appreciated that to be suffered from myocardial infarction by diagnose, the experimenter of arrhythmia or myocardial hypertrophy might not suffer from DCM (dilated cardiomyopathy) or the myocardial contractility reduction.

The inhibitor of CaMKII can be chemical compound, compositions or the medicament that any inhibition CaMKII is active or express (as quantity or pathogenic effect).This chemical compound can be peptide or non-peptide medicament, for example comprises the nucleic acid of the inhibitor peptides of having encoded.In addition, this medicament can be to suppress the antisensenucleic acids that CaMKII expresses in the heart (see GenBank, be numbered L13407, Hoch et al., Circ Res 1999, isomer δ 3 and δ 2 shown in Figure 9 in its description) ⁵" inhibition " means restriction, stops or lowers.Therefore, inhibitor is passable, for example lowers the medicament of enzymatic activity and/or expression of enzymes amount.Inhibitory action can be reversible or irreversible.Intravital CaMKII activity of experimenter or CaMKII total amount can for example, be determined by ultrasoundcardiogram or other clinical parameter according to the detection of its functional response or measurement are easily determined.This literary composition provides measures the active ad hoc approach of CaMKII in the inhuman animal model.Therefore, the evaluation to the chemical compound that suppresses CaMKII is a normal experiment.

An example of CaMKII inhibitor is a kind of peptide, comprises the peptide of SEQ ID NO:2, and AC3-I also be called in this literary composition.This inhibitor of the present invention can be made of the peptide of SEQ ID NO:2.

Another example of CaMKII inhibitor is a kind of peptide, comprises the peptide of SEQ ID NO:4, i.e. CaM-KIIN.This inhibitor can be a fragment of total length CaM-KIIN (SEQ ID NO:4) and/or this full-length peptide; This fragment is called as CaM-KIINtide (SEQ ID NO:6).Among Chang et al.PNAS (USA) 199895:10890-10895 CaMKIIN and CaM-KIINtide have been described, herein with its complete being incorporated herein by reference.

Can suppress CaMKII because these peptides all show, therefore can expect that containing other peptide and the polypeptide that also comprise non essential amino acid outside these peptides also will have similar activity.Non essential amino acid is not influence the function of this peptide or the aminoacid that this peptide is finished the approach of its function (as its secondary structure or the active final result of this peptide).The example of non essential amino acid of the present invention includes but are not limited to and contains GFP, but promptly a kind of labelling and identification of protein or peptide are to carry out the peptide-labeled aminoacid of purification.

The present invention considers that CaMKII has other multiple inhibitor, and one of them is KN-93.The Nonpeptide inhibitors of this CaMKII of KN-93 has been described among the WO98/33491, herein with its complete introducing with reference to its theory about KN-93 and CaMKII inhibitor.

The method of heart dysfunction after having the present invention further provides a kind of treatment or having prevented experimenter's myocardial infarction, this method comprises the CaMKII inhibitor that gives experimenter's effective dose, gives the heart dysfunction that the experimenter could be treated or prevent to this inhibitor.The contractile function that " heart dysfunction " means blood pump pressure chamber lowers, and this situation can cause the clinical condition of heart failure.The method of heart dysfunction is well-known to those skilled in the art after the diagnosis experimenter myocardial infarction.

In the method for heart dysfunction, this CaMKII inhibitor can be a kind of peptide after treatment or prevention experimenter myocardial infarction, and for example this peptide comprises the peptide of SEQ ID NO:2, or is made up of the peptide of SEQ ID NO:2.This CaMKII inhibitor can be a kind of peptide, and it comprises the peptide of SEQ ID NO:4, or is made up of the peptide of SEQ ID NO:4.In addition, this inhibitor can be a kind of peptide, and it comprises the peptide of SEQ ID NO:6, or is made up of the peptide of SEQ ID NO:6.This inhibitor can be a Nonpeptide inhibitors, for example, contains the inhibitor of KN-93 active region, perhaps itself is exactly KN-93.

That the present invention also provides is that a kind of treatment or prevention experimenter's DCM (dilated cardiomyopathy) or arbitrary reason cause, have the method for any heart disease of heart chamber expansion phenotype, this method comprises the CaMKII inhibitor that gives experimenter's effective dose, gives DCM (dilated cardiomyopathy) or minimizing ventricular dilatation that this inhibitor can be treated the experimenter.Diagnosis DCM (dilated cardiomyopathy) experimenter's method is well-known to those skilled in the art.

In the method for treatment or prevention DCM (dilated cardiomyopathy), this CaMKII inhibitor can be a kind of peptide, and for example it comprises the peptide of SEQ ID NO:2, or is made up of the peptide of SEQ ID NO:2.This CaMKII inhibitor can be a kind of peptide, and it comprises the peptide of SEQ ID NO:4, or is made up of the peptide of SEQ ID NO:4.In addition, this inhibitor can be a kind of peptide, and it comprises the peptide of SEQ ID NO:6, or is made up of the peptide of SEQ ID NO:6.This inhibitor can be a Nonpeptide inhibitors, for example, contains the inhibitor of KN-93 active region, perhaps itself is exactly KN-93.

The invention provides a kind of the diagnosis and suffering from DCM (dilated cardiomyopathy) at quilt, or among arbitrary reason any heart disease experimenter that cause, that have heart chamber expansion or contractile function reduction phenotype, improve the method for its myocardial contractility, this method comprises the CaMKII inhibitor that gives experimenter's effective dose, gives the myocardial contractility that this inhibitor can improve the experimenter.Measure the technology of cardiac contractility, for example ultrasoundcardiogram, RAG, and nuclear magnetic resonance all is well-known to those skilled in the art." myocardial contractility " used herein refers to that is weighed a myocardial contraction, more specifically, is to weigh the yardstick that left ventricle is shunk.As shown in Figure 7, AC3-I and KN-93 have improved myocardial contractility (positive inotropic action) and have not caused cardiac hypertrophy.

In addition, the present invention also provides a kind of method that improves its myocardial contractility in the experimenter who is suffered from myocardial infarction by diagnosis, and this method comprises the CaMKII inhibitor that gives experimenter's effective dose, gives the myocardial contractility that this inhibitor can improve the experimenter.

It is a kind of after being suffered from myocardial infarction by diagnosis among the experimenter of heart dysfunction that the present invention also provides, improve the method for its myocardial contractility, this method comprises the CaMKII inhibitor that gives experimenter's effective dose, gives the myocardial contractility that this inhibitor can improve the experimenter.

In the method that improves myocardial contractility, the CaMKII inhibitor can be a kind of peptide, and for example it comprises the peptide of SEQ ID NO:2, or is made up of the peptide of SEQ ID NO:2.This CaMKII inhibitor can be a kind of peptide, and it comprises the peptide of SEQ ID NO:4, or is made up of the peptide of SEQ ID NO:4.In addition, this inhibitor also can be a kind of peptide, and it comprises the peptide of SEQ ID NO:6, or is made up of the peptide of SEQ ID NO:6.This inhibitor can be a Nonpeptide inhibitors, for example, contains the inhibitor of KN-93 active region, perhaps itself is exactly KN-93.

The CaMKII inhibitor can be used to improve the contractility of cardiac muscle, and does not cause myocardial hypertrophy, thereby can treat by diagnosis and suffer from the heart dysfunction after myocardial infarction, the myocardial infarction and/or the experimenter of DCM (dilated cardiomyopathy).

The invention provides a kind of evaluation and can treat the method for the chemical compound of structural heart disease, this method comprises: the cardiac contractility of a) measuring the animal that suffers from structural heart disease; B) this chemical compound is given animal in the step a); C) cardiac contractility of animal determination step b); D) determine to compare with the cardiac contractility of animal in the step a), the increase of the cardiac contractility of animal in the step b), the increase that detects this cardiac contractility just can identify the chemical compound that can treat structural heart disease.In this method of the present invention, the animal that suffers from structural heart disease can be the transgenic animal of expressing AC3-C.The method of measuring cardiac contractility is well-known to those skilled in the art, includes but are not limited to ultrasoundcardiogram.Assay method comprises RAG, nuclear magnetic resonance, and left ventricular angiography.

The invention provides a kind of evaluation and can treat the method for the chemical compound of structural heart disease, this method comprises: the brain natriuretic peptide of a) measuring the animal that suffers from structural heart disease; B) this chemical compound is given animal in the step a); C) brain natriuretic peptide of animal determination step b); D) determine to compare with the brain natriuretic peptide of animal in the step a), the increase of the brain natriuretic peptide of animal in the step b), the increase that detects this brain natriuretic peptide just identifies the chemical compound that this can treat structural heart disease.In this method of the present invention, the animal that suffers from structural heart disease can be the transgenic animal of expressing AC3-C.

Heart failure is a kind of clinical syndrome, comprises because the tolerance of exercise that the attenuating of heart contraction and tissue oxygenation causes reduces, ⁷Therefore the blood plasma brain natriuretic peptide level of picked-up of the tissue oxygen of the exercise test that is undertaken by bicycle or bicycle ergonometry, reduction or rising is the sign of the heart failure order of severity.The numerical value of indication myocardial contraction extreme and moderate lesion, motor capacity, maximal oxygen consumption and circulation brain natriuretic peptide level is all described to some extent in treatment heart failure field and is known by the technical staff. ⁷Heart dysfunction, myocardial contractility that the example of structural heart disease includes but are not limited to after myocardial infarction, the myocardial infarction reduce, and DCM (dilated cardiomyopathy).

The invention provides the method for heart dysfunction after a kind of experimenter's of treatment myocardial infarction, the myocardial infarction and/or DCM (dilated cardiomyopathy), this method comprises a kind of said method compounds identified of passing through that gives experimenter's effective dose, the giving to treat of this chemical compound suffers from the experimenter of heart dysfunction after the myocardial infarction and/or DCM (dilated cardiomyopathy), or the patient with symptoms such as heart failure, heart chamber expansion and the reductions of left ventricle contractility.

The invention provides the transgenic animal of the nucleic acid of expressing coding CaMKII inhibitor." transgenic animal " refer to that all cells of its health all contains a kind of animal of exogenous nucleic acid.In the example of transgenic animal of the present invention, this transgenic specifically expressing and is started by the heart cell specific promoter in myocardial cell.This mice adopts specific heart α myoglobulin heavy chain promoter (GenBank numbers U71441) to design.The method for preparing transgenic animal is well-known to those skilled in the art, and is able to concrete illustration in this literary composition.

The CaMKII inhibitor of transgenic animal expression in vivo of the present invention can be a kind of peptide, and it comprises the peptide of SEQ ID NO:2.In addition, transgenic animal of the present invention can be expressed the peptide of being made up of the peptide of SEQID NO:2.Nearest report finds to have at least 4 kinds of different CaMKII δ isomers in heart, ⁵And owing to the conservative in the fixed adjusting territory of target, AC3-I (SEQ IDNO:2) can suppress all CaMKII isomers. ³The territory that AC3-I target in the CaMKII is fixed and other CaMK type (be CaMKI and CaMKIV, Fig. 1) the similar functions territory difference in, and in fact lack activity at protein kinase A and C. ³

The CaMKII inhibitor of transgenic animal expression in vivo of the present invention can be a kind of peptide, and it comprises the peptide of SEQ ID NO:4.In addition, these transgenic animal of the present invention can be expressed the peptide of being made up of the peptide of SEQ ID NO:4.

The present invention further provides the transgenic animal of the nucleic acid of expressing a kind of peptide of coding in myocardial cell, this peptide comprises SEQ ID NO:8, is also referred to as the peptide of AC3-C.Shown in Figure 4 and 5, AC3-I has similar transgene expression with the AC3-C mice, but the AC3-C mice shows cardiomyopathy, has the inhibiting AC3-I mice of CaMKII and then acts normally.As shown in Figure 6, with suffer from myocardiac patient in viewed result the same, the AC3-C mice has high total CaMKII activity.Therefore, these transgenic animal can be applied to identifying in the inventive method of a kind of chemical compound that can treat structural heart disease.Because AC3-C is considered to non-activity, so these the possibility of result need be by the explanation that is used for of the genetically modified green fluorescent protein of AC3-C-GFP (GFP) part. ⁶This mice is that the basic methods of abideing by preparation AC3-I transgenic mice obtains.

The present invention also provides a kind of dual transgenic animal (AC3-I+/CAN+) of expressing the nucleic acid of the nucleic acid of coding AC3-I and the calcineurin of encoding in myocardial cell.This calcineurin (CAN+) transgenic mice is to generally acknowledge the model of suffering from serious DCM (dilated cardiomyopathy). ¹⁰Known CAN antagonist can ' be saved ' the cardiomyopathy phenotype that occurs in the multiple mouse model, ¹⁶But CaMKII suppresses never can be improved by expection the cardiac function or the structure of these or any other cardiomyopathy model.As Fig. 8 institute illustration, the left ventricular function of also expressing these dual transgenic animal of CaMKII inhibitor except that CAN improves, and with the CAN+ animal model relatively the time, demonstrates left ventricle dilatation and plump symptom also is eased.Therefore, the intravital CaMKII of this animal suppresses to treat DCM (dilated cardiomyopathy).

In the method for the present invention, can give CaMKII inhibitor by known approach.In the particular instance, inhibitor peptides is made into cell membrane permeability.Cell " membrane permeation " means can be by opening on the film or gap ¹⁶This method has adopted the peptide sequence that is added in the peptide for inhibiting, but the Fructus Amomi Rotundus acyl groupization is to make peptide pass through the another kind of method of cell membrane.

Compositions of the present invention also can be contained in and is given in the pharmaceutical acceptable carrier in the body." pharmacy can be accepted " means a kind of material that meets biology or others needs, promptly this material can be given the experimenter together with component, and do not cause any biological effect of taking place of not wishing, perhaps can be with deleterious mode and any other component interaction that comprises its pharmaceutical composition.As well-known to those skilled in the art, this carrier should make any palliating degradation degree of effective ingredient minimize, and minimizes the generation of harmful side effect in the subject.

Although local intranasal administration or be typical preferred administering mode by inhalation still can give said composition by modes such as oral, parenteral (as intravenous injection, intramuscular injection, intrathecal injection, intra-arterial injection and peritoneal injection), percutaneous, external, parts." local intranasal administration " used herein means via one or two nostril said composition to be sent and passs to nostril and nasal meatus, can comprise by aerosol apparatus or liquid-drop machine, perhaps passs by sending of realizing of nebulae inhalation agent.It also can be any part (as trachea, bronchus and lung) that directly arrives lower respiratory tract by intubate that the sending of said composition passed.According to the order of severity of experimenter's ethnic group, age, body weight and general body state, the situation for the treatment of, used particular composition, its mode of administration etc., the accurate amount of different experimenter's desired compositions is all inequality.Therefore, can not specify the accurate amount of every kind of compositions.But, can pass through a kind of ordinary skill in this area, only utilize the normal experiment of method mentioned herein just can determine appropriate amount.

If adopt parenteral to give said composition, its administration feature is generally injection.The injectable thing conventionally form be can be prepared into,, the solid form or the Emulsion of in liquid, being dissolved into suspension before injecting be applicable to as liquid solution or suspension.The parenteral method of revision relates to the application of slow release or lasting delivery systme recently, can keep constant dosage.See for example U.S.Patent No.3,610,795, introduced the document herein with for referencial use.

This material can be dissolved in solution, the suspension (for example being impregnated in microgranule, liposome or cell).Can these material targets are fixed on particular cell types by means of antibody, receptor or receptors ligand.Following document be utilize this technology with the specified protein target fixed to tumor tissues example (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br.J.Cancer,60:275-281, (1989); Bagshawe, et al., Br.J.Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol.Immunother., 35:421-425, (1992); Pietersz andMcKenzie, Immunolog.Reviews, 129:57-80, (1992); And Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)).Carrier is as the liposome (lipid that comprises mediation treatment colon cancer medicine) of " stealth " liposome and other coupling antibody, via the fixed receptor of the ligand-mediated DNA target of cell-specific, target is decided the tumor lymphatic cell, and target is decided the high specific therapy sexual inversion record virus of neuroglial cytoma in the murine body.Following document be utilize this technology with the specified protein target fixed to tumor tissues example (Hughes et al., Cancer Research, 49:6214-6220, (1989); AndLitzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).Usually, receptor is included in the endocytosis approach inductively by composing type or by part.These receptors in the alveole of clathrin bag quilt bunch enter in the cell acidified endosome by means of the vesicles of clathrin bag quilt, receptor is classified in this acidify endosome, then or be recycled to cell surface, be stored in the cell, perhaps in lysosome, be degraded.The internalization approach has multiple function, as the removing of the picked-up of nutrition, activation of protein, macromolecular removing, virus and the opportunistic invasion of toxin, the separation and the degraded of part, and the adjusting of acceptor levels.Many receptors participate in surpassing one the interior approach of cell, and this depends on this cell type, acceptor density, part type, part valency and ligand concentration.The molecule of receptor-mediated endocytosis and celelular mechanism are existing comments (Brown and Greene, DNA and Cell Biology10:6,399-409 (1991)).

Compositions of the present invention can comprise the nucleic acid of a kind of inhibitor of encoding, and perhaps can comprise the CaMKII antisensenucleic acids.Can the disclosure compositions be sent by number of ways and pass to target cell.For example, can pass through electroporation, fat transfection, perhaps the calcium phosphate precipitation method is sent and is passed said composition.Selected sending passed mechanism partly according to the type of target cell, and whether this send to pass and take place, for example in body or external.

Therefore, except disclosed compositions or carrier, said composition for example can also contain, such as liposome, as the lipid of cationic-liposome (as DOTMA, DOPE, DC-cholesterol) or anionic liposome.If necessary, liposome can further contain protein, is beneficial to target and decides specific cells.The administration of compositions comprises chemical compound and cationic-liposome is administered in the blood, passs to Target organ to send, and perhaps it is sucked in the respiratory tract, to send the target cell of passing to respiratory tract.About liposome, see for example Brigham et al.Am.J.Resp.Cell.Mol.Biol.1:95-100 (1989); Felgner et al Proc.Natl.Acad.Sci USA 84:7413-7417 (1987); U.S.Pat.No.4,897,355.In addition, can be with of the component administration of this chemical compound as a kind of microcapsule, this microcapsule can be fixed on particular cell types by target, as macrophage, perhaps with this chemical compound from this microcapsule diffusion or send to pass and be designed to special speed or dosage.

Give foreign DNA and it sent to pass to the intracellular method of experimenter (being gene transfer or transfection) above-mentioned comprising, can said composition be sent by means of multiple mechanism and pass to cell.One of them example is to send by means of liposome to pass compositions, promptly adopt commercial available Liposomal formulation, as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc.Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), and other is according to the liposome of the operation standard research and development of this area.In addition, send in can body by electroporation and to pass nucleic acid of the present invention or carrier, can adopt Genetronics, Inc. (San Diego, method CA), and by SONOPORATION machinery approach (ImaRx Pharmaceutical Corp., Tucson AZ) uses this technology.

Sent and pass, and the nucleic acid that will be integrated in the host cell gene group typically contains integration sequence to cell.These sequences are generally viral correlated series, especially when the system that adopts based on virus.These viral integrase systems also can be integrated into and await adopting non-based on nucleic acid, in the nucleic acid that send the system of passing to be sent to pass as liposome, thereby make this send the nucleic acid that comprises in the system of passing to be integrated in the host genome." nucleic acid " used herein comprises strand or duplex molecule, i.e. the DNA that may be made up of nucleotide base A, T, C, G, the perhaps RNA that is made up of base A, U (substituting T), C and G.This nucleic acid can be represented coding strand or its complementary strand.The sequence of this nucleic acid may be identical with the part of the sequence that exists naturally, perhaps may comprise optional codon, these codons encoded with exist naturally in the sequence the identical aminoacid of the aminoacid of finding.In addition, as known to those skilled in the art, nucleic acid can comprise the conservative codon that replaces of represented amino acid.

Other current techique that is used to be integrated into host genome comprises, for example is designed to can promote to carry out with host genome the system of homology reorganization.These systems typically rely on the flanking sequence for the treatment of express nucleic acid, and the abundant homology of target sequence in this sequence and the host cell gene group reorganization of vector nucleic acid and target nucleic acid has taken place, and caused sending the nucleic acid of passing to be integrated in the host genome.Known these systems of those skilled in the art and method are for promoting that homologous recombination is essential.

As mentioned above, can give said composition by means of pharmaceutical acceptable carrier, and by multiple mechanism well-known to those skilled in the art (as the picked-up of naked NDA, liposome merge, by means of the DNA intramuscular injection of particle gun, endocytosis etc.) with in the body and/or external mode said composition sent passed to experimenter's cell.If what use is in vitro method, can cell or tissue be shifted out and maintain external according to standard operating instructions well-known to those skilled in the art.By means of any gene transfer mechanism, for example gene delivery, electroporation, microinjection or the proteoliposome method of calcium phosphate mediation all can be with in the said composition transfered cells.Then can via standard method this transducer cell injection (as at pharmaceutical acceptable carrier) or equipotential be transplanted back in the subject according to the cell or tissue type.Various kinds of cell is transplanted or injected the intravital standard method of experimenter is known.

This inhibitor can effectively suppress the dosed administration that CaMKII is active or measure arbitrarily.As noted above, the detection of the minimizing of or amount active to CaMKII is in working doctor's skill.More specifically, the dosage of this inhibitor is that the about 0.05mg of per kilogram of body weight is to about 5.0mg.Optionally, the dosage of this inhibitor is that the about 0.3mg of per kilogram of body weight is to about 3.0mg.

The description of following embodiment is to provide complete disclosure for those of ordinary skills; and how to prepare and evaluate the claimed compositions and/or the description of method herein; these embodiment only are illustration of the present invention, the category of the present invention that the present inventor assert are not construed as limiting.The inventor has endeavoured to ensure the accuracy of numeral (as quantity, temperature etc.), but still has some sum of errors deviation.More specifically described the present invention among the following embodiment, because the many places that wherein occur change and variation will be apparent and understandable to those skilled in the art, so these embodiment only have illustrative.

Embodiment

The AC3-I transgenic mice: this AC3-I mice is to be that the mini-gene that carries out on the basis is synthesized into (Fig. 1) with preparation with the peptide sequence that is used for AC3-I.' mini-gene ' of coding AC3-I (KKALHRQEAVDCL), ²The CaMKII peptide for inhibiting all is to adopt these complementary oligonucleotides to make up:

(SEQ?ID?NO：1)

GATCAAAAAAGCCCTTCACCGCCAGGAGGCAGTTGACTGCCTTGCTTTTTTC

GGGAAGTGGCGGTCCTCCGTCAACTGACGGAACGCTAG，

The following complementary oligonucleotide of same employing makes up the mini-gene that is used for relevant non-activity control peptide AC3-C (KKALHAQERVDCL):

(SEQ?ID?NO：7)

GATCAAAAAAGCCCTTCACGCACAGGAGCGCGTTGACTGCCTTGC

TTTTTTCGGGAAGTGCGTGTCCTCGCGCAACTGACGGAACGCTAG

With being inserted in the BspEI site of pEGFP-C1 (Clontech) in this mini-gene and the EGFP frame, make EGFP be positioned at the N-terminal of this peptide.This AC3-I mini-gene comprises Kozak consensus sequence translation initiation site.Then check order and in the HEK293 cell, express, demonstrate green fluorescence.Research in the early time points out that the AC3-I-GST-MTS fusogenic peptide has kept synthetic CaMKII substrate (AC3-I-GST-MTS IC ₅₀=0.4 μ M; AC3-I IC ₅₀0.5 whole CaMKII μ M) suppress to render a service, and show that AC3-I-GFP protein has also kept CaMKII and suppressed active.Then adopt pcr amplification 800bp AC3-I-GFP sequence, amplified production is carried out purification and its sub-clone entered to contain the SalI site of the pBluescript carrier of α-MHC promoter vector (GenBank numbers U71441) and human growth hormone (HGH) polyadenylic acid afterbody (by the Dr.J.Robbins research and development).By checking order and in murine atrium tumor cell line (HL1), expressing and demonstrate green fluorescence and just can verify this construct.In Vanderbilt transgenic mice core facility, the murine embryonic stem cell is advanced in the pseudo-pregnant female mice body of B6D2 together with linearizing DNA (2ng/ml) injection and transplanting.

When microscopically is inspected tissue slice, AC3-I links to each other with green fluorescent protein (GFP) and can demonstrate the homology expression that spreads all over heart.These AC3-I mices survive, and have normal basic cardiac dimensions and function (Fig. 2).But, total heart CaMKII that heart had of these mices is active significantly to have reduced (Fig. 3 (A)), and compare with the contrast of the brood birth of wild type mice, after the myocardial infarction experiment, the heart function injury of these mices less significantly (Fig. 3 (B)).These discoveries show that the CaMKII activity is a new signal of heart dysfunction after the myocardial infarction, and also are the bases that our claimed passing through suppresses CaMKII treatment myocardial infarction experimenter's method.

AC3-I+/CAN+ transgenic mice: adopt the dual transgenic mice of method step outbreeding shown in Figure 9.For the first generation is carried out gene type, carry out the afterbody biopsy in mice during 3 ages in week, and in 55 ℃, overnight incubation in the solution that contains 0.5Mg/ml E.C. 3.4.21.64,50mM Tris (pH8.0), 100mM EDTA and 0.5%SDS.After adopting phenol/chloroform/isoamyl alcohol (25: 24: 1) to carry out extracted twice, adopt isopyknic isopropanol precipitating genomic DNA.Contain dissolving DNA in 1/10TE (pH8.0) solution of 2.5 μ g RNA enzyme A in 50 μ l.Adopt the genomic DNA of EcoRI catapepsis 15 μ g, and in 1xTAE, adopt 0.8% agarose gel to separate the DNA that has digested.Behind the electrophoresis, the incubation gel is 30 minutes in 0.25N HCl, carries out 15 minutes neutralization reaction twice in 0.5M NaOH-1.5M NaCl, and in 0.5M Tris-1.5M NaCl twice of balance each 15 minutes.(Micron separations Inc.Westborought spends the night the gel trace on MA), and carries out UV-crosslinked to this filter membrane at MSI nylon transfer membrane.By random oligonucleotide caused (Stratagene, La Jolla, CA) ³²The GFP-AC3I of P labelling or GFP-AC3C dna fragmentation are as probe.Under the condition that 50% Methanamide, 5xSSPE, 5xDenhardt solution, 0.1%SDS and 100 μ g/ml degeneration, broken salmon sperm dna exist, hybridize in 42 ℃ and to spend the night.In 65 ℃, the washing filter membrane is 30 minutes in 0.5xSSC-0.1%SDS, and then in 65 ℃, washing is 20 minutes twice in 0.1%SSC-0.1%SDS.Expose this filter membrane to Kodak autoradiography egative film and make its development.

Routine screening to the transgenic mice after the first generation is the (see figure 9) of being undertaken by PCR.There are two primers to be applicable to that amplification is positioned at the 442-bp district of human growth hormone (hGH) gene 3 ' end.The sequence of these two primers is 5 '-Hgh (5 '-(SEQ ID NO:9) GTCTATTCGGGAACCAAGCT-3 ') and 3 '-Hgh (5 '-(SEQ ID NO:10) ACAGGCATCTACTGAGTGGACC-3 ').The purified genomic dna of 100ng is mixed with every kind of primer of 200pM, and increase according to following step: 95 ℃ following 5 minutes, then carry out 30 and comprise 95 ℃ following 45 seconds, 50 ℃ following 45 seconds, 72 ℃ following circulations of 1 minute, under 72 ℃, carry out last 7 minutes extension at last.Under 0.5 μ g/ml ethidium bromide existence condition, all samples is splined on carries out electrophoresis on 1.5% agarose gel, and in the irradiation under ultraviolet ray painted DNA band of range estimation down.New primer sets is to research and develop at the dual transgenic mice (Fig. 9) of interbreeding, and can be used for distinguishing dual and single transgenic animal.

Ultrasoundcardiogram: ultrasoundcardiogram is to adopt the Hewlett Packard Sonos5500 (fully at murine research) and the 12MHz probe of especial manufacture to carry out.Cardiac dimensions obtains the M mode image from 2 dimension guiding, and adopts the forward position method, gets minor axis and keel length axial plane off-line reading by sealing, independent reader.(15mg/Kg i.p.) just can easily make the animal calmness to adopt pentobarbital; But also can need not anesthesia ground and measure, wherein mice need experience of short duration ' training ' phase to adapt to whole process.About 90% mice can be trained to such an extent that can bear ultrasoundcardiogram research, and not produce the heart depression effect of anesthesia.To beat continuously from LV rear wall (LVPW), interventricular septum (IVS) and LV internal diameter (LVID) 3 times and average the acquisition measured value.Ejection fraction shortens (FS) and is used to the evaluate cardiac contractile function, and can calculate according to formula F S=(LVID diastole-LVID paradoxical expansion)/LVID relaxing period x100.

The myocardial infarction surgical operation: this surgical operation carries out in Vanderbilt mice physiology core laboratory, and with other report basically identical of having delivered ^{18 8 14}In brief, anesthetized mice (pentobarbital 33 μ g/g and ketamine 33 μ g/g, i.p.), be placed on rodent respiratory organ (tidal volume 0.5ml, 120 breaths/min of speed) on, adopt left parasternal thoracotomy that its thoracic cavity is opened, and adopt the 8-0 stitching thread that a left front tremulous pulse central region of falling is sewed up.Closed-chest (5-0 stitching thread), and with this animal dialysis respiratory organ.Wet by the pulmonary that increases: dry weight can determine that heart failure typically appears in wild-type mice.The ligation step is omitted in sham-operation.About 75% surviving animals has successfully had the antetheca of infraction by surgical operation.

The CaMKII activity analysis: the CaMKII activity is to adopt our disclosed method before ¹, in the contrast of AC3-I mice and brood birth mice, adopt to have CaMKII comparison CaMKIV is surpassed about 50 times of syntide 2 this synthetic substrates optionally, from complete heart (ventricle) homogenate, analyze acquisition.

DCM (dilated cardiomyopathy): in the prevention of DCM (dilated cardiomyopathy) being carried out by inhibition CaMKII, obtained noticeable data.Prepared the contrast transgenic mice of expressing the GFP that links to each other with AC3-C (a kind of non-activity congener of AC3-I).Non-existent quite serious DCM (dilated cardiomyopathy) in the AC3-I-GFP mice has just appearred in this mice, and has very high-caliber CaMKII activity (Fig. 5).

Run through the application, with reference to multiple publication.In order more intactly to describe the situation in field under the present invention, in this literary composition complete introducing this application of disclosure, as a reference with these publications.

To those skilled in the art, should be understood that the present invention can carry out multiple change and variation, does not depart from category of the present invention and essence.Consider description and practice that the present invention is disclosed herein, other embodiment of the present invention will be apparent and understandable to those skilled in the art.This description and embodiment only are illustration, and true spirit of the present invention and essence are indicated by following claim.

List of references

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2.Braun?A.，Schulman?H.A?non-selective?cation?current?activated?via?themultifunctional?Ca(2+)-calmodulin-dependent?protein?kinase?in?human?epithelialcells.J?Physiol?1995；488：37-55.

3.Braun?A.，Schulman?H.The?multifunctional?calcium/calmodulin-dependentprotein?kinase：from?form?to?function.Ann?Rev?Physiol?1995；57：417-445.

4.Gottlieb?S.，McCarter?R.，Vogel?R.Effect?of?beta-blockade?on?mortality?amonghigh-risk?and?low-risk?patients?after?myocardial?infarction.[see?comments].NewEngland?Journal?of?Medicine?1998；339：489-497.

5.Hoch?B.，Meyer?R.，Hetzer?R.，Krause?E.，Karczewski?P.Identification?andexpression?of?delta-isoforms?of?the?multifunctional?Ca2+/calmodulin-dependentprotein?kinase?in?failing?and?nonfailing?human?myocardium.Circulation?Research1999；84：713-721.

6.Huang?W.，Aramburu?J.，Douglas?P.，Izumo?S.Transgenic?expression?of?greenfluorescence?protein?can?cause?dilated?cardiomyopathy.Nat?Med?2000；6：482-483.

7.Hunt?S.，Baker?D.，Chin?M.，Cinquegrani?M.，Feldman?A.，Francis?G.，Ganiats?T.，Goldstein?S.，Gregoratos?G.，Jessup?M.，Noble?R.，Packer?M.，Silver?M.，Stevenson?L.，Gibbons?R.，Antman?E.，Alpert?J.，Faxon?D.，Fuster?V.，Jacobs?A.，Hiratzka?L.，Russell?R.，Smith?S.，Jr.，American?College?of?Cardiology/AmericanHeart?Association.ACC/AHA?guidelines?for?the?evaluation?and?management?ofchronic?heart?failure?in?the?adult：executive?summary.A?report?of?the?AmericanCollege?of?Cardiology/American?Heart?Association?Task?Force?on?PracticeGuidelines(Committee?to?revise?the?1995?Guidelines?for?the?Evaluation?andManagement?of?Heart?Failure).Journal?of?the?American?College?of?Cardiology2001；38：2101-2113.

8.Kinugawa?S.，Tsutsui?H.，Hayashidani?S.，Ide?T.，Suematsu?N.，Satoh?S.，UtsumiH.，Takeshita?A.Treatment?with?dimethylthiourea?prevents?left?ventricularremodeling?and?failure?after?experimental?myocardial?infarction?in?mice：role?ofoxidative?stress.Circulation?Research?2000；87：392-398.

9.Miyano?O.，Kameshita?I.，Fujisawa?H.Purification?and?characterization?of?abrain-specific?multifunctional?calmodulin-dependent?protein?kinase?from?ratcerebellum.J?Biol?Chem?1992；267：1198-1203.

10.Molkentin?J.，Lu?J.，Antos?C.，Markham?B.，Richardson?J.，Robbins?J.，Grant?S.，Olson?E.A?calcineurin-dependent?transcriptional?pathway?for?cardiachypertrophy.Cell?1998；93：215-228.

11.Pfeffer?J.，Fischer?T.，Pfeffer?M.Angiotensin-converting?enzyme?inhibition?andventricular?remodeling?after?myocardial?infarction..Ann?Rev?Physiol1995；57：805-826.

12.Pitt?B.，Zannad?F.，Remme?W.，Cody?R.，Castaigne?A.，Perez?A.，Palensky?J.，Wittes?J.The?effect?of?spironolactone?on?morbidity?and?mortality?in?patients?withsevere?heart?failute.Randomized?Aldactone?Evaluation?Study?Investigators..New?England?Journal?of?Medicine?1999；341：709-717.

13.Rhoads?A.，Friedberg?F.Sequence?motifs?for?calmodulin?recognition..FASEB1997；11：331-340.

14.Sam?F.，Sawyer?D.，Chang?D.，Eberli?F.，Ngoy?S.，Jain?M.，Amin?J.，Apstein?C.，Colucci?W.Progressive?left?ventricular?remodeling?and?apoptosis?late?aftermyocardial?infarction?in?mouse?heart.American?Journal?of?Physiology-Heart&Circulatory?Physiology?2000；279：H422-H428.

15.Spencer?F.，Meyer?T.，Goldberg?R.，Yarzebski?J.，Hatton?M.，Lessard?D.，Gore?J.Twenty?year?trends(1975-1995)in?the?incidence，in-hospital?and?long-term?deathrates?associated?with?heart?failure?complicating?acute?myocardial?infarction：acommunity-wide?perspective.J?Am?Coll?Cardiol?1999；34：1378-1387.

16.Sussman?M.，Lim?H.，Gude?N.，Taigen?T.，Olson?E.，Robbins?J.，Colbert，MC，Gualberto?A.，Wieczorek?D.，Molkentin?J.Prevention?of?cardiac?hypertrophy?inmice?by?calcineurin?inhibition[see?comments].Science?1998；281：1690-1693.

17.Tokumitsu?H，Brickey?D.，Glod?J.，Hidaka?H.，Sikela?J.，Soderling?T.Activationmechanisms?for?Ca2+/calmodulin-dependent?protein?kinase?IV.Identification?of?abrain?CaM-kinase?IV?kinase.J?Biol?Chem?1994；269：28640-28647.

18.Trueblood?N.，Xie?Z.，Communal?C.，Sam?F.，Ngoy?S.，Liaw?L.，Jenkins?A.，WangJ.，Sawyer?D.，Bing?O.，Apstein?C.，Colucci?W.，Singh?K.Exaggerated?leftventricular?dilation?and?reduced?collagen?deposition?after?myocardial?infarction?inmice?lacking?osteopontin.Circulation?Research?2001；88：1080-1087.

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Sequence table

＜110〉Vanderbilt University

M. Anderson

＜120〉with the myocardial dysfunction in the cam kinase II inhibitor for treating structural heart disease

<130>22000.0117P1

<140>PCT/US02/31496

<141>2002-10-01

<150>60/328,010

<151>2001-10-08

<150>60/326,576

<151>2001-10-01

<160>10

<170>FastSEQ?for?Windows?Version?4.0

<210>1

<211>90

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note; Note=

Synthetic construct

<400>1

gatcaaaaaa?gcccttcacc?gccaggaggc?agttgactgc?cttgcttttt?tcgggaagtg 60

gcggtcctcc?gtcaactgac?ggaacgctag 90

<210>2

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note; Note=

Synthetic construct

<400>2

Lys?Lys?Ala?Leu?His?Arg?Gln?Glu?Ala?Val?Asp?Cys?Leu

1 5 10

<210>3

<211>255

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note; Note=

Synthetic construct

<400>3

cggctcccct?gctgagctag?ggccggtccg?gcagtcagcc?tctgcccgtg?ccccgccgca 60

gtccctagcc?cgcccggtgc?ccgccgcctg?caggacacca?actccttctt?cgctggcaac 120

caggccaagc?ggccccccaa?gctgggccag?atcggccgag?ccaagagagt?ggtgatcgag 180

gatgaccgga?tagacgacgt?gctgaagggg?atgggggaga?agcctccgtc?cggagtgtag 240

acgcgccggc?tctgg 255

<210>4

<211>79

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note; Note=

Synthetic construct

<400>4

Met?Ser?Glu?Ile?Leu?Pro?Tyr?Gly?Glu?Asp?Lys?Met?Gly?Arg?Phe?Gly

1 5 10 15

Ala?Asp?Pro?Glu?Gly?Ser?Asp?Leu?Ser?Phe?Ser?Cys?Arg?Leu?Gln?Asp

20 25 30

Thr?Asn?Ser?Phe?Phe?Ala?Gly?Asn?Gln?Ala?Lys?Arg?Pro?Pro?Lys?Leu

35 40 45

Gly?Gln?Ile?Gly?Arg?Ala?Lys?Arg?Val?Val?Ile?Glu?Asp?Asp?Arg?Ile

50 55 60

Asp?Asp?Val?Leu?Lys?Gly?Met?Gly?Glu?Lys?Pro?Pro?Ser?Gly?Val

65 70 75

<210>5

<211>129

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note; Note=

Synthetic construct

<400>5

aagcggcccc?ccaagctggg?ccagatcggc?cgagccaaga?gagtggtgat?cgaggatgac 60

cggatagacg?acgtgctgaa?ggggatgggg?gagaagcctc?cgtccggagt?gtagacgcgc 120

cggctctgg 129

<210>6

<211>27

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note; Note=

Synthetic construct

<400>6

Lys?Arg?Pro?Pro?Lys?Leu?Gly?Gln?Ile?Gly?Arg?Ala?Lys?Arg?Val?Val

1 5 10 15

Ile?Glu?Asp?Asp?Arg?Ile?Asp?Asp?Val?Leu?Lys

20 25

<210>7

<211>90

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note; Note=

Synthetic construct

<400>7

gatcaaaaaa?gcccttcacg?cacaggagcg?cgttgactgc?cttgcttttt?tcgggaagtg 60

cgtgtcctcg?cgcaactgac?ggaacgctag 90

<210>8

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence note; Note=

Synthetic construct

<400>8

Lys?Lys?Ala?Leu?His?Ala?Gln?Glu?Arg?Val?Asp?Cys?Leu

1 5 10

<210>9

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note; Note=

Synthetic construct

<400>9

gtctattcgg?gaaccaagct 20

<210>10

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note; Note=

Synthetic construct

<400>10

acaggcatct?actgagtgga?cc 22

Claims (2)

1. the purposes of cam kinase II inhibitor in identifying the chemical compound to treat structural heart disease.

2. the purposes of claim 1, wherein said structural heart disease can be at least a in DCM (dilated cardiomyopathy) or the myocardium shrinkage function obstacle.

Images