Interaction of HILPDA and PHB1 and application thereof

Abstract

The invention relates to an interaction between HILPDA and PHB1 and an application of the HILPDA and PHB1. Specifically, the invention provides application of HILPDA as a target spot in screening potential substances for preventing or treating myocardial contraction function impairment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the interaction between HILPDA and PHB1 and their applications. Background Art

[0002] Dilated cardiomyopathy (DCM), as one of the most common types of cardiomyopathy, is a major cause of heart failure. If not intervened in the early stage of the disease, irreversible pathological changes such as myocardial cell death and fibrosis will occur in patients, and ultimately lead to arrhythmia, heart failure and sudden cardiac death. Although some influencing factors are known, the current clinical treatment for dilated cardiomyopathy is still limited to improving symptoms, reducing complications and preventing sudden death, and there is no effective radical cure. Therefore, further exploring the mechanism of the occurrence and development of dilated cardiomyopathy and finding new therapeutic targets have become the current research hotspots.

[0003] Hypoxia-inducible Lipid Droplet-Associated protein (HILPDA), formerly known as Hypoxia-Inducible Gene 2 / HIG-2, was cloned from human cervical epithelial cell cDNA in a hypoxic environment by Denko N. et al. The HILPDA protein is only 7 kDa. It has been found that it can bind to the extracellular domain of the FZD10 receptor as an autocrine growth factor to activate the pro-cancer activity of the Wnt pathway; subsequently, it has been identified as a poor prognostic biomarker for pancreatic cancer, glioma, uterine and ovarian cancer, lung adenocarcinoma, etc. After 2010, due to multiple studies supporting its expression in subcellular organelles such as the cytoplasm or lipid droplets and its participation in the regulation of lipid droplet formation and lipid metabolism, it was named hypoxia-inducible lipid droplet-associated protein. So far, no study has clarified the regulatory function of the lipid droplet-associated protein HILPDA in cardiomyocytes. Whether the change in the expression level of the HILPDA protein will cause abnormal myocardial function remains to be further studied. Summary of the Invention

[0004] The present invention first provides an hESC cell with down-regulated expression or activity of HILPDA.

[0005] In one or more embodiments, the down-regulation of the expression or activity of HILPDA is achieved by any of the following groups of methods: (1) Introducing siRNA specifically targeting HILPDA or its expression vector, antisense RNA or its expression vector, ribozyme or its expression vector, gene editing vector, such as a CRISPR gene editing vector or a TALEN gene editing vector, into hESC cells; (2) introducing an anti-HILPDA antibody or its expression vector into hESC cells; (3) introducing a small molecule inhibitor into hESC cells; (4) introducing a homologous recombination vector containing a nucleotide sequence encoding a mutant inactive or attenuated HILPDA variant into hESC cells.

[0006] In one or more embodiments, (1) the siRNA, antisense RNA, or gene editing vector targets the second exon of the HILPDA-encoding gene. The second exon is nucleotides 33 - 1250 of the genomic sequence of HILPDA.

[0007] In one or more embodiments, the CRISPR gene editing vector expresses a gRNA as shown in SEQ ID NO:1.

[0008] In one or more embodiments, step (1) results in a deletion mutation at amino acids 1 - 172 of the wild-type amino acid sequence of HILPDA.

[0009] In one or more embodiments, step (1) results in a mutation at amino acids 16 - 18 of the wild-type amino acid sequence of HILPDA. In one or more embodiments, the mutation is a deletion, substitution, or insertion mutation.

[0010] In one or more embodiments, the inactive or attenuated HILPDA variant is selected from: an HILPDA variant with a deletion mutation at amino acids 1 - 172 of the wild-type amino acid sequence of HILPDA, and / or an HILPDA variant with a mutation at amino acids 16 - 18 of the wild-type amino acid sequence of HILPDA; the mutation is, for example, a deletion, substitution, or insertion mutation.

[0011] The present invention also provides a cardiomyocyte with downregulated expression or activity of HILPDA, which is differentiated from hESC cells according to any one of the embodiments herein.

[0012] In one or more embodiments, the differentiation step includes: (1) culturing hESC cells in a medium containing a WNT agonist, (2) culturing hESC cells in a medium containing a WNT antagonist, (3) culturing hESC cells in a medium without a WNT agonist and a WNT antagonist.

[0013] In one or more embodiments, the WNT agonist includes CHIR-99021.

[0014] In one or more embodiments, the concentration of the WNT agonist is 1 - 20 mM, preferably 3 - 15 mM, more preferably 10 - 15 mM, such as 12 mM.

[0015] In one or more embodiments, the WNT antagonist includes IWR-1.

[0016] In one or more embodiments, the concentration of the WNT agonist is 1 - 20 mM, preferably 3 - 15 mM, more preferably 4 - 10 mM, such as 5 mM.

[0017] In one or more embodiments, the culture medium in step (1) and / or (2) is selected from DMEM / F12, RPMI1640, and GlutaMAX. In one or more embodiments, the culture medium contains B-27. In one or more embodiments, the culture medium does not contain insulin.

[0018] In one or more embodiments, the culture in step (1) is for at least 1 day or at least 2 days.

[0019] In one or more embodiments, the culture in step (2) is for at least 1 day or at least 2 days.

[0020] In one or more embodiments, the culture medium in step (3) is DMEM / F12, RPMI1640, and GlutaMAX. In one or more embodiments, the culture medium contains BSA, and its concentration is preferably 10 - 1000 g / ml. In one or more embodiments, the culture medium contains ascorbic acid, and its concentration is preferably 10 - 500 g / ml.

[0021] In one or more embodiments, the method includes the steps of: (1) Culturing hESC cells for 1 - 2 days in RPMI1640 medium supplemented with B-27 but without insulin and containing 1 - 20 mM CHIR-99021; (2) Culturing the cells obtained in (1) for 1 - 2 days in RPMI1640 medium containing B-27 and without insulin; (3) Culturing the cells obtained in (2) for 1 - 2 days in RPMI 1640 medium supplemented with B-27 but without insulin and containing 1 - 20 mM IWR-1; (4) Treating the cells obtained in (3) (for example, for at least 1 day) with a culture medium containing BSA (such as 500 g / ml) and ascorbic acid (such as 213 g / ml) to obtain cardiomyocytes.

[0022] In one or more embodiments, the hESCs are obtained by the following method: culturing hESCs using a culture medium (selected from DMEM / F12, RPMI1640, GlutaMAX, and mTeSR-1), and adhering them to the surface of a medium (such as a culture plate coated with Matrigel). When the cell density reaches at least 70%, subculture is performed using a cell digestive solution (such as).

[0023] The present invention also provides a nucleic acid construct comprising a promoter and a nucleic acid sequence encoding HILPDA.

[0024] In one or more embodiments, the amino acid sequence of HILPDA is as shown in SEQ ID NO:2.

[0025] In one or more embodiments, the nucleic acid sequence of HILPDA is as shown in SEQ ID NO:3.

[0026] In one or more embodiments, the promoter is a CMV promoter or the original promoter of HILPDA on the genome.

[0027] In one or more embodiments, the nucleic acid construct further comprises a terminator.

[0028] In one or more embodiments, the nucleic acid construct further comprises a tag sequence, such as a FLAG sequence.

[0029] In one or more embodiments, the nucleic acid construct is a recombinant vector, a cloning vector, or an expression vector.

[0030] The present invention also provides a host cell comprising the nucleic acid construct described in any one of the embodiments herein.

[0031] In one or more embodiments, the cell is a cardiomyocyte, such as the human cardiomyocyte AC16.

[0032] The present invention also provides a method for reducing the interaction between HILPDA and PHB1 or a method for reducing the transfer of PHB1 to mitochondria, comprising:

[0033] (1) causing a deletion mutation in amino acids 1-172 of the wild-type amino acid sequence of HILPDA, and / or

[0034] (2) mutating amino acids 16-18 of the wild-type amino acid sequence of HILPDA, and / or

[0035] (3) mutating amino acids 14-16 of the wild-type amino acid sequence of PHB1.

[0036] In one or more embodiments, the mutation is a deletion, substitution, or insertion mutation.

[0037] In one or more embodiments, the wild-type amino acid sequence of HILPDA is as shown in SEQ ID NO:2.

[0038] In one or more embodiments, the wild-type amino acid sequence of PHB1 is as shown in SEQ ID NO:4.

[0039] The present invention also provides the use of HILPDA as a target in screening for potential substances for preventing or treating impaired myocardial contractile function, wherein a candidate substance that enhances the interaction between HILPDA and PHB1 is a potential substance for treating impaired myocardial contractile function.

[0040] In one or more embodiments, the candidate substance is a candidate substance that mutates HILPDA or PHB1, and the mutations include: (1) causing a deletion mutation in amino acids 1-172 of the wild-type amino acid sequence of HILPDA, and / or (2) mutating amino acids 16-18 of the wild-type amino acid sequence of HILPDA, and / or (3) mutating amino acids 14-16 of the wild-type amino acid sequence of PHB1.

[0041] In one or more embodiments, the wild-type amino acid sequence of HILPDA is as shown in SEQ ID NO:2.

[0042] In one or more embodiments, the wild-type amino acid sequence of PHB1 is as shown in SEQ ID NO:4.

[0043] In one or more embodiments, the impaired myocardial contractile function is dilated cardiomyopathy.

[0044] The present invention also provides a method for screening potential substances for preventing or treating impaired myocardial contractile function or its symptoms, including: (1) contacting a candidate substance with a system containing HILPDA and PHB1, and (2) detecting changes in the interaction between HILPDA and PHB1, wherein a substance that enhances the interaction between HILPDA and PHB1 is a potential substance for preventing or treating impaired myocardial contractile function. In one or more embodiments, the impaired myocardial contractile function is dilated cardiomyopathy.

[0045] In one or more embodiments, the symptoms are selected from one or more of the following: increased cardiomyocyte volume, disordered arrangement of myofilament structural proteins, weakened cardiomyocyte contractility, abnormal calcium recycling and release processes, enlarged ventricular cavity, thinning of the left ventricular wall, decreased cardiac function, increased fibrosis, increased mitochondrial fission in cardiomyocytes, obvious disappearance of cristae, swelling, and vacuolization, etc., changes in the number, size, shape, and structure of mitochondria, and changes in mitochondrial respiration, oxygen consumption, and energy metabolism.

[0046] The present invention also provides a method for promoting the aggregation of PHB1 in mitochondria, including expressing wild-type HILPDA in cells.

[0047] In one or more embodiments, the method includes: (1) introducing the nucleic acid construct described herein into the cell, and (2) incubating the cell under conditions of HILPDA expression.

[0048] In one or more embodiments, the nucleic acid construct is a recombinant vector, a cloning vector, or an expression vector.

[0049] In one or more embodiments, the cell is a human cardiomyocyte cell line, such as AC16.

[0050] Advantages of the present invention: The applicant found that the deletion of HILPDA can lead to damage to cardiac function and pathological remodeling. There has been no report on the relationship between HILPDA and the occurrence and development of dilated cardiomyopathy. This study will provide a theoretical basis for HILPDA as a potential future therapeutic target for dilated cardiomyopathy. Brief Description of the Drawings

[0051] Figure 1 , mechanism hypothesis diagram.

[0052] Figure 2 , after knocking out the potential pathogenic gene HILPDA, the volume, expression of myofilament structural proteins, and myocardial contractility of hESCs-derived cardiomyocytes were all affected, but the efficiency of myocardial directed differentiation was not disturbed. A: Design a CRISPR-CAS9 system guide RNA targeting the second exon of HILPDA, and select two hESCs clones H7-15KO and H7-18KO with silenced HILPDA expression; B: Immunofluorescence staining shows that the cardiomyocytes differentiated from the HILPDA-KO-hESCs have an increased volume, and the distribution of myofilament structural proteins ACTN2 (green fluorescence) and cTNT (red fluorescence) is disordered; C: Flow cytometry shows that the proportion of cTNT-positive cardiomyocytes selected before and after HILPDA knockout has no significant difference; D: After HILPDA knockout, the contraction amplitude of cardiomyocytes is weakened and the contraction frequency becomes slower; E: Calcium transient results suggest that the calcium handling ability of cardiomyocytes is disordered after HILPDA knockout.

[0053] Figure 3 , Whole-transcriptome sequencing analysis of cardiomyocytes derived from HILPDA knockout hESCs. A: Volcano plot showing genes with significantly different expressions; B: Heatmap showing genes with significantly different expressions; C: GO analysis of enriched pathways of differentially expressed genes; D: Bubble plot showing enriched pathways of differentially expressed genes by KEGG analysis.

[0054] Figure 4 , The expression of HILPDA was significantly decreased in the myocardial tissues of patients with dilated cardiomyopathy and TNNT2-delK210 mice. A-B: Single-cell data showed that HILPDA was expressed in multiple cardiac cells; and it was significantly decreased mainly in cardiomyocytes of DCM patients; C: GEO data found that the expression level of HILPDA was significantly decreased in the myocardial tissues of patients with dilated cardiomyopathy.

[0055] Figure 5 , HILPDA global knockout mice showed phenotypes of dilated cardiomyopathy. A: Doppler ultrasound was used to monitor the changes in cardiac function of 7-week and 14-week HILPDA global knockout mice. The results showed that after 14 weeks of birth, the cardiac cavities of these mice were enlarged, and the ejection fraction EF and fractional shortening FS were significantly decreased; B: HE, Masson, and WGA staining showed that the cardiac cavities of 14-week KO mice were enlarged, the degree of fibrosis increased, and the volume of cardiomyocytes became larger.

[0056] Figure 6 , HILPDA knockout did not disrupt myocardial triglyceride metabolism and did not affect the morphology of lipid droplets, but mitochondrial damage occurred. A: The levels of triglyceride TG, free fatty acid FFA, and glycerol Glycerol in the myocardial tissues of HILPDA knockout mice did not change significantly; B: The expression levels of catalytic enzymes related to triglyceride metabolism in the myocardial tissues of HILPDA knockout mice; C: Transmission electron microscopy showed mitochondrial damage such as vacuolization, but there was no significant difference in the size of lipid droplets (indicated by black arrows); D: Statistical chart of lipid droplet area, showing no significant difference before and after HILPDA knockout; E: The ATP production in the myocardial tissues of HILPDA knockout was significantly reduced.

[0057] Figure 7 , HILPDA interacts with mitochondrial protein PHB1. A: Overexpression of HILPDA-Flag protein in the AC16 human cardiomyocyte cell line, and proteins interacting with HILPDA were screened by immunoprecipitation-mass spectrometry; B: Co-IP verified the interaction between the two; C-D: Interaction between truncated PHB1 and HILPDA; E: AlphaFold2 predicted the binding sites between HILPDA and the target protein.

[0058] Figure 8, HILPDA knockout affects the translocation of PHB1 to mitochondria. A: Before and after HILPDA knockout, the total expression level of PHB1 in cardiomyocytes did not change significantly; B: After HILPDA knockout, the expression of PHB1 in mitochondria decreased significantly; C: Ad-COX8a-GFP virus was used to trace mitochondria (red) in cardiomyocytes. After HILPDA knockout, the localization of PHB1 (green) in mitochondria decreased significantly.

[0059] Figure 9 , HILPDA regulates the translocation and aggregation of PHB1 to mitochondria in AC16 cells and affects the expression of respiratory chain complexes. A: The expression of HILPDA was interfered with by siRNA in AC16 cells; in the isolated mitochondrial proteins, the expression level of PHB1 decreased significantly after the expression of HILPDA was interfered; B: HILPDA was overexpressed in AC16 cells, and the expression changes of PHB1 in mitochondria and cytoplasm; C: The changes in the expression levels of mitochondrial respiratory chain complexes after HILPDA knockdown (AC16: siRNA) or overexpression (AC16: OE). Detailed implementation manners

[0060] In this article, the inventors used the CRISPR-Cas9 technology to construct models such as HILPDA knockout hESCs and HILPDA cardiomyocyte-specific knockout mice to study the regulation of cardiac function by HILPDA protein; and the method of regulating the expression level of PHB1 by regulating the expression level of HILPDA includes finding the binding sites of the interaction between HILPDA and PHB1.

[0061] The present invention first provides a cell with down-regulated expression or activity of HILPDA and / or PHB1. In this cell, the interaction between HILPDA and PHB1 is weakened, and the aggregation of PHB1 in mitochondria decreases. This cell can also be used as a cell for detecting mitochondrial function. In this article, cardiac mitochondrial function includes the stability and activity of the oxidative respiratory chain complex, mitochondrial respiratory function and energy metabolism, ATP generation concentration, and mitochondrial oxidative stress injury index.

[0062] Reducing the expression level or activity of HILPDA and / or PHB1 in a cell by a reagent that can reduce the expression and / or activity of HILPDA and / or PHB1. As used herein, a reagent that reduces the expression and / or activity of HILPDA and / or PHB1 protein includes administering an inhibitor of HILPDA and / or PHB1, which includes, but is not limited to, proteins, polypeptides, nucleic acids, and small molecule compounds. For example, the protein can be an anti-HILPDA antibody, preferably a monoclonal antibody. The nucleic acid can be siRNA or its expression vector, antisense RNA or its expression vector, ribozyme or its expression vector, and gene editing vectors, such as CRISPR-CAS9 gene editing vector or TALEN gene editing vector. In some embodiments, the sgRNA used in the CRISPR technique contains the sequence shown in SEQ ID NO:1. Methods for constructing gene editing vectors containing the sgRNA sequence are well known in the art. Small molecule compounds include small molecule compounds well known in the art that can inhibit the enzymatic activity of HILPDA and / or PHB1. In some embodiments, the reagent that inhibits the expression of HILPDA and / or PHB1 is a homologous recombination vector that contains a nucleotide sequence encoding a mutant inactive or hypoactive HILPDA variant and / or PHB1 variant, and knocks out the wild-type HILPDA and / or PHB1 gene in a host cell to express the mutant inactive or hypoactive HILPDA variant and / or PHB1 variant. Nonsense mutations or mutations that reduce the activity of HILPDA and / or PHB1 can be determined by those skilled in the art using conventional techniques, and these mutations can cause the loss or reduction of the activity of HILPDA and / or PHB1. Therefore, the protein activity can be inhibited by expressing such mutant HILPDA and / or PHB1 in the cells of a subject through homologous recombination technology.

[0063] In certain embodiments, the complete knockout of HILPDA and / or PHB1 genes can be achieved by administering a targeting vector, thereby inhibiting or reducing the expression of HILPDA and / or PHB1 genes. In other embodiments, the expression of HILPDA and / or PHB1 genes can be inhibited or its expression level can be reduced by administering siRNA of the HILPDA and / or PHB1 genes. In other embodiments, the expression of HILPDA and / or PHB1 proteins in a subject can be reduced by gene editing techniques, such as administering a reagent that uses ZFN, TALEN, or CRISPR technology to knockout or knockdown the HILPDA and / or PHB1 genes, such as sgRNA.

[0064] The present invention also provides a method for reducing the interaction between HILPDA and PHB1 or a method for reducing the transfer of PHB1 to mitochondria, including: (1) causing a deletion mutation in amino acids 1-172 of the wild-type amino acid sequence of HILPDA, and / or (2) causing a mutation in amino acids 16-18 of the wild-type amino acid sequence of HILPDA, and / or (3) causing a mutation in amino acids 14-16 of the wild-type amino acid sequence of PHB1. Herein, the mutation includes deletion, substitution or insertion mutation. The present invention also provides a method for increasing the transfer of PHB1 to mitochondria, including the step of enhancing the interaction between HILPDA and PHB1.

[0065] The present invention also provides a method for upregulating the expression or activity of HILPDA and / or PHB1 in cells. The expression level or activity of HILPDA and / or PHB1 in cells is reduced by a reagent that can increase the expression and / or activity of HILPDA and / or PHB1. For example, by introducing a nucleic acid construct capable of expressing HILPDA and / or PHB1 into the cells, the nucleic acid construct comprising a promoter and a nucleic acid sequence encoding HILPDA and / or PHB1. The interaction between HILPDA and PHB1 in the cells is enhanced, and the aggregation of PHB1 in mitochondria increases.

[0066] Herein, the protein sequence of wild-type HILPDA (NCBI: NM_001098786.2) has the sequence shown in SEQ ID NO: 2 or a variant having at least 80%, at least 90%, at least 95% or at least 99% sequence identity thereto. The nucleic acid sequence of wild-type HILPDA has the sequence shown in SEQ ID NO: 3 or a variant having at least 80%, at least 90%, at least 95% or at least 99% sequence identity thereto.

[0067] Herein, the protein sequence of wild-type PHB1 (NCBI: Gene ID 5245) has the sequence shown in SEQ ID NO: 4 or a variant having at least 80%, at least 90%, at least 95% or at least 99% sequence identity thereto. The nucleic acid sequence of wild-type PHB1 has the sequence shown in SEQ ID NO: 5 or a variant having at least 80%, at least 90%, at least 95% or at least 99% sequence identity thereto.

[0068] Variants of the amino acid sequences of the present invention have at least 95%, 96%, 97%, 98% or 99% identity with their source sequences and retain the biological functions of the source sequences (for example, the RD3L protein variants have the functions of the RD3L protein). Variants of the nucleic acid sequences of the present invention have at least 95%, 96%, 97%, 98% or 99% identity with their source sequences, and the amino acid sequences encoded by the variants of the nucleic acid sequences are the same as or different from but retain the biological functions of the amino acid sequences encoded by the source sequences (for example, the proteins encoded by the variants of the nucleic acid sequences of the RD3L protein are the same as or different from but have the functions of the RD3L protein). The sequence identity described in the present invention can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, such as BLASTP or BLASTN.

[0069] The full-length nucleic acid sequences of the present invention or fragments thereof can generally be obtained by PCR amplification, recombination or artificial synthesis. Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination. This is usually done by cloning them into a vector, then transferring them into cells, and then isolating the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0070] Then the DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. Methods well known to those skilled in the art can be used to construct recombinant vectors, see, for example, the techniques described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory), Ausubel et al. (1989, Short Protocols in Molecular Biology, Wiley) or other standard textbooks. Alternatively, the polynucleotide and the vector can be reconstituted into liposomes for delivery to target cells. Vectors containing the polynucleotides of the present invention can be transferred into host cells by well-known methods, which vary according to the type of cell host. For example, calcium chloride transfection is usually used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook et al. (see above).

[0071] As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a vast number of nucleic acids can be prepared, all of which encode the fusion polypeptides of the present invention. Thus, in the case where a specific amino acid sequence has been identified, those skilled in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. Accordingly, the present invention also relates to polynucleotides that hybridize to the above-described polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides described in the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0072] The nucleic acid construct may further comprise one or more regulatory sequences operably linked to the sequence. The polynucleotides described in the present invention can be manipulated in various ways to ensure the expression of the polypeptide or protein. The nucleic acid construct can be manipulated according to the difference or requirement of the expression vector before being inserted into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0073] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is generally operably linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3'-end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, the untranslated region of the mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5'-end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention.

[0074] The nucleic acid construct described herein can be an expression cassette, which, according to different expression situations, can consist of a CMV promoter, the coding sequence of the HILPDA protein, a terminator, and a FLAG sequence.

[0075] In certain embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a homologous recombination vector. The polynucleotides of the present invention can be cloned into many types of vectors, such as plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors can be used to provide the coding sequences of the fusion proteins of the present invention. Expression vectors can be provided to cells in the form of viral vectors. Generally, expression of the polynucleotides of the present invention is achieved by operably linking the polynucleotides of the present invention to a promoter and incorporating the construct into an expression vector. The vector can be suitable for replication and integration in eukaryotic cells. Typical expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Homologous recombination vectors are used to integrate the expression cassettes described herein into the host genome. Vectors that can effectively express proteins in mammalian cells, particularly hESCs and cardiomyocytes, are preferably used in the present invention.

[0076] Expression vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) generally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multiple cloning region for inserting the nucleic acid encoding the protein to be expressed, and optional marker elements.

[0077] The vector can optionally contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the fusion polypeptide; the oligonucleotide sequence encodes polyhistidine (such as 6His) or another "tag", such as FLAG, HA, or myc. This tag typically fuses with the polypeptide when the polypeptide is expressed and can serve as a means for affinity purification or detection of the protein from the host cell. Affinity purification can be accomplished, for example, by column chromatography using an antibody against this tag as an affinity matrix. The tag can optionally be subsequently removed from the purified protein by various means such as using certain peptidases for cleavage.

[0078] The flanking sequences can be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), chimeric (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Similarly, the source of the flanking sequences can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences function in the host cell machinery and can be activated by the host cell machinery.

[0079] Methods for introducing genes into cells and expressing genes in cells are known in the art. Polynucleotides can be easily introduced into host cells by any method in the art, such as mammalian, bacterial, yeast, or insect cells. Generally, polynucleotides can be transferred into host cells by physical, chemical, or biological means. Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing polynucleotides of interest into host cells include transfection using DNA, RNA vectors, viral vectors, or mRNA. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0080] As used herein, a host cell refers to a cell capable of receiving and accommodating the polynucleotides described herein. Ideal host cells should meet the two conditions of being easily accessible and proliferating. The "host cells" of the present invention can be prokaryotic cells and eukaryotic cells, including bacterial cells, yeast cells, insect cells, and mammalian cells, etc. The host cells are preferably various cells that are conducive to the expression of gene products or fermentation production, and such cells are well-known and commonly used in the art. The host cells can constitutively express the proteins described herein, can also express the proteins described herein under certain induction conditions, and can also specifically express the proteins described herein in different host cell types. Methods for enabling host cells to constitutively express, induce expression, or specifically express the proteins of the present invention are well-known in the art. In certain embodiments, the present invention provides a genetically modified cell (such as hESC or cardiomyocyte) that contains the polynucleotides described herein, or contains the nucleic acid constructs described herein, or is prepared by the methods described herein, or stably expresses the proteins described herein.

[0081] A gene knock-in vector is used to knock in the polynucleotide sequence described herein into the region of interest in the genome. Generally, in addition to the polynucleotide sequence, the gene knock-in vector may also contain a 5' homologous arm and a 3' homologous arm required for genomic homologous recombination. In certain embodiments, the nucleic acid construct herein contains a 5' homologous arm, the polynucleotide sequence described herein, and a 3' homologous arm. When using a gene knock-in vector, the CRISPR technique can be used simultaneously to homologous recombine the polynucleotide sequence to the desired position. The CRISPR technique guides the Cas9 nuclease to modify the genome at the insertion position by designing a guide RNA targeting the target gene, resulting in an increase in the homologous recombination efficiency of the gene modification region, and homologous recombining the target fragment contained in the gene knock-in vector to the target site.

[0082] The present invention also provides the use of HILPDA as a target in screening for potential substances for preventing or treating impaired myocardial contractile function, wherein a candidate substance that enhances the interaction between HILPDA and PHB1 is a potential substance for treating impaired myocardial contractile function. The candidate substance is a candidate substance that causes mutations in HILPDA or PHB1, and the mutations include: (1) causing a deletion mutation in amino acids 1-172 of the wild-type amino acid sequence of HILPDA, and / or (2) mutating amino acids 16-18 of the wild-type amino acid sequence of HILPDA, and / or (3) mutating amino acids 14-16 of the wild-type amino acid sequence of PHB1.

[0083] The present invention also provides a method for screening potential substances for preventing or treating impaired myocardial contractile function or its symptoms, comprising: (1) contacting a candidate substance with a system containing HILPDA and PHB1, and (2) detecting changes in the interaction between HILPDA and PHB1, wherein a substance that enhances the interaction between HILPDA and PHB1 is a potential substance for preventing or treating impaired myocardial contractile function. In one or more embodiments, the impaired myocardial contractile function is dilated cardiomyopathy.

[0084] In some embodiments, if the interaction between HILPDA and PHB1 in the test group is statistically higher (preferably significantly higher, such as more than 20% higher, more preferably more than 50% higher; even more preferably more than 80% higher) than that in the control group, it indicates that the candidate substance is a potential substance for preventing or treating impaired myocardial contractile function. The system selected from: cell systems (such as cells expressing HILPDA, PHB1), cell culture systems, subcellular systems, solution systems, tissue systems, organ systems or animal systems.

[0085] As used herein, the "solution system" is a liquid having the function of storing or preserving cells, for example, cell culture medium, which contains elements required for cell growth, including but not limited to amino acids, vitamins, carbohydrates, inorganic ions. The amino acids include essential amino acids: L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-valine; the vitamins include fat-soluble vitamins and water-soluble vitamins, such as vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, biotin, nicotinamide; the carbohydrates mainly include glucose, ribose, deoxyribose, sodium pyruvate and acetic acid, etc.; the inorganic ions include basic inorganic ions such as sodium, potassium, magnesium, calcium, phosphorus, etc.

[0086] "Cell cryopreservation solution" refers to a solution that can preserve cells for a long time without affecting the growth status of cells, and it includes cryoprotectants and culture media. "Cell preservation solution" is a solution that maintains and fixes the original morphological structure of cells and is composed of potassium chloride, sodium chloride, ethanol, anticoagulants, etc.

[0087] In this article, the symptoms of impaired myocardial contractile function are selected from one or more of the following: increased myocardial cell volume, disordered arrangement of myofilament structural proteins, weakened myocardial cell contractility, abnormal calcium recycling and release processes, enlarged ventricular cavity, thinning of the left ventricular wall, decreased cardiac function, increased fibrosis, increased myocardial mitochondrial fission, obvious disappearance of cristae, swelling and vacuolization, etc., changes in the number, size, shape and structure of mitochondria, and changes in mitochondrial respiration, oxygen consumption and energy metabolism.

[0088] The present invention also provides the use of a reagent for upregulating or downregulating the expression and / or activity of HILPDA and / or PHB1 in the preparation of a drug for treating impaired myocardial contractile function or its symptoms. The present invention also provides the use of a reagent for enhancing or weakening the interaction between HILPDA and PHB1 in the preparation of a drug for treating impaired myocardial contractile function or its symptoms. Reagents targeting HILPDA for reducing the expression and / or its activity applicable to the uses and methods described herein include various preparations described above. For example, siRNA or its expression vector, antisense RNA or its expression vector, ribozyme or its expression vector, gene editing vector such as CRISPR-CAS9 gene editing vector or TALEN gene editing vector that inhibit the expression and / or its activity of HILPDA; anti-HILPDA antibody or its expression vector; small molecule inhibitor; homologous recombination vector containing a nucleotide sequence encoding a mutant inactive or weakly active HILPDA variant. In a particularly preferred embodiment of the above uses and methods, the reagent includes the sgRNA shown in SEQ ID NO:1 or its expression vector. In a particularly preferred embodiment, the present invention provides the use of an sgRNA or its expression vector targeting HILPDA in the preparation of a drug for treating or preventing DCM.

[0089] The present invention provides an hESC cell with downregulated expression or activity of HILPDA. The present invention also provides a myocardial cell with downregulated expression or activity of HILPDA, and the myocardial cell is differentiated from an hESC cell with downregulated expression or activity of HILPDA.

[0090] Typically, hESCs need to be pre-treated to achieve a relatively high density and good cell viability for subsequent differentiation steps. The culture methods for pre-treatment are common knowledge in the art. Exemplarily, the hESCs are obtained by the following method: culturing hESCs using a culture medium (selected from DMEM / F12, RPMI1640, GlutaMAX, and mTeSR-1), and adhering them to the surface of a substrate (such as a culture plate coated with Matrigel). When the cell density reaches at least 70%, passage culture is performed using a cell digestive solution (such as).

[0091] The method for differentiating cardiomyocytes from hESC cells is a conventional technique in the art. Exemplary differentiation methods include (1) culturing hESC cells using a culture medium containing a WNT agonist, (2) culturing hESC cells using a culture medium containing a WNT antagonist, and (3) culturing hESC cells using a culture medium without a WNT agonist and a WNT antagonist. The concentration of the WNT agonist is 1-20 mM, preferably 3-15 mM, more preferably 10-15 mM, such as 12 mM. The concentration of the WNT antagonist is 1-20 mM, preferably 3-15 mM, more preferably 4-10 mM, such as 5 mM.

[0092] WNT agonists and antagonists have their ordinary meanings in the art. Exemplary WNT agonists include CHIR-99021, and exemplary WNT antagonists include IWR-1.

[0093] The culture medium in step (1) and / or (2) can be selected from any culture medium suitable for hESC cell culture or cardiomyocyte culture, such as DMEM / F12, RPMI1640, and GlutaMAX. In one or more embodiments, the culture medium contains B-27 and does not contain insulin. The culture in step (1) is for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days. The culture in step (2) is for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days.

[0094] The culture medium in step (2) can be selected from any culture medium suitable for hESC cell culture or cardiomyocyte culture, such as DMEM / F12, RPMI1640, and GlutaMAX. The culture medium can contain BSA, and its concentration is preferably 10-1000 g / ml. The culture medium can contain ascorbic acid, and its concentration is preferably 10-500 g / ml.

[0095] In one or more embodiments, a method for differentiating hESC cells into cardiomyocytes by downregulating the expression or activity of HILPDA described herein includes the steps of: (1) culturing hESC cells in RPMI 1640 medium supplemented with B-27 but without insulin and containing 1-20 mM CHIR-99021 for 1-2 days; (2) culturing the cells obtained in (1) in RPMI 1640 medium containing B-27 and without insulin for 1-2 days; (3) culturing the cells obtained in (2) in RPMI 1640 medium supplemented with B-27 but without insulin and containing 1-20 mM IWR-1 for 1-2 days; (4) treating the cells obtained in (3) with a medium containing BSA (e.g., 500 μg / ml) and ascorbic acid (e.g., 213 μg / ml) (e.g., for at least 1 day) to obtain cardiomyocytes.

[0096] The present invention also provides a pharmaceutical composition, comprising a pharmaceutically acceptable excipient and: HILPDA or its coding sequence or promoter, and / or PHB1 or its coding sequence or promoter. As described herein, the pharmaceutical composition can be formulated by any method known or developed in the field of pharmacology, including but not limited to contacting the active ingredient (e.g., viral particles or recombinant vectors) with excipients or other auxiliary components, and dividing or packaging the product into dosage units. As used herein, the term "pharmaceutically acceptable excipient" encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, and emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents, excipients, stabilizers, preservatives, and viscosity-inducing agents. Examples of excipients can be found in Martin (1975) Remington's Pharm. Sci., 15th ed. (Mack Publ. Co., Easton).

[0097] The present invention will be described below by way of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are conventional materials and methods in the art unless otherwise specified.

[0098] Examples

[0099] The present invention uses CRISPR-Cas9 technology to construct models such as HILPDA knockout hESCs and HILPDA cardiomyocyte-specific knockout mice, and further deeply studies the mechanism of action of HILPDA protein in regulating cardiac function. The applicant proposes that HILDPA may bind to the scaffold protein PHB1, import PHB1 into mitochondria, and then maintain the stability of mitochondrial respiratory chain complexes, ensuring mitochondrial electron transport and oxidative phosphorylation functions; a decrease in the expression level of this gene will cause the occurrence of DCM. Cardiomyocyte-targeted overexpression of HILPDA may delay the occurrence and development of heart failure by improving mitochondrial respiratory function ( Figure 1).

[0100] Example 1: Study on the regulatory effect of HILPDA on myocardial function

[0101] 1.1 Study on the effect of HILPDA on the contractility of cardiomyocytes

[0102] 1) Beatable cardiomyocytes can be obtained by in vitro directed differentiation: Use the CRISPR / Cas9 technology to construct HILPDA knockout hESC cell lines H7-15KO and H7-18KO (design the CRISPR-CAS9 system guide RNA (SEQ ID NO:1) targeting the second exon of HILPDA (positions 33 to 1250 of the genomic sequence of HILPDA), and select two hESCs clones H7-15KO and H7-18KO with silenced HILPDA expression); Use mTeSR-1 medium (Stem Cell TM # 85850) to culture hESCs and seed them on culture plates coated with Matrigel (Corning Catalog #354277). When the cell density reaches 90%, passage the cells at a ratio of 1:4 to 1:5 using Accutase (GIBCO, A1110501). Use the WNT agonist CHIR-99021 and antagonist IWR-1 (Selleck, S2924; Sigma, I0161) to induce the differentiation of hESCs in chronological order. On day 0 and day 1, activate the WNT signal in the cells using RPMI1640 / B-27 insulin-free medium containing 12 mM CHIR-99021. On day 2, change the medium to RPMI1640 / B-27 insulin-free medium. On day 3 and day 4, change to RPMI 1640 / B-27 insulin-free medium containing 5 mM IWR-1. Starting from day 5, treat the differentiated cells with medium containing RPMI1640 / BSA (500 g / ml) / ascorbic acid (213 g / ml), and change the medium every other day. Wild-type cardiomyocytes start to beat on day 7 or day 8 of differentiation.

[0103] The results showed that compared with wild-type cardiomyocytes, their cell volume was significantly larger, and the arrangement of the filament structural proteins ACTN2 (green fluorescence) and cTNT (red fluorescence) was disordered ( Figure 2 , B). However, the flow cytometry results indicated that knockout of HILPDA did not have a significant effect on the efficiency of myocardial directed differentiation of hESCs ( Figure 2 , C). Further data showed that the cell contractility was weakened, and the calcium recycling and release process was also abnormal ( Figure 2 , D-E).

[0104] 2) Overexpress full-length HILPDA on day 15 of cardiomyocyte differentiation from wild-type or HILPDA-knockout hESCs. On day 30, observe the arrangement of myofilament proteins such as troponin cTnT and sarcomeric protein α-actinin, as well as the sarcomere width, by transmission electron microscopy and immunofluorescence staining techniques to determine whether the myofilament structure disorder is restored.

[0105] 3) Detect the expression changes of cardiomyocyte fiber-related proteins such as TNNT2, TNNI, ACTN2, MYH6, and MYH7 by qPCR and Western Blot.

[0106] 4) Use a multi-electrode microarray MEA system to record the changes in field potential during the spontaneous contraction of cardiomyocytes: Attach the differentiated cardiomyocyte clusters to the Matrigel-treated MEA chip. After 3 days of culture, the cardiomyocytes maintain rhythmic contraction. Use the McRack acquisition and analysis recording software to record the changes in the field potential of cardiomyocytes to detect the electrophysiological properties of cardiomyocytes in different groups of wild-type, knockout, and overexpression, including their action potential waveforms, rhythms, and reactivity to electrophysiological drugs such as epinephrine, metoprolol, verapamil, etc.

[0107] 5) Use the Cal-520 calcium probe to detect the spontaneous calcium transient activity of cardiomyocytes in each group and evaluate the ability to handle calcium ion release and recovery: Digest the cardiomyocytes on day 30 of differentiation into single cells with collagenase and trypsin, and then inoculate them on a Matrigel-coated 15 mm round glass dish. When the cells survive and beat spontaneously, add a tabletop solution containing 5 μM Cal-520AM and 0.02% Pluronic F-127 for staining and incubate at 37°C for 15 min. Record the calcium signal using the line scan mode (10 ms) of an LSM-710 laser scanning confocal microscope at 37°C and 5% CO2, and analyze it using MATLAB software.

[0108] 6) Use the Felix GX cell edge motility detection system to evaluate the changes in the contractility of cardiomyocytes: Digest the cardiomyocytes on day 30 of differentiation into single cells with collagenase and trypsin, and then inoculate them on a Matrigel-coated 15 mm round glass dish. When the cells survive and beat spontaneously, use the motility edge detection system to detect single-beating cardiomyocytes. Use the VideoSarcomere Length software to record the movement trajectories of cardiomyocytes. Use the FelixGX 4.2.2 software to record and analyze the contraction data in real time. All tests are carried out under the conditions of 37°C and 5% CO2. The Y-axis voltage indirectly reflects the relative contractility of beating cardiomyocytes, and the number of peaks within a certain period indirectly reflects the relative contraction rate.

[0109] 1.2 To study whether the myocardial-specific knockout of HILPDA (Hilpda cKO ) in mice fed a normal diet will show pathological changes of dilated cardiomyopathy

[0110] 1) At 6, 12, 18, 24, and 30 weeks, perform echocardiography on wild-type mice and Hilpda cKO mice for comparison, and detect their ejection fraction EF, fractional shortening FS, left ventricular end-systolic diameter LVIDs, left ventricular end-diastolic diameter LVIDd, etc.; use B-Mode images to calculate indexes related to myocardial stress changes, including global longitudinal strain, radial strain, radial strain rate, longitudinal strain, longitudinal strain rate, and time to peak, etc., so as to judge whether there are changes in the cardiac systolic and diastolic functions.

[0111] 2) Take transverse sections of the hearts of mice at the above time points for histological frozen sections and paraffin sections, stain them using HE staining and Masson staining, calculate, analyze, and compare indexes such as ventricular wall thickness and ventricular cavity size in each group through ImageJ software; perform WGA immunofluorescence staining, and statistically analyze whether the size of individual cardiomyocytes has changed to further clarify whether there are pathological changes such as cardiac dilation.

[0112] 3) Use qPCR and Western Blot to detect the expression changes of cardiomyocyte fiber-related proteins in mouse myocardial tissues, such as TNNT2, TNNI, ACTN2, MYH6, MYH7, etc.

[0113] 4) Use transmission electron microscopy and IF staining to observe the arrangement of cardiomyocytes and the width of sarcomeres in myocardial tissues.

[0114] 1.3 To study the effects of HILPDA on the morphology, size, and number of cardiomyocyte mitochondria; as well as mitochondrial respiratory function, oxygen consumption, and ATP synthesis ability.

[0115] 1) Observe the changes in the number, size, morphology, and structure of mitochondria in cardiomyocytes of the wild-type control group, HILPDA knockout group, and HILPDA rescue group: After fixing the cardiomyocytes, prepare ultrathin sections and observe the ultrastructure of cardiomyocytes using transmission electron microscopy; after transfecting the cardiomyocytes of each experimental group with Ad-COX8a-GFP virus for 48 hours, observe the dynamic changes of mitochondria under a confocal microscope and take pictures for recording. Use MiNA to analyze the changes in the number, size, morphology, and structure of mitochondria.

[0116] 2) JC-1 staining shows mitochondrial membrane potential: Single cardiomyocytes were seeded on Matrigel-coated 15-mm round dishes. After corresponding treatments, the mitochondrial membrane potential of cardiomyocytes was detected using a JC-1 mitochondrial membrane potential detection kit. The culture medium was aspirated, the cells were washed once with PBS, and 1 ml of cell culture medium was added. 1 ml of JC-1 staining working solution was added and mixed well. The cells were incubated at 37 °C in a cell culture incubator for 20 minutes. After the incubation at 37 °C was completed, the supernatant was aspirated, and the cells were washed twice with JC-1 staining buffer. Then 2 ml of cell culture medium was added, and the cells were observed under a fluorescence microscope or a laser scanning confocal microscope. Observe whether the restoration of full-length HILPDA can reverse the mitochondrial membrane potential.

[0117] 3) Detect the expression of key genes such as Mfn2 and Drp1 that mediate mitochondrial fusion and fission by qPCR and other methods.

[0118] 4) Use the XF24 Seahorse analyzer to detect mitochondrial respiration, oxygen consumption, and energy metabolism. The order of adding drugs is as follows: For electron flow measurement: 10 mM pyruvate, 2 mM malate, and 4 μM FCCP. For coupled measurement: 40 mM ADP, 25 μg / ml oligomycin, 40 μM FCCP, and 40 μM antimycin. For electron transfer measurement: 20 μM rotenone, 100 mM succinate, 40 μM antimycin, 100 mM ascorbic acid, and 1 mM TMPD.

[0119] 5) Observe the comprehensive ATP production capacity of mitochondria by detecting ATP concentration: The ATP level was detected using an ATP bioluminescence detection kit. The cells were lysed thoroughly, the supernatant was collected, the protein concentration was determined by the BCA method, and the luminescence intensity was measured using a microplate reader. A standard curve was generated by gradient dilution of the standard solution, and the ATP level was calculated according to the standard curve and normalized to the standard protein concentration.

[0120] 6) Take the heart tissue of Hilpda cKO mice, and use experiments such as WB and IF to detect whether the expression level of PHB1 in mitochondria has changed; use transmission electron microscopy to observe the changes in the number, size, morphology, and structure of mitochondria in Hilpda cKO cardiomyocytes of mice; detect the expression of key genes such as Mfn2 and Drp1 that mediate mitochondrial fusion and fission by qPCR and other methods; detect the activities of the five mitochondrial respiratory chain complexes and the changes in ATP production.

[0121] Example 2: Whole transcriptome sequencing analysis suggests that after HILPDA knockout, the expression of genes related to myofilament structure and mitochondrial function has changed significantly.

[0122] To clarify the effect of HILPDA deficiency on cardiomyocyte function, the inventors performed transcriptome sequencing analysis on cardiomyocytes differentiated from HILPDA- / - hESCs in vitro for 15 days. The results showed that the genes with significant expression changes were mainly concentrated in aspects related to myofilament structure, myocardial contractility, ATP generation, mitochondrial electron transport chain, etc. Figure 3 )

[0123] Example 3: The expression of HILPDA is significantly reduced in the myocardial tissue of end-stage heart failure patients with DCM

[0124] Through the CELLxGENE Discover single-cell database, it was found that HILDPA is widely expressed in various cardiac cells ( Figure 4 , A), but the expression of HILDPA in the myocardial tissue of DCM does not change in all cell types. Only the expression level of HILPDA in cardiomyocytes is significantly reduced ( Figure 4 , B). Further analysis of GEO transcriptome data (GSE3585 and GSE57345) found that the expression of HILPDA in the myocardial tissue of end-stage heart failure patients with DCM is indeed reduced ( Figure 4 , C). This suggests that changes in the expression level of HILPDA may lead to myocardial function damage.

[0125] Example 4: Systemic knockout of HILPDA in mice results in a DCM phenotype

[0126] Hilpda systemic knockout mice were purchased, and their cardiac function at 7 weeks and 14 weeks was detected by Doppler ultrasound to track their development and cardiac function changes. The results showed that systemic knockout of Hilpda did not cause embryonic lethality or early developmental abnormalities; however, their cardiac function progressively declined 14 weeks after birth, accompanied by an enlarged left ventricular cavity, a thinner left ventricular wall, an increased volume of cardiomyocytes, and an increase in fibrosis ( Figure 5 , B), which is consistent with the relevant phenotypes of DCM.

[0127] Example 5: Knockout of HILPDA does not disrupt myocardial triglyceride metabolism in mice, the morphology of lipid droplets shows no obvious change, but mitochondria show obvious damage

[0128] Although HILPDA, as a lipid droplet-related protein, plays an important regulatory role in triglyceride metabolism, under normal diet conditions, the levels of triglyceride TG, free fatty acid FFA, and glycerol Glycerol in the myocardial tissue of HILPDA systemic knockout mice do not change significantly ( Figure 6 , A), and at the same time, the expression of the HILPDA homologous structural analogue G0S2 shows compensatory increase, while the expression levels of other related catalytic enzymes in triglyceride metabolism do not change significantly ( Figure 6, B). Transmission electron microscopy showed that there were no significant differences in the size and morphology of lipid droplets in the myocardium after HILPDA knockout compared with wild-type myocardium, but mitochondrial damage such as cristae disappearance, swelling, and vacuolization occurred ( Figure 6 , C-D). At the same time, the ability of the HILPDA-knockout myocardial tissue to produce ATP also decreased significantly ( Figure 6 , E). These results suggest that HILPDA may play an important function beyond regulating triglyceride metabolism, and HILPDA deficiency can lead to myocardial mitochondrial damage.

[0129] Example 6: Co-immunoprecipitation combined with mass spectrometry analysis found that HILPDA may interact with the scaffold protein PHB1, and the two may bind to each other through the N-terminal domain

[0130] The pCMV-HILPDA-FLAG overexpression plasmid was transfected into AC16 human cardiomyocytes, and target proteins interacting with HILPDA were screened by FLAG antibody co-immunoprecipitation combined with mass spectrometry identification. After two independent IP-MS experiments, the results showed that in addition to the previously reported ATGL protein, the scaffold protein Prohibitin 1 (PHB1) was identified as a potential interacting protein ( Figure 7 ). Further CO-IP experiments confirmed the interaction between HILPDA and PHB1 ( Figure 7 , B). According to the conserved domains of PHB1, it was divided into three truncated PHB1s: the N-terminal PHB domain (amino acids 1-172), the coiled-coil domain (amino acids 177-272), and the C-terminal nuclear export sequence (amino acids 257-270), which were cloned into the pCMV-HA vector. The three truncated PHB vectors were co-transfected with the pCMV-HILPDA-Flag vector into 293T cells respectively; after 48 h, the proteins were collected and CO-IP experiments were performed with HA and FLAG antibodies respectively to find the region where the binding site of Hilpda and PHB is located. The existing results suggest that the N-terminal PHB domain may be the region that binds to HILPDA, while the coiled-coil domain does not interact with HILPDA ( Figure 7 , C-D).

[0131] Construct the expression vectors of HILPDA with deletion of leucine at position 16 to serine at position 18 (contained within the second exon) (HILPDA p.Leu16_Ser18del) and PHB1 with deletion of leucine at position 14 to leucine at position 16 (PHB1 p.Leu14_Leu16del) respectively. The expression vector is pcDNA3.1. Transfect the HILPDA p.Leu16_Ser18del mutant expression vector and the full-length PHB1 expression vector, as well as the PHB1 p.Leu14_Leu16del and the full-length HILPDA expression vector into 293 cells respectively, and perform CO-IP experiments to verify whether the binding ability between the two has changed.

[0132] Bimolecular Fluorescence Complementation (BiFC) analysis technology is a method for detecting protein-protein interactions. Using Venus (a mutant of EYFP) as the fluorescent protein indicating protein-protein interactions, it is cut at the 155th amino acid to form a non-fluorescent N-terminal peptide segment and C-terminal peptide segment. The expression vectors pBiFC-VN173 (Plasmid #22010) and pBiFC-VC155 (Plasmid #22011) of the two are purchased from Addgene. HILPDA and PHB1 are cloned into the above expression vectors to form fusion proteins. Observe whether due to the interaction between HILPDA and PHB1, the originally non-fluorescent N segment and C segment are pulled by the target protein to approach each other spatially, and a complete and active Venus fluorescent protein is reconstituted, and fluorescence is emitted under the excitation light of this fluorescent protein. The effect of the mutant expression vector on the reconstruction of Venus fluorescent protein is verified.

[0133] Example 7: After HILPDA knockout, the total protein level of PHB1 did not change, but its expression level in mitochondria decreased significantly

[0134] Considering that PHB1, as an inner mitochondrial membrane protein, plays an important role in maintaining mitochondrial homeostasis and function, the inventor isolated mitochondrial proteins and detected the expression level of PHB1 in mitochondria before and after HILPDA knockout. As Figure 8 shown, after HILPDA knockout, the total expression level of PHB1 in cardiomyocytes did not change significantly, but the amount of PHB1 protein entering mitochondria decreased significantly. Co-staining with mitochondria (red) in cardiomyocytes traced by Ad-COX8a-GFP virus, it was found that after HILPDA knockout, the localization of PHB1 (green) in mitochondria decreased significantly.

[0135] Example 8: The aggregation level of PHB1 in mitochondria is affected by the expression level of HILPDA, and it affects the stability of mitochondrial respiratory chain complexes.

[0136] Like the vast majority of mitochondrial proteins, PHB1 is synthesized in the cytoplasm. Such nuclear-encoded mitochondrial proteins rely on the N-terminal presequence of the precursor protein or internal signals hidden in the mature protein to be imported into mitochondria. For example, the N-terminus of the human PHB2 precursor protein has a typical mitochondrial targeting sequence, which can be recognized by the TOM complex of the outer mitochondrial membrane translocase and enter the inner mitochondrial membrane through the inner membrane translocase TIM23

[30] . Mitochondrial precursor proteins containing internal signals may be delivered to the TOM complex receptor TOM70 with the cooperation of cytoplasmic chaperones (such as HSP70 or HSP90, etc.), interact with zinc finger proteins such as TIM9, and translocate through the inner membrane translocase TIM22 complex in a Δψ-dependent manner to be imported into the inner mitochondrial membrane

[38] .

[0137] It is worth noting that although PHB1 is highly homologous to PHB2, the N-terminus of PHB1 lacks positively charged amino acids that can form amphiphilic α-helices and has no hydrophobic segment domain that forms a transmembrane domain, and no typical mitochondrial targeting sequence is seen. Collect and isolate cardiomyocytes derived from HILPDA-knockout hESCs on the 30th day of differentiation and control sample cells. Use a cell mitochondrial isolation kit (Beyotime, C3601) to isolate mitochondria from cardiomyocytes. Add 1 ml of mitochondrial isolation reagent to 20 million cells, gently suspend the cells, and place them in an ice bath for 10 - 15 minutes. Transfer the cell suspension to a glass homogenizer of an appropriate size and homogenize 20 times. Centrifuge the cell homogenate at 600g for 10 minutes at 4°C, carefully transfer the supernatant to another centrifuge tube, and centrifuge at 11000g for 10 minutes at 4°C. Carefully remove the supernatant. The precipitate is the isolated mitochondria. Determine the protein concentration of the lysed protein sample by the BCA method. Use methods such as WB and IF to clarify the expression levels of HILPDA and PHB1 in the cytoplasm and mitochondria. It was initially found in the AC16 cell line that after interfering with HILPDA, the expression of PHB1 in mitochondria was significantly reduced ( Figure 9 , A); after overexpressing HILPDA, more PHB1 was transferred from the cytoplasm into mitochondria, which could significantly promote the aggregation of PHB1 in mitochondria ( Figure 9 , B). It can be seen that the expression level of PHB1 in mitochondria is closely related to HILPDA.

[0138] Use myocardial tissues of HILPDA cardiomyocyte-specific knockout mice to verify whether the expression levels of PHB1 in the cytoplasm and mitochondria have changed by WB, IF, etc.

[0139] Meanwhile, the expression levels of its important downstream target, mitochondrial respiratory complex, are also closely associated with HILPDA expression. The inventors detected the expression levels of respiratory chain complexes I-V and found that they changed with the alteration of HILPDA expression levels ( Figure 9 , C).

[0140] Example 9: Influence of the binding of HILPDA to PHB1 on the function of PHB1

[0141] 1) Collect approximately 5×10⁶ cardiomyocytes differentiated from HILPDA knockout hESCs on day 30 of differentiation and sample cells of the control group, extract mitochondria, and detect the activity of mitochondrial respiratory chain complexes. Based on the fact that mitochondrial respiratory chain complex I can catalyze the dehydrogenation of NADH to generate NAD⁺, directly read the oxidation rate of NADH at 340 nm, and thus calculate the change in the activity of complex I after HILPDA knockout. The catalytic product of complex II, reduced coenzyme Q, can further reduce 2,6-dichlorophenolindophenol, which has a characteristic absorption peak at 605 nm. The change in the activity of complex II can be calculated by detecting the reduction rate of 2,6-dichlorophenolindophenol. Mitochondrial respiratory chain complex III transfers the hydrogen of reduced CoQ to cytochrome C to generate reduced cytochrome C. Different from oxidized cytochrome C, reduced cytochrome C has a characteristic light absorption at 550 nm. Detecting the increase rate of light absorption at 550 nm can reflect the enzyme activity of mitochondrial respiratory chain III. Mitochondrial respiratory chain complex IV can catalyze the generation of oxidized cytochrome C from reduced cytochrome C. Therefore, the decrease rate of light absorption at 550 nm can also reflect the enzyme activity of mitochondrial respiratory chain complex IV. Complex V can hydrolyze ATP to produce ADP and Pi, and then the activity of mitochondrial respiratory chain complex V can be detected by measuring the increase rate of Pi.

[0142] 2) Construct hESCs cells carrying mutations at the key binding sites of HILPDA and PHB1 through gene editing technology. Collect approximately 5×10 6 cardiomyocytes differentiated on day 30 of differentiation and sample cells of the control group, and detect the ATP level using an ATP bioluminescence detection kit. Use an XF24 Seahorse analyzer to detect mitochondrial respiration, oxygen consumption, and energy metabolism.

[0143] 3) Take the myocardial tissues of HILPDA cardiomyocyte-specific knockout mice to verify the stability and activity of mitochondrial respiratory chain complexes; detect the ATP level using an ATP bioluminescence detection kit; detect the expression of key genes such as Mfn2 and Drp1 that mediate mitochondrial fusion and fission by qPCR.

[0144] Example 10: Complementation of HILPDA improves mitochondrial respiratory function and delays the development of DCM.

[0145] On HILPDA-knockout hESCs-CM, full-length or mutant HILPDA was re-supplemented to study whether the expression level of PHB1 in mitochondria could be restored and whether mitochondrial morphology and function could return to normal.

[0146] On HILPDA-knockout cardiomyocytes, full-length HILPDA and mutant vector p.Leu16_Ser18del were re-supplemented respectively, and they were divided into three groups: HILPDA knockout group, full-length HILPDA re-supplementation group, and mutant HILPDA re-supplementation group. The Seahorse experiment was used to detect the respiratory capacity of mitochondria; the concentration of ATP generated was measured to observe the comprehensive energy production capacity of mitochondria; the activities of five complexes of the mitochondrial respiratory chain were detected; the content of ROS (reactive oxygen species) was detected by fluorescence tracing method; JC-1 staining was used to show the mitochondrial membrane potential.

[0147] Related sequences

[0148]

[0149]

Claims

1. hESC cells with down-regulated expression or activity of HILPDA.

2. The hESC cells according to claim 1, characterized in that, The down-regulation of the expression or activity of HILPDA is achieved by any of the following methods: (1) Introducing into hESC cells siRNA or its expression vector, antisense RNA or its expression vector, ribozyme or its expression vector, gene editing vector specifically targeting HILPDA, such as CRISPR gene editing vector or TALEN gene editing vector; (2) Introducing into hESC cells anti-HILPDA antibody or its expression vector; (3) Introducing into hESC cells small molecule inhibitors; (4) Introducing into hESC cells a homologous recombination vector containing a nucleotide sequence encoding a mutant inactive or hypoactive HILPDA variant, Preferably, the siRNA, antisense RNA, gene editing vector in method (1) target the second exon of the HILPDA-encoding gene, and / or method (1) causes a deletion mutation in amino acids 1 - 172 of the wild-type amino acid sequence of HILPDA, and / or method (1) causes a mutation in amino acids 16 - 18 of the wild-type amino acid sequence of HILPDA, and / or the inactive or hypoactive HILPDA variant is selected from: an HILPDA variant with a deletion mutation in amino acids 1 - 172 of the wild-type amino acid sequence of HILPDA, and / or, an HILPDA variant with a mutation in amino acids 16 - 18 of the wild-type amino acid sequence of HILPDA.

3. Cardiomyocytes with down-regulated expression or activity of HILPDA, said cardiomyocytes being differentiated from the hESC cells of claim 1, Preferably, the differentiation steps include: (1) Culturing hESC cells in a medium containing a WNT agonist, (2) Culturing hESC cells in a medium containing a WNT antagonist, (3) Culturing hESC cells in a medium without a WNT agonist and a WNT antagonist.

4. The cardiomyocyte according to claim 3, characterized in that, The WNT agonist includes CHIR-99021, and / or, the WNT antagonist includes IWR-1.

5. A nucleic acid construct comprising a promoter and a nucleic acid sequence encoding HILPDA, Preferably, the amino acid sequence of HILPDA is as shown in SEQ ID NO:2, and / or the nucleic acid sequence of HILPDA is as shown in SEQ ID NO:3, and / or the promoter is a CMV promoter or the original promoter of HILPDA on the genome, and / or the nucleic acid construct further comprises a terminator, and / or the nucleic acid construct further comprises a tag sequence, such as a FLAG sequence, and / or the nucleic acid construct is a recombinant vector, a cloning vector or an expression vector.

6. A host cell comprising the nucleic acid construct of claim 5, Preferably, the cell is a cardiomyocyte, such as human cardiomyocyte AC16.

7. A method for reducing the interaction between HILPDA and PHB1 or a method for reducing the transfer of PHB1 to mitochondria, comprising: (1) Deleting amino acids 1 - 172 of the wild - type amino acid sequence of HILPDA, and / or (2) Mutating amino acids 16 - 18 of the wild - type amino acid sequence of HILPDA, and / or (3) Mutating amino acids 14 - 16 of the wild - type amino acid sequence of PHB1, Preferably, The mutation is a deletion, substitution or insertion mutation, and / or The wild - type amino acid sequence of HILPDA is as shown in SEQ ID NO:2, and / or The wild - type amino acid sequence of PHB1 is as shown in SEQ ID NO:

4.

8. Use of HILPDA as a target in screening for potential substances for preventing or treating impaired myocardial contractile function, wherein, A candidate substance that enhances the interaction between HILPDA and PHB1 is a potential substance for treating impaired myocardial contractile function. Preferably, The candidate substance is a candidate substance that mutates HILPDA or PHB1. The mutations include: (1) deleting amino acids 1 - 172 of the wild - type amino acid sequence of HILPDA, and / or (2) mutating amino acids 16 - 18 of the wild - type amino acid sequence of HILPDA, and / or (3) mutating amino acids 14 - 16 of the wild - type amino acid sequence of PHB1, and / or The wild - type amino acid sequence of HILPDA is as shown in SEQ ID NO:2, and / or The wild - type amino acid sequence of PHB1 is as shown in SEQ ID NO:4, and / or The impaired myocardial contractile function is dilated cardiomyopathy.

9. A method for screening potential substances for preventing or treating impaired myocardial contractile function or its symptoms, comprising: (1) Contacting a candidate substance with a system containing HILPDA and PHB1, and (2) detecting changes in the interaction between HILPDA and PHB1. Among them, a substance that enhances the interaction between HILPDA and PHB1 is a potential substance for preventing or treating impaired myocardial contractile function. Preferably, The impaired myocardial contractile function is dilated cardiomyopathy, and / or The symptoms are selected from one or more of the following: increased myocardial cell volume, disordered arrangement of myofilament structural proteins, weakened myocardial cell contractility, abnormal calcium recycling and release processes, enlarged ventricular cavity, thinner left ventricular wall, decreased cardiac function, increased fibrosis, increased myocardial mitochondrial fission, obvious disappearance of cristae, swelling and vacuolization, etc., changes in the number, size, shape and structure of mitochondria, and changes in mitochondrial respiration, oxygen consumption and energy metabolism.

10. A non - therapeutic or diagnostic method for promoting the aggregation of PHB1 in mitochondria in vitro, including expressing wild - type HILPDA in cells. Preferably, the method includes: (1) Introducing the nucleic acid construct as claimed in the claim into the cells, and (2) incubating the cells under the condition of HILPDA expression. More preferably, The nucleic acid construct is a recombinant vector, a cloning vector or an expression vector, and / or The cells are a human myocardial cell line, such as AC16.

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