JP5344671B2 - Heart failure drug - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substance binding to phospholamban and a cardiac failure-treating agent which contains the substance and corrects the abnormality of Ca<SP>2+</SP>dynamics in a cardiac muscle cell in order to provide a cardiac failure-treating agent which is not accompanied by gene manipulation or gene expression. <P>SOLUTION: Disclosed is the cardiac failure-treating agent which targets a fusion protein comprising cytoplasmic domain of phospholamban and contains a phospholamban aptamer which is any one of a base sequence comprising a specific sequence obtained by SELEX method, a base sequence in which one or several bases of the base sequence comprising a specific sequence are deleted, substituted or added, or a base sequence in which homology to the base sequence comprising a specific sequence is 90% or more. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a DNA or RNA aptamer that specifically binds to phospholamban and inhibits phospholamban function, and a therapeutic agent for heart failure containing the aptamer.

It has been clarified that Ca ²⁺ in cardiomyocytes plays a central role in controlling the contractile force of the heart. Cardiomyocyte intracellular Ca ²⁺ is regulated mainly by Ca ²⁺ transport to the myocardial endoplasmic reticulum, which is the intracellular Ca ²⁺ storage site, and Ca ²⁺ release. Myocardial relaxation occurs when the cytoplasmic Ca ²⁺ concentration decreases due to Ca ²⁺ transport to the myocardial endoplasmic reticulum, and contraction occurs when the cytoplasmic Ca ²⁺ concentration increases due to the release of Ca ²⁺ from the myocardial endoplasmic reticulum. Conventionally, as a therapeutic agent for heart failure, mainly a cardiotonic agent, a screening method using as an index the enhancement of myocardial contractility by administration to an animal or administration experiment to an extracted beating heart has been used. The cardiotonic drugs obtained by this method are related to the sympathetic nervous system and the renin-angiotensin system, have the effect of increasing Ca ²⁺ influx from the outside of the cell into the cell, and increase the intracellular Ca ²⁺ kinetics. It is not something to control. For this reason, the cytoplasmic Ca ²⁺ concentration at the time of relaxation does not sufficiently decrease, and sufficient relaxation cannot be obtained, so that it works in the direction of worsening severe heart failure.

The myocardial intracellular ^{Ca 2+} dynamics, and ^{Ca 2+} pump ATPase (SERCA) protein of myocardial vesicles that controls the intracellular ^{Ca 2+,} phospholamban the regulatory proteins are important. Phospholamban acts as an inhibitor of SERCA that governs Ca ²⁺ transport in the myocardial endoplasmic reticulum (Non-patent Document 1), and it is revealed that Ca ²⁺ transport is promoted when phospholamban dissociates from SERCA (Non-patent Document 2) ) And the binding sites of both proteins were also identified (Non-patent Document 3). When the Ca ²⁺ transport of the myocardial endoplasmic reticulum is promoted, the myocardial contractile force is finally enhanced.

The safety and effectiveness of heart failure treatment based on such a mechanism is based on experiments in mice deficient in phospholamban by genetic manipulation (Non-patent Document 4) and in large amounts of inactive phospholamban using viruses. This is shown in an experiment of an expressed rat (Non-patent Document 5). In these hearts, the SERCA activity of the myocardial endoplasmic reticulum is markedly promoted, and abnormal Ca ²⁺ kinetics are corrected. However, at present, it is extremely difficult to use these gene manipulations and gene expression by viruses as therapeutic strategies in clinical terms from the viewpoint of safety and technology.

In Patent Document 1, two peptide conjugates and a phospholamban mutant protein are used as a therapeutic agent for heart failure by inhibiting the interaction between phospholamban and sarcoplasmic reticulum SERCA in cardiomyocytes, thereby enhancing contractility in failing hearts. It is revealed. In addition, as a screening method for a therapeutic agent for heart failure, a useful substance is selected using a Ca ²⁺ pump gene recombinant protein containing a binding site with phospholamban and a phospholamban gene recombinant protein containing a binding site with Ca ²⁺ pump. Although a method (Patent Document 2) has been proposed, it is necessary to screen a huge number of compound libraries, and no such drug has yet been found.

Aptamers, which are short single-stranded DNAs and RNAs that bind to enzymes and receptors, are attracting attention in various fields because of their potential as pharmaceuticals. Aptamers that bind to specific proteins can be found relatively easily by using the SELEX (Systematic evolution of ligands by exponential enrichment) method. As aptamer reports obtained by the SELEX method, in addition to RNA aptamers that specifically bind to prion proteins (Patent Document 3), vitronectin aptamers (Patent Document 4), and aptamers that specifically bind to HGF (hepatocyte growth factor) (Patent Document 5), and their respective uses as diagnostic agents for prion diseases, anticancer agents, and cancer metastasis inhibitors have been clarified. However, no aptamer is known that acts on phospholamban in the heart and promotes SERCA activity of the myocardial endoplasmic reticulum.
Inui, M.M. et al. , J .; Biol. Chem. 261: 1794-1800, 1986 James, P.M. et al. , Nature 342: 90-92, 1989. Sasaki, T .; et al. , J .; Biol, Chem. 267: 1674-1679, 1992 Minamisawa, S .; et al. Cell 99: 312-322, 1999 Luo, W .; et al. , Circ. Res. 75: 401-409, 1994 Iwanaga, Y. et al. et al. , J .; Clin. Invest. 113: 727-736, 2004 Special Table 2002-528512 JP 2002-62296 A JP 2006-042645 A JP 2002-58491 A JP 2006-149302 A

In order to provide a therapeutic agent for heart failure that does not involve genetic manipulation or gene expression, the present invention provides a substance that binds to phospholamban and a therapeutic agent for cardiac failure that corrects abnormal Ca2 ⁺ kinetics in cardiomyocytes containing the substance. This is the main issue.

The present inventors have found that a substance that binds to phospholamban, which acts as an inhibitor of SERCA that controls Ca ²⁺ transport in the myocardial endoplasmic reticulum, inhibits the action of phospholamban, and has completed the present invention.

That is, the present invention provides the following (1) to (3) .

(1) A single-stranded DNA or RNA in which a phospholamban aptamer selected by a SELEX method targeting a fusion protein containing a cytoplasmic domain of phospholamban has any one of the following base sequences (a) to (c) An aptamer having a specific binding property to phospholamban.
(A) A nucleotide sequence represented by any one of SEQ ID NOs: 2 to 12 (excluding SEQ ID NO: 6).
(B) A base sequence in which one or several bases of the base sequence represented by any one of SEQ ID NOs: 2 to 12 (excluding SEQ ID NO: 6) are deleted, substituted, or added.
(C) A base sequence having a homology of 90% or more with the base sequence represented by any of SEQ ID NOs: 2 to 12 (excluding SEQ ID NO: 6) .

(2) E Suhoranban aptamers according to (1) labeled protein is one that was bound to 26 amino acid portion from the first fusion protein phospholamban.

(3) A therapeutic agent for heart failure containing the phospholamban aptamer according to (1) or (2 ) above.

The nucleic acid aptamer having a specific binding property to phospholamban of the present invention improves the cardiac Ca2 ⁺ kinetics during heart failure and improves the contractile function of the heart, and thus is an effective therapeutic agent for patients with severe heart failure. .

The aptamer of the present invention has a specific binding property to phospholamban which acts on SERCA of myocardial endoplasmic reticulum. Phospholamban is a small molecule protein present in the slow and smooth muscles of the heart, consisting of 52 amino acids as shown in SEQ ID NO: 1, and the cytoplasmic domain is a functional binding between phospholamban and Ca ²⁺ -ATPase. It is said to be an indispensable area. Phospholamban, which is a SERCA inhibitor of myocardial endoplasmic reticulum, inhibits SERCA activity by binding to SERCA, and activates SERCA by dissociating from SERCA as necessary. When SERCA of the myocardial endoplasmic reticulum is activated, Ca ²⁺ transport to the myocardial endoplasmic reticulum is increased, myocardial relaxation is promoted, and the subsequent myocardial contractile force is enhanced.

  The aptamer of the present invention is a substance that has a specific binding property to phospholamban, can dissociate phospholamban from SERCA, and promote SERCA activity, and can be obtained by the SELEX method.

  The SELEX method is an operation method for obtaining a substance that specifically binds to a target such as a protein. In the SELEX method performed in the present invention, a DNA or RNA pool having a random sequence is selected from those having a sequence that binds to a target, and a PCR (Polymerase chain reaction) method or an RT-PCR (Reverse Transcription Polymer Reaction Reaction): Amplification by the reverse transcription polymerase chain reaction method is performed, and a sequence that binds again is selected from single-stranded DNA or transcribed RNA based on the amplification. By repeating this cycle, the sequences to be combined are converged. In each cycle, it is possible to obtain a sequence with high binding specificity by stricter binding conditions such as the concentration ratio of protein and DNA or RNA pool and competitors during selection.

  As a target substance used in the SELEX method of the present invention, a selected portion of the cytoplasmic domain of phospholamban can be used, but in order to increase the accuracy in the SELEX method, it is suitable for a substance having the amino acid sequence of the domain selection portion. It is desirable to use a fusion protein to which a protein is bound. The amino acid sequence of the selected portion of the cytoplasmic domain of phospholamban is a sequence consisting of amino acids from the first M (methionine) to the 26th Q (glutamine) of the phospholamban protein shown in SEQ ID NO: 1. An appropriate protein that binds to the sequence via a linker may be any protein that has a labeling function that can be easily selected during SELEX, such as Myc, His6, HA, GST, and FLAG. Can do. These proteins can be bound to an appropriate linker that binds to the cytoplasmic external position of phospholamban to form a fusion protein, which can be used as a target substance used in the SELEX method. A suitable linker is one that binds easily to the cytoplasmic domain selection portion of phospholamban and to the labeled protein, and can be any as long as it can add a molecular weight that can be confirmed by SDS electrophoresis as a fusion protein. Well, for example, a PDZ domain can be used.

  The phospholamban aptamer obtained by the SELEX method of the present invention can be selected from a random library of DNA or RNA having 20 to 80 bases, preferably 20 to 60 bases, for example. The random library can be obtained by chemically synthesizing randomly 20 to 80 bases, preferably 20 to 60 bases, from 4 types of oligonucleotides. In this case, an apparatus for DNA synthesis can be used. Sequences for amplification by PCR are added to both ends of DNA or RNA. In the case of RNA, a sequence for RNA synthesis is further added to the 5 'end. In the case of DNA aptamer, a single-stranded DNA library; in the case of RNA aptamer, RNA synthesized from a DNA library is immobilized on beads. Recombinant fusion protein not containing the cytoplasmic domain of phospholamban (control fusion protein) To remove non-specifically binding DNA or RNA. DNA or RNA that did not bind to the control fusion protein is mixed with beads immobilized with a recombinant fusion protein containing the phospholamban cytoplasmic domain. After washing, bound DNA or RNA is eluted and collected. In the case of a DNA aptamer, double-stranded DNA is amplified from the recovered DNA by PCR, and further converted into single-stranded DNA. In the case of an RNA aptamer, double-stranded DNA is amplified by RT-PCR, and RNA is synthesized using this as a template. Based on these single-stranded DNAs or RNAs, the cycle of removing nonspecific binding by the control fusion protein, binding to the fusion protein, and amplification by PCR or RT-PCR is repeated. The amplification cycle is 7 to 20 cycles, preferably 9 to 15 cycles.

  Double-stranded DNA amplified by PCR or RT-PCR is inserted into a cloning vector, and the aptamer sequence is determined. An aptamer can be obtained by synthesizing single-stranded DNA or RNA from the obtained sequence. The ability of an aptamer to bind to a recombinant fusion protein containing the cytoplasmic domain of phospholamban is determined by adding biotin to the 5 'or 3' end of the aptamer and quantifying the bound biotin-labeled aptamer using enzyme-labeled avidin. Can be sought.

  In addition, the effect of these aptamers on SERCA activity should be measured using myocardial ER follicles prepared from dog heart and compared with the effect of aptamers on sarcoplasmic reticulum follicles in rabbit skeletal muscle without phospholamban. Can be obtained. Aptamers dissociate phospholamban from SERCA by binding to phospholamban in myocardial endoplasmic reticulum follicles and promote SERCA activity. In the sarcoplasmic reticulum follicle of skeletal muscle, the SERCA activity does not change, and the extent of the effect of the aptamer on the SERCA activity can be determined by determining the ratio of both SERCA activities.

  Examples of DNA aptamers that specifically bind to phospholamban selected by the SELEX method include DNAs having the nucleotide sequences of SEQ ID NOs: 2 to 12. The present invention is not limited to these DNAs. In addition to RNA that specifically binds to phospholamban, one or several bases of the base sequence represented by SEQ ID NOs: 2 to 12 are deleted or substituted. Alternatively, a base sequence having 90% or more homology with the added base sequence or the base sequences represented by SEQ ID NOs: 2 to 12 is also included.

  The aptamer of the present invention can be formulated into various forms suitable for oral administration, parenteral administration or topical administration. In this case, excipients, binders, lubricants, disintegrants, preservatives, tonicity agents, stabilizers, dispersants, antioxidants, colorants, flavoring agents that are usually used for formulation Additives such as buffers can be used.

  The therapeutic agent for heart failure of the present invention is a solid form such as tablets, hard capsules, soft capsules, granules, powders, fine granules, pills, troches, or semi-solid forms such as suppositories and ointments, injections It can be formulated as a pharmaceutical form such as a liquid form such as an agent, emulsion, suspension, lotion, spray. In these preparation forms, pharmaceutically acceptable additives preferable for the dosage form are used. For example, lactose, glucose, lactose, fructose, maltose, magnesium carbonate, talc, magnesium stearate, methylcellulose, carboxymethylcellulose, gum arabic , Polyethylene glycol, p-hydroxybenzoic acid alkyl ester, syrup, ethanol, propylene glycol, glycerin, petrolatum, lipid components such as hard oil, sodium chloride, sodium sulfite, sodium phosphate, citric acid and the like.

  In the therapeutic agent for heart failure of the present invention, the aptamer content varies depending on the dosage form, but is generally 0.01 to 10% by weight in the solid and semi-solid forms, and 0 in the liquid form. It is desirable to contain in the density | concentration of 0.001 to 1 weight%. The dose varies depending on the administration method, patient age and weight, patient condition, etc., but is 0.01 to 10 mg / kg body weight per day when administered orally, and per day when administered parenterally. The dose can be 0.001-1 mg / kg body weight. The dose can be administered once or divided into several times a day.

  Hereinafter, examples will be given to describe the present invention in more detail, but the present invention is not limited thereto.

<Selection of phospholamban aptamer by SELEX method>

Phospholamban aptamer was selected by the following method. PLN ^1-26 -PDZ-Myc-His ₆ or PDZ-Myc-His ₆ fusion protein was prepared by the method of Kimura et al. (Kimura, Y. et al., Mol. Pharmacol. 61: 667-673, 2002). I went there. Preparation of Ni-NTA beads conjugated with PLN ^1-26 -PDZ-Myc-His ₆ or PDZ-Myc-His ₆ was carried out by changing Ni-NTA beads into PBS-T (phosphate buffer containing 0.05% Tween 20). After equilibration with (solution), 200 μl of PLN ^1-26 -PDZ-Myc-His ₆ or PBS-T containing 10 μg of PDZ-Myc-His ₆ was added to 5 μl of beads and shaken at 4 ° C. for 30 minutes. The reacted beads were washed 3 times with 1 ml of PBS-T. As a single-stranded DNA library, one of 80 bases in which PCR amplification sequences P1 (SEQ ID NO: 13) and P2 (SEQ ID NO: 14) are added to the 5 ′ end and 3 ′ of a 40 base mixed sequence, respectively. Strand DNA was synthesized. 1 nmol of single-stranded DNA library was denatured in 98 μl of PBS-T for 3 minutes at 98 ° C. and immediately cooled at 4 ° C. In the first selection, the single-stranded DNA library was added to 10 ml of PBS-T containing 5 μl of PDZ-Myc-His6-binding Ni-NTA beads and 1 μg / ml of BSA. After shaking for 30 minutes at room temperature, the mixture was centrifuged at 3,000 rpm for 5 minutes, and the supernatant was mixed with 5 μl of PLN ^1-26 -PDZ-Myc-His _6- conjugated Ni-NTA beads for 30 minutes at room temperature. . The beads were washed 3 times with 1 ml PBS-T. The aptamer bound to the protein was eluted in 50 μl of 0.5 mM imidazole-HCl (pH 7.4). The eluate was extracted with a phenol-chloroform mixture, and single-stranded DNA was precipitated with ethanol. After washing with 70% ethanol, the single-stranded DNA was 10 μl of 10 × PCR buffer, 8 μl of 4dNTPs (each 2.5 mM), 100 pmol of P1 primer (SEQ ID NO: 13) and 5 ′ end biotinylated P2 primer (sequence) No. 14) It was dissolved in 100 μl of a PCR mixture containing 2.5 U of 5% DMSO and Ex Taq DNA polymerase (Takara Bio). In the PCR reaction, denaturation was performed at 95 ° C. for 2 minutes. 20 cycles of heat denaturation at 95 ° C. for 30 seconds, annealing at 58 ° C. for 30 seconds, and extension reaction at 72 ° C. for 15 seconds were performed, and the final extension reaction was performed at 72 ° C. for 2 minutes.

A part of the PCR product was electrophoresed on a 4% agarose gel, and it was confirmed that double-stranded DNA having the correct length was amplified. 90 μl of the PCR product and 23 μl of 5M NaCl were mixed with 5 μl of Neuroavidin beads (Pierce) at room temperature for 10 minutes. After washing 3 times with 1 ml of PBS-T, single-stranded DNA not biotinylated is shaken in 50 μl of 0.1N NaOH for 5 minutes, then centrifuged at 3,000 rpm for 5 minutes, and then the DNA is removed from the Neuroavidin beads. Recovered to Qing. In order to adjust the pH of the resulting supernatant, 5 μl of 1M HCl was added to the supernatant. Single-stranded DNA was precipitated with ethanol, washed with 70% ethanol, and dissolved in 100 μl of PBS-T. Using this single-stranded DNA, the second and subsequent phospholamban aptamers were selected in the same manner as the first selection, but in the second and subsequent selections, PLN ^1-26 -PDZ- bound to Ni-NTA beads was used. The amount of Myc-His ₆ was 5 μg per 5 μl of beads.

  After the ninth selection, the obtained single-stranded DNA was amplified by PCR using a P1 primer (SEQ ID NO: 13) and a non-biotinylated P2 primer (SEQ ID NO: 14). The double-stranded DNA obtained by PCR was inserted into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA), introduced into E. coli (TOP 10: Invitrogen), and transformed. Forty-five colonies were arbitrarily selected from the grown colonies of E. coli, and the plasmid was purified from E. coli. The inserted DNA sequence was determined from the purified plasmid using T7 promoter and M13 reverse transcription primer, and the aptamer DNA sequence was determined. As a result, 11 types of DNA aptamers were obtained. The 11 types of sequences were as shown in Table 1.

<Effect of phospholamban aptamer on myocardial ER SERCA activity>

Myocardial endoplasmic reticulum follicles prepared from canine heart (final concentration 50 μg / ml) or sarcoplasmic reticulum follicles prepared from rabbit skeletal muscle (final concentration 50 μg / ml) were mixed with 20 mM imidazole-HCl (pH 6.9), 100 mM. Suspended in 50 μl of a reaction solution containing KCl, 2 mM MgCl 2, 5 mM NaN 3, 0.1 mM ATP, 0.35 μM Ca ²⁺ (Ca-EGTA buffer: 0.5 mM CaCl 2 and 1.3 mM EGTA) and synthesized. ATPase activity was measured at 37 ° C. for 4 minutes in the presence and absence of each of 11 types of phospholamban aptamers (10 μM). For the activity measurement, the method of Sasaki et al. (Sasaki, T. et al., J. Biol, Chem. 267: 1674-1679, 1992) was used. Also measured in the same manner ATPase activity ^{Ca 2+} absence plus 2mM of EGTA instead of 0.35MyuM Ca ^2+, was obtained from the difference between ^{Ca 2+} dependent ATPase activity (SERCA activity). In order to see the effect of aptamer on SERCA activity, the ratio of SERCA activity in the presence and absence of aptamer was determined.

  FIG. 1 shows the results of determining the ratio of SERCA activity in the presence and absence of 11 types of phospholamban aptamers for myocardial and skeletal muscle endoplasmic reticulum. The vertical axis represents the degree of promotion of SERCA activity by the aptamer. Aptamers had little effect on SERCA activity in skeletal sarcoplasmic reticulum (black) without phospholamban. On the other hand, in the myocardial endoplasmic reticulum (white), promotion of SERCA activity by the aptamer was recognized. In the comparison between skeletal muscle endoplasmic reticulum and myocardial endoplasmic reticulum in the presence of 10 μM aptamer, aptamer no. 10 types of aptamers except 5 significantly promoted SERCA activity of myocardial ER. These results indicate that the aptamer acts on phospholamban to release the suppression of SERCA and promote SERCA activity. The data shows the mean and standard deviation of 3-5 measurements. Statistical significance of comparison between skeletal muscle endoplasmic reticulum and myocardial endoplasmic reticulum was determined by T test. *, **, and *** indicate P <0.05, P <0.01, and P <0.001, respectively.

<Concentration dependence of the effect of phospholamban aptamer on myocardial ER SERCA activity>

  Synthetic aptamer Nos. Of various concentrations were prepared in myocardial ER follicles (final concentration 50 μg / ml) prepared from dog heart. 9 (0-40 μM) was added, SERCA activity was measured in the same manner as in Example 2, and the ratio to the absence of aptamer was determined.

  FIG. 2 shows an example of the experimental results. Phospholamban aptamer (aptamer No. 9) promoted myocardial ER SERCA activity in a concentration-dependent manner. The ED50 for this promoting effect was 15 μM. This result shows that the aptamer acts on phospholamban in a concentration-dependent manner to promote SERCA activity.

<Effect of phospholamban aptamer on Ca2 ⁺ concentration dependence of myocardial ER SERCA activity>

A synthesized aptamer No. 5 was prepared from a myocardial ER follicle (final concentration 50 μg / ml) prepared from a dog heart. 9 (30 μM) was added, and SERCA activity was measured in the same manner as in Example 2 in the presence of various Ca ²⁺ (0.11 to 16.3 μM as free Ca ²⁺ ). The dependency of SERCA activity on Ca ²⁺ concentration was expressed as a percentage with the maximum SERCA activity as 100%.

FIG. 3 shows phospholamban aptamer no. The Ca ²⁺ concentration dependence of SERCA activity in the absence (black circle) and presence (white circle) of 9 was shown. Aptamer No. 9 promoted myocardial endoplasmic reticulum SERCA activity, particularly at a Ca ²⁺ concentration of 0.11-2 μM / ml, and shifted the concentration-dependent curve to the left. This effect means that the inhibitory effect of phospholamban on SERCA has been lifted. Each data point represents the average value and standard deviation of experiments performed in triplicate. Statistical significance of the comparison between the presence and absence of aptamer was performed by T test. *, **, and *** indicate P <0.05, P <0.01, and P <0.001, respectively.

  The phospholamban aptamer of the present invention provides a new therapeutic means for severe heart failure patients who are difficult to treat, and its industrial utility value is extremely large.

It is the figure which showed the result of having compared the Ca2 ⁺ dependence ATPase activity (SERCA activity) of 11 types of phospholamban aptamers with a myocardial endoplasmic reticulum and a skeletal muscle endoplasmic reticulum. It is the figure which showed the result which the phospholamban aptamer (aptamer No. 9) accelerated | stimulated the myocardial endoplasmic reticulum SERCA activity concentration-dependently. It is the figure which showed the effect which the phospholamban aptamer (aptamer No. 9) exerts on the Ca ²⁺ concentration dependency of the myocardial ER SERCA activity.

Claims (3)

A phospholamban aptamer selected by a SELEX method targeting a fusion protein containing a phospholamban cytoplasmic domain is a single-stranded DNA or RNA having any of the following base sequences (a) to (c): An aptamer having a specific binding property to phospholamban.
(A) A nucleotide sequence represented by any one of SEQ ID NOs: 2 to 12 (excluding SEQ ID NO: 6).
(B) A base sequence in which one or several bases of the base sequence represented by any one of SEQ ID NOs: 2 to 12 (excluding SEQ ID NO: 6) are deleted, substituted, or added.
(C) A base sequence having a homology of 90% or more with the base sequence represented by any of SEQ ID NOs: 2 to 12 (excluding SEQ ID NO: 6) . Ho Suhoranban aptamers according to claim 1 fusion protein in which the labeled protein is bound to the 26 amino acid portion from the first phospholamban. A therapeutic agent for heart failure comprising the phospholamban aptamer according to claim 1 or 2.

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