JPWO2015045333A1 - Cardiomyocyte proliferation inducer, pharmaceutical composition containing the same, and method for producing cardiomyocytes - Google Patents

Abstract

As a novel drug having an activity of inducing cardiomyocyte proliferation, a cardiomyocyte proliferation inducer and pharmaceutical composition containing any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof: provide. This pharmaceutical composition can be suitably used for the treatment and / or prevention of heart disease, particularly ischemic heart disease. (1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. (2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of inducing cardiomyocyte proliferation. (3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of inducing cardiomyocyte proliferation.

Description

  The present invention relates to a cardiomyocyte proliferation inducer, a pharmaceutical composition containing the same, and a method for producing cardiomyocytes. More specifically, the present invention relates to a cardiomyocyte proliferation inducing agent containing a muskrin protein and / or a partial peptide thereof.

  Acute loss of cardiomyocytes after myocardial infarction reduces cardiac function and leads to heart failure. When a myocardial infarction occurs, cardiomyocytes rapidly decrease due to apoptosis and necrosis, the interstitial vascular and non-vascular components are almost destroyed by ischemic damage, and the cardiomyocytes in the infarcted region are fibrotic non-muscular It is gradually replaced by cells and becomes scar tissue. The amount of cardiomyocyte loss after myocardial infarction is a factor that determines the severity of cardiac dysfunction. In order to reduce heart failure after myocardial infarction, it is an important issue to recover the infarcted myocardial region.

  Traditionally, cardiomyocytes were thought to lose the ability to terminally differentiate and divide after birth. However, cardiomyocytes have been reported to divide under certain pathological conditions or physiological conditions (see, for example, Non-Patent Document 1). Subsequently, studies showing the division or regeneration of cardiomyocytes derived from cardiomyocytes resident immediately after myocardial infarction, stem cells circulating in the blood, or both, have been reported in the human heart (eg, non- Patent Document 2).

  At present, after myocardial infarction, reperfusion and administration of a chemical agent at an early stage have been attempted to reduce myocardial cell death and reduce the infarcted myocardial region. However, these treatments fail to regenerate damaged cardiomyocytes and cannot restore the damaged function of the heart.

  Here, in connection with the present invention, musclin will be described. Musculin was originally reported as a secreted protein derived from skeletal muscle under the name of osteocrin (see Non-Patent Documents 3 and 4). Patent Document 1 discloses a method for increasing bone formation using muskline. Subsequently, muskrin has a natriuretic peptide (NP) homology region and is reported to bind to natriuretic peptide receptor 3 competitively with atrial natriuretic peptide (ANP). (See Non-Patent Document 5). Patent Document 2 discloses a screening system for agonists and / or antagonists of muskline and its receptor.

  None of the prior art documents relating to these musklines has reported any association between muskulin and cardiomyocytes.

US Patent Publication No. 2007/0049521 US Patent Publication No. 2011/0104705

J. Circ. Res., 1998, 83, 1-14 N. Eng. J. Med., 2001, 344, 1750-1757 J. Bio. Chem., 2004, 279, 19391-19395 J. Bio. Chem., 2003, 278, 50563-50571 J. Endocrinol., 2009, 201, 287-95

  In order to regenerate damaged cardiomyocytes after myocardial infarction and restore the function of the heart, a technique for inducing cardiomyocyte division and proliferation is required. Therefore, the main object of the present invention is to provide a novel drug having an activity of inducing proliferation of cardiomyocytes.

The present inventors have found that muskrin protein and / or a partial peptide thereof has an activity to induce cardiomyocyte proliferation. Based on this finding, the present invention provides a cardiomyocyte proliferation inducing agent and a pharmaceutical composition containing any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof. This pharmaceutical composition can be suitably used for the treatment and / or prevention of heart disease, particularly ischemic heart disease.
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of inducing cardiomyocyte proliferation.
(3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of inducing cardiomyocyte proliferation.

The pharmaceutical composition according to the present invention may contain any one or more of the following polypeptides (4) to (6).
(4) A polypeptide consisting of the 83rd to 112th amino acid sequence of the amino acid sequence described in SEQ ID NO: 1.
(5) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3.
(6) A polypeptide comprising the 80th to 130th amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 3.

The present invention relates to a heart disease, particularly ischemic, comprising a procedure for administering to a subject (including humans and animals) any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof: A method of treating and / or preventing heart disease is also provided.
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of inducing cardiomyocyte proliferation.
(3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of inducing cardiomyocyte proliferation.

  The present invention also provides the following method for producing cardiomyocytes.

A method for producing cardiomyocytes, comprising a step of treating the separated cardiomyocytes and / or precursor cells thereof with any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof.
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of inducing cardiomyocyte proliferation.
(3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of inducing cardiomyocyte proliferation.

Furthermore, the present inventors have found that muskrin protein and / or partial peptides thereof have an activity of lowering blood pressure. Based on this finding, the present invention provides a blood pressure lowering agent containing any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof. Therefore, the pharmaceutical composition according to the present invention can be suitably used for the treatment and / or prevention of hypertension and hypertension-related diseases in addition to the treatment and / or prevention of ischemic heart disease.
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of lowering blood pressure.
(3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of lowering blood pressure.

The present invention relates to hypertension and hypertension-related diseases, including a procedure for administering to a subject (including humans and animals) any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof: Also provided are methods of treatment and / or prevention.
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of lowering blood pressure.
(3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of lowering blood pressure.

  In the present invention, heart diseases particularly include ischemic heart diseases, and examples of ischemic heart diseases include impending myocardial infarction, myocardial infarction, ischemic cardiomyopathy, angina pectoris, and heart failure after myocardial infarction. . Furthermore, the heart disease in the present invention widely includes heart diseases accompanied by myocardial cell death, such as congestive cardiomyopathy, hypertrophic obstructive cardiomyopathy, hypertrophic non-occlusive cardiomyopathy, idiopathic cardiomyopathy, It includes myocarditis, chronic heart failure, valvular disease, coronary artery disease, post-heart syndrome, post-myocardial infarction syndrome (Dressler syndrome), and the like. Hypertension-related diseases include stroke, congestive heart failure; cardiac hypertension; cardiac fibrosis; hypertensive heart disease; post-infarction cardiomyopathy; nephropathy, angiopathy, neuropathy, etc. Disease; coronary vascular disease; restenosis after angioplasty; elevated intraocular pressure; glaucoma; abnormal vascular proliferation; hyperaldosteronism; anxiety and cognitive impairment.

  The present invention provides a novel drug having an activity of inducing cardiomyocyte proliferation.

Alignment of amino acid sequences of human, bovine, mouse and rat musculin. It is a photograph which shows the expression analysis result of a musculin in a myocardial injury model. It is a graph which shows the measurement result of the number of cardiomyocytes in the zebrafish embryo which micro-injected musculin mRNA. It is a graph which shows the measurement result of the number of cardiomyocytes in muskulin expression transgenic zebrafish. It is a graph which shows the result of having evaluated the proliferation induction activity with respect to the culture | cultivation cardiomyocyte of a musculin. It is a graph which shows the result of having evaluated the blood pressure lowering effect of a muskline. It is a graph which shows the result of administering a musculin to a mouse | mouth myocardial infarction model and evaluating the change of survival rate. It is a photograph which shows the macroscopic findings of the heart of the mouse | mouth myocardial infarction model which administered musculin. (A) shows the results of the sham operation group, B shows the results of the control group (distilled water administration group), and C shows the results of the test group (musculin administration group). It is a photograph which shows the tissue staining image of the heart of the mouse | mouth myocardial infarction model which administered musculin. (A) shows the results of the sham operation group, B shows the results of the control group (distilled water administration group), and C shows the results of the test group (musculin administration group).

  Hereinafter, preferred embodiments for carrying out the present invention will be described. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

1. Cardiomyocyte proliferation inducer The present inventors have found that the gene transfer of musculin or administration of a partial peptide thereof induces the division and proliferation of cardiomyocytes (see Examples), and the muskrin protein and / or part thereof. It was revealed that the peptide is useful as a cardiomyocyte proliferation inducer.

[Polypeptide]
The polypeptide which is an active ingredient in the cardiomyocyte proliferation inducing agent according to the present invention may be a human musculin protein consisting of the amino acid sequence set forth in SEQ ID NO: 1.

  Further, the polypeptide which is an active ingredient consists of an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1, and has an activity of inducing proliferation of cardiomyocytes. It may be a polypeptide. The number of amino acids to be deleted, substituted, inserted or added is not particularly limited as long as the polypeptide can maintain cardiomyocyte proliferation inducing activity.

More specifically, the polypeptide as the active ingredient may be a polypeptide having the following amino acid sequence.
(I) an amino acid sequence in which 1 to 20 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids are deleted in the amino acid sequence described in SEQ ID NO: 1;
(Ii) an amino acid sequence in which 1 to 20 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids of the amino acid sequence described in SEQ ID NO: 1 are substituted with other amino acids,
(Iii) an amino acid sequence in which 1 to 20 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids are added to the amino acid sequence of SEQ ID NO: 1,
(Iv) an amino acid sequence in which 1 to 20 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids are inserted into the amino acid sequence set forth in SEQ ID NO: 1,
(V) An amino acid sequence in which the deletion, substitution, insertion or addition described in (i) to (iv) above is combined.

  When one or several amino acids in the amino acid sequence are substituted, conservative substitution between similar amino acid residues is preferred. For example, amino acids are classified into hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl group-containing side chain (S, T, Y), sulfur Amino acids having atom-containing side chains (C, M), amino acids having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), aromatic It is classified into amino acids (H, F, Y, W) having a containing side chain. It is known that the amino acids classified into each group are likely to maintain the activity of the polypeptide when substituted with each other, and such mutual substitution of amino acids is preferred. Examples thereof include substitution between glycine and proline, glycine and alanine or valine, leucine and isoleucine, glutamic acid and glutamine, aspartic acid and asparagine, cysteine and threonine, threonine and serine or alanine, and lysine and arginine.

  Furthermore, the polypeptide which is an active ingredient consists of an amino acid sequence having 80% or more sequence identity to the amino acid sequence described in SEQ ID NO: 1, and may be a polypeptide having an activity of inducing proliferation of cardiomyocytes. Good. The sequence identity is not particularly limited as long as the peptide can maintain cardiomyocyte proliferation-inducing activity, but may be 50% or more, preferably 60% or more, more preferably 70% or more, and particularly preferably 90%. More particularly, it may be 95% or more, most preferably 98% or more.

  Therefore, the polypeptide which is an active ingredient in the cardiomyocyte proliferation inducing agent according to the present invention includes, in addition to the human musculin protein represented by SEQ ID NO: 1, a mouse musculin protein, rat musculin protein, bovine musk represented by SEQ ID NO: 3. Includes phosphoproteins. FIG. 1 shows the amino acid sequences of human, bovine, mouse and rat musculin proteins. In addition, the polypeptide which is an active ingredient includes a musculin homolog protein in various organisms such as monkeys, dogs, pigs and rabbits.

[Partial peptide]
The active ingredient in the cardiomyocyte proliferation inducing agent according to the present invention may be a partial peptide of the above polypeptide. The amino acid sequence of the partial peptide can be the amino acid sequence of any part of the polypeptide as long as it has cardiomyocyte proliferation inducing activity.

  Preferable amino acid sequences of the partial peptide include the 83rd to 112th amino acid sequences of the amino acid sequence described in SEQ ID NO: 1 and the 80th to 130th amino acid sequences of the amino acid sequence described in SEQ ID NO: 3 (implementation). See example).

  FIG. 1 shows a polypeptide (humanM (83-112)) consisting of the 83rd to 112th amino acid sequence of the amino acid sequence described in SEQ ID NO: 1, and the 80th to 130th amino acid sequence of the amino acid sequence described in SEQ ID NO: 3. The amino acid sequence of a polypeptide consisting of (mouse M (80-130)) is shown. The amino acid sequence of “KKKR” present on the N-terminal side of humanM (83-112) and the amino acid sequence of “KRR” present on the C-terminal side are cleavage sites by proteases.

  Musculin is presumed to be a secreted protein, and the musculin protein may exhibit cardiomyocyte proliferation-inducing activity in a state where it is cleaved by a protease at the above cleavage site into a partial peptide. Therefore, as a partial peptide that can show activity, first, a polypeptide consisting of the 83rd to 133rd amino acid sequences of the amino acid sequence described in SEQ ID NO: 1 (humanM (83-133)) and the amino acid sequence described in SEQ ID NO: 3 are used. Examples include polypeptides consisting of amino acid sequences 80 to 130 (mouse M (80-130)), and human M (83-112) and human M (116-133) and mouse M (80 which are generated by cleavage of these polypeptides. -109) and mouseM (113-130) peptides (see FIG. 1).

  Further, the partial peptide which is an active ingredient consists of an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the partial peptide of the above polypeptide and has an activity of inducing cardiomyocyte proliferation. It may be a peptide. The number of amino acids to be deleted, substituted, inserted or added is not particularly limited as long as the peptide can maintain cardiomyocyte proliferation inducing activity.

More specifically, the partial peptide which is an active ingredient may be a polypeptide having the following amino acid sequence.
(I) In the amino acid sequence of humanM (83-112), humanM (116-133), humanM (83-133), mouseM (80-109), mouseM (113-130) or mouseM (80-130), 1 An amino acid sequence in which -15 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids have been deleted;
(Ii) 1 to the amino acid sequence of humanM (83-112), humanM (116-133), humanM (83-133), mouseM (80-109), mouseM (113-130) or mouseM (80-130) An amino acid sequence in which 15 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids are substituted with other amino acids,
(Iii) 1 to the amino acid sequence of humanM (83-112), humanM (116-133), humanM (83-133), mouseM (80-109), mouseM (113-130) or mouseM (80-130) An amino acid sequence to which 15 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids are added;
(Iv) 1 to the amino acid sequence of humanM (83-112), humanM (116-133), humanM (83-133), mouseM (80-109), mouseM (113-130) or mouseM (80-130) An amino acid sequence in which 15 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids are inserted;
(V) An amino acid sequence in which the deletion, substitution, insertion or addition described in (i) to (iv) above is combined.

  Further, the active polypeptide may be humanM (83-112), humanM (116-133), humanM (83-133), mouseM (80-109), mouseM (113-130) or mouseM (80-130). And a polypeptide having an activity of inducing proliferation of cardiomyocytes. The sequence identity is not particularly limited as long as the peptide can maintain cardiomyocyte proliferation-inducing activity, but may be 50% or more, preferably 60% or more, more preferably 70% or more, and particularly preferably 90%. More particularly, it may be 95% or more, most preferably 98% or more.

  Accordingly, partial peptides which are active ingredients in the cardiomyocyte proliferation inducing agent according to the present invention include humanM (83-112), humanM (116-133), humanM (83-133), mouseM (80-109), mouseM. In addition to (113-130) and mouseM (80-130), a partial peptide of musculin homologue in various organisms such as monkeys, dogs, cows, pigs, rats and rabbits, comprising humanM (83-112), Peptides consisting of amino acid sequences corresponding to humanM (116-133), humanM (83-133), mouseM (80-109), mouseM (113-130) and mouseM (80-130) are included.

  The musculin protein and its partial peptide which are active ingredients in the cardiomyocyte proliferation inducing agent according to the present invention may be modified with a fatty acid or polyethylene glycol. As long as an improvement in medicinal effect and safety is recognized, the length and degree of unsaturation of the modified fatty acid or the molecular weight and degree of branching of polyethylene glycol are not limited. Preferably, a fatty acid having 10 to 20 carbon atoms or polyethylene glycol having a molecular weight of 4000 to 20000 is used.

2. Antihypertensive agent The present inventors have found that administration of a partial peptide of musculin protein lowers blood pressure (see Examples), and that musculin protein and / or its partial peptide are useful as a hypotensive agent. Was revealed.

  The active ingredient in the antihypertensive agent according to the present invention is the same as the polypeptide of the above-mentioned cardiomyocyte proliferation inducer and its partial peptide.

3. Pharmaceutical composition (1) Therapeutic / preventive agent for ischemic heart disease The pharmaceutical composition according to the present invention effectively uses the above-mentioned cardiomyocyte proliferation inducer (or the polypeptide contained in the cardiomyocyte proliferation inducer and its partial peptide). Contains as a component. Since this pharmaceutical composition is capable of inducing cardiomyocyte division and proliferation, it is useful for regenerating cardiomyocytes damaged by heart disease, particularly ischemic heart disease, and restoring cardiac function. It can be suitably used for the treatment and / or prevention of myocardial infarction and the like.

(2) Antihypertensive and Hypertensive Related Disease Treatment / Prevention Agent In addition, in another aspect, the pharmaceutical composition according to the present invention is the above-described antihypertensive agent (or a polypeptide and a partial peptide thereof contained in the antihypertensive agent). Is contained as an active ingredient. Since this pharmaceutical composition is capable of lowering blood pressure, it can be suitably used for the treatment and / or prevention of hypertension and hypertension related diseases (stroke, heart disease, kidney disease, etc.).

  The pharmaceutical composition according to the present invention comprises a cardiomyocyte proliferation inducer or a blood pressure lowering agent (or a polypeptide and a partial peptide thereof) alone or mixed with a solubilizing agent, a bulking agent, an excipient or a carrier. Then, it can be used as preparations such as injections, tablets, granules, fine granules, powders, capsules, solutions and sustained-release agents. As the additive such as a solubilizer, a bulking agent, an excipient or a carrier, a pharmaceutically acceptable one is selected, and the type and composition thereof can be appropriately determined depending on the administration route and the administration method. For example, in the case of injections, sugars such as sodium chloride, glucose and mannitol are generally desirable. In the case of an oral preparation, starch, lactose, crystalline cellulose, magnesium stearate and the like are desirable.

  The administration route of the pharmaceutical composition according to the present invention is systemically administered orally or parenterally by injection. Further, it may be administered remotely through a catheter or the like, or the drug may be bound to a stent, a graft, or an integrated stent graft. Furthermore, it may be administered in sustained release by indwelling in the body. In particular, oral administration is preferred.

  The dosage of the pharmaceutical composition according to the present invention is determined in consideration of the type of dosage form, the administration method, the age and weight of the administration subject (including humans and animals), symptoms, etc. The amount is sufficient to cure or at least partially block symptoms such as ischemic heart disease or hypertension. The administration period can be appropriately selected depending on the age and symptoms of the patient.

  The pharmaceutical composition according to the present invention may contain, as an active ingredient, a drug that has already been clinically applied for the treatment of myocardial infarction or hypertension or a drug that is in the research stage. Clinically applied myocardial infarction drugs include β-blockers, renin / angiotensin / aldosterone inhibitors (ACE inhibitors, angiotensin receptor antagonists, aldosterone antagonists, renin inhibitors), antiplatelet drugs, anticoagulants Drugs, lipid metabolism abnormality improvers (hyperlipidemic drugs, cholesterol-lowering drugs, HDL-elevating drugs), diabetes drugs, nitrate drugs, nicorandil, antiarrhythmic drugs and cardiotonic drugs, angiotensin receptor antagonists and neutral endopeptidases Inhibitor compounding agents and the like. Antihypertensive drugs include renin / angiotensin / aldosterone system inhibitors (ACE inhibitors, angiotensin receptor antagonists, aldosterone antagonists, renin inhibitors), β-blockers, calcium antagonists, diuretics, etc. .

  As another embodiment, the pharmaceutical composition according to the present invention may be combined with the above-mentioned drugs already applied clinically or drugs in research stage. Here, “combined” means that when two or more medicaments are administered at different times, when each medicament is administered at the same time, one kind of pharmaceutical composition comprising two or more medicaments is administered. If you mean. In the former two cases, each of two or more medicaments may be administered by the same route of administration, or may be administered by different routes of administration. Each medicament is administered in an amount sufficient to cure or at least partially block symptoms to the patient. The administration period can be appropriately selected depending on the age and symptoms of the patient.

4). 2. Method for producing cardiomyocytes (1) Peptide treatment A cardiomyocyte proliferation-inducing agent containing muskulin protein and / or a partial peptide thereof can be used for producing cardiomyocytes in vitro.

  The cardiomyocyte production method according to the present invention includes, as one aspect, the above-described cardiomyocyte proliferation inducer (or the polypeptide contained in the cardiomyocyte proliferation inducer and a portion thereof) on the isolated cardiomyocytes and / or precursor cells thereof. Peptide).

  The said process can be performed by culturing the cell isolate | separated from the biological body by a conventional method, and adding a musculin protein and / or its partial peptide in a culture solution. In this step, the cells divide and proliferate by a cardiomyocyte division-inducing action such as muskulin protein, and a large number of cardiomyocytes are obtained.

  Here, the “cardiomyocyte progenitor cell” is not particularly limited, but cells having the ability to differentiate into cardiomyocytes such as embryonic stem cells (ES cells), inducible stem cells (iPS cells), and mesenchymal stem cells may be used. included.

  The obtained cardiomyocytes can be used as a transplant material to the heart in the form of cell dispersion, cell sputum and cell sheet, etc., and can be used for various tests such as drug screening and toxicity evaluation using cardiomyocytes. it can.

(2) Gene transfer The cardiomyocyte production method according to the present invention includes, as one aspect, the above-described cardiomyocyte proliferation inducer (or the cardiomyocyte proliferation inducer) on the isolated cardiomyocytes and / or precursor cells thereof. It may include a step of introducing a gene encoding a polypeptide to be contained and a partial peptide thereof in an expressible manner.

  The nucleotide sequences of genes encoding human musculin protein and mouse musculin protein are shown in SEQ ID NOs: 2 and 4, respectively. Musculin gene introduction may be carried out by introducing DNA comprising the nucleotide sequences described in SEQ ID NOs: 2 and 4 into cells using a commonly used vector or gene introduction reagent. Alternatively, a partial peptide of musculin can also be expressed by gene transfer by modifying the base sequence of DNA consisting of the base sequences described in SEQ ID NOs: 2 and 4 by a known technique.

<Material>
1. Zebrafish Lines The following lines of zebrafish were used in the experiments.
(1) AB strain (wild-type zebrafish)
(2) Tg (Cmlc2: mVenus-zGeminin (1/100)) line and Tg (mCherry-zCdt1 (1/190)) line
A transgenic line that expresses Fucci (fluorescent ubiquitination-based cell cycle indicator) downstream of the Cmlc2 (Cardiac myosin-light chain 2) promoter in a cardiomyocyte-specific manner.
(3) Tg (Cmlc2: NLS-mCherry) line A transgenic line that expresses NLS (nuclear localization signal) -mCherry in the nucleus of cardiomyocytes.
(4) Tg (Cmlc2: Fucci) strain (cell cycle visualization zebrafish)
A transgenic line produced by crossing Tg (Cmlc2: mVenus zGeminin (1/100)) and Tg (Cmlc2: mCherry-zCdt1 (1/190)). The Tg (Cmlc2: Fucci) line expresses mVenus-geminin (green) and mCherry-Cdt1 (red) in cardiomyocytes. Since Geminin degrades in G0 / G1 phase and Cdt1 degrades in S / G2 / M phase, in Tg (Cmlc2: Fucci) line, cardiomyocytes in S / G2 / M phase (dividing phase) are green and G0 / G1 (stationary) cells are visualized in red.

  The Tol2 transposon system was used for the establishment of the transgenic lines (2) and (3) above. A sequence expressing mVenus-zGeminin (1/100), mCherry-zCdt1 (1/190), or NLS-mCherry was incorporated into the Tol2 transposon vector downstream of the Cmlc2 promoter. Using a manipulator, 30 pg of plasmid DNA and 25 pg of Tol2 transferase mRNA were injected into the blastomere of 1 cell stage embryo of AB strain.

<Test Example 1: Separation of dividing cardiomyocytes>
Dividing cardiomyocytes in S / G2 / M phase were separated from the heart of cell cycle visualization zebrafish (Tg (Cmlc2: Fucci) strain) by the following procedure.

Hearts were removed with tweezers from a cell cycle visualization zebrafish of a tricaine-anesthetized 84 hour embryo. Hearts were collected in HBSS on ice using a Pipetteman. After washing twice with PBS, 1 ml of enzyme solution (5 mg / ml trypsin, 1 mM EDTA / PBS, pH 8.0) was added to separate the cells. Incubating at 37 ° C. for 60 min while pipetting every 30 min. Thereafter, 200 μl of a reaction stop solution (30% FBS, 6 mM CaCl ₂ / PBS) was added to stop the enzyme reaction. It was washed with PBS and suspended in 200 μl of a suspension (1% FBS, 0.8 mM CaCl ₂ , 50 U / ml penicillin, 0.05 mg / ml streptomycin, DMEM, 50 ml). In order to remove dead cells, SYTOX Blue dead cell stain (invitrogen) was added and incubated at room temperature for 15 min to stain dead cells.

  Separated cardiomyocytes were separated using FACSAria III (BD). From the viable cell population of SYTOX Blue dead cell stain (-), the S / G2 / M phase cell population (mVenus (+) · mCherry (-)) and the G0 / G1 phase cell population (mVenus (-) · mCherry ( +)) Were separated and each recovered in suspension.

  In order to extract mRNA using NucleoSpin RNA XS (Takara), the collected cells are washed twice with PBS, then PBS is thoroughly removed, dissolved in RA1 solution with TECP added, and stored at -20 ° C. did.

<Test Example 2: Identification of a gene expressed in dividing cardiomyocytes>
By extracting mRNA from the dividing cardiomyocytes isolated in Test Example 1 and performing sequencing, genes expressed in the dividing cardiomyocytes were comprehensively identified.

  All samples for RNASequence were prepared according to the kit method. After recovering 5,000 cells or more, Total RNA was extracted from the cells using NucleoSpin RNA XS (Takara). The RNA was reverse-transcribed from RNA to DNA using cDNA Synthesis Kits-SMARTer Ultra Low Input RNA for Illumina Sequencing (Clontech). Using the Advantage 2 PCR Kit (Takara), the reverse-transcribed DNA was amplified and a cDNA library was prepared. The cDNA was fragmented to 200 bp using Covaris S220 (MS Instruments Co., Ltd.). Tags were added to the cDNA library using NEBNext DNA Library Preparation (NEW ENGLAND BioLabs) and NEBNext Multiplex oligos For Illumina (NEW ENGLAND BioLabs), and the cDNA library (with Tag) was analyzed using Mi-seq (Illumina).

  As a result of the analysis, muskrin was identified as a gene whose expression is increased in dividing cardiomyocytes and a gene other than a cell cycle-related factor. The expression level of musculin in mitotic cells was 5.67 times that of quiescent cells.

<Test Example 3: Expression analysis of muskline in a myocardial injury model>
The expression of musculin in the heart of a myocardial injury model was analyzed by the following procedure.

  A 4-12 month zebrafish was opened under tricaine anesthesia, the heart was pushed out, and a 0.5 mm diameter copper wire cooled with liquid nitrogen was applied to the apex to cause necrosis. The heart was removed on the 3rd, 7th, and 14th days after the operation, and fixed with 4% PFA / PBS.

  The fixed tissue was treated with 10% sucrose / PBS at 4 ° C. for 4 hr and 20% sucrose / PBS at 4 ° C. overnight, then embedded and frozen in liquid nitrogen. A 14 μm-thick section was prepared using a cryostat (CM 1850, Leica) and attached to a slide glass. It dried with the dryer (cold air) and preserve | saved at -80 degreeC.

The frozen section slide was washed with RNase-free water and then permeabilized with proteinase K (20 μg / ml). After adding 4% PFA / PBS and re-fixing at room temperature for 15 min, it was washed well with PBST. After prehybridization with hybridization solution at 65 ° C for 1 hr, add DIG (digoxigenin) -labeled RNA probe dissolved in hybridization solution and hybridize overnight at 65 ° C with parafilm on top to prevent drying. did. The next day, samples were washed with Wash Solution I (75% formamide, 2 x SSC), Wash Solution II (50% formamide, 2 x SSC), Wash Solution III (25% formamide, 2 x SSC), Wash Solution IV (2 x SSC). Wash once at 65 ° C for 10 min, then use washing solution V (0.2 x SSC) twice at 65 ° C for 30 min, wash solution VI (0.1 x SSC) at room temperature, 10 min Washed once under the conditions of

  The section slides were washed with maleic acid solution (0.1M maleic acid, pH 7.5) for 15 min at room temperature, and blocking solution (0.1M maleic acid, pH 7.5, 5% sheep serum, 2% blocking reagent (Roche) ) For 60 min, followed by addition of anti-DIG antibody conjugated with alkaline phosphatase (Roche) and shaking at 4 ° C. overnight. On the next day, the plate was washed 5 times with PBST, and staining reaction was started using BM-purple AP substarate (Roche). At the end of staining, the cells were washed with PBST and fixed with 4% PFA / PBS. After washing with PBST, it was gradually replaced with glycerol and photographed with a stereomicroscope (MVX10, Olympus).

  The above DIG-labeled RNA probe was prepared as follows. A primer corresponding to the target gene sequence was prepared, and a pCR4blunt TOPO vector (Invitrogen) incorporating the full length of the zMusclin cDNA sequence (SEQ ID NO: 5) was prepared. Cleavage was performed using a restriction enzyme site present only on the upstream 5 ′ end side of the gene sequence, and the sample was precipitated with ethanol. To 3 μg of DNA, add 1 μl of DIG mix (Roche), 1 μl of RNase inhibitor (Roche), and 1 μl of T3 or T7 promoter RNA polymerase (Roche) located downstream of the 3 ′ end, 37 ° C, 120 min Reacted. 1 μl of DNase was added and reacted at 37 ° C. for 15 min. The sample was precipitated with lithium chloride and 25 μl of RNase free water was added. After confirming that a probe was formed by electrophoresis using an agarose gel, a hybridization solution was added and stored at −20 ° C.

  The results are shown in FIG. In the apex of the lesion, the expression of musculin was observed from the third day onward, and it was revealed that the expression of musculin increased in the dividing cardiomyocytes after the injury. In addition, a control | contrast shows the result of the apex part which has not added the disorder | damage | failure.

<Example 1: Evaluation of cardiomyocyte proliferation-inducing action of muskline by in vivo gene transfer>
Zebrafish musculin mRNA was introduced into embryos of the developing zebrafish by microinjection, and the proliferation-inducing activity of musculin on cardiomyocytes was evaluated.

  Using a manipulator, 200 pg of mRNA was injected into the blastomere of 1 cell stage embryo of Tg (Cmlc2: NLS-mCherry) strain. mRNA was synthesized using mMESSAGE mMACHINE kit (Ambion). A cDNA sequence encoding the full length of the molecule to be synthesized was incorporated into pCS2 + vector (Clontech) and sequenced to confirm the sequence. After digestion with the restriction enzyme NotI, proteinase K (100 mg / ml) /0.5% SDS treatment was performed, and the inactivated RNase was precipitated with ethanol and dissolved in RNase free-water. MRNA was synthesized from the cut template DNA using SP6 RNA polymerase. The concentration of RNA was measured with a NanoPhotometer (IMPLEN), and it was confirmed that the RNA was not degraded by electrophoresis using an agarose gel.

  Two days after the injection of mRNA, the number of cardiomyocytes exhibiting red fluorescence was significantly increased (see FIG. 3). From these results, it was revealed that muskline has an action of promoting or inducing proliferation of cardiomyocytes in the developing zebrafish.

  Next, transgenic zebrafish expressing musculin downstream of the Cmlc2 promoter was prepared, and the effect of increasing the number of cardiomyocytes by musculin gene transfer was examined. Establishment of the transgenic line was carried out by the method described above.

  The results are shown in FIG. A significant increase in cardiomyocytes was confirmed in the transgenic line (M in the figure) expressing muskrin as compared to the wild type line ((-)).

<Example 2: Evaluation of cardiomyocyte proliferation-inducing action of muskline by in vitro peptide treatment>
The cultured cardiomyocytes were treated with a partial peptide of musculin protein, and the proliferation-inducing activity of musculin on cardiomyocytes was evaluated.

A mouse neonatal cardiomyocyte kit (Primary Cell) was used. The attached fibronectin solution was added to the dish and allowed to stand overnight in a 37 ° C. incubator for coating. The dish was washed twice with sterile water immediately before use. Cardiomyocytes are seeded in the center of the dish using 200 μl of the attached culture medium so that the density becomes 1.3 × 10 ⁵ cells / dish (first time) and 9 × 10 ⁴ cells / dish (second day). The cells were cultured in a 37 ° C. incubator in the presence of% CO ₂ . At 6 hr after sowing, 1 ml of culture medium was added to the dish. In addition, the culture medium was exchanged 16 hours after seeding to remove cells that did not adhere. The culture medium was changed 88 hours after sowing only the samples treated for 6 days.

The addition of the test substance was started 24 hours after seeding, and more reagents were added every 24 hours. The following substances were used as test substances. Two dishes were prepared for each test substance.
(1) FGF2 (human): final concentration 25ng / ml
(2) mMusclin (80-130) peptide: peptide consisting of amino acid sequence 80 to 130 of mouse amino acid sequence (SEQ ID NO: 3) (Phoenix Pharmaceuticals), final concentrations 1 nM, 10 nM, 100 nM
(3) hMusclin (83-112) Peptide: Peptide consisting of the 83rd to 112th amino acid sequence of human mucrine amino acid sequence (SEQ ID NO: 1) (Phoenix Pharmaceuticals), final concentrations of 1 nM, 10 nM, 100 nM

  Cells synthesized with DNA were detected using Click-iT EdU Alexa Fluor 647 Imaging kit (Invitrogen). EdU (5-ethynyl-2′-deoxyuridine) was added to a final concentration of 10 μM 6 hr after the start of the test substance treatment. In the case of 6-day stimulation, EdU was added 78 hours after the start of stimulation. From the start of the test substance treatment, the cells were washed twice with PBS at 72 hr or 144 hr, 4% PFA / PBS was added, and the cells were fixed at room temperature for 20 min.

  The fixed sample was stained with EdU according to the method of Click-iT EdU Alexa Fluor 647 Imaging kit (Invitrogen), and then immunofluorescent stained. 0.5% Triton X-100 / PBS was added and permeabilization was performed at room temperature for 20 min. Reaction cocktail was added and reacted at room temperature for 30 min, and Alexa Fluor 647 aside was added to EdU. 4% BSA treated at room temperature for 1 hr for blocking, anti-MF20 antibody (IOWA hybridoma bank) as a primary antibody diluted 200-fold, anti-Phospho-Histone H3 (Ser10) antibody (Millipore) diluted 100-fold for 1 hr . After washing with PBS, dilute Alexa Fluor 488 goat anti-rabbit IgG conjugate highly cross-absorbed (Invitrogen) and Alexa Fluor 546 goat anti-mouse IgG conjugate highly cross-absorbed (Invitrogen) as the secondary antibodies, respectively. The reaction was performed for 1 hr. After washing with PBS, the cells were incubated in 0.5 ng / ml DAPI for 30 min and observed with an inverted confocal laser microscope (fv-1000, Olympus). Eight fields of view were taken for each sample, the number of MF20 positive cardiomyocytes and the number of EdU positive cells in the cardiomyocytes were counted, and the average value of the ratio of EdU positive cardiomyocytes was determined.

  The results are shown in FIG. A shows the percentage of EdU-positive cardiomyocytes when stimulated with mMusclin (80-130) peptide for 3 days. B shows the percentage of EdU positive cardiomyocytes when stimulated with mMusclin (80-130) peptide or hMusclin (83-112) peptide for 6 days. The vertical axis represents the average value of the ratio of dividing cardiomyocytes (EdU positive cardiomyocytes).

  As shown in A, the treatment with Musclin 100 nM was able to induce the proliferation of cardiomyocytes to the same extent as the FGF treatment. In addition, as shown in B, the proliferation-inducing action of cardiomyocytes was higher with the mMusclin (80-130) peptide than with the hMusclin (83-112) peptide.

<Example 3: Evaluation of blood pressure-lowering effect of muskline by in vivo peptide administration>
Mucrine protein partial peptides were intravenously administered to mice, and the blood pressure lowering effect of musculin was evaluated.

  12 week old male C57BL / 6J mice were used. After inhalation anesthesia with isoflurane, endotracheal intubation was performed and continuous anesthesia was performed with 1.5% isoflurane. The mice were placed on a heat pad to monitor rectal temperature and kept at 37 ° C. during the experiment. After peeling off the left jugular vein, a 1.2Fr polyurethane catheter filled with heparinized physiological saline was inserted to make a drug administration line. Next, after exfoliating the right carotid artery, a 1Fr pressure / volume measuring catheter (PVR-1045) manufactured by Millar was inserted to monitor blood pressure. The pressure data was continuously displayed as a waveform on a notebook computer via PowerLab 4/26 (ADI INSTRUMENTS).

  When ANP was administered intravenously at 30 pmol, a rapid decrease in blood pressure of about 20 mmHg was observed in systolic blood pressure (see FIG. 6A). Blood pressure recovered approximately 30 seconds after administration. Next, when 30 μmol intravenously administered mMusclin (80-130), a rapid decrease in blood pressure similar to that of ANP was observed (see FIG. 6B). Similar to ANP, recovery of blood pressure was observed approximately 30 seconds after administration.

<Example 4: Evaluation of cardiomyocyte proliferation-inducing action of muskline by in vivo peptide administration>
Using a mouse myocardial infarction model, the proliferation-inducing activity of musculin on cardiomyocytes and the therapeutic effect of myocardial infarction were evaluated.

  Eight week old male C57BL / 6J mice (20 mice) were used. On the day before myocardial infarction preparation, an osmotic pump (with an intravenous administration catheter) filled with muskline was implanted subcutaneously under pentobarbital anesthesia, and the catheter was placed in the blood vessel from the left jugular vein. In the test group (10 animals), mMusclin (80-130) dissolved in distilled water was administered at 0.05 μg / kg body weight / min in a total of about 50 to 100 μg. Distilled water was administered to the control group (10 animals).

  The next day, after inhalation anesthesia with isoflurane, endotracheal intubation was performed and continuous anesthesia was performed with 1.5% isoflurane. Mice were placed on a heat pad to monitor rectal temperature and kept at 37 ° C throughout the experiment. The left intercostal space was opened and the adipose tissue around the heart was removed. The coronary artery was ligated with 6-0 nylon thread just under the left atrial appendage. After confirming whitening of the myocardial tissue and ST elevation in the limb lead (aVL) electrocardiogram, the wound was sutured.

  Survival rate was determined with an observation period of 2 weeks, and surviving mice were evaluated for cardiac function by echocardiography, and the heart weight after experimental killing was measured. After preparing the tissue section, Masson trichrome staining was performed, and the positive staining site was evaluated as an infarct.

  The results are shown in FIGS. FIG. 7 shows the survival rate change (2 weeks) of the control group and the test group. FIGS. 8 and 9 show the macroscopic findings and tissue staining images of the hearts of the group (A), the control group (B), and the test group (C) subjected to sham operation (sham operation). In the control group, 80% of the mice died in 2 weeks, whereas half of the test group survived (see FIG. 7). In the control group, cardiac expansion (see FIG. 8B) and extensive infarct (see solid line arrow in FIG. 9B) were observed accompanying myocardial infarction due to coronary artery ligation. On the other hand, in the test group, cardiac expansion was reduced (see FIG. 8C), and infarct was also reduced (see broken line arrow in FIG. 9C). These results indicated that musculin is useful for the treatment of myocardial infarction.

Claims (9)

A pharmaceutical composition comprising any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof.
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of inducing cardiomyocyte proliferation.
(3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of inducing cardiomyocyte proliferation. The pharmaceutical composition according to claim 1, comprising any one or more of the following polypeptides (4) to (6).
(4) A polypeptide consisting of the 83rd to 112th amino acid sequence of the amino acid sequence described in SEQ ID NO: 1.
(5) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3.
(6) A polypeptide comprising the 80th to 130th amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 3.   The pharmaceutical composition according to claim 1 for the treatment and / or prevention of heart disease.   The pharmaceutical composition according to claim 3, wherein the heart disease is ischemic heart disease. A cardiomyocyte proliferation inducer containing any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof:
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of inducing cardiomyocyte proliferation.
(3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of inducing cardiomyocyte proliferation. An antihypertensive agent comprising any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof.
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of lowering blood pressure.
(3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of lowering blood pressure. A method for producing cardiomyocytes, comprising a step of treating the separated cardiomyocytes and / or precursor cells thereof with any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof.
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of inducing cardiomyocyte proliferation.
(3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of inducing cardiomyocyte proliferation. A method for treating and / or preventing a heart disease, comprising a procedure of administering any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof to a subject.
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of inducing cardiomyocyte proliferation.
(3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of inducing cardiomyocyte proliferation. A method for treating and / or preventing hypertension and a hypertension-related disease, comprising a procedure of administering any one or more of the following polypeptides (1) to (3) and / or partial peptides thereof to a subject.
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of lowering blood pressure.
(3) A polypeptide that exhibits 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and has an activity of lowering blood pressure.

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