Cardiac dilatation function improving agent, and method for screening for cardiac dilatation function improving agent

Abstract

A cardiac dilatation function improving agent according to an embodiment of the present invention includes a polynucleotide encoding a reprogramming factor polypeptide Gata4.

Description

Cardiac diastolic function improving agent and screening method for cardiac diastolic function improving agent

[0001] The present invention relates to cardiac diastolic function improving agents and methods for screening such agents.

[0002] Heart failure is classified into two types based on the ejection fraction (EF): heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF). Decreased cardiac diastolic function is known to be a common pathological feature of heart failure. Effective drug and non-drug treatments have been discovered for HFrEF. While HFpEF is considered a complex pathological condition involving fibrosis, cardiac hypertrophy, and inflammation, the detailed pathogenesis remains unclear, creating a problem of a lack of effective treatments.

[0003] For example, Patent Document 1 discloses direct myocardial reprogramming, which induces cardiomyocytes from fibroblasts in vivo. Also, Non-Patent Document 1 discloses that fibrosis is suppressed by introducing the GATA4 gene into fibroblasts after myocardial infarction.

[0004] International Publication No. 2011 / 139688

[0005] J Thorac Cardiovasc Surg. 2017 Nov;154(5):1601-1610

[0006] Patent Document 1 discloses a technique for inducing fibroblasts isolated from the hearts of α-myosin heavy chain-green fluorescent protein (αMHC-GFP) mice or Isl1-yellow fluorescent protein (Isl1-YFP) mice to develop into cardiomyocytes by transducing them with reprogramming genes including Gata4, Mef2c, and Tbx5. However, it does not teach the effect of Gata4 on cardiac diastolic function in individuals. For direct myocardial reprogramming, it is necessary to introduce three or more direct myocardial reprogramming genes into fibroblasts. While a technique for simultaneously introducing genes into fibroblasts or the development of vectors capable of carrying multiple genes is necessary, this has not yet been realized.

[0007] The technique disclosed in Non-Patent Document 1 confirms the state of fibrosis in the heart of a myocardial infarction model rat after death, but does not improve the reduced cardiac diastolic function in living individuals. Therefore, further research and development to improve the reduced cardiac diastolic function is desired.

[0008] One aspect of the present invention aims to provide techniques for improving cardiac diastolic function.

[0009] An agent for improving cardiac diastolic function according to one embodiment of the present invention comprises a polynucleotide encoding the reprogramming factor polypeptide Gata4.

[0010] In one aspect of the present invention, the reprogramming factor polypeptide Gata4 comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. In one aspect of the present invention, the polynucleotide comprises a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0011] A cardiac diastolic function improving agent according to one embodiment of the present invention is an agent for improving heart failure. The cardiac diastolic function improving agent according to one embodiment of the present invention comprises a factor that upregulates expression of the Gata4 gene. The cardiac diastolic function improving agent according to one embodiment of the present invention comprises a factor that upregulates expression of the Gata4 gene. The cardiac diastolic function improving agent according to one embodiment of the present invention comprises a factor that increases the expression level of the Gata4 gene in cardiac fibroblasts.

[0012] A screening method for cardiac diastolic function improving agents according to one embodiment of the present invention includes a contacting step of contacting a test substance with fibroblasts, and an evaluation step of evaluating the expression of the Gata4 gene in the fibroblasts.

[0013] According to one aspect of the present invention, a technique for improving cardiac diastolic function can be provided.

[0014] 1. A diagram relating to Tcf21 iCre mice. 2. A diagram relating to CAG-CAT-MGTH2A mice. 3. A diagram relating to tdTomato mice. 4. A diagram of cardiac immunostained sections from myocardial direct reprogramming mice. 5. A diagram showing the results of quantitative PCR for Gata4. Dots indicate individual sample values. 6. A diagram showing the results of quantitative PCR for Mef2c. 7. A diagram showing the results of quantitative PCR for Tbx5. 8. A diagram showing the results of quantitative PCR for Hand2. 9. A diagram showing the experimental protocol for HFpEF model mice. 10. A diagram showing the results of systolic blood pressure measurements in the normal diet group, HFpEF group, and reprogramming group. 11. A diagram showing the results of body weight measurements in the normal diet group, HFpEF group, and reprogramming group. 12. A diagram showing the results of echocardiography (M-mode) in the normal diet group, HFpEF group, and reprogramming group. 13. A diagram showing the results of echocardiography (Doppler mode) in the normal diet group, HFpEF group, and reprogramming group. 14. A diagram showing the time course of EF. 15. A diagram showing the time course of E / A. 16. A diagram showing the time course of E / E'. 17. A diagram showing the results of mouse catheterization (left ventricular pressure waveform). 18. A diagram showing the results of EDP measurement. 1 shows the results of a mouse catheterization test (PV loop waveform). 2 shows the results of EDPVR measurement. 3 shows the state of mouse treadmill evaluation. 4 shows the running distance of each group. 5 shows the results of immunostaining of mouse heart sections. The photograph in the upper right box is an enlarged photograph of the area surrounded by a square in the center of the photograph. The scale bar is 50 μm. 6 shows the myocardial induction efficiency of each group. 7 shows macroscopic images of the heart and immunostained images of heart sections. 8 shows the results of measurement of the heart weight / femur length ratio. 9 shows the results of measurement of cardiomyocyte area. 10 shows the results of Sirius red staining of heart sections. 11 shows the fibrosis area of ​​each group. 12 shows the results of scRNA-seq of cardiac non-cardiomyocytes. 13 shows the results of interaction analysis between cardiac non-cardiomyocytes. 14 shows the vertical axis showing comprehensive scores for pathological genes whose expression is increased in HFpEF. 15 shows a heat map showing the expression of representative genes that are activated when the heart is damaged. 16 shows the experimental flow of ATAC-seq analysis. 17 shows the experimental flow of ATAC-seq analysis.1 is a diagram showing, as a heat map, the signal intensity of regions where significant differences occurred in peak waveform height in ATAC-seq. FIG. 1 is a diagram showing Gata4 ChIP-seq data. FIG. 2 is a diagram showing Gata4 ChIP-seq data in a heat map. FIG. 3 is a diagram showing the results of GO analysis. FIG. 4 is a diagram showing an overview of genetically modified mice. FIG. 5 is a diagram showing the results of quantitative PCR for Gata4, Mef2c, Tbx5, and Hand2. FIG. 6 is a diagram showing the results of quantitative PCR for Col1a2, Fn1, and Postn. FIG. 7 is a diagram showing the results of FACS. FIG. 8 is a diagram showing an overview of genetically modified mice. FIG. 9 is a diagram showing the experimental protocol. FIG. 10 is a diagram showing changes in body weight, systolic blood pressure (SBP), and diastolic blood pressure (DBP) over time. FIG. 11 is a diagram showing the results of echocardiography. FIG. 12 is a diagram showing the distance traveled in a treadmill test. FIG. 13 is a diagram showing the results of cardiac catheterization. FIG. 14 is a diagram showing the results of Sirius red staining of heart sections. FIG. 15 is a diagram showing the area of ​​fibrotic regions in each group. FIG. 16 is a diagram showing the results of quantitative PCR for Col1a1, Col3a1, Nppd, and Tgfb1. FIG. 17 is a diagram summarizing the results of this example.

[0015] [Definition of Terms, etc.] In this specification, "polynucleotide" can be alternatively referred to as "nucleic acid" or "nucleic acid molecule," and refers to a polymer of nucleotides. Furthermore, "base sequence" can be alternatively referred to as "nucleic acid sequence" or "nucleotide sequence," and unless otherwise specified, polynucleotides can exist in the form of RNA or DNA. An example of the RNA form is mRNA. An example of the DNA form is cDNA or genomic DNA. DNA may be double-stranded or single-stranded.

[0016] As used herein, the term "protein" can also be referred to as "polypeptide."

[0017] The protein described herein may be a polypeptide formed by peptide bonds between amino acids, but is not limited thereto, and may also include structures other than polypeptides. Examples of structures other than polypeptides referred to here include, but are not limited to, sugar chains and isoprenoid groups.

[0018] In this specification, "A and / or B" is a concept that includes both A and B and A or B, and can be rephrased as "at least one of A and B."

[0019] As used herein, "improving cardiac diastolic function" includes reducing or alleviating a decline in cardiac diastolic function or the risk of such decline, delaying the progression of a decline in cardiac diastolic function, curing a decline in cardiac diastolic function, and improving cardiac diastolic function. As used herein, "comprise" is a concept that also encompasses "consist essentially of" and "consist only of." In the numerical ranges described herein, the upper or lower limit of the range may be replaced with a value shown in the examples or a value that can be unambiguously derived from the examples. Furthermore, in this specification, a numerical value connected with "to" means a numerical range that includes the numbers before and after "to" as the lower and upper limits.

[0020] [Cardiac Diastolic Function Improving Agent] One embodiment of the cardiac diastolic function improving agent of the present invention comprises a polynucleotide encoding the programming factor polypeptide Gata4 (hereinafter, sometimes referred to as a "Gata4 polynucleotide"). The myocardial infarction rat described in Non-Patent Document 1 is a model of ischemic heart disease different from HFpEF, and the pattern of cardiac fibrosis differs from that revealed in this study. Prior to the filing date of this application, it was impossible to infer from the disclosure of Non-Patent Document 1 whether Gata4 would have a therapeutic effect on cardiac fibrosis in this completely different model animal. Furthermore, it has been suggested that cardiac diastolic function dysfunction is caused by a variety of factors, including not only cardiac fibrosis but also cardiomyocyte hypertrophy, endothelial damage, and inflammation. However, it was unclear whether improving these causes would lead to improved diastolic function. The present inventors have discovered that intervention in fibrotic cells results in an improvement in cardiac diastolic function that could not be predicted by conventional anti-fibrotic inhibition.

[0021] (Gata4) Gata4 polypeptide is a member of the GATA family of zinc finger transcription factors that recognizes and binds to the GATA motif present in the promoter region of many genes (e.g., by recognizing and binding to the consensus sequence 5'-AGATAG-3'). Gata4 is described, for example, in Huang et al., Gene, 1995, 155(2):219-23. Amino acid sequences for Gata4 polypeptides from various species and nucleotide sequences encoding Gata4 polypeptides are known in the art. Examples of amino acid sequences for Gata4 polypeptides and accession numbers for nucleotide sequences encoding Gata4 polypeptides are listed below.

[0022] <Examples of amino acid sequences related to Gata4 polypeptide> NP_002043 (Homo sapiens; SEQ ID NO: 1) NP_0321188 (Mus musculus; SEQ ID NO: 3) NP_653331 (Rattus norvegicus) ABI63575 (Danio rerio) AAH71101 (Xenopus laevis)

[0023] <Examples of nucleotide sequences encoding Gata4 polypeptides> - CDS sequence in NM_002052 (Homo sapiens) (SEQ ID NO: 2) - CDS sequence in NM_008092 (Mus musculus) (SEQ ID NO: 4) - CDS sequence in NM_144730 (Rattus norvegicus) - CDS sequence in DQ886664 (Danio rerio) - CDS sequence in BC071107 (Xenopus laevis)

[0024] In some embodiments, a Gata4 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a Gata4 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. Gata4 polypeptides are biologically active, for example, by recognizing and binding to a GATA motif present in a promoter (e.g., recognizing and binding to the consensus sequence 5'-AGATAG-3') and activating transcription of a gene operably linked to a promoter containing a GATA motif.

[0025] In some embodiments, the polynucleotide encoding Gata4 comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the polynucleotide encoding Gata4 comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4.

[0026] In some embodiments, a polypeptide functionally equivalent to a Gata4 polypeptide (or a nucleotide sequence encoding such a functional equivalent) is used. For example, in some embodiments, a Gata5 polypeptide (or a nucleotide sequence encoding a Gata5 polypeptide) is used. In other embodiments, a Gata6 polypeptide (or a nucleotide sequence encoding a Gata6 polypeptide) is used.

[0027] The amino acid sequence of a Gata5 polypeptide and the nucleotide sequence encoding the Gata5 polypeptide are known in the art. Examples of the accession numbers for the amino acid sequence of a Gata5 polypeptide and the nucleotide sequence encoding the Gata5 polypeptide are shown below.

[0028] <Examples of amino acid sequences related to Gata5 polypeptide> NP_536721 (Homo sapiens) NP_032119 (Mus musculus) NP_001019487 (Rattus norvegicus)

[0029] <Examples of nucleotide sequences encoding Gata5 polypeptide> ・CDS sequence in NM_080473 (Homo sapiens) ・CDS sequence in NM_008093 (Mus musculus) ・CDS sequence in NM_001024316 (Rattus norvegicus)

[0030] The amino acid sequence of the Gata6 polypeptide and the nucleotide sequence encoding the Gata6 polypeptide are known in the art. Examples of the accession numbers for the amino acid sequence of the Gata6 polypeptide and the nucleotide sequence encoding the Gata6 polypeptide are shown below.

[0031] <Examples of amino acid sequences related to Gata6 polypeptide> NP_005248 (Homo sapiens) NP_034388 (Mus musculus) NP_062058 (Rattus norvegicus)

[0032] <Examples of nucleotide sequences encoding Gata6 polypeptide> ・CDS sequence in NM_005257 (Homo sapiens) ・CDS sequence in NM_010258 (Mus musculus) ・CDS sequence in NM_019185 (Rattus norvegicus)

[0033] In some embodiments, a suitable functional equivalent of a Gata4 polypeptide is a polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of a Gata5 polypeptide or a Gata6 polypeptide.

[0034] In some embodiments, a nucleotide sequence encoding a functional equivalent of a Gata4 polypeptide comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% nucleotide sequence identity to a nucleotide sequence encoding a Gata5 polypeptide or a Gata6 polypeptide.

[0035] The method for obtaining (isolating) the Gata4 polynucleotide is not particularly limited, and may be synthesized according to a nucleic acid synthesis method such as the phosphoramidite method.

[0036] Another method for obtaining a Gata4 polynucleotide is to use a nucleic acid amplification method such as PCR. For example, primers are prepared from the 5' and 3' sequences (or their complementary sequences) of the cDNA of the polynucleotide, and these primers are used to perform PCR or other amplification using genomic DNA or cDNA as a template to amplify the DNA region between the primers. This allows for the large-scale production of DNA fragments containing the polynucleotide of the present invention.

[0037] An embodiment of the present invention also includes a cardiac diastolic function improving agent containing a Gata4 polynucleotide (e.g., DNA). In a preferred embodiment of the cardiac diastolic function improving agent, the Gata4 polynucleotide (e.g., DNA) is inserted into a vector. The type of vector may be, for example, an autonomously replicating vector (e.g., a plasmid), or may be a vector that, when introduced into a host cell, is integrated into the genome of the host cell and replicates together with the chromosome into which it has been integrated.

[0038] The vector is preferably an expression vector, in which the Gata4 polynucleotide is operably linked to elements necessary for transcription (e.g., promoter, enhancer, ribosome binding site, splice signal, terminator, etc.).

[0039] Examples of vectors include viral vectors such as retroviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV vectors), Sendai viral vectors, and lentiviral vectors; and non-viral vectors such as plasmid vectors, bacterial vectors, phage vectors, phagemid vectors, and cosmid vectors.

[0040] The vector can be constructed using, for example, known genetic engineering techniques. In one example, the vector expresses a Gata4 polynucleotide specifically in cardiac fibroblasts.

[0041] An embodiment of the present invention also includes an agent for improving cardiac diastolic function, comprising fibroblasts transfected with a Gata4 polynucleotide (e.g., mRNA) or a vector containing a Gata4 polynucleotide. By administering Gata4-expressing cells to a subject, cardiac diastolic function can be improved.

[0042] An example of a fibroblast is a cardiac fibroblast. Fibroblasts can be obtained from a living individual, for example, from tissue collected from a living individual. Fibroblasts can be isolated from tissue using known techniques. For example, the method described in Ieda et al., Dev Cell., 2009, 16(2), 233-244 or the method described in the Examples can be used. The collected fibroblasts may be allogeneic cells (autologous cells) collected from the individual to be administered, or heterogeneic cells (allogeneic cells) collected from an individual different from the individual to be administered.

[0043] To introduce a Gata4 polynucleotide or a vector into which a Gata4 polynucleotide has been inserted into fibroblasts, methods that can be used include, for example, electroporation, calcium phosphate, lipofection, microinjection, liposome-based introduction, gene gun-based introduction, and cationic polymers (e.g., DEAE dextran, polyethyleneimine, polyethylene glycol).

[0044] Another embodiment of the present invention includes a cardiac diastolic function improving agent containing a factor that upregulates Gata4 gene expression. Examples of factors that upregulate Gata4 gene expression include the Gata4-binding factors RbAp46 (retinoblastoma protein-associated protein 46) and RbAp48 (retinoblastoma protein-associated protein 48). Another embodiment of the present invention includes a cardiac diastolic function improving agent containing a factor that increases the expression level of the Gata4 gene in cardiac fibroblasts. Examples of factors that increase the expression level of the Gata4 gene include the Gata4-binding factors RbAp46 (retinoblastoma protein-associated protein 46) and RbAp48 (retinoblastoma protein-associated protein 48).

[0045] (Other Components) The cardiac diastolic function improving agent according to one embodiment of the present invention may further contain other components in addition to the Gata4 polynucleotide, the factor that upregulates Gata4 gene expression, or the factor that increases the expression level of the Gata4 gene in cardiac fibroblasts. These other components are not particularly limited, but include, for example, pharmaceutically acceptable carriers, lubricants, preservatives, stabilizers, humectants, emulsifiers, salts for adjusting osmotic pressure, buffers, stabilizers, preservatives, excipients, antioxidants, viscosity adjusters, colorants, flavorings, and sweeteners. When the cardiac diastolic function improving agent is formulated as an aqueous solution, pure water (sterile water), physiological saline, phosphate-buffered physiological saline, or the like may be used as the carrier. When the cardiac diastolic function improving agent is formulated as another suitable solution, organic esters that can be introduced into the body, such as glycol, glycerol, or olive oil, may be used as the carrier. A cardiac diastolic function improving agent containing a Gata4 polynucleotide, a factor that upregulates the expression of the Gata4 gene, or a factor that increases the expression level of the Gata4 gene in cardiac fibroblasts, and one or more other ingredients is also called a composition for improving cardiac diastolic function.

[0046] The cardiac diastolic function improving agent according to one embodiment of the present invention may be housed in a container, pack, dispenser, or the like together with instructions for use.

[0047] (Subjects and Administration of Cardiac Diastolic Function Improving Agent) Subjects to which the cardiac diastolic function improving agent according to one embodiment of the present invention is administered include, for example, humans and non-human animals, more specifically, vertebrates such as birds and mammals. Mammals include laboratory animals such as mice, rats, rabbits, guinea pigs, and primates other than humans; pets such as dogs and cats; livestock such as pigs, cows, goats, sheep, and horses; and humans.

[0048] The cardiac diastolic function improving agent according to one embodiment of the present invention is preferably used to improve heart failure, and the cardiac diastolic function improving agent can be an agent for improving heart failure. Examples of heart failure include heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF). The cardiac diastolic function improving agent can be used to improve both HFrEF and HFpEF.

[0049] The administration route or method is not particularly limited, and may be administered directly to the heart or its surroundings, or indirectly. Examples of administration routes include oral, intravenous, intramuscular, subcutaneous, intraventricular, intraperitoneal, and transdermal routes, as well as local administration and methods using a gene gun.

[0050] The dose and frequency of administration can be appropriately selected depending on the severity of symptoms, age, sex, body weight, administration form, etc.

[0051] [Method for Improving Cardiac Diastolic Function] One embodiment of the present invention also includes a method for improving cardiac diastolic function, comprising administering a Gata4 polynucleotide to a subject in need of such improvement. Another embodiment of the present invention also includes a method for improving cardiac diastolic function, comprising administering a factor that upregulates Gata4 gene expression or a factor that increases the expression level of Gata4 gene in cardiac fibroblasts to a subject in need of such improvement.

[0052] The above method may further comprise a step of introducing a Gata4 polynucleotide into fibroblasts prior to the administration step. The fibroblasts may be isolated from the same individual as the subject to whom the Gata4 polynucleotide is administered, or a different individual of the same species, or the fibroblasts may be cultured cells derived from the same individual as the subject to whom the Gata4 polynucleotide is administered, or a different individual of the same species. The subject to whom the Gata4 polynucleotide is administered is as described above for the subject to whom the cardiac diastolic function improving agent is administered. The Gata4 polynucleotide may be introduced in the form of a vector. Therefore, one aspect of the present invention also includes a method for producing fibroblasts for introduction into a subject, comprising a step of introducing a Gata4 polynucleotide into fibroblasts. The above method may further comprise a step of introducing into fibroblasts a factor that upregulates Gata4 gene expression or a factor that increases the expression level of the Gata4 gene in cardiac fibroblasts. The fibroblasts may be isolated from the same individual as the subject to which the Gata4 polynucleotide is administered or from a different but identical individual, or the fibroblasts may be cultured cells derived from the same individual as the subject to which the Gata4 polynucleotide is administered or from a different but identical individual. The factor that upregulates Gata4 gene expression or increases the expression level of the Gata4 gene in cardiac fibroblasts may be introduced in the form of a vector. Therefore, one aspect of the present invention also includes a method for producing fibroblasts for introduction into a subject, which comprises the step of introducing into fibroblasts a factor that upregulates Gata4 gene expression or increases the expression level of the Gata4 gene in cardiac fibroblasts.

[0053] [Method of screening for cardiac diastolic function improving agents] A method of screening for cardiac diastolic function improving agents according to one embodiment of the present invention includes a contacting step and an evaluation step.

[0054] (Contacting Step) The contacting step involves contacting fibroblasts with a test substance. Examples of fibroblasts include cardiac fibroblasts. The cells can be obtained from a living individual, for example, from tissue collected from a living individual. The living individual may be an individual with normal cardiac diastolic function or an individual with reduced cardiac diastolic function. Fibroblasts can be isolated from tissue using known techniques.

[0055] The screening method may further comprise the step of introducing a Gata4 polynucleotide into fibroblasts prior to the contacting step. The Gata4 polynucleotide may be introduced in the form of a vector. In this case, contacting the fibroblasts with the test substance comprises contacting the fibroblasts into which the Gata4 polynucleotide has been introduced or a vector containing the fibroblasts with the test substance. Specific methods of introduction include those described in the section on "cardiac diastolic function improving agents."

[0056] (Evaluation step) In the evaluation step, the expression of the Gata4 gene in fibroblasts is evaluated after the contact step. Specifically, the expression level of the Gata4 gene or the amount of the Gata polypeptide in fibroblasts after the contact step is measured. Examples of measurement methods that can be used include RT-PCR, hybridization analysis, and molecular biology methods.

[0057] The screening method may include a comparison step of comparing the expression level (e.g., expression amount) of the Gata4 gene in fibroblasts after the contacting step with the expression level of the Gata4 gene in fibroblasts before the contacting step. If the expression of the Gata4 gene in fibroblasts after the contacting step is upregulated compared to the expression of the Gata4 gene in fibroblasts before the contacting step, the test substance may be a candidate for an agent for improving cardiac diastolic function. The test substance may be selected as a candidate for an agent for improving cardiac diastolic function. Alternatively, the screening method may include a comparison step of comparing the expression level (e.g., expression amount) of the Gata polypeptide in fibroblasts after the contacting step with the expression level of the Gata polypeptide in fibroblasts before the contacting step. If the expression of the Gata polypeptide in fibroblasts after the contacting step is upregulated compared to the expression of the Gata polypeptide in fibroblasts before the contacting step, the test substance may be a candidate for an agent for improving cardiac diastolic function and may be selected.

[0058] [Summary] A cardiac diastolic function improving agent according to Aspect 1 of the present invention comprises a polynucleotide encoding a reprogramming factor polypeptide Gata4. A cardiac diastolic function improving agent according to Aspect 2 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 1. A cardiac diastolic function improving agent according to Aspect 3 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, the polypeptide recognizing and binding to a GATA motif present in a promoter. A cardiac diastolic function improving agent according to Aspect 4 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. A cardiac diastolic function improving agent according to Aspect 5 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 3. A cardiac diastolic function improving agent according to Aspect 6 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polynucleotide encoding a polypeptide that recognizes and binds to a GATA motif present in a promoter, the polynucleotide comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 3. A cardiac diastolic function improving agent according to Aspect 7 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3.A cardiac diastolic function improving agent according to Aspect 8 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polynucleotide comprising a nucleotide sequence having at least 90% nucleotide sequence identity with the nucleotide sequence shown in SEQ ID NO: 2. A cardiac diastolic function improving agent according to Aspect 9 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polynucleotide comprising a nucleotide sequence having at least 90% nucleotide sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, and wherein the polynucleotide encodes a polypeptide that recognizes and binds to a GATA motif present in a promoter. A cardiac diastolic function improving agent according to Aspect 10 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises the nucleotide sequence shown in SEQ ID NO: 2. A cardiac diastolic function improving agent according to Aspect 11 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polynucleotide comprising a nucleotide sequence having at least 90% nucleotide sequence identity with the nucleotide sequence shown in SEQ ID NO: 4. A cardiac diastolic function improving agent according to Aspect 12 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a nucleotide sequence having at least 90% nucleotide sequence identity with the nucleotide sequence shown in SEQ ID NO: 4, and encodes a polypeptide that recognizes and binds to a GATA motif present in a promoter. A cardiac diastolic function improving agent according to Aspect 13 of the present invention is the cardiac diastolic function improving agent according to Aspect 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises the nucleotide sequence shown in SEQ ID NO: 4.

[0059] A cardiac diastolic function improving agent according to a fourteenth aspect of the present invention comprises a factor that upregulates the expression of the Gata4 gene.

[0060] A cardiac diastolic function improving agent according to Aspect 15 of the present invention comprises a factor that increases the expression level of Gata4 gene in cardiac fibroblasts.

[0061] A cardiac diastolic function improving agent according to Aspect 16 of the present invention is the cardiac diastolic function improving agent according to any one of Aspects 1 to 3 of the present invention, which is a cardiac failure improving agent.

[0062] A seventeenth aspect of the present invention provides the cardiac diastolic function improving agent according to the fourth aspect of the present invention, wherein the heart failure is heart failure with preserved ejection fraction (HFpEF).

[0063] A screening method for cardiac diastolic function improving agents according to aspect 18 of the present invention includes a contacting step of contacting a test substance with fibroblasts, and an evaluation step of evaluating the expression of the Gata4 gene in the fibroblasts.

[0064] The following examples are provided to further explain the embodiments of the present invention. It goes without saying that the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. Furthermore, all of the documents cited in this specification are incorporated by reference.

[0065] All experiments described below were conducted with the approval of the Animal Experiment Ethics Committee of the University of Tsukuba.

[0066] Materials and Methods: (Mice) Tcf21 iCre / tdTomato mice were obtained by crossing Tcf21 iCre mice (Figure 1) with R26 tdTomato reporter mice (Figure 3). Cre-responsive MGTH2A (a gene sequence consisting of Mef2c, Gata4, Tbx5, and Hand2 linked via the self-cleaving peptide 2A) transgenic mice were constructed by modifying a CAG-CAT-Z plasmid (Figure 2, CAG-CAT-MGTH2A mice). This plasmid contains a CMV (cytomegalovirus) enhancer and a CAG (chicken beta actin) promoter linked to a CAT (chloramphenicol acetyltransferase) gene surrounded by loxp sequences. The full-length mouse MGTH2A gene sequence was constructed by ligating T2A and Hand2 to the Mef2c-Gata4-Tbx5 gene sequence and inserted into the Z site of the CAG-CAT-Z plasmid. The constructed plasmid was purified, linearized, and microinjected into the pronuclei of fertilized egg blastocysts from FVBN mice. The presence of CAT was verified by DNA PCR. Triple transgenic mice were generated by crossing CAG-CAT-MGTH2A mice with Tcf21 iCre / tdTomato mice.

[0067] To generate single-factor mice for Gata4, Mef2c, Tbx5, and Hand2 (hereafter abbreviated as G, M, T, and H), we used a CAG-LSL (loxp-Stop-loxp)-Z plasmid containing a CAG promoter and SV40pA surrounded by loxp sequences within a sequence homologous to the Rosa26 locus. Plasmids containing the G, M, T, and H gene sequences were inserted into the Z site of CAG-LSL-Z. The constructed plasmids were purified, linearized, and then transfected into ICR mouse fertilized eggs. The target gene sequences were inserted into the Rosa26 locus using homologous recombination. DNA PCR was used to confirm the successful introduction of the inserted gene into Rosa26 and the integration of genes into other loci. Experiments were performed using mice with the gene integrated only into the target Rosa26 locus. Double transgenic mice were generated by crossing CAG-LSL-Single factor (G, M, T, H) mice with Tcf21 iCre mice.

[0068] No immunodeficiency or other health problems were observed in the transgenic mice, and they had been kept free of other experiments and drugs up until the start of this study. All animals were group-housed in a dedicated specific pathogen-free (SPF) facility with a 12-hour / 12-hour light / dark cycle, had free access to food and water, and were checked daily. Their health was checked regularly to maintain their SPF grade.

[0069] (HFpEF model mice) HFpEF (heart failure preserved ejection fraction) model mice were generated by continuous administration of L-NAME (Nω-nitro-L-arginine methyl ester, Sigma, N5751) and a high-fat diet (CLEA Japan, HFD32) following a previous report (Schiattarella GG, et al. Nature 2019). L-NAME was dissolved in tap water at a concentration of 1.0 g / L and mice were allowed to drink ad libitum from a water bottle. L-NAME and the high-fat diet were replaced every 2–3 days.

[0070] Tamoxifen (Sigma, T5648) was administered intraperitoneally to mice at 2 mg / day for 5 consecutive days starting from the fifth week of the experiment. Tamoxifen was dissolved in peanut oil (90%; P2144 Sigma) and ethanol (10%) at a concentration of 50 mg / mL.

[0071] Mouse embryonic fibroblasts (MEFs) were isolated as follows: Embryos isolated from 12.5-day-old pregnant mice were washed with phosphate-buffered saline (PBS), followed by careful removal of the head and visceral tissues. Each remaining embryo was washed with fresh PBS, minced with scissors, and transferred to a 0.25% trypsin / ethylenediaminetetraacetic acid solution (Gibco, 25200-072) and incubated at 37°C for 15 minutes. After trypsinization, an equal volume of fetal bovine serum (FBS, Thermo Scientific, SV30014.03) was added and the tissue was dissociated by pipetting several times. The lysate was transferred to a new tube, and the cells were collected by centrifugation. They were then resuspended in DMEM / 10% FBS (Dulbecco's Modified Eagle's Medium with 10% FBS, FUJIFILM, 044-29765) and cultured at 37°C in 5% CO2. The genotype of MEFs was determined by DNA PCR using head and visceral tissues.

[0072] (Echocardiography) Transthoracic echocardiography was performed using a Visual Sonics Vevo 2100 system. Mice were anesthetized with 1.0-2.0% low-dose isoflurane for echocardiography. During echocardiography, body temperature was controlled with a heating pad, and electrocardiograms were monitored with limb electrodes. Cardiac contractility was assessed by measuring the left ventricular end-diastolic diameter (LVDd) and left ventricular end-systolic diameter (LVDs) using M-mode echocardiography at the papillary muscle level in the left ventricular short-axis view, and left ventricular ejection fraction (LVEF) was assessed. Furthermore, mitral valve blood flow velocity waveforms were measured using apical four-chamber Doppler echocardiography, and the peak early diastolic velocity (E') of the mitral annulus was measured using tissue Doppler. LVEF was calculated using the Teichholz equation as follows: EF = (left ventricular end-diastolic volume - left ventricular end-systolic volume) / left ventricular end-diastolic volume × 100 (%) (Equation 1).

[0073] The left ventricular end-diastolic volume and left ventricular end-systolic volume were calculated using the following formulas 2 and 3, respectively: Left ventricular end-diastolic volume = [{7.0 / (2.4 + LVDd)} × (LVDd) 3 ] ... (Equation 2) Left ventricular end-systolic volume = [{7.0 / (2.4 + LVDs)} × (LVDs) 3 ]...(Formula 3)

[0074] (Blood Pressure Measurement) Systolic and diastolic blood pressure were measured noninvasively in conscious mice using a mouse tail blood pressure measuring device (CODA, Kent Scientific). Mice were placed in individual holders on a temperature-controlled platform (37°C), and recordings were performed under steady state conditions. Before testing, all mice were trained to acclimate to short-term restraint. Blood pressure was measured at least 10 times, and the average of the obtained measurements was used.

[0075] (Cardiac catheterization) Cardiac catheterization was performed via the right internal carotid artery approach using a mouse conductance catheter (Millar, SPR-839) and a pressure-volume system (Millar, MPVS-400). Mice were anesthetized with isoflurane and placed on a warmer. Electrocardiograms were monitored using limb electrodes. The right internal carotid artery was exposed by incision, and a conductance catheter was inserted. The tip was inserted into the left ventricle, and the steady-state left ventricular pressure waveform was recorded. Next, a small incision was made in the upper abdomen, and the inferior vena cava was identified. By compressing the inferior vena cava with a cotton swab, the pressure-volume loop (PV loop) waveform during inferior vena cava occlusion was recorded. Analysis was performed using LabChart (ADInstruments).

[0076] (Mouse treadmill) Mouse treadmills were performed using a belt-type forced running device (MELQUEST, TMS-4N). For two days prior to the actual test, mice were acclimatized to the treadmill with a 0°, 10 m / min, and 10 min run schedule. For the actual test, mice were run at a warm-up speed of 5 m / min for 4 min on a 20° incline, followed by a 2-min run at 14 m / min. The speed was then increased by 2 m / min every 2 min until the mice reached exhaustion, and the total distance traveled was measured. Exhaustion was defined as the inability to resume running within 10 s of contact with the rear electrical stimulation grid.

[0077] To detect cTnT expression by fluorescence-activated cell sorting (FACS), cells were fixed with 4% PFA for 15 minutes, permeabilized with saponin (Sigma Aldrich, 47036-250G-F), stained with anti-cTnT (Thermo Scientific, MS-295-P1) antibody, and incubated with a secondary antibody conjugated to Alexa Fluor 488 (Invitrogen, A11001). Cells were then analyzed using a FACS instrument (Beckman Coulter, CytoFLEX S) and FlowJo software (Tomy Digital Biology). A BD FACSAria™ was used to collect non-cardiomyocytes and Tomato-positive cells. TM IIIu (BD) and MoFlo XDP (Beckman Coulter) were used.

[0078] (Histological Examination) After euthanasia, the hearts were perfused apically with PBS and then 0.4% paraformaldehyde (PFA). The hearts were rapidly removed and fixed overnight in 4% PFA. For immunostaining using frozen sections, the tissue was replaced with 20% sucrose, embedded in OCT compound, and frozen in liquid nitrogen. Using a microtome, 7-μm sections were cut perpendicularly to visualize both ventricles. The frozen sections were stained with a primary antibody against α-actinin (Sigma, A7811) and secondary antibodies conjugated with Alexa488 and DAPI. Wheat germ agglutinin (WGA) staining was performed using an Alexa488-conjugated antibody. Confocal microscopy was performed using an LSM800 microscope (Carl Zeiss). The ratio of α-Actinin+ / Tomato+ cells was measured by counting in 10 randomly selected fields from five or more different sections in each mouse.

[0079] For paraffin section preparation, paraffin-embedded sections were prepared from fixed hearts and then stained with Sirius Red or wheat germ agglutinin (WGA). WGA staining was performed using an Alexa488-conjugated antibody (Thermo Scientific, W11261). Fibrosis area and cardiomyocyte cross-sectional area were measured using Image J (NIH). All measurements and calculations were performed in a blinded manner.

[0080] (DNA PCR and qRT-PCR) Genotyping of transgenic mice was performed by standard PCR using the primers listed in Table 1. Total RNA was extracted from in vitro transduced fibroblasts, hearts from both ventricles, and isolated Tomato-positive cells using standard protocols. qRT-PCR was performed using the StepOnePlus Real-Time PCR system with the primers and TaqMan probes (Applied Biosystems) listed in Tables 2 and 3. Table 2 shows the details of the TaqMan gene expression assay (Applied Biosystems, Thermo Fischer Scientific), and Table 3 shows the details of the Universal Probe Library System (Roche). Target mRNA expression was normalized by Gapdh expression.

[0081]

[0082]

[0083]

[0084] (Isolation and Collection of Non-Cardiomyocytes and Tomato-Positive Cells) After euthanasia by CO2 inhalation, the heart was immediately cannulated and perfused with chilled PBS (50 mL). After removal of the heart, the atria and valves were dissected to separate the ventricles. The ventricular myocardium was cut into approximately 1 mm pieces on a sterile dish on ice. The pieces were transferred to a 10 mL tube containing 3 mL of enzyme solution and incubated in a 37°C water bath for 45 minutes, with pipetting every 15 minutes. The enzyme solution contained 2 mg / mL collagenase type IV (Worthington Biochemical, CLS-4) and 1.2 U / mL Dispase II (Sigma, 255-914-4). After the final pipetting, the cell suspension was filtered through a 40 μm cell strainer. After removing debris by centrifugation using Debris Removal Solution (Miltenyi Biotec, 130-109-398), red blood cells were removed using RBC lysis buffer (pluriSelect, 60-00050-11). The cell suspension was stained with Live / Dead (Invitrogen, L34975, 1:1000) and Calcein Violet Working Solution (BioLegend, 425203, 0.1 μM). Cells with intact membranes and metabolic activity (Live / Dead-, Calcein+) were collected using a FACS instrument with minimal pressure on the cells. The FACS was performed using a BD FACSAria. TM IIIu (BD) and MoFlo XDP (Beckman Coulter) were used. When collecting Tomato-positive cells, the cells were collected by FACS without staining.

[0085] (Single-cell RNA sequencing) Single-cell RNA sequencing (scRNA-seq) was performed using two samples per group. An scRNA-seq library from non-cardiomyocytes was generated using a Chromium controller from 10X Genomics. Approximately 10,000 cells were loaded into each channel of a dedicated plate and processed using the Chromium Single Cell 3' v3.1 reagent kit (10X Genomics). Sequencing was performed using a NovaSeq 6000 (Illumina) system operated by the Omics Bioinformatics Center, Graduate School of Frontier Sciences, The University of Tokyo.

[0086] Sequencing reads were processed using the 10X Genomics Cell Ranger v.5.0.0 pipeline to generate fastq files. Briefly, demultiplexed fastq files were aligned to a custom reference genome consisting of the mm10 / GRCm38 reference genome plus tdTomato sequences, and a gene expression matrix was generated using the Cellranger count pipeline. After Cellranger count processing, scRNA-seq data were analyzed using Seurat version 4.0.1 (Cell, 2021) under R version 4.0.3. To exclude low-quality cells, cells with fewer than 200 or more than 5,000 gene expression profiles or with more than 10% of reads mapped to mitochondria were excluded. After excluding low-quality cells, expression values ​​were normalized (NormalizeData function; scale.factor = 10,000). The IntegratedData function was used to generate an integrated Seurat object that compensated for technical differences between datasets. Dimensionality reduction was performed using the UMAP (Uniform Manifold Approximation and Projection) algorithm with the RunPCA and RunUMAP functions implemented in the Seurat package. After dimensionality reduction of the integrated Seurat object with dims = 1:30 and resolution = 0.25, the cell type of each cluster was identified based on the expression of known marker genes. Three clusters with extremely low numbers of expressed genes (red blood cell cluster, platelet cluster, and cluster suspected to be dead cells) were excluded for downstream analysis. The DimPlot function was used to visualize clusters after dimensionality reduction. The CellChat (Nature communications, 2021) package was used to analyze intercellular interactions. The FindMarkers function was used to compare gene expression differences between subclusters.Genes in fibroblast clusters that were significantly elevated in the HFpEF group compared to the normal diet group were selected based on a Bonferroni adjusted p-value of less than 0.05 and an average logFC of 0.2 or greater. The AddModuleScore function was used to calculate the average score across multiple gene groups, and the VlnPlot function was used for visualization. The DoHeatmap function was used to compare and visualize the expression levels of representative genes activated by cardiac damage between samples.

[0087] (ATAC Sequencing) ATAC sequencing was performed in triplicate in each group. Tomato-positive cells were isolated and collected from in vivo hearts using the method described above. To prepare cell nuclei, 50,000 cells were counted from the Tomato-positive cells and centrifuged at 500g for 5 minutes. Then, the cells were washed with chilled PBS and centrifuged again at 500g for 5 minutes. Cells were lysed using a lysis solution containing 0.1% NP40, 0.1% Tween 20, and 0.01% digitonin. Immediately after lysis, the nuclei were centrifuged at 500g for 10 minutes in a refrigerated centrifuge. After discarding the supernatant, the nuclear pellet was resuspended in a transposase reaction mix (25µL 2xTD buffer, 2.5µL transposase, and 22.5µL nuclease-free water). The transposase reaction was carried out at 37°C for 30 minutes. Immediately after transposase, samples were purified using the MinElute kit (QIAGEN, 28004). Following purification, library fragments were amplified using 1x NEBnext PCR Master Mix and 1.25 μM custom Nextera PCR primers. To reduce GC and size bias in PCR, PCR reactions were monitored using qPCR and stopped before saturation. For this qPCR, the complete library was amplified for five cycles, after which 5 μl of the PCR reaction product was taken and 10 μl of PCR cocktail containing 0.6x Syber Green was added. This reaction was run for 20 cycles to determine the number of additional cycles required for the remaining 45 μL reaction. The library was purified using the MinElute kit. Finally, double size selection was performed using SPRIselect (BECKMAN COULTER, B23317) to remove fragments >1000 bp and <100 bp. Library sequencing was performed using a HiSeq X Ten (Illumina). Adapter sequences were removed using fastp software (Chen et al. 2018). Reads were first aligned to the mm10 genome using Bowtie2 with the following parameters: --no-mixed --no-discordant -X 2000.Duplicate reads were removed using picard (https: / / broadinstitute.github.io / picard / ). Bam files were converted to bigwig files using deeptools bamCoverage with the following parameters: -bs 1 -of bigwig --normalizeUsing CPM (Ramirez et al., 2016). Peak calling was performed using MACS2 (Zhang et al., 2008) with the parameters "--nomodel --shift -50 --extsize 100." Peaks detected in two replicates out of three samples were defined as reliable peaks and used for further analysis. The coverage of each peak was calculated using featureCounts (Liao et al., 2014). To detect peaks whose accessibility was altered by transcription factor processing or overexpression, a likelihood ratio test was performed using edgeR, with significance determined at p<0.01. Motifs enriched in altered peaks were detected using HOMER (Heinz et al. 2010). The closest genes to each peak were identified using HOMER, and gene ontology analysis of the listed genes was performed using Metascape (Zhou et al. 2019). Visualization was performed using an original Python script and deeptools. To infer direct Gata4 binding sites among the altered peaks, overlap with published Gata4 ChIP-seq data from fibroblasts (Hashimoto et al. 2019. GSM3067561) was analyzed.

[0088] (Cell culture and retroviral vector infection) To construct the pMXs retroviral vector, the Cre coding region was amplified by PCR and subcloned into the pMXs vector for transfection into Plat-E cells using Fugene 6 (Promega, E2691). Fibroblasts were transduced with the newly generated pMX-Cre vector. 24 hours after infection, the medium was replaced with 20% FBS-supplemented DMEM / M199 (11150-059; Gibco) and cultured at 37°C in 5% CO2.

[0089] (Statistical analysis) Statistical significance was determined using Student's t-test for comparisons between two groups, and one-way analysis of variance (ANOVA) with Turkey or Dunnett's post hoc test for tests involving three or more groups. Two-way analysis of variance (Two-way ANOVA) with Turkey's post hoc test was used for tests involving time-dependent changes, such as echocardiography. Differences between groups were considered significant at p < 0.05. Statistical analysis was performed using GraphPad Prism software.

[0090] [Results] (Development of mice capable of freely reprogramming and labeling cardiac fibroblasts) We crossbred three types of mice: Tcf21 iCre mice, CAG-CAT-MGTH2A mice, and R26 tdTomato mice, to develop triple transgenic mice capable of controlling cardiac direct reprogramming in cardiac fibroblasts in vivo. The Tcf21 iCre mice are shown in Figure 1, the CAG-CAT-MGTH2A mice in Figure 2, and the R26 tdTomato mice in Figure 3.

[0091] Tcf21 iCre mice express Cre protein in Tcf21 (Transcription Factor 21), which is specifically expressed in cardiac fibroblasts. MeRCreMer in Figure 1 is the sequence of a mutated estrogen receptor (MER) bound to Cre recombinase.

[0092] CAG-CAT-MGTH2A mice express the reprogramming factors MGTH (four factors: Mef2c, Gata4, Tbx5, and Hand2) upon Cre expression. MGTH in Figure 2 is arranged in the order Mef2c, Gata4, Tbx5, and Hand2. CAG is a structure consisting of a cytomegalovirus enhancer and a chicken β-actin promoter. CAT is chloramphenicol acetyltransferase.

[0093] The R26 tdTomato mouse expresses the fluorescent protein Tomato when Cre is expressed. Rosa26 (R26) in Figure 3 is a gene region on mouse chromosome 6 that is easy to insert genes into and allows for constitutive expression of the inserted protein. tdTomato (tandem dimer Tomato) is a sequence in which two red fluorescent proteins, Tomato, are joined in tandem.

[0094] When tamoxifen was administered to mice with direct myocardial reprogramming, Cre was expressed throughout the cardiac fibroblasts, and reprogramming factors and Tomato were expressed. Therefore, tamoxifen treatment triggered the induction of some cardiac fibroblasts into red fibroblasts and some into red cardiomyocytes. Using these mice, we aimed to treat HFpEF and elucidate its molecular biological mechanisms.

[0095] (Confirmation of the expression of fluorescent proteins and reprogramming factors in cardiac fibroblasts) One week after administering tamoxifen to mice undergoing direct myocardial reprogramming, immunostaining was performed on the hearts of the mice. The results of the immunostained cardiac sections are shown in Figure 4. Fibroblasts present in the myocardial interstitium were labeled red.

[0096] In addition, hearts of mice undergoing direct myocardial reprogramming were treated with tamoxifen and then enzymatically isolated one week later. Tomato-positive cardiac fibroblasts isolated from the mice were then collected using a fluorescence activated cell sorter (FACS), and RNA was extracted and quantitative PCR was performed. The results are shown in Figures 5-8. The control group expressed only the Tomato protein, but no reprogramming factors. These results are from RNA samples of Tomato-positive cells collected from Tcf21 iCre / R26 Tomato mice.

[0097] As shown in Figures 5 to 8, administration of tamoxifen to mice with direct myocardial reprogramming resulted in Cre expression throughout the cardiac fibroblasts, as well as the expression of the four reprogramming factors and Tomato. Note that in Figures 5 to 8, 18, 20, 22, 24, 26, 29, 43 (right panel), 48, 49, 51, and 52, the dots indicate the individual values ​​of the samples.

[0098] (Establishment of a HFpEF Model and Examination of Therapeutic Effects) A HFpEF model was established using direct myocardial reprogramming mice and the therapeutic effects were examined. The HFpEF model was established by providing free access to L-NAME and a continuous high-fat diet. Direct myocardial reprogramming mice were used as the treatment group, while R26 tdTomato mice, which express only fluorescent proteins, were used as the control group. A comparison was made between the normal diet control group and the HFpEF stress group, and the effects of the reprogramming treatment group, in which reprogramming begins after the onset of HFpEF, were examined. Hereafter, the normal diet control group will be referred to as "normal diet," the HFpEF stress control group as "HFpEF," and the HFpEF stress direct myocardial reprogramming group as "reprogramming." The experimental protocol is shown in Figure 9. Reprogramming gene expression in the tamoxifen-induced reprogramming group was monitored under continuous HFpEF stress. Reprogramming genes are continuously expressed after tamoxifen administration.

[0099] The systolic blood pressure measurement results for the normal diet group, HFpEF group, and reprogramming group are shown in Figure 10, and the body weight measurement results are shown in Figure 11. "ns" indicates non-significant, and "****" indicates p<0.0001. Systolic blood pressure and body weight increased equally in the HFpEF group and the reprogramming group, and no significant difference was observed between the two groups.

[0100] Figure 12 shows the results of echocardiography (M-mode) of left ventricular contraction over time. Figure 13 also shows the results of echocardiography (Doppler mode) of mitral orifice blood flow velocity waveforms. The waveform typically consists of two peaks, the first half of which is the E wave (early diastolic), and the second half is the A wave (atrial contraction). E / A, the ratio of E wave height to A wave height, is an index of left ventricular diastolic function; in mice, the higher the ratio, the worse the diastolic function. E / E', calculated by dividing the E wave height by the tissue Doppler mitral annular early diastolic velocity E', also indicates left ventricular diastolic function. An increase in the E / E' value indicates a decline in left ventricular diastolic function.

[0101] The time course of ejection fraction (EF), E / A, and E / E' are shown in Figures 14, 15, and 16, respectively. "ns" indicates non-significant. As shown in Figure 14, there was no decline in EF from baseline in all groups. Furthermore, as shown in Figures 15 and 16, HFpEF load worsened left ventricular diastolic function in both the HFpEF group and the programming group, but the reprogramming group showed improvement compared to the HFpEF group from the start of drug administration.

[0102] (Improvement of Diastolic Function by Reprogramming) The results of mouse catheterization 15 weeks after the start of the experiment are shown in Figures 17 to 20. Figure 17 shows the left ventricular pressure waveform, with the arrow indicating EDP (End Diastolic Pressure). Figure 18 shows a bar graph of EDP values. Elevated EDP is the gold standard for heart failure. Figure 19 shows the PV loop (ressure-volume loop) waveform. The arrow points to the EDPVR (End Diastolic Pressure-Volume Relationship) curve. The slope of this curve reflects left ventricular diastolic function; a higher slope indicates a greater increase in pressure relative to an increase in volume, indicating poorer left ventricular diastolic function. Figure 20 shows a bar graph of EDPVR values. In Figures 18 and 20, "ns" indicates non-significant, "**" indicates p<0.01, "***" indicates p<0.001, and "****" indicates p<0.0001.

[0103] As shown in Figures 18 and 20, EDP and EDPVR were significantly elevated in the HFpEF group compared to the normal diet group, while EDP and EDPVR were improved in the reprogramming group.

[0104] (Improvement of exercise tolerance through reprogramming) The exercise tolerance of mice was evaluated 15 weeks after the start of the experiment. Figure 21 shows the evaluation using a mouse treadmill. Mice ran on an inclined conveyor belt, gradually increasing their speed over time, and exercise tolerance was evaluated based on the total distance traveled.

[0105] Figure 22 shows the running distance for each group. In Figure 22, "**" indicates p<0.01, and "****" indicates p<0.0001. As shown in Figure 22, the running distance, which was reduced in the HFpEF group, improved in the reprogramming group, confirming the effect of reprogramming treatment on improving exercise tolerance. This indicates that the improvement in cardiac diastolic function in the reprogramming group resulted in improved exercise tolerance.

[0106] (Evaluation of Cardiomyocyte Induction Efficiency) At 15 weeks after the start of the experiment, the efficiency of myocardial induction was evaluated by immunostaining mouse heart sections. The immunostaining results are shown in Figure 23. As shown in Figure 23, in the reprogramming group, expression of myocardial-specific proteins was observed in some Tomato-positive cells, confirming regeneration into cardiomyocytes. Furthermore, in the reprogramming group, clear striations were formed in cells that co-expressed Tomato and α-actinin (αAct).

[0107] The results of quantifying the percentage of cells induced from fibroblasts to cardiomyocytes are shown in Figure 24. "ns" indicates non-significant, and "**" indicates p<0.01. As shown in Figure 24, approximately 1% of fibroblasts in the reprogramming group were reprogrammed to cardiomyocytes.

[0108] (Improvement of myocardial hypertrophy by reprogramming) The top row of Figure 25 shows a macroscopic image of the heart 15 weeks after the start of the experiment. The bottom row shows an immunostained image of a heart section 15 weeks after the start of the experiment. By using wheat germ agglutinin (WGA) to specifically stain the cell membrane green, the outline of cardiomyocytes is clarified, making it possible to measure the myocardial cross-sectional area. The macroscopic image of the heart shows that cardiac hypertrophy occurred in the HFpEF group, while cardiac hypertrophy was improved in the reprogramming group. Furthermore, the immunostained image shows that cardiomyocyte size increased in the HFpEF group, while cardiomyocyte size improved in the reprogramming group.

[0109] Figure 26 shows the results of measuring the heart weight-femur length ratio. "ns" indicates non-significant, "***" indicates p<0.001, and "****" indicates p<0.0001. Heart weight, an indicator of cardiac hypertrophy, was corrected for femur length to reduce individual variability among mice. As shown in Figure 26, heart weight significantly increased in the HFpEF group, while the increase in heart weight was improved in the reprogramming group.

[0110] Figure 27 shows the results of measuring cardiomyocyte area. "****" indicates p<0.0001. The cross-sectional area of ​​approximately 100 randomly selected cardiomyocytes from each individual was measured and displayed as a violin plot. Overall, the cross-sectional area of ​​cardiomyocytes in the HFpEF group increased, while the increase in area was improved in the reprogramming group. One of the causes of impaired cardiac diastolic function is cardiomyocyte hypertrophy, and the results in Figures 25-27 indicate that suppressing cardiomyocyte hypertrophy is associated with improved cardiac diastolic function.

[0111] (Reduction of fibrosis by reprogramming) Figure 28 shows the results of Sirius red staining of heart sections 15 weeks after the start of the experiment. Sirius red staining stains fibrotic areas red, while normal tissue is stained yellow. For example, the arrowheads in Figure 28 indicate fibrotic areas. While it was found that fibrotic foci in parts of the interstitium and around blood vessels worsened in HFpEF, the fibrotic areas decreased in the reprogramming group.

[0112] The fibrotic area, which is the ratio of the red-stained fibrotic area to the total area of ​​the observed tissue section, was analyzed using software and the results are shown in Figure 29. "**" indicates p<0.01, "***" indicates p<0.001, and "****" indicates p<0.0001. The fibrotic area in the reprogramming group was significantly reduced compared to the HFpEF group.

[0113] Direct reprogramming of cardiac fibroblasts in HFpEF model mice was found to improve HFpEF by improving cardiac hypertrophy and reducing fibrosis. A breakdown of the direct reprogramming showed that approximately 1% of disease-activated fibroblasts were induced to become cardiomyocytes. However, changes throughout the heart were diffuse, and we hypothesized that the therapeutic effect of reprogramming may be due not only to the induction of the 1% of fibroblasts into cardiomyocytes, but also to the quenching of the remaining 99% of fibroblasts that were not induced into cardiomyocytes. Further analysis was conducted to determine the potential anti-fibrotic effects of direct myocardial reprogramming.

[0114] (scRNA-seq) The results of single-cell RNA sequencing (scRNA-seq) of cardiac non-cardiomyocytes are shown in Figure 30. Each dot represents the information of a single cell, which is classified into multiple clusters based on differences in its gene expression patterns. Cell types were identified by confirming representative gene expression in each cluster. Figure 30 shows that non-cardiomyocytes within the heart, including fibroblasts, were uniformly collected.

[0115] Figure 31 is a diagram analyzing the ligand-receptor interactions between each cell cluster shown in Figure 30. The lines in Figure 31 indicate interactions between cells, with thicker lines indicating stronger interactions. Figure 31 shows that cardiac fibroblasts have a significant influence on other cells and also exhibit strong interactions among fibroblasts. Therefore, we decided to conduct a detailed analysis of fibroblasts that underwent gene expression therapeutic intervention.

[0116] (Improvement of pathological gene expression changes in fibroblasts by reprogramming) The results of extracting and analyzing gene expression changes in the fibroblast group alone are shown in Figure 32. In Figure 32, the central violin plot shows the pathological genes whose expression is elevated in HFpEF compared to the normal diet group as a comprehensive score on the vertical axis. In the reprogramming group, the pathological gene expression changes that were elevated in HFpEF were improved.

[0117] The expression of representative genes activated upon cardiac injury is shown as a heat map in Figure 33. In the reprogramming group, the expression of many fibrosis-related genes and cardiac hypertrophy signaling-related genes such as Il6 and Tgfb was improved.

[0118] To analyze the effect of expressed reprogramming genes on the therapeutic efficacy, we performed ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing). This analysis uses Tn5 transposase to specifically fragment open chromatin regions and simultaneously construct a tagged sequencing library. This method allows us to evaluate the open / closed state of chromatin and speculate on upstream regulatory mechanisms of gene expression.

[0119] The experimental flow of ATAC-seq analysis is shown in Figures 34 and 35. As shown in Figure 34, ATAC-seq was performed in two groups, the HFpEF group and the reprogramming group, to evaluate the therapeutic mechanism. Tomato-positive fibroblasts were collected from the hearts of live mice 5 weeks after tamoxifen administration (10 weeks after the start of the experiment). To further explore the effect of direct binding of reprogramming factors, we also performed an analysis integrating published ChIP-seq data (Figure 35). ChIP-seq (chromatin immunoprecipitation sequencing) is a method that immunoprecipitates DNA and transcription factor complexes with antibodies, then sequences the precipitated DNA fragments to analyze which regions of the DNA interact with the transcription factor.

[0120] Figure 36 shows a heat map of signal intensity for regions where significant differences in peak waveform height occurred in ATAC-seq (ATAC peak change). n = 3 for both the HFpEF and reprogramming groups. ATAC peak change included 1,890 open regions, where peaks were higher in the reprogramming group, and 1,045 close regions, where peaks were lower. Motif analysis was performed to determine the enrichment of known gene sequences that can bind transcription factors in each region. Gata4 binding motifs were found in both open and close regions. No motifs for Mef2c, Tbx5, or Hand2 were found, suggesting that Gata4 may be an important reprogramming factor involved in the impact of treatment efficacy.

[0121] The ATAC-peak changes analyzed in this study were classified into two regions: those with significant peaks (Gata4-dependent) and those without (Gata4-independent) in the published fibroblast Gata4 ChIP-seq data. Figure 37 shows all peak heights aggregated and divided into Gata4-dependent and Gata4-independent regions.

[0122] Figure 38 shows the intensity of each peak signal in a heat map. Among the ATAC peak changes, there are 561 Gata4-dependent peaks and 2374 Gata4-independent peaks.

[0123] GO analysis was performed on the genes near each peak. GO analysis (Gene ontology analysis) is an analysis that annotates gene function by focusing on the known biological processes and molecular functions of the input gene group. The analysis results are shown in Figure 39. As shown in Figure 39, all of the genes were found to be related to fibrosis. This suggests that Gata4 is directly and indirectly involved in the therapeutic effect.

[0124] (Generation of genetically modified mice expressing only a single reprogramming factor) Based on the results of previous experiments, we investigated whether HFpEF treatment using a single reprogramming factor (single factor) is possible. Figure 40 shows an overview of the genetically modified mice. Four strains of single factor mice were generated, each containing a loxp-enclosed STOP sequence downstream of the CAG promoter and one gene each for Gata4, Mef2c, Tbx5, and Hand2. In addition to these four types of single factor mice, we also analyzed the myocardial direct reprogramming mice we had been using, designated 4F mice.

[0125] We collected fetal fibroblasts from these genetically engineered mice and analyzed the changes that occurred by forcing Cre expression of each target factor on cell plates using the retroviral vector pMx-Cre. Figure 41 shows the results of quantitative PCR analysis of RNA extracted from fetal fibroblasts (wild-type, each of the four single factor mouse strains, and 4F mouse fibroblasts) one week after infection with pMx-Cre. While the 4F mice show forced expression of all factors (Gata4, Mef2c, Tbx5, and Hand2), the single factor groups show forced expression of only the respective factors. Importantly, as indicated by the arrows, the expression of each factor was enhanced, without enhancing the expression of other endogenous factors.

[0126] Figure 42 shows a graph of quantitative PCR performed on RNA extracted from fetal fibroblasts one week after infection with pMx-Cre. Quantitative PCR was performed on well-known fibrotic genes Col1a2, Fn1, and Postn. Compared to wild-type fibroblasts, only Gata4 single factor and 4F, as indicated by the arrows, showed a decrease in all of these fibrotic genes.

[0127] Figure 43 shows FACS results one week after infection of fetal fibroblasts with pMx-Cre. The positive rate was determined by immunostaining for cTnT (troponin T), a myocardial-specific protein. Only 4F cells were positive for cTnT, indicating that expression of a single factor, including Gata4, alone does not result in cardiomyocyte induction. The "**" in the graph indicates p<0.01.

[0128] When direct myocardial reprogramming is performed using four or more reprogramming factors, it has been reported that reprogramming using a polycistronic vector carrying multiple reprogramming factors as a single gene results in higher quality myocardial regeneration than reprogramming using a vector carrying each reprogramming factor individually (Kohei Inagawa et al., Circ Res. 2012 Oct 12; 111(9):1147-56). This report suggests that for efficient treatment, four or more genes must be introduced uniformly.

[0129] Meanwhile, reports have also been published on the size of genes that can be loaded into safe vectors suitable for clinical use in humans for gene therapy (Kenneth Lundstrom, Diseases 2018, 6, 42; Clare E. Thomas et al., Nat Rev Genet. 2003 May, 4(5):346-58; Takehiro Ura et al., Vaccines 2014, 2, 624-641). The four or more reprogramming factors described above are larger than the size of genes that can be loaded into safe vectors suitable for clinical use in humans, and the development of safe polycistronic vectors capable of uniformly delivering four or more genes has yet to be realized.

[0130] Given this background technology, it is desirable to develop a treatment using a single gene that is small enough to be safely loaded onto a vector, so we decided to examine the therapeutic effect of a single reprogramming factor on HFpEF.

[0131] (Investigation of the therapeutic effect of Gata4 alone on HFpEF) To investigate the therapeutic effect of Gata4 alone on HFpEF, an in vivo experiment was performed on mice. Figure 44 shows an overview of the genetically modified mice used. Tcf21 expressing Cre in Tcf21 specifically expressed in cardiac fibroblasts. iCre We crossed mice with single factor mice, which express a single reprogramming factor upon Cre expression, or 4F mice, which express four reprogramming factors (MGTH). To confirm that the effects of single factor mice are not solely due to the expression of Gata4, we also used Mef2c single factor mice, which have been reported to be an important reprogramming factor in myocardial induction. The mice we generated express a single factor specifically in cardiac fibroblasts.

[0132] Figure 45 shows the experimental protocol. iCre Mice with only Tcf21 gene expression were divided into two groups: a control group (Ctrl) fed a normal diet and a HFpEF-stressed group (Ctrl-HF). iCre / Single factor (Gata4 or Mef2c) mice (SF-HF (Gata4-HF or Mef2c-HF)), and Tcf21 iCre Both groups of 4F / 4F mice (4F-HF) continued to be subjected to HFpEF. Treatment with the factor was initiated by administering tamoxifen at 5 weeks after the start of the experiment, and analysis was performed at a total of 10 weeks.

[0133] Figure 46 shows the time course of body weight, systolic blood pressure (SBP), and diastolic blood pressure (DBP), which are indicators of HFpEF stress. The horizontal axis is weeks. "ns" indicates non-significant. It can be seen that there were no differences in body weight, SBP, or DBP in the group that underwent HFpEF stress.

[0134] Figure 47 shows the results of echocardiography. "ns" indicates non-significant, and "**" indicates p<0.01. EF (ejection fraction), an index of cardiac contractility, remained unchanged in all groups. Left ventricular diastolic function indices E / A and E / E' deteriorated in all groups exposed to HFpEF, whereas Gata4-HF and 4F-HF showed improvement from the start of drug administration. Importantly, Mef2c alone did not show significant improvement.

[0135] Figure 48 shows the treadmill test distance (m), an index of exercise tolerance, at 10 weeks after the start of the experiment. "*" indicates p<0.05, and "**" indicates p<0.01. HFpEF loading reduced exercise tolerance, but Gata4-HF and 4F-HF showed significant improvement.

[0136] Figure 49 shows the cardiac catheterization results at 10 weeks after the start of the experiment. "*" indicates p<0.05, and "**" indicates p<0.01. Left ventricular end-diastolic pressure (LVEDP), an index of heart failure, and end-diastolic pressure-volume relationship (EDPVR), an index of left ventricular diastolic function, worsened with HFpEF, but showed significant improvement only in the Gata4-HF and 4F-HF groups.

[0137] The results of Sirius red staining of heart sections at 10 weeks after the start of the experiment are shown in Figure 50. Compared to Ctrl, Ctrl-HF and Mef2c-HF showed an increase in the fibrotic area, whereas Gata4-HF and 4F-HF showed a decrease in the fibrotic area.

[0138] A graph analyzing the fibrotic area is shown in Figure 51. "*" indicates p<0.05, and "**" indicates p<0.01. The fibrotic area, which increased in HFpEF, was significantly improved in Gata4-HF and 4F-HF.

[0139] At 10 weeks after the start of the experiment, mouse hearts were excised, and RNA was extracted from both ventricles. The results of quantitative PCR are shown in Figure 52. Expression of Col1a1 and Col3a1, which indicate cardiac fibrosis, Nppb, an indicator of heart failure, and Tgfb1, a representative signaling factor for fibrosis and hypertrophy, was elevated with HFpEF. Expression of these genes improved in Gata4-HF and 4F-HF, but not in Mef2c-HF.

[0140] [Summary] Figure 53 summarizes the results of this example. Fibroblast activation was observed in HFpEF, and direct reprogramming was found to result in 1% of fibroblasts regenerating into myocardium. Furthermore, taking into account the results of recent gene expression analysis, it was found that the remaining 99% of fibroblasts that do not regenerate into myocardium act as anti-fibrotic agents, and these factors collectively improve HFpEF. Analysis of single factors demonstrated that while GMTH4 is required for myocardial regeneration, Gata4 alone can also exert anti-fibrotic therapeutic effects and improve cardiac diastolic function in HFpEF. This example demonstrates that gene therapy using Gata4 alone could be a potential treatment for HFpEF.

[0141] The present invention can be used in treatments such as gene therapy for heart failure (particularly HFpEF).

Claims

1. An agent for improving cardiac diastolic function, comprising only a polynucleotide encoding the reprogramming factor polypeptide Gata4 as a reprogramming factor.

2. The cardiac diastolic function improving agent according to claim 1, which is used as a single agent.

3. The cardiac expansion function improving agent of claim 1, wherein the reprogramming factor polypeptide Gata4 comprises an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:

3.

4. The cardiac diastolic function improving agent according to claim 1, wherein the polynucleotide comprises a nucleotide sequence having at least 90% nucleotide sequence identity with the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO:

4.

5. The cardiac diastolic function improving agent according to any one of claims 1 to 4, which is a cardiac failure improving agent.

6. The cardiac diastolic function improving agent according to claim 5, wherein the heart failure is heart failure with preserved ejection fraction (HFpEF).

7. (delete)

8. a contacting step of contacting a test substance with fibroblasts; A screening method for an agent for improving cardiac diastolic function, comprising: an evaluation step of evaluating the expression of only the Gata4 gene in fibroblasts as a reprogramming factor.