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

Abstract

A diastolic function improving agent according to the present embodiment comprises a polynucleotide encoding a reprogramming factor polypeptide Gata4.

Description

Technical Field

[0001] The present invention relates to an agent for improving cardiac diastolic function and a method for screening an agent for improving cardiac diastolic function. Background Art

[0002] Heart failure is classified into two types: heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF) according to the ejection fraction (EF). A decrease in cardiac diastolic function is known as a common pathological condition in heart failure. Drug therapies or non-drug therapies effective for HFrEF have been discovered. HFpEF is considered to be a complex pathological condition involving fibrosis, cardiac hypertrophy, and inflammation. On the other hand, regarding the detailed pathological condition, there is a problem that no effective treatment method has been elucidated.

[0003] For example, in Patent Document 1, direct cardiac reprogramming for inducing cardiomyocytes from fibroblasts in vivo is disclosed. In addition, Non-Patent Document 1 discloses that fibrosis is suppressed by introducing the GATA4 gene into fibroblasts after myocardial infarction.

[0004] Prior Art Documents Patent Documents Patent Document 1: WO 2011 / 139688 Non-Patent Documents Non-Patent Document 1: J Thorac Cardiovasc Surg. 2017 Nov;154(5):1601-1610 Summary of the Invention Technical Problem to be Solved by the Invention Patent Document 1 discloses a technique for inducing fibroblasts into cardiomyocytes by transducing reprogramming genes including Gata4, Mef2c, and Tbx5 into fibroblasts isolated from the hearts of α-myosin heavy chain-green fluorescent protein (αMHC-GFP) mice or Isl1-yellow fluorescent protein (Isl1-YFP) mice, but does not teach the effect of Gata4 on cardiac diastolic function in individuals. For direct cardiac reprogramming, it is necessary to introduce three or more cardiac direct reprogramming genes into fibroblasts. A technique for simultaneously introducing genes into fibroblasts or a vector capable of carrying multiple genes needs to be developed, but has not been realized.

[0005] The technology disclosed in Non-Patent Document 1 confirmed a fibrotic state in the heart after the death of rats with a myocardial infarction model, but did not improve the reduction in cardiac diastolic function in living individuals. Therefore, in order to improve the reduction in cardiac diastolic function, further research and development are expected.

[0006] An object of one aspect of the present invention is to provide a technology for improving cardiac diastolic function.

[0007] Technical solutions for solving technical problems The cardiac diastolic function improver according to one aspect of the present invention contains a polynucleotide encoding the reprogramming factor polypeptide Gata4.

[0008] In the cardiac diastolic function improver according to one aspect of the present invention, the reprogramming factor polypeptide Gata4 contains an amino acid sequence having at least 90% amino acid sequence identity with the amino acid shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0009] In the cardiac diastolic function improver according to one aspect of the present invention, the polynucleotide contains 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.

[0010] The cardiac diastolic function improver according to one aspect of the present invention is a heart failure improver.

[0011] In the cardiac diastolic function improver according to one aspect of the present invention, it contains a factor that upregulates the expression of the Gata4 gene.

[0012] The cardiac diastolic function improver according to one aspect of the present invention contains a factor that upregulates the expression of the Gata4 gene.

[0013] The cardiac diastolic function improver according to one aspect of the present invention contains a factor that increases the expression level of the Gata4 gene in cardiac fibroblasts.

[0014] The screening method of the cardiac diastolic function improver according to one aspect 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 fibroblasts.

[0015] Advantages of the invention According to one aspect of the present invention, a technology for improving cardiac diastolic function can be provided. Description of the drawings

[0016] Figure 1 It is a figure regarding Tcf21 iCre mice.

[0017] Figure 2It is a figure about CAG-CAT-MGTH2A mice.

[0018] Figure 3 It is a figure about tdTomato mice.

[0019] Figure 4 It is a figure of cardiac immunostaining sections of directly reprogrammed cardiomyocytes mice.

[0020] Figure 5 It is a figure showing the results of quantitative PCR for Gata4. The dots represent the individual values of the samples.

[0021] Figure 6 It is a figure showing the results of quantitative PCR for Mef2c.

[0022] Figure 7 It is a figure showing the results of quantitative PCR for Tbx5.

[0023] Figure 8 It is a figure showing the results of quantitative PCR for Hand2.

[0024] Figure 9 It is a figure showing the experimental protocol of HFpEF model mice.

[0025] Figure 10 It is a figure showing the measurement results of systolic blood pressure of the normal diet group, HFpEF group, and reprogramming group.

[0026] Figure 11 It is a figure showing the measurement results of body weight of the normal diet group, HFpEF group, and reprogramming group.

[0027] Figure 12 It is a figure showing the results of echocardiogram (M-mode) of the normal diet group, HFpEF group, and reprogramming group.

[0028] Figure 13 It is a figure showing the results of echocardiogram (Doppler mode) of the normal diet group, HFpEF group, and reprogramming group.

[0029] Figure 14 It is a figure showing the time-course change of EF.

[0030] Figure 15 It is a figure showing the time-course change of E / A.

[0031] Figure 16 It is a figure showing the time-course change of E / E'.

[0032] Figure 17 It is a figure showing the results of mouse catheterization (left ventricular pressure waveform).

[0033] Figure 18 It is a figure showing the measurement results of EDP.

[0034] Figure 19 It is a figure showing the results of mouse catheter examination (PV loop waveform).

[0035] Figure 20 It is a figure showing the measurement results of EDPVR.

[0036] Figure 21 It is a figure showing the situation of the evaluation using a mouse treadmill.

[0037] Figure 22 It is a figure showing the walking distances of each group.

[0038] Figure 23 It is a figure showing the immunostaining results of mouse heart sections. The photo in the upper right frame is a magnified photo of the part surrounded by a quadrilateral in the center of the photo. The scale bar is 50 μm.

[0039] Figure 24 It is a figure showing the myocardial induction efficiency of each group.

[0040] Figure 25 It is a figure showing the macroscopic image of the heart and the immunostaining image of the heart section.

[0041] Figure 26 It is a figure showing the measurement results of the heart weight to femur length ratio.

[0042] Figure 27 It is a figure showing the measurement results of the myocardial cell area.

[0043] Figure 28 It is a figure showing the Sirius red staining results of the heart section.

[0044] Figure 29 It is a figure showing the fibrotic area of each group.

[0045] Figure 30 It is a figure showing the results of scRNA-seq of non-myocardial cells in the heart.

[0046] Figure 31 It is a figure showing the results of the analysis of the interaction between non-myocardial cells in the heart.

[0047] Figure 32 It is a figure with the morbid genes with increased expression in HFpEF shown on the vertical axis as an inclusive score.

[0048] Figure 33 It is a figure showing the expression of representative gene sets activated during heart injury as a heat map.

[0049] Figure 34 It is a figure showing the experimental process of ATAC-seq analysis.

[0050] Figure 35 It is a figure showing the experimental process of ATAC-seq analysis.

[0051] Figure 36 It is a figure representing the intensity of signals in regions with significantly different peak waveform heights in ATAC-seq as a heatmap.

[0052] Figure 37 It is a figure showing Gata4 ChIP-seq data.

[0053] Figure 38 It is a figure representing Gata4 ChIP-seq data as a heatmap.

[0054] Figure 39 It is a figure showing the results of GO analysis.

[0055] Figure 40 It is a figure showing the overview of genetically modified mice.

[0056] Figure 41 It is a figure showing the measurement results of quantitative PCR for Gata4, Mef2c, Tbx5, and Hand2.

[0057] Figure 42 It is a figure showing the measurement results of quantitative PCR for Col1a2, Fn1, and Postn.

[0058] Figure 43 It is a figure showing the results of FACS.

[0059] Figure 44 It is a figure showing the overview of genetically modified mice.

[0060] Figure 45 It is a figure showing the experimental protocol.

[0061] Figure 46 It is a figure showing the time-course changes in body weight, systolic blood pressure (SBP), and diastolic blood pressure (DBP).

[0062] Figure 47 It is a figure showing the results of echocardiography.

[0063] Figure 48 It is a figure showing the walking distance in treadmill tests.

[0064] Figure 49 It is a figure showing the results of cardiac catheterization.

[0065] Figure 50 It is a figure showing the Sirius red staining result of a heart section.

[0066] Figure 51 It is a figure showing the fibrotic area area of each group.

[0067] Figure 52 It is a figure showing the results of quantitative PCR for Col1a1, Col3a1, Nppd, and Tgfb1.

[0068] Figure 53 It is a figure summarizing the results of this example. Detailed implementation mode

[0069] 〔Definition of terms, etc.〕 In this specification, "polynucleotide" may also be referred to as "nucleic acid" or "nucleic acid molecule", and refers to a polymer of nucleotides. In addition, "base sequence" may also be referred to as "nucleic acid sequence" or "nucleotide sequence". Unless otherwise specified, the polynucleotide may exist in the form of RNA or DNA. The form of RNA is, for example, mRNA. The form of DNA is, for example, cDNA or genomic DNA. DNA can be double-stranded or single-stranded.

[0070] In this specification, "protein" may also be referred to as "polypeptide".

[0071] The protein described in this specification only needs to be a polypeptide formed by peptide bonding of amino acids, but is not limited thereto, and may also include structures other than polypeptides. Examples of structures other than polypeptides mentioned here include sugar chains and isoprenyl groups, etc., and there is no particular limitation.

[0072] In this specification, "A and / or B" is a concept including A and B and both A or B, and may also be referred to as "at least one of A and B".

[0073] In this specification, "improvement of cardiac diastolic function" includes alleviating or mitigating the reduction of cardiac diastolic function or the risk of such reduction, delaying the progression of the reduction of cardiac diastolic function, curing the reduction of cardiac diastolic function, and improving cardiac diastolic function, etc.

[0074] In this specification, "comprise" is a concept including "consist essentially of" and "consist of".

[0075] In the numerical ranges described in this specification, the upper limit value or the lower limit value of the numerical range can be replaced with the value shown in the examples or the value unambiguously derived from the examples. Further, in this specification, the numerical values connected by "~" refer to the numerical range including the numerical values before and after "~" as the lower limit value and the upper limit value.

[0076] Cardiac diastolic function improving agent The cardiac diastolic function improving agent according to one aspect of the present invention contains a polynucleotide encoding a reprogramming factor polypeptide Gata4 (hereinafter, sometimes referred to as "Gata4 polynucleotide").

[0077] The myocardial infarction rats in Non-Patent Document 1 are a model of ischemic heart disease different from HFpEF, and the mode of cardiac fibrosis is different from the mode clarified in this study. Before the filing date of this application, based on the disclosure of Non-Patent Document 1, it was impossible to analogize whether Gata4 has a therapeutic effect on cardiac fibrosis in model animals with completely different modes.

[0078] In addition, although it is revealed that the causes of cardiac diastolic dysfunction involve not only cardiac fibrosis but also multiple aspects such as cardiomyocyte hypertrophy, endothelial dysfunction, and inflammation, it is not clear whether the improvement of these causes will lead to the improvement of diastolic function.

[0079] The present inventors have found that the intervention in fibrotic cells brings an improvement effect on cardiac diastolic function that cannot be predicted in the conventional inhibition of fibrosis.

[0080] (Gata4) The Gata4 polypeptide is a member of the GATA family zinc finger transcription factors that recognize and bind to the GATA motif present in the promoter regions of many genes (for example, recognize and bind to the consensus sequence 5'-AGATAG-3'). Regarding Gata4, it is described, for example, in Huang et al., Gene, 1995, 155(2):219-23. The amino acid sequences of Gata4 polypeptides from various species and the nucleotide sequences encoding Gata4 polypeptides are known in the art. An example of the accession numbers of the amino acid sequences of Gata4 polypeptides and the nucleotide sequences encoding Gata4 polypeptides is shown below.

[0081] <An example of the amino acid sequence of the 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) <An example of the nucleotide sequence encoding the Gata4 polypeptide> ·The CDS sequence (SEQ ID NO: 2) in NM_002052 (Homo sapiens) ·The CDS sequence (SEQ ID NO: 4) in NM_008092 (Mus musculus) ·The CDS sequence in NM_144730 (Rattus norvegicus) ·The CDS sequence in DQ886664 (Danio rerio) ·The CDS sequence in BC071107 (Xenopus laevis) In some embodiments, the 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 acids shown in SEQ ID NO: 1. In some embodiments, the 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 shown in SEQ ID NO: 3. The Gata4 polypeptide is biologically active, for example, recognizing and binding to the GATA motif present in the promoter (e.g., recognizing and binding to the consensus sequence 5'-AGATAG-3'), and activating the transcription of a gene operably linked to a promoter containing the GATA motif.

[0082] 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 shown 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 shown in SEQ ID NO: 4.

[0083] In some embodiments, a polypeptide that is functionally equivalent to the Gata4 polypeptide (or a nucleotide sequence encoding such a functional equivalent) is used. For example, in some embodiments, the Gata5 polypeptide (or a nucleotide sequence encoding the Gata5 polypeptide) is used. In another embodiment, the Gata6 polypeptide (or a nucleotide sequence encoding the Gata6 polypeptide) is used.

[0084] The amino acid sequence of the Gata5 polypeptide and the nucleotide sequence encoding the Gata5 polypeptide are known in the art. An example of the accession number for the amino acid sequence of the Gata5 polypeptide and the nucleotide sequence encoding the Gata5 polypeptide is shown below.

[0085] <An example of the amino acid sequence of the Gata5 polypeptide> ·NP_536721 (Homo sapiens) ·NP_032119 (Mus musculus) ·NP_001019487 (Rattus norvegicus) <An example of the nucleotide sequence encoding the Gata5 polypeptide> ·The CDS sequence in NM_080473 (Homo sapiens) ·The CDS sequence in NM_008093 (Mus musculus) ·The CDS sequence in NM_001024316 (Rattus norvegicus) The amino acid sequence of the Gata6 polypeptide and the nucleotide sequence encoding the Gata6 polypeptide are known in the art. An example of the accession number for the amino acid sequence of the Gata6 polypeptide and the nucleotide sequence encoding the Gata6 polypeptide is shown below.

[0086] <An example of the amino acid sequence of the Gata6 polypeptide> ·NP_005248 (Homo sapiens) ·NP_034388 (Mus musculus) ·NP_062058 (Rattus norvegicus) <An example of the nucleotide sequence encoding the Gata6 polypeptide> ·The CDS sequence in NM_005257 (Homo sapiens) ·The CDS sequence in NM_010258 (Mus musculus) ·The CDS sequence in NM_019185 (Rattus norvegicus) In some embodiments, suitable functional equivalents of the Gata4 polypeptide are polypeptides 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 with the amino acid sequence of the Gata5 polypeptide or the Gata6 polypeptide.

[0087] In some embodiments, the nucleotide sequence encoding a functional equivalent of the 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 with the nucleotide sequence encoding the Gata5 polypeptide or the Gata6 polypeptide.

[0088] The method for obtaining (isolating) the Gata4 polynucleotide is not particularly limited. For example, it can be synthesized according to nucleic acid synthesis methods such as the phosphoramidite method.

[0089] In addition, as a method for obtaining the Gata4 polynucleotide, a method using a nucleic acid amplification method such as PCR can be cited. For example, primers are prepared from the 5'-side and 3'-side sequences (or their complementary sequences) in the cDNA of the polynucleotide, and PCR or the like is performed using these primers with genomic DNA or cDNA as a template to amplify the DNA region sandwiched between the two primers. Thus, a DNA fragment containing the polynucleotide of the present invention can be obtained in large quantities.

[0090] A diastolic function improver containing the Gata4 polynucleotide (e.g., DNA) is also included in one aspect of the present invention. In a preferred aspect of the diastolic function improver, the Gata4 polynucleotide (e.g., DNA) is inserted into a carrier. The types of such carriers can be, for example, autonomously replicating carriers (e.g., plasmids, etc.), or can also be carriers that are integrated into the genome of the host cell when introduced into the host cell and replicated together with the integrated chromosome.

[0091] The above-mentioned carrier is preferably an expression vector. In the expression vector, the Gata4 polynucleotide is functionally linked to elements required for transcription (such as promoters, enhancers, ribosome binding sites, splicing signals, and terminators, etc.).

[0092] Examples of the carrier include viral vectors such as retroviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV vectors), Sendai virus vectors, and lentiviral vectors; non-viral vectors such as plasmid vectors, bacterial vectors, phage vectors, phagemid vectors, and cosmid vectors, etc.

[0093] The construction of the carrier can be carried out, for example, using known genetic engineering methods. In one example, the carrier specifically expresses the Gata4 polynucleotide in cardiac fibroblasts.

[0094] A diastolic function improver containing fibroblasts into which the Gata4 polynucleotide (e.g., mRNA) has been introduced or a carrier into which the Gata4 polynucleotide has been inserted is also included in one aspect of the present invention. By administering cells expressing Gata4 to a subject, the diastolic function can be improved.

[0095] As an example of fibroblasts, cardiac fibroblasts can be cited. Fibroblasts can be obtained from a living individual, for example, from tissues collected from a living individual. A method for isolating fibroblasts from tissues can be carried out using a known method. 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 can be syngeneic cells (autologous cells) collected from an individual as the administration subject, or allogeneic cells (heterologous cells) collected from an individual different from the administration subject.

[0096] When introducing a Gata4 polynucleotide or a vector inserted with a Gata4 polynucleotide into fibroblasts, for example, electroporation, calcium phosphate method, lipofection, microinjection, introduction method using liposomes, introduction method using a gene gun, introduction method using a cationic polymer (e.g., DEAE dextran, polyethyleneimine, polyethylene glycol), etc. can be used.

[0097] A cardiac diastolic function improver containing a factor that upregulates the expression of the Gata4 gene is also included in one aspect of the present invention. As examples of the factor that upregulates the expression of the Gata4 gene, RbAp46 (retinoblastoma protein-associated protein 46) and RbAp48 (retinoblastoma protein-associated protein 48), which are Gata4 binding factors, etc. can be cited. In addition, a cardiac diastolic function improver containing a factor that increases the expression level of the Gata4 gene in cardiac fibroblasts is also included in one aspect of the present invention. As examples of the factor that increases the expression level of the Gata4 gene, RbAp46 (retinoblastoma protein-associated protein 46) and RbAp48 (retinoblastoma protein-associated protein 48), etc. can be cited.

[0098] (Other components) One aspect of the present invention relates to a diastolic function improving agent for the heart, which may further contain components other than 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. These other components are not particularly limited, and examples thereof include pharmaceutically acceptable carriers, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for osmotic pressure adjustment, buffering agents, stabilizers, preservatives, excipients, antioxidants, viscosity regulators, coloring agents, flavoring agents, and sweetening agents. When the diastolic function improving agent for the heart is in the form of an aqueous solution, pure water (sterilized water), physiological saline, phosphate buffered saline, or the like can be used as the carrier. When the diastolic function improving agent for the heart is in the form of other suitable solutions, organic esters that can be introduced into the living body, such as glycols, glycerol, or olive oil, can be used as the carrier. A diastolic function improving agent for the heart 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 components is also referred to as a composition for improving diastolic function of the heart.

[0099] One aspect of the present invention relates to a diastolic function improving agent for the heart, which can be stored together with an instruction manual in a container, packaging, dispenser, or the like.

[0100] (Subjects and Administration of Diastolic Function Improving Agent for the Heart) Examples of the subjects to which the diastolic function improving agent for the heart according to one aspect of the present invention is administered include humans and non-human animals. More specifically, examples include vertebrates such as birds and mammals. Examples of mammals include experimental animals such as mice, rats, rabbits, guinea pigs, and non-human primates; pet animals (pets) such as dogs and cats; livestock such as pigs, cows, goats, sheep, and horses; or humans.

[0101] One aspect of the present invention relates to a diastolic function improving agent for the heart, which is preferably used to improve heart failure, and the diastolic function improving agent can be a heart failure improving agent. Examples of heart failure include heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF). The diastolic function improving agent can be used to improve either HFrEF or HFpEF.

[0102] The route or method of administration is not particularly limited and may be administered directly to the heart or its periphery of the subject or indirectly. Examples of the route of administration include oral, intravenous, intramuscular, subcutaneous, intraventricular, intraperitoneal, and transdermal routes. Other examples include topical administration and methods using a gene gun.

[0103] The dosage and frequency of administration can be appropriately selected according to the severity of symptoms, age, sex, body weight, administration method, and the like.

[0104] [Methods to improve cardiac diastolic function] One embodiment of the present invention also includes a method for improving cardiac diastolic function comprising the step of administering a Gata4 polynucleotide to a subject in need of improvement in cardiac diastolic function.

[0105] One embodiment of the present invention also includes a method for improving cardiac diastolic function comprising administering a factor that upregulates Gata4 gene expression or increases Gata4 gene expression in cardiac fibroblasts to a subject in need of improvement in cardiac diastolic function.

[0106] In the above method, before the administration step, a step of introducing Gata4 polynucleotide into fibroblasts may also be included. The fibroblasts may be fibroblasts isolated from the same individual as the subject to be administered with the Gata4 polynucleotide or a different but homologous individual, or the fibroblasts may be cultured cells derived from the same individual as the subject to be administered with the Gata4 polynucleotide or a different but homologous individual. The subject to be administered with the Gata4 polynucleotide is as described above with respect to the subject to be administered with the cardiac diastolic function improver. The Gata4 polynucleotide may also be introduced in the form of a vector. Therefore, a method for producing fibroblasts for introducing the subject comprising the following steps is also included in one embodiment of the present invention: a step of introducing the Gata4 polynucleotide into fibroblasts.

[0107] In the above method, it may further include a step of introducing 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 into the fibroblasts. The fibroblasts may be fibroblasts isolated from the same individual as the subject to which the Gata4 polynucleotide is administered or from a different but same-species 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 same-species individual. The factor that upregulates the expression of the Gata4 gene or the factor that increases the expression level of the Gata4 gene in cardiac fibroblasts may also be introduced in the form of a vector. Therefore, a method for manufacturing fibroblasts for introduction into a subject, which includes the following steps, is also included in one aspect of the present invention: a step of introducing 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 into the fibroblasts.

[0108] 〔Screening method for cardiac diastolic function improver〕 A screening method for a cardiac diastolic function improver according to one aspect of the present invention includes a contacting step and an evaluating step.

[0109] (Contacting step) The above contacting step includes contacting a test substance with fibroblasts. Examples of the fibroblasts include cardiac fibroblasts. The cells can be obtained from a living individual, for example, from a 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. A known method can be used to isolate fibroblasts from the tissue.

[0110] The above screening method may further include a step of introducing a Gata4 polynucleotide into the fibroblasts before the above contacting step. The Gata4 polynucleotide can be introduced in the form of a vector. In this case, contacting the test substance with the fibroblasts includes contacting the test substance with the fibroblasts into which the Gata4 polynucleotide has been introduced or a vector containing the fibroblasts. Specific introduction methods include those described in 〔Cardiac diastolic function improver〕.

[0111] (Evaluating step) In the above evaluating step, after the above contacting step, the expression of the Gata4 gene in the fibroblasts is evaluated. Specifically, the expression level of the Gata4 gene or the amount of polypeptide Gata in the fibroblasts after the contacting step is measured. As examples of the measuring methods, RT-PCR, hybridization analysis, and molecular biological methods can be used.

[0112] The above screening method may include a comparison step of comparing the expression level (such as the 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. When 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 can be a candidate for a diastolic function improving agent. The test substance can be selected as a candidate for a diastolic function improving agent.

[0113] Alternatively, the above screening method may include a comparison step of comparing the expression level (such as the expression amount) of polypeptide Gata in fibroblasts after the contacting step with the expression level of polypeptide Gata in fibroblasts before the contacting step. When the expression of polypeptide Gata in fibroblasts after the contacting step is upregulated compared to the expression of polypeptide Gata in fibroblasts before the contacting step, the test substance can be a candidate for a diastolic function improving agent and can be selected.

[0114] 〔Summary〕 The diastolic function improving agent according to Mode 1 of the present invention comprises a polynucleotide encoding a reprogramming factor polypeptide Gata4.

[0115] The diastolic function improving agent according to Mode 2 of the present invention is the diastolic function improving agent of Mode 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 shown in SEQ ID NO: 1.

[0116] The diastolic function improving agent according to Mode 3 of the present invention is the diastolic function improving agent of Mode 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 shown in SEQ ID NO: 1 and recognizing and binding to a GATA motif present in a promoter.

[0117] The diastolic function improving agent according to Mode 4 of the present invention is the diastolic function improving agent of Mode 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 1. The diastolic function improving agent according to Mode 5 of the present invention is the diastolic function improving agent of Mode 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 shown in SEQ ID NO: 3.

[0118] The cardiac diastolic function improver according to Mode 6 of the present invention is the cardiac diastolic function improver of Mode 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 shown in SEQ ID NO: 3 and recognizing and binding to the GATA motif present in the promoter.

[0119] The cardiac diastolic function improver according to Mode 7 of the present invention is the cardiac diastolic function improver of Mode 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polypeptide containing the amino acid sequence shown in SEQ ID NO: 3. The cardiac diastolic function improver according to Mode 8 of the present invention is the cardiac diastolic function improver of Mode 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polynucleotide containing a nucleotide sequence having at least 90% nucleotide sequence identity with the nucleotide sequence shown in SEQ ID NO: 2.

[0120] The cardiac diastolic function improver according to Mode 9 of the present invention is the cardiac diastolic function improver of Mode 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 encoding a polypeptide that recognizes and binds to the GATA motif present in the promoter.

[0121] The cardiac diastolic function improver according to Mode 10 of the present invention is the cardiac diastolic function improver of Mode 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises the nucleotide sequence shown in SEQ ID NO: 2.

[0122] The cardiac diastolic function improver according to Mode 11 of the present invention is the cardiac diastolic function improver of Mode 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises a polynucleotide containing a nucleotide sequence having at least 90% nucleotide sequence identity with the nucleotide sequence shown in SEQ ID NO: 4.

[0123] The cardiac diastolic function improver according to Mode 12 of the present invention is the cardiac diastolic function improver of Mode 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 and encoding a polypeptide that recognizes and binds to the GATA motif present in the promoter.

[0124] The cardiac diastolic function improver according to Mode 13 of the present invention is the cardiac diastolic function improver of Mode 1 of the present invention, wherein the polynucleotide encoding the reprogramming factor polypeptide Gata4 comprises the nucleotide sequence shown in SEQ ID NO: 4.

[0125] The cardiac diastolic function improver according to Mode 14 of the present invention comprises a factor that upregulates the expression of the Gata4 gene.

[0126] The cardiac diastolic function improver according to Mode 15 of the present invention comprises a factor that increases the expression level of the Gata4 gene in cardiac fibroblasts.

[0127] The cardiac diastolic function improver according to Mode 16 of the present invention is the cardiac diastolic function improver according to any one of Modes 1 to 3 of the present invention, and it is a heart failure improver.

[0128] In Mode 17 of the present invention, for the cardiac diastolic function improver, in Mode 4 of the present invention, the heart failure is heart failure with preserved ejection fraction (HFpEF).

[0129] The screening method for the cardiac diastolic function improver according to Mode 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 fibroblasts.

[0130] Examples are shown below to further illustrate the embodiments of the present invention in more detail. Of course, it is not limited to the following examples of the present invention, and various methods can be adopted for details. Moreover, the present invention is not limited to the above embodiments, and various changes can be made within the scope shown in the claims. Embodiments obtained by appropriately combining the separately disclosed technical means are also included in the technical scope of the present invention. In addition, all the documents described in this specification are incorporated by reference.

[0131] Examples All the experiments shown below were carried out with the approval of the Animal Experiment Ethics Committee of the University of Tsukuba.

[0132] [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 formed by linking Mef2c, Gata4, Tbx5, and Hand2 with a self-cleaving peptide 2A) transgenic mice were constructed by modifying a plasmid with the CAG-CAT-Z structure ( Figure 2, CAG-CAT-MGTH2A mice). This plasmid contains the CMV (Cytomegalovirus) enhancer and the CAG (Chicken beta actin) promoter, which is ligated to the CAT (Chloramphenicol acetyltransferase) gene surrounded by loxp sequences. T2A and Hand2 were ligated to the Mef2c-Gata4-Tbx5 gene sequence to generate the full-length mouse MGTH2A gene sequence, which was inserted into the Z site of the CAG-CAT-Z structural plasmid. The constructed plasmid was purified and linearized, and then introduced into the pronucleus of the fertilized egg blastocyst of FVBN mice by microinjection. Transgenic mice were verified for the presence of CAT by DNA PCR. The CAG-CAT-MGTH2A mice were crossed with Tcf21 iCre / tdTomato mice to generate triple-transgenic mice.

[0133] When generating Single factor mice for Gata4, Mef2c, Tbx5, and Hand2 (hereinafter, also abbreviated by their first letters as G, M, T, and H), plasmids with the CAG-LSL (loxp-Stop-loxp)-Z structure containing the CAG promoter and SV40pA surrounded by loxp sequences in the Rosa26 locus homologous sequence were used for construction. Plasmids with the gene sequences of G, M, T, and H inserted into the Z site of CAG-LSL-Z were generated respectively. The constructed plasmids were purified and linearized, and then introduced into the fertilized eggs of ICR mice. Using the homologous recombination method, the target gene sequence was inserted into the Rosa26 locus. DNA PCR was used to confirm the integration of the inserted gene into Rosa26 and the insertion of the gene into other loci, and individuals with the gene inserted only at the target locus of Rosa26 were used for the experiment. The CAG-LSL-Single factor (G, M, T, H) mice were crossed with Tcf21 iCre mice to generate double-transgenic mice.

[0134] No immunodeficiency or other health problems were observed in the transgenic mice, and they were maintained in a state without other experiments and drugs until the start of this experiment. All animals were group-housed in a dedicated SPF (Specific pathogen free) breeding facility with a 12-hour / 12-hour light / dark cycle, had free access to food and water, and were examined daily. To maintain the SPF level, the health status was regularly checked.

[0135] (HFpEF model mice) The HFpEF (Heart failure preserved ejection fraction) model mice were prepared following past reports (Schiattarella GG, et al. Nature 2019), by continuously administering L-NAME (Nω-nitro-L-arginine methyl ester, Sigma, N5751) and High fat diet (CLEA Japan, HFD32). L-NAME was dissolved in tap water at a concentration of 1.0 g / L and freely provided for drinking in the water supply bottles. L-NAME and High fat diet were replaced every 2 or 3 days.

[0136] (Induction of Cre based on tamoxifen administration) From the fifth week of the experiment, tamoxifen (Sigma, T5648) was administered to the mice. Tamoxifen was administered intraperitoneally to the mice at a dose of 2 mg / day for 5 consecutive days. Tamoxifen was dissolved in peanut oil (90%; P2144 Sigma) and ethanol (10%) at a concentration of 50 mg / mL for use.

[0137] (Mouse fibroblasts) Mouse embryonic fibroblasts (MEF) were isolated as follows. The embryos isolated from pregnant mice on day 12.5 were washed with phosphate-buffered saline (PBS), and then the head and visceral tissues were carefully removed. The remaining parts of the embryos were washed with fresh PBS, chopped with scissors, transferred to a 0.25% trypsin / ethylenediaminetetraacetic acid solution (Gibco, 25200-072), and incubated at 37 °C for 15 minutes. After trypsin treatment, 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 and resuspended in DMEM / 10% FBS (Dulbecco's modified Eagle's medium DMEM containing 10% FBS, FUJIFILM, 044-29765), and cultured at 37 °C and 5% CO2. The genotype of MEF was determined by DNA PCR using the head and visceral tissues.

[0138] (Echocardiography) Transthoracic echocardiography was analyzed using a Visual Sonics, Vevo 2100 system. For echocardiography examination, mice were anesthetized with a low dose of isoflurane at 1.0 - 2.0%. During echocardiography imaging, electrocardiogram monitoring was performed using limb electrodes while the body temperature was adjusted with a warming pad. Regarding cardiac contractility, the left ventricular end-diastolic diameter (LVDd), left ventricular end-systolic diameter (LVDs) were measured using M-mode echocardiography at the level of the papillary muscles in the left ventricular short-axis view, and the left ventricular ejection fraction (LVEF) was evaluated. In addition, the mitral valve blood flow velocity waveform was measured from the apical four-chamber view using Doppler echocardiography, and the peak early diastolic velocity (E') of the mitral annulus was measured using tissue Doppler. LVEF was calculated using Teicholz's formula according to Equation 1 below.

[0139] EF = (left ventricular end-diastolic volume - left ventricular end-systolic volume) / left ventricular end-diastolic volume × 100 (%)... (Equation 1) The left ventricular end-diastolic volume and left ventricular end-systolic volume were calculated according to Equation 2 and Equation 3 below, respectively.

[0140] 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 ... (Equation 3) (Blood pressure measurement) Systolic blood pressure and diastolic blood pressure were measured non-invasively in conscious mice using a mouse tail blood pressure measurement device (CODA, Kent Scientific). Mice were placed on individual holders on a temperature-controlled platform (37°C), and recordings were made in a steady state. Before the experiment, all mice were trained to adapt to short-term restraint. Blood pressure was measured at least 10 times or more, and the average value of the obtained measurement values was used.

[0141] (Cardiac catheterization) Cardiac catheterization was performed via the right internal carotid artery using a conductance catheter for mice (Millar, SPR-839) and a Pressure-Volume System (Millar, MPVS-400). The mice were anesthetized with isoflurane while kept on a warmer. Electrocardiogram monitoring was observed using limb electrodes. The right internal carotid artery was exposed by incision, and the conductance catheter was inserted. The tip was inserted into the left ventricle, and the waveform of the steady-state left ventricular pressure was recorded. Subsequently, a small incision was made in the upper abdomen to identify the inferior vena cava. The inferior vena cava was compressed with a cotton swab, and the Pressure-Volume loop (PV loop) waveform in the state of inferior vena cava occlusion was recorded. Analysis was performed using LabChart (ADInstruments).

[0142] (Mouse treadmill) The mouse treadmill was used with a belt-driven forced walking device (MELQUEST, TMS-4N). Two days before the start of the formal experiment, the mice were habituated to treadmill exercise according to a schedule of 0°, 10 m / min, and 10 min. In the formal experiment, under the condition of a 20° uphill slope, after walking at a warm-up speed of 5 m / min for 4 minutes, the speed was increased to 14 m / min and walked for 2 minutes. Then, the speed was increased by 2 m / min every 2 minutes until the mice were exhausted, and the total walking distance was measured. Exhaustion was defined as the inability to start walking again within 10 seconds after contact with the electric shock grid at the rear.

[0143] (Fluorescence-activated cell sorting) To detect cTnT expression by fluorescence-activated cell sorting (FACS), the cells were fixed with 4% PFA for 15 minutes, permeabilized with saponin (Sigma Aldrich, 47036-250G-F), stained with an anti-cTnT (Thermo Scientific, MS-295-P1) antibody, and incubated with a secondary antibody conjugated to Alexa Fluor 488 (Invitrogen, A11001). Then, using a FACS device (Beckman Coulter, CytoFLEX S), the cells were analyzed using FlowJo software (Tomy DigitalBiology). Non-myocardial cells and Tomato-positive cells were collected using BD FACSAria TM IIIu (BD), MoFlo XDP (Beckman Coulter).

[0144] (Histological examination) After euthanizing the mice, the heart was perfused successively from the apex with PBS and 0.4% paraformaldehyde (PFA). The heart was quickly removed and fixed in 4% PFA overnight. In the case of immunostaining using cryosections, after replacing with 20% sucrose solution, it was embedded in OCT compound, and a cryo-block was made using liquid nitrogen. Using a microtome, 7-μm sections were cut vertically in a way that both ventricles of the heart could be seen. The cryosections were stained with a primary antibody against α-Actinin (Sigma, A7811) and a secondary antibody conjugated with Alexa488 and DAPI. Wheat germ agglutinin (WGA) staining was performed using an antibody conjugated with Alexa488. Confocal microscopy was carried out using an LSM800 microscope (Carl Zeiss). The ratio of α-Actinin+ / Tomato+ cells was counted and measured in 10 regions randomly selected from more than 5 different sections in each mouse.

[0145] In the case of making paraffin sections, after making paraffin-embedded sections from the fixed heart, Sirius red staining or wheat germ agglutinin (WGA) staining was performed. WGA staining was carried out using an antibody conjugated with Alexa488 (Thermo Scientific, W11261). Measurement of the fibrotic area and measurement of the cross-sectional area of cardiomyocytes were performed using Image J (NIH). All determinations and calculations were carried out under blind conditions.

[0146] (DNA PCR and qRT-PCR) Genotyping of transgenic mice was performed by standard PCR using the primers shown in Table 1. Total RNA was extracted from in vitro-transduced fibroblasts, hearts from both ventricles, and isolated Tomato-positive cells by a standard protocol. qRT-PCR was carried out using a StepOnePlus real-time PCR system, using the primers and TaqMan probes shown in Tables 2 and 3 (Applied Biosystems). 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). The expression of the target mRNA was corrected by the expression of Gapdh.

[0147] [Table 1] Table 1 [Table 2] Table 2 [Table 3] Table 3 (Isolation and collection of non - cardiomyocytes and Tomato - positive cells) After euthanizing the mice by inhaling CO2, the heart was immediately cannulated and perfused with chilled PBS (50 mL). After removing the heart, the atria and valves were cut and separated to isolate the ventricles. The ventricular myocardium was cut into pieces about 1 mm in size on a sterile petri dish on ice. It was 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), RBC lysis solution (pluriSelect, 60 - 00050 - 11) was used to remove red blood cells. The cell suspension was stained with Live / Dead (Invitrogen, L34975, 1:1000), Calcein Violet Working solution (BioLegend, 425203, 0.1 μM). Using a FACS device, while minimizing the pressure on the cells, cells without cell membrane damage and maintaining metabolic activity (Live / Dead -, Calcein+) were collected. FACS used BDFACSAria TM IIIu (BD), MoFlo XDP (Beckman Coulter). When collecting Tomato - positive cells, Tomato - positive cells were collected by FACS without staining the cells.

[0148] (Single - cell RNA sequencing) Single-cell RNA sequencing (scRNA-seq) was analyzed using a sample size of n = 2 for each group. The scRNA-seq libraries from non-myocardial cells were prepared using the 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 on a NovaSeq 6000 (Illumina) system operated by the Omics·Bioinformatics Center, Graduate School of Frontier Sciences, The University of Tokyo.

[0149] Sequence reads were processed using the 10X Genomics Cell Ranger v.5.0.0 pipeline to generate fastq files. Briefly, the deduplicated fastq files were aligned to a custom reference genome with the tdTomato sequence added in the mm10 / GRCm38 reference genome, and the Cellranger count pipeline was used to generate a gene expression matrix. The scRNA-seq data after Cellranger count processing was analyzed using Seurat version 4.0.1 (Cell, 2021) under R version 4.0.3. To exclude low-quality cells, cells with gene expression below 200 or above 5000, or with more than 10% of the reads mapped to mitochondria were excluded. After excluding low-quality cells, the expression values were normalized (NormalizeData function; scale.factor = 10000). To generate an integrated Seurat object with technical differences between datasets corrected, the IntegratedData function was used. Dimension reduction was performed using the UMAP (Uniform Manifold Approximation and Projection) algorithm, using the RunPCA and RunUMAP functions installed in the Seurat package. After dimension reduction of the integrated Seurat object with dims = 1:30 and resolution = 0.25, the cell types of each cluster were identified based on the gene expression of known markers. Three clusters with extremely low numbers of expressed genes (erythrocyte cluster, platelet cluster, and cluster of suspected dead cells) were excluded for downstream analysis. Visualization of the clusters after dimension reduction was performed using the DimPlot function. Analysis of cell-cell interactions used the CellChat (Nature communications, 2021) package. The FindMarkers function was used to compare gene expression differences between subclusters. In the fibroblast cluster, based on the criteria of an adjusted p value less than 0.05 after Bonferroni multiple correction and an average logFC of 0.2 or more, gene sets that were significantly upregulated in the HFpEF group compared to the normal diet group were selected. Calculation of the average scores of multiple gene sets used the AddModuleScore function, and visualization used the VlnPlot function. To visualize and compare the expression values of representative gene sets activated due to heart injury among samples, the DoHeatmap function was used.

[0150] (ATAC sequencing) ATAC sequencing was performed with n = 3 for each group. Tomato-positive cells were isolated and collected from the heart in vivo using the above method. To prepare the cell nuclei, 50,000 cells were counted from the Tomato-positive cells and centrifuged at 500 g for 5 minutes. Then, they were washed with chilled PBS and centrifuged again at 500 g for 5 minutes. The 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 500 g for 10 minutes using a refrigerated centrifuge. After discarding the supernatant, the nuclear pellet was resuspended in a transferase reaction mixture (25 μL of 2× TD buffer, 2.5 μL of transferase, 22.5 μL of nuclease-free water). The transferase reaction was carried out at 37 °C for 30 minutes. Immediately after the transferase, the samples were purified using the MinElute kit (QIAGEN, 28004). After purification, the library fragments were amplified using 1× NEBnext PCR Master Mix and 1.25 μM custom Nextera PCR primers. To reduce GC and size biases in PCR, the PCR reaction was monitored using qPCR and amplification was stopped before saturation. For this qPCR, after amplifying the complete library for 5 cycles, 5 μl was taken from the PCR reaction product and 10 μl of a PCR mixture containing Syber Green at a final concentration of 0.6× was added. This reaction was carried out for 20 cycles to determine the number of additional cycles required for the remaining 45 μL of the reaction. The library was purified using the MinElute kit. Finally, by using dual-size selection with SPRIselect (BECKMAN COULTER, B23317), fragments larger than 1000 bp and smaller than 100 bp were removed. Sequencing of the library was performed using HiSeq X Ten (Illumina). Adapter sequences were removed using the 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 / ). The Bam file was converted to a bigwig file using bamCoverage of deeptools 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".In a sample with N = 3, peaks detected by two replicates were defined as reliable peaks for further analysis. The coverage rate of each peak was calculated using featureCounts (Liao et al., 2014). To detect peaks with changes in accessibility due to the treatment or overexpression of transcription factors, a likelihood ratio test was performed using edgeR, and significance was judged by p < 0.01. Motifs enriched in the changed peaks were detected using HOMER (Heinz et al. 2010). The closest gene to each peak was detected using HOMER, and Metascape (Zhou et al., 2019) was used for gene ontology analysis of the listed genes. Visualization was performed using the original python script and deeptools. To infer the direct binding sites of Gata4 in the changed peaks, the overlap with publicly available Gata4 ChIP-seq (Hashimoto et al. 2019. GSM3067561) data of fibroblasts was analyzed.

[0151] (Cell culture and retroviral vector infection) To construct the pMXs retroviral vector, the coding region of Cre was amplified by PCR and subcloned into the pMXs vector for transfection of Plat-E cells using Fugene 6 (Promega, E2691) to generate retroviruses. The newly generated pMX-Cre vector was transduced into fibroblasts. After 24 hours of infection, the medium was replaced with DMEM / M199 (11150-059; Gibco) supplemented with 20% FBS, and the cells were cultured at 37°C and 5% CO2.

[0152] (Statistical analysis) Student's t-test was used for comparison between two groups, and one-way ANOVA and Turkey's or Dunnett's post hoc test were used for tests with three or more groups to study statistical significance. For tests with time-course changes such as echocardiogram, two-way ANOVA and Turkey's post hoc test were used. Differences between groups were considered significant when the p-value < 0.05. Statistical analysis was performed using GraphPad Prism software.

[0153] [Results] (Development of a labelable mouse capable of freely reprogramming cardiac fibroblasts) Crossbreed three types of mice, namely Tcf21 iCre mice, CAG-CAT-MGTH2A mice, and R26 tdTomato mice, to develop a direct cardiac reprogramming mouse, which is a triple-transgenic mouse capable of controlling direct cardiac reprogramming in cardiac fibroblasts in vivo. The Tcf21 iCre mouse is shown in Figure 1 , the CAG-CAT-MGTH2A mouse is shown in Figure 2 , and the R26 tdTomato mouse is shown in Figure 3 .

[0154] The Tcf21 iCre mouse is a mouse that expresses Cre protein with Tcf21 (transcription factor 21) specifically expressed in cardiac fibroblasts. Figure 1 The MeRCreMer in is a sequence formed by binding the modified estrogen receptor (MER: mutated estrogen receptor) to the Cre recombinase.

[0155] The CAG-CAT-MGTH2A mouse is a mouse that expresses the reprogramming factors MGTH (four factors of Mef2c, Gata4, Tbx5, and Hand2) when Cre is expressed. Figure 2 The MGTH in is arranged in the order of Mef2c, Gata4, Tbx5, and Hand2. CAG is a structure in which a cytomegalovirus enhancer is linked to a chicken β-actin promoter. CAT is chloramphenicol acetyltransferase.

[0156] The R26 tdTomato mouse is a mouse that expresses the fluorescent protein Tomato when Cre is expressed. Figure 3 The Rosa26 (R26) in is a gene region located on mouse chromosome 6, which can be easily inserted with genes and can constantly express the inserted protein. tdTomato (tandem dimer Tomato) is a sequence formed by tandemly connecting two red fluorescent proteins Tomato.

[0157] If tamoxifen is administered to the direct cardiac reprogramming mouse, Cre will be expressed in all cardiac fibroblasts, and the reprogramming factors and Tomato will be expressed. Therefore, with the administration of tamoxifen as an inducement, a part of the cardiac fibroblasts will become red fibroblasts, and a part will be induced into red cardiomyocytes. Using this mouse, the goal is to treat HFpEF and clarify the molecular biological mechanism.

[0158] (Confirmation of the expression of fluorescent proteins and reprogramming factors in cardiac fibroblasts) The heart of a mouse with direct reprogramming of cardiomyocytes was immunostained 1 week after administration of tamoxifen. The observation results of the heart immunostaining sections are shown in Figure 4 . Fibroblasts present in the myocardial interstitium were labeled red.

[0159] In addition, the heart of a mouse with direct reprogramming of cardiomyocytes was enzymatically treated 1 week after administration of tamoxifen to isolate cells. Subsequently, fluorescence protein Tomato-positive cardiac fibroblasts isolated from the mouse with direct reprogramming of cardiomyocytes were collected using FACS (Fluorescence activated cell sorter), and quantitative PCR was performed after RNA extraction. The results are shown in Figures 5 - 8 . The control group only expressed fluorescence protein Tomato and did not express reprogramming factors, which was the result of the RNA sample of Tomato-positive cells collected from Tcf21 iCre / R26 Tomato mice.

[0160] From Figures 5 - 8 it can be confirmed that if tamoxifen is administered to a mouse with direct reprogramming of cardiomyocytes, Cre, four reprogramming factors, and Tomato are expressed in the overall cardiac fibroblasts.

[0161] It should be noted that in the Figures 5 - 8 , Figure 18 , Figure 20 , Figure 22 , Figure 24 , Figure 26 , Figure 29 , Figure 43 , the right figures of Figure 48 , Figure 49 , Figure 51 , Figure 52 , the dots represent the individual values of the samples.

[0162] (Study on the establishment of HFpEF model and treatment effect) Using mice with direct reprogramming of cardiomyocytes, a study on the establishment of HFpEF model and treatment effect was conducted. The HFpEF model was established by continuously providing free drinking water of L-NAME and a High Fat Diet. For the treatment group, mice with direct reprogramming of cardiomyocytes were used, and for the control group, R26 tdTomato mice that only expressed fluorescence protein were used. A comparison was made between the control group on a normal diet and the HFpEF-loaded group, and the effect of the reprogramming treatment group that started reprogramming after the onset of HFpEF was studied. Subsequently, the normal diet control group was denoted as "Normal diet", the HFpEF-loaded control group was denoted as "HFpEF", and the HFpEF-loaded direct reprogramming of cardiomyocytes group was denoted as "Reprogramming". The experimental protocol is as shown in Figure 9As shown. The reprogramming gene expression in the reprogramming group given the agent tamoxifen was carried out under the condition of continuous HFpEF load. The reprogramming genes were continuously expressed after administration of tamoxifen.

[0163] The measurement results of systolic blood pressure in the normal diet group, HFpEF group, and reprogramming group are shown in Figure 10 , and the measurement results of body weight are shown in Figure 11 . "ns" indicates non-significant, and " " indicates p < 0.0001. In the HFpEF group and the reprogramming group, systolic blood pressure and body weight increased equally, and no significant differences were found between the two groups.

[0164] The observation results of the time-course contraction of the left ventricle in echocardiogram (M-mode) are shown in Figure 12 . In addition, the observation results of the mitral valve orifice blood flow velocity waveform using echocardiogram (Doppler type) are shown in Figure 13 . The waveform is usually composed of two wave peaks. The first half is the E wave (early diastole), and the second half is the A wave (atrial contraction). The ratio of the E wave height to the A wave height, that is, E / A, is an index representing left ventricular diastolic function. In mice, the higher it rises, the worse the diastolic function. In addition, E / E', which is the E wave height divided by the early diastolic velocity E' of the mitral annulus in tissue Doppler, also represents left ventricular diastolic function. The higher the E / E' value, the lower the left ventricular diastolic function.

[0165] The time-course changes of ejection fraction (EF), E / A, and E / E' are shown respectively in Figure 14 , Figure 15 , Figure 16 . "ns" indicates non-significant. As Figure 14 shown, it can be seen that there is no decrease from the EF baseline in all groups. In addition, as Figure 15 and Figure 16 shown, due to HFpEF load, the left ventricular diastolic function in the HFpEF group and the reprogramming group deteriorated, but improvement was shown in the reprogramming group compared with the HFpEF group starting from the administration of the agent.

[0166] (Improvement of diastolic function based on reprogramming) The results of the catheter examination of mice 15 weeks after the start of the experiment are shown in Figures 17 - 20 . Figure 17 represents the left ventricular pressure waveform, and the arrow indicates EDP (End diastolic pressure: left ventricular end-diastolic pressure). Figure 18 is a graph showing the value of EDP in a bar chart. The increase in EDP is the gold standard for indicating heart failure. Figure 19Represents the PV loop (pressure-volume loop) waveform. The arrow indicates the curve of the EDPVR (End-diastolic pressure-volume relationship). The slope of this curve reflects the left ventricular diastolic function. The steeper the slope, the greater the pressure increase relative to the volume increase, indicating poorer left ventricular diastolic function. Figure 20 Is a graph showing the values of EDPVR in a bar chart. In Figure 18 and Figure 20 ,"ns" indicates not significant, " " indicates p < 0.01, " " indicates p < 0.001, " " indicates p < 0.0001.

[0167] As Figure 18 and Figure 20 show, in the HFpEF group, EDP and EDPVR were significantly higher than those in the normal diet group. On the other hand, in the reprogramming group, EDP and EDPVR were improved.

[0168] (Improvement in exercise tolerance based on reprogramming) Evaluated the exercise tolerance of mice at 15 weeks after the start of the experiment. Figure 21 Shows the situation of the evaluation using a mouse treadmill. The mouse walks on an inclined conveyor belt, and the speed is gradually increased over time. The exercise tolerance is evaluated by the total walking distance.

[0169] Figure 22 Shows the walking distances of each group. Figure 22 In it, " " indicates p < 0.01, " " indicates p < 0.0001. As Figure 22 shows, the reduced walking distance in the HFpEF group was improved in the reprogramming group, and the improvement effect of exercise tolerance based on reprogramming treatment was confirmed. This indicates that the cardiac diastolic ability was improved in the reprogramming group, and as a result, the exercise tolerance was improved.

[0170] (Evaluation of myocardial induction efficiency) Evaluated the myocardial induction efficiency by immunostaining of mouse heart sections at 15 weeks after the start of the experiment. The immunostaining results are shown in Figure 23 . As Figure 23 shows, in the reprogramming group, the expression of myocardial-specific proteins was observed in some Tomato-positive cells, confirming the regeneration into cardiomyocytes. In addition, it was found that in the reprogramming group, clear cross striations were formed in the cells co-expressing Tomato and α-actin (αAct).

[0171] The results of the proportion of cells that induced fibroblasts to cardiomyocytes quantitatively are shown in Figure 24 . "ns" indicates not significant, " " indicates p < 0.01. As Figure 24 shown, in the reprogramming group, approximately 1% of fibroblasts were reprogrammed into cardiomyocytes.

[0172] (Improvement of cardiomyocyte hypertrophy based on reprogramming) Figure 25 The upper part of

[0173] Figure 26 shows the macroscopic image of the heart 15 weeks after the start of the experiment. The lower part shows the immunostaining image of the heart section 15 weeks after the start of the experiment. By using wheat germ agglutinin (WGA), the cell membrane is specifically stained green, thus clarifying the outline of cardiomyocytes and enabling the measurement of the cross-sectional area of cardiomyocytes. In the macroscopic image of the heart, it can be seen that cardiac hypertrophy occurred in the HFpEF group. On the other hand, cardiac hypertrophy was improved in the reprogramming group. In addition, in the immunostaining image, it can be seen that the size of cardiomyocytes increased in the HFpEF group. On the other hand, the size of cardiomyocytes was improved in the reprogramming group. " indicates p < 0.001, " " indicates p < 0.0001. Regarding the heart weight, which is an index of cardiac hypertrophy, in order to reduce the individual differences of each mouse, it was corrected with the femur length. As Figure 26 shown, in the HFpEF group, the heart weight increased significantly. On the other hand, in the reprogramming group, the increase in heart weight was improved.

[0174] Figure 27 shows the measurement results of the cardiomyocyte area. " " indicates p < 0.0001. The cross-sectional areas of randomly selected cardiomyocytes, about 100 cells in each individual, were measured and presented as violin plots. Overall, the cross-sectional area of cardiomyocytes in the HFpEF group increased. On the other hand, the increase in area was improved in the reprogramming group.

[0175] As one of the causes of reduced cardiac diastolic function, cardiomyocyte hypertrophy can be cited, but Figures 25 - 27 the results show that the inhibition of cardiomyocyte hypertrophy is related to the improvement of cardiac diastolic function.

[0176] (Reduction of fibrosis based on reprogramming) The Sirius red staining results of the heart sections at 15 weeks after the start of the experiment are shown in Figure 28 . By Sirius red staining, the fibrotic area is stained red and the normal tissue is stained yellow. For example, Figure 28The arrows in it indicate the fibrotic regions. In HFpEF, it was determined that some interstitial and perivascular fibrotic lesions deteriorated. On the other hand, the fibrotic regions decreased in the reprogramming group.

[0177] The area of the fibrotic region was analyzed using software, that is, the ratio of the fibrotic area after red staining to the area of the entire observed tissue section, and the results are shown in Figure 29 ." " indicates p < 0.01, " " indicates p < 0.001, " " indicates p < 0.0001. In the reprogramming group, the fibrotic area was significantly reduced compared with the HFpEF group.

[0178] For HFpEF model mice, it was determined that HFpEF was improved due to the improvement of cardiac hypertrophy and the reduction of fibrosis if direct reprogramming of cardiac fibroblasts was performed. Observing the details of direct reprogramming showed that approximately 1% of the fibroblasts activated by the disease were induced into cardiomyocytes. On the other hand, the changes in the whole heart were diffuse, and the following hypothesis was proposed: In addition to the 1% myocardial induction, the therapeutic effect during reprogramming also originated from the quiescence of the remaining 99% of the fibroblasts that were not induced into cardiomyocytes. Further analysis was performed on the antifibrotic effect that direct reprogramming of the myocardium could have.

[0179] (scRNA-seq) The results of single-cell RNA sequencing (scRNA-seq) of non-myocardial cells in the heart are shown in Figure 30 . One dot represents the information of one cell, and according to different gene expression patterns, they are classified into multiple clusters. Cell types were identified by confirming representative gene expression in each cluster. It can be seen from Figure 30 that non-myocardial cells in the heart including fibroblasts can be collected equally.

[0180] Figure 31 is a graph analyzing the ligand-receptor interactions between the cell clusters shown in Figure 30 . The lines in Figure 31 indicate the existence of cell-cell interactions, and the thicker the line, the stronger the interaction. It can be seen from Figure 31 that cardiac fibroblasts have a great influence on other cells, and the interaction between fibroblasts themselves is also strong. Therefore, detailed analysis was performed on the fibroblasts that had undergone gene expression therapy intervention.

[0181] (Improvement of pathological gene expression changes in fibroblasts based on reprogramming) The results obtained by separately extracting and analyzing the gene expression changes of only the fibroblast group are shown in Figure 32 .Figure 32 It is a graph with the vertical axis showing an inclusive score of morbid genes whose expression increases in HFpEF compared with the normal diet group, presented as a central violin plot. In the reprogramming group, the changes in the expression of morbid genes that increase in HFpEF were improved.

[0182] The expression of a representative genomic set activated during heart injury was made into a heatmap and shown in Figure 33 It can be seen that in the reprogramming group, the genomic sets related to a large amount of fibrosis, the expression related to heart hypertrophy signals such as Il6 and Tgfb, etc. were improved.

[0183] (ATAC-seq) To analyze the effect of the reprogrammed genes on the treatment effect, ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) was performed. In this analysis, the Tn5 transposase was used to specifically fragment the open chromatin regions, and at the same time, a tagged sequence library was constructed. By this method, the open / closed state of chromatin can be evaluated, and the upstream control mechanism of gene expression can be inferred.

[0184] The experimental procedure of the ATAC-seq analysis is shown in Figure 34 and Figure 35 As Figure 34 shown, ATAC-seq was used for evaluating the treatment mechanism. It was evaluated using two groups, the HFpEF group and the reprogramming group. At 5 weeks from the administration of tamoxifen (10 weeks from the start of the experiment), Tomato-positive fibroblasts were collected from the hearts of mouse organisms and analyzed. Additionally, as Figure 35 shown, in order to further explore the role of the direct binding of the reprogramming factors, an analysis integrating the publicly available ChIP-seq data was also performed. ChIP-seq (Chromatin Immunoprecipitation Sequencing) is a method of immunoprecipitating the complex of DNA and transcription factors with an antibody, sequencing the precipitated DNA fragments, and analyzing which regions of the DNA interact with the transcription factors.

[0185] The intensity of the signals in the regions with significant differences in the peak waveform height in ATAC-seq (ATAC peak changes) was made into a heatmap and shown in Figure 36。Both the HFpEF group and the reprogramming group had n = 3. In the ATAC peak changes, there were 1,890 peaks in the open regions where the peaks became higher and 1,045 peaks in the closed regions where the peaks became lower in the reprogramming group. In each region, when performing motif analysis, that is, analyzing to what extent the known gene sequences that can bind transcription factors are concentrated, the Gata4 binding motif was confirmed in both the open / closed regions. No motifs of Mef2c, Tbx5, and Hand2 were found, suggesting that Gata4 among the reprogramming factors might be an important factor affecting the treatment effect.

[0186] In the ATAC-peak changes analyzed in this study, two regions classified as having significant peaks (Gata4-dependent) or no significant peaks (Gata4-independent) in the publicly available fibroblast Gata4 ChIP-seq data were analyzed. Figure 37 The heights of all the peaks were aggregated and represented separately as Gata4-dependent and Gata4-independent.

[0187] Figure 38 The intensity of each peak signal was represented by a heatmap. In the ATAC peak change, there were 561 Gata4-dependent peaks and 2,374 Gata4-independent peaks.

[0188] GO analysis of the genes near each peak was performed. GO analysis (gene ontology analysis) is an analysis that annotates gene functions by focusing on the known biological processes and molecular functions of the input genome. The analysis results are shown in Figure 39 。As Figure 39 shown, it can be seen that they are all genomes related to fibrosis. This indicates that Gata4 is involved in the treatment effect both directly and indirectly.

[0189] (Production of Genetically Modified Mice Expressing Only a Single Reprogramming Factor) Based on the experimental results so far, it was investigated whether HFpEF treatment using a single reprogramming factor (Singlefactor) could be carried out. Figure 40 This is an overview of the genetically modified mice. Four systems of single-factor mice were produced, each with a gene in which a STOP sequence flanked by loxp sequences, Gata4, Mef2c, Tbx5, or Hand2 was arranged downstream of the CAG promoter. In addition to these four single-factor mice, the previously used cardiomyocyte direct reprogramming mice were named 4F mice and analyzed simultaneously.

[0190] Fetal fibroblasts were collected from these genetically modified mice, and the expression of each target factor was induced by forced Cre expression on the cell dish using the retrovirus pMx-Cre, and the resulting changes were analyzed. Figure 41This is a graph showing the results of infecting fetal fibroblasts (wild-type, fibroblasts from single-factor mice of each of the 4 systems, and 4F mice) with pMx-Cre, extracting RNA 1 week later, and performing quantitative PCR. It can be seen that all factors of Gata4, Mef2c, Tbx5, and Hand2 in 4F are forcibly expressed. On the other hand, in the single-factor groups, only each factor is forcibly expressed. Importantly, as indicated by the arrows, only the expression of each factor is enhanced, and the expression of other factors is not enhanced endogenously.

[0191] Figure 42 This is a graph showing the results of infecting fetal fibroblasts with pMx-Cre, extracting RNA 1 week later, and performing quantitative PCR. Quantitative PCR was performed on Col1a2, Fn1, and Postn, which are well-known as fibrosis genes. Compared with wild-type fibroblasts, only Gata4 single factor and 4F as indicated by the arrows showed a decrease in all these fibrosis genes.

[0192] Figure 43 This is a FACS of fetal fibroblasts 1 week after infection with pMx-Cre, immunostaining for cTnT (troponin T), which is a myocardial-specific protein, and observing the positive rate. Only 4F was positive for cTnT intracellularly, indicating that including the Gata4 single factor, induction into cardiomyocytes does not occur when only a single factor is expressed. The " " in the figure indicates p < 0.01.

[0193] It has been reported that in the case of direct cardiac reprogramming using more than 4 reprogramming factors, compared with reprogramming based on vectors carrying each reprogramming factor, reprogramming based on a polycistronic vector carrying multiple reprogramming factors as 1 gene can achieve high-quality cardiac regeneration (Kohei Inagawa et al., Circ Res. 2012 Oct 12; 111(9):1147-56). According to this report, for highly efficient treatment, it is necessary to uniformly introduce more than 4 genes.

[0194] On the other hand, during gene therapy, the sizes of genes that can be carried by safe vectors clinically applicable to humans have also been reported (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 above four or more reprogramming factors are larger than the sizes of genes that can be carried by safe vectors clinically applicable to humans described in these reports, and the technological development of a safe polycistronic vector capable of uniformly introducing four or more genes has not been achieved.

[0195] Based on such a background technology, the development of a therapy based on a single gene with a size that can be carried by a safe vector is expected, and thus, a study on the therapeutic effect of HFpEF under one reprogramming factor was conducted.

[0196] (Study on the therapeutic effect of HFpEF under a single factor of Gata4) To study the therapeutic effect of HFpEF under a single factor of Gata4, experiments were conducted in mice. Figure 44 Shows an overview of the genetically modified mice used. Tcf21 mice that express Cre in a heart fibroblast-specific manner were used, and were crossed with single factor mice that express a reprogramming single factor or 4F mice that express four reprogramming factors (MGTH) when Cre is expressed. To confirm that in addition to Gata4, the single factor mice are not only affected by the expression of simple factors, Mef2c single factor mice, which are reported to be important reprogramming factors in myocardial induction, were also used in the experiment. In the mice produced, a single factor was specifically expressed in heart fibroblasts. iCre Shows the experimental protocol. In two groups, a control group (Ctrl) based on a normal diet and a HFpEF-loaded group (Ctrl-HF), mice with only the gene expression of Tcf21 were used. Tcf21 / Single factor (Gata4 or Mef2c) mice (SF-HF (Gata4-HF or Mef2c-HF)) and Tcf21 / 4F mice (4F-HF) were both continuously loaded with HFpEF. Factor-based therapy was carried out by administering tamoxifen at 5 weeks from the start of the experiment and analyzed at a total of 10 weeks.

[0197] Figure 45 Shows the experimental protocol. In two groups, a control group (Ctrl) based on a normal diet and a HFpEF-loaded group (Ctrl-HF), mice with only the gene expression of Tcf21 were used. iCre Tcf21 iCre / Single factor (Gata4 or Mef2c) mice (SF-HF (Gata4-HF or Mef2c-HF)) and Tcf21 iCre / 4F mice (4F-HF) were both continuously loaded with HFpEF. Factor-based therapy was carried out by administering tamoxifen at 5 weeks from the start of the experiment and analyzed at a total of 10 weeks.

[0198] Figure 46 Shows the time-course changes in body weight, systolic blood pressure (SBP), and diastolic blood pressure (DBP) as HFpEF load indicators. The horizontal axis represents weeks. "ns" indicates not significant. It can be seen that there are no differences in body weight, SBP, and DBP in the group with HFpEF load.

[0199] Figure 47 Shows the results of echocardiography. "ns" indicates not significant, " " indicates p < 0.01. EF (ejection fraction), as an index of cardiac contractility, did not change in all groups. E / A and E / E', as indices of left ventricular diastolic function, deteriorated in all groups receiving HFpEF load. On the other hand, Gata4-HF and 4F-HF showed improvement starting from the administration of the drug. Importantly, single Mef2c did not show significant improvement.

[0200] Figure 48 Shows the treadmill test walking distance (m) as an index of exercise tolerance at 10 weeks after the start of the experiment. " " indicates p < 0.05, " " indicates p < 0.01. Exercise tolerance decreased with HFpEF load, but significant improvement was shown in Gata4-HF and 4F-HF.

[0201] Figure 49 Shows the cardiac catheterization results at 10 weeks after the start of the experiment. " " indicates p < 0.05, " " indicates p < 0.01. LVEDP (Left ventricular end-diastolic pressure), as an index of heart failure, and EDPVR (End-diastolic pressure-volume relationship), as an index of left ventricular diastolic function, deteriorated due to HFpEF load. On the other hand, only the two groups of Gata4-HF and 4F-HF showed significant improvement.

[0202] The Sirius red staining results of cardiac sections at 10 weeks after the start of the experiment are shown in Figure 50 . In Ctrl-HF and Mef2c-HF, an increase in the fibrotic area was confirmed compared to Ctrl. On the other hand, the fibrotic area decreased in Gata4-HF and 4F-HF.

[0203] The figure analyzing the fibrotic area is shown in Figure 51 ". " " indicates p < 0.05, " " indicates p < 0.01. The fibrotic area increased by HFpEF was significantly improved in Gata4-HF and 4F-HF.

[0204] The mouse hearts at 10 weeks from the start of the extraction experiment were taken out, RNA was extracted from both ventricles, and the results of quantitative PCR are shown in Figure 52 . The gene expressions of Col1a1 and Col3a1 indicating cardiac fibrosis, Nppb as an indicator of heart failure, and Tgfb1 as a representative of fibrotic and hypertrophic signals increased under HFpEF load. These gene expressions were improved in Gata4-HF and 4F-HF. On the other hand, no improvement was confirmed in Mef2c-HF.

[0205] 〔Summary〕 The figure summarizing the results of the examples is shown in Figure 53 . Activation of fibroblasts was confirmed in HFpEF, and it was found that if direct reprogramming was performed, 1% of the fibroblasts were regenerated into cardiomyocytes. Moreover, considering the results of the latest gene expression analysis, it was found that the remaining 99% of the fibroblasts that were not regenerated into cardiomyocytes played an anti-fibrotic role, and they comprehensively improved HFpEF. According to the analysis under a single factor, cardiomyocyte regeneration requires the GMTH4 factor as known. On the other hand, the therapeutic effect of anti-fibrosis is possible even with a single Gata4, showing improvement in the diastolic function of the heart in HFpEF. This example shows that gene therapy based on a single Gata4 can be a treatment means for HFpEF.

[0206] Industrial Applicability The present invention can be used for treatments such as gene therapy for heart failure (especially HFpEF), etc.

Claims

1. A cardiac diastolic function improver, characterized in that, A polynucleotide encoding the reprogramming factor polypeptide Gata4.

2. The diastolic function improving agent according to claim 1, wherein, The reprogramming factor polypeptide Gata4 comprises an amino acid sequence having at least 90% amino acid sequence identity with the amino acids shown in SEQ ID NO: 1 or SEQ ID NO:

3.

3. The 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 sequences shown in SEQ ID NO: 2 or SEQ ID NO:

4.

4. The cardiac diastolic function improver according to any one of claims 1 to 3, wherein, The diastolic function improving agent is a heart failure improving agent.

5. The diastolic function improving agent according to claim 4, wherein, The heart failure is heart failure with preserved ejection fraction, i.e., HFpEF.

6. A cardiac diastolic function improver, characterized in that, Comprising a factor that upregulates the expression of the Gata4 gene.

7. A diastolic function improving agent for the heart, characterized in that, Comprising a factor that increases the expression level of the Gata4 gene in cardiac fibroblasts.

8. A screening method for a cardiac diastolic function improver, characterized in that, Including the following steps: A contacting step, in which a test substance is contacted with fibroblasts; and An evaluating step, in which the expression of the Gata4 gene in fibroblasts is evaluated.

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