A cell model for inducing human cardiomyocyte hypertrophy by esrra and a construction method and application thereof

Abstract

This invention relates to the field of cell biology, and discloses a cell model for inducing human cardiomyocyte hypertrophy via ESRRA, its construction method, and its applications. This human cardiomyocyte hypertrophy cell model is obtained through ESRRA induction. This invention uses a single regulatory factor, ESRRA, as a clearly defined core driving factor to directly induce and construct a cell model for human cardiomyocyte hypertrophy. The induction conditions are simple, the target is clear, and the reproducibility is good, making it more conducive to humanization research and applicable to a wider range of scenarios.

Description

Technical Field

[0001] This invention relates to the field of cell biology, specifically to a cell model for inducing hypertrophy of human cardiomyocytes via ESRRA, its construction method, and its application. Background Technology

[0002] Cardiomyocyte hypertrophy is one of the most common cell phenotypes in cardiac remodeling, mainly characterized by an increase in the area and volume of individual cardiomyocytes. Current research on cardiomyocyte hypertrophy largely focuses on pathological stimulation models, such as induction by angiotensin, norepinephrine, phenylephrine, mechanical stretching, or pressure loading. While these methods can achieve the hypertrophic phenotype to some extent, they still have the following limitations: First, existing methods mostly rely on exogenous stimuli, have complex induction conditions, poor batch stability, and low reproducibility between different laboratories.

[0003] Second, most existing models are based on mouse cardiomyocytes, primary animal cardiomyocytes, or tumor-like cell lines, lacking a stable construction system based on human cardiomyocytes.

[0004] Third, existing technologies primarily induce cardiomyocyte hypertrophy through overall stimulation of the environment, rather than directly constructing a hypertrophic phenotype through a specific single regulatory factor. Therefore, unclear targets are often encountered in mechanism analysis and subsequent drug screening.

[0005] Currently, there is a lack of technical solutions that use a single regulatory factor as the core driving factor to directly induce the hypertrophic phenotype in human cardiomyocyte systems. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing cardiomyocyte hypertrophy models, such as inconsistent sources, complex induction conditions, unclear targets, and insufficient reproducibility, and to provide a cell model for directly inducing human cardiomyocyte hypertrophy using a single regulatory factor, ESRRA, as a clearly defined core driving factor.

[0007] To achieve the above objectives, a first aspect of the present invention provides a cell model of human cardiomyocyte hypertrophy obtained by ESRRA induction.

[0008] A second aspect of the present invention provides a method for constructing a cell model of human cardiomyocyte hypertrophy, the method comprising: introducing a viral vector containing a human ESRRA coding sequence into human cardiomyocytes, wherein ESRRA is stably and highly expressed in human cardiomyocytes, thereby inducing a cell model of human cardiomyocyte hypertrophy.

[0009] A third aspect of the present invention provides a method for constructing a cell model of human cardiomyocyte hypertrophy, the method comprising: introducing a human ESRRA coding sequence into human cardiomyocytes by plasmid transfection or mRNA transfection, wherein ESRRA is stably and highly expressed in human cardiomyocytes, thereby inducing a cell model of human cardiomyocyte hypertrophy.

[0010] A fourth aspect of the present invention provides a method for constructing a cell model of human cardiomyocyte hypertrophy, the method comprising: introducing a human ESRRA coding sequence into human cardiomyocytes using CRISPR activation technology, wherein ESRRA is stably and highly expressed in human cardiomyocytes, thereby inducing a human cardiomyocyte model.

[0011] The fifth aspect of the present invention provides a cell model of human cardiomyocyte hypertrophy prepared by the method of the second aspect, the method of the third aspect, or the method of the fourth aspect.

[0012] The sixth aspect of the present invention provides the application of the cell model of human cardiomyocyte hypertrophy described in the first or fifth aspect in engineered exosomes.

[0013] The seventh aspect of the present invention provides the use of the human cardiomyocyte hypertrophy cell model described in the first or fifth aspect in screening candidate molecules, drugs, small interfering RNAs, nucleic acid preparations or protein factors that regulate cardiomyocyte hypertrophy.

[0014] The technical solution of the present invention has at least the following advantages: This invention establishes a method for constructing human cardiomyocyte hypertrophy with ESRRA as the clearly defined core factor, and applies it to different human cardiomyocyte models such as hiPSC-CM (human induced pluripotent stem cell-derived cardiomyocytes) cells and AC16 cells, in order to overcome the problems of inconsistent model sources, complex induction conditions, unclear targets and insufficient reproducibility in the existing technology.

[0015] The human cardiomyocyte hypertrophy cell model provided by this invention directly uses ESRRA as the core driving factor, resulting in a clearer technical route and more defined intervention points. Furthermore, using human cardiomyocytes as the target avoids the species differences associated with relying solely on animal-derived cardiomyocytes, making it more conducive to humanized research and expanding its application scenarios. Attached Figure Description

[0016] Figure 1 It is the transfection plasmid vector sequence of Example 1; Figure 2 This is a graph showing the lentivirus transfection efficiency test for validating the GFP reporter gene. Figure 3 This is a graph showing the mRNA expression level of ESRRA detected by qPCR. Figure 4 This is a Western blot (WB) test result of ERRα protein expression level. Figure 5 Immunofluorescence staining was used to observe the size of hiPSC-CM cardiomyocytes overexpressing ESRRA; Figure 6 Immunofluorescence staining was used to observe the size of AC16 cardiomyocytes overexpressing ESRRA. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] The ESRRA mentioned in this invention refers to the nucleotide sequence encoding estrogen receptor-associated protein α (ERRα); the ESRRA mRNA is the messenger RNA transcribed from this gene.

[0019] The inventors of this invention discovered in their research that after lentiviral infection and overexpression of ESRRA in hiPSC-CM, ESRRA was successfully upregulated at the molecular level, and an increase in cell area was observed morphologically. At the same time, a consistent hypertrophy trend was also observed in AC16 cells, indicating that the effect of ESRRA in inducing hypertrophy in human cardiomyocytes has a certain degree of cross-model reproducibility.

[0020] As previously stated, a first aspect of the present invention provides a cell model of human cardiomyocyte hypertrophy obtained by ESRRA induction.

[0021] As previously stated, a second aspect of the present invention provides a method for constructing a cell model of human cardiomyocyte hypertrophy, the method comprising: introducing a viral vector containing a human ESRRA coding sequence into human cardiomyocytes, wherein ESRRA is stably and highly expressed in human cardiomyocytes, thereby inducing a cell model of human cardiomyocyte hypertrophy.

[0022] The human ESRRA coding sequence described in this invention refers to a nucleotide sequence that encodes estrogen receptor-associated protein α (ERRα).

[0023] In some embodiments, the viral vector is selected from at least one of lentivirus, adenovirus, and adeno-associated virus. Preferably, the viral vector is lentivirus.

[0024] In some embodiments, the human cardiomyocytes are selected from at least one of human induced pluripotent stem cell-derived cardiomyocytes, human cardiomyocyte-like cell lines, primary human cardiomyocytes, or human heart organoid-derived cardiomyocytes.

[0025] In a preferred embodiment, the human cardiomyocytes are hiPSC-CM cells and / or AC16 cells.

[0026] According to a preferred embodiment, the specific operations of the method include: (1) The human ESRRA coding sequence was inserted into the plasmid vector using the standard restriction endonuclease cloning method to obtain the ESRRA expression plasmid; (2) In the presence of the transfection reagent, the packaging plasmid, the envelope plasmid, and the ESRRA expression plasmid were co-transfected into packaging cells. Viral supernatant was collected at 45-50 h and 70-75 h after transfection to obtain viral fluid; the titer of the viral fluid was 0.8 × 10⁻⁶. 8 -1.2×10 8 TU mL - ¹; (3) The viral solution was added to a human cardiomyocyte culture system containing polybrene and incubated for 12-16 h to obtain a cell model of human cardiomyocyte hypertrophy.

[0027] According to one specific embodiment, the packaging cells are HEK293T cells.

[0028] In some embodiments, the method in step (3) further includes: screening the incubated cells with puromycin for 48-72 h to obtain the human cardiomyocyte hypertrophy cell model; the screening concentration of puromycin is 1-2 μg / mL. - ¹.

[0029] A third aspect of the present invention provides a method for constructing a cell model of human cardiomyocyte hypertrophy, the method comprising: introducing a human ESRRA coding sequence into human cardiomyocytes by plasmid transfection or mRNA transfection, wherein ESRRA is stably and highly expressed in human cardiomyocytes, thereby inducing a cell model of human cardiomyocyte hypertrophy.

[0030] A fourth aspect of the present invention provides a method for constructing a cell model of human cardiomyocyte hypertrophy, the method comprising: introducing a human ESRRA coding sequence into human cardiomyocytes using CRISPR activation technology, wherein ESRRA is stably and highly expressed in human cardiomyocytes, thereby inducing a cell model of human cardiomyocyte hypertrophy.

[0031] In this invention, for mRNA transfection technology, the human ESRRA coding sequence refers to the mRNA molecule that encodes human estrogen receptor-associated protein α (ERRα), which can be translated into ERRα protein after being introduced into cells.

[0032] The fifth aspect of the present invention provides a cell model of human cardiomyocyte hypertrophy prepared by the method described in the second aspect, the third aspect, or the fourth aspect.

[0033] The human cardiomyocyte hypertrophy cell model provided by this invention has simple induction conditions, clear target, and can be stably produced with good reproducibility.

[0034] According to one optional specific implementation, the presence or absence of hypertrophic growth in human cardiomyocytes is evaluated using at least one of the following methods: F-actin immunofluorescence, ImageJ single-cell area analysis, WGA staining, high-content imaging, membrane dye labeling, or real-time live-cell imaging.

[0035] According to a preferred embodiment, F-actin immunofluorescence combined with ImageJ single-cell area analysis is used to evaluate whether human cardiomyocytes exhibit hypertrophic growth. This determination method is clear in operation, provides intuitive results, and is easy to standardize.

[0036] The sixth aspect of the present invention provides the application of the cell model of human cardiomyocyte hypertrophy described in the first or fifth aspect in engineered exosomes.

[0037] In this invention, engineered exosomes refer to natural exosomes that have been artificially modified, including targeted modification, content loading, and / or membrane surface modification, to endow, enhance, or optimize their specific biological functions, enabling them to serve as drug delivery carriers, diagnostic tools, or therapeutic bioactive agents. The human cardiomyocyte hypertrophy cell model provided by this invention, applied in the field of engineered exosomes, can be used to treat diseases including (but not limited to) heart failure, cardiomyopathy, and myocardial infarction, improving cardiac function in these patients by enhancing cardiomyocyte function.

[0038] The seventh aspect of the present invention provides the use of the human cardiomyocyte hypertrophy cell model described in the first or fifth aspect in screening candidate molecules, drugs, small interfering RNAs, nucleic acid preparations or protein factors that regulate cardiomyocyte hypertrophy.

[0039] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, the raw materials used are all commercially available products.

[0040] Example 1 1. Culture of hiPSC cells and myocardial differentiation First, hiPSC cells were cultured in vitro and then myocardial-directed differentiation was performed using a combination of small molecule induction and growth factor induction to form hiPSC-CM cells.

[0041] 1.1 Experimental Materials RPMI 1640 (Thermo Fisher Scientific, 11875093); BMP4, 4 ng / mL - ¹(R&D Systems, 314-BP); activin A, 5 ng mL - ¹(eBioscience, 14-8788-80); CHIR99021, 2 μM (Axon Medchem, 1386); IWP-2, 2 μM (Santa Cruz Biotechnology, sc-215596); B-27 Supplement (Thermo Fisher Scientific, 17504001); Ascorbic acid, 256 μg mL - ¹(Sigma-Aldrich, A4403).

[0042] 1.2 Experimental Methods hiPSC cells were cultured in RPMI 1640 medium with BMP4, activin A, CHIR99021, and B-27 supplements added during the early differentiation phase. Subsequently, the culture was switched to an RPMI system containing IWP-2 and B-27 for further induction. From day 5 onwards, the cells were maintained in RPMI medium containing ascorbic acid and B-27. Spontaneous cardiomyocyte contraction was observed around day 10, which was used for subsequent experiments. hiPSC cells induced by CHIR99021 and IWP-2 formed a cardiomyocyte lineage and exhibited spontaneous beating.

[0043] 2. Culture of AC16 cells 2.1 Experimental Reagents AC16 cells, EMD Millipore, catalog number SCC109; DMEM / F-12 medium, Sigma-Aldrich, catalog number D8437; Fetal bovine serum (FBS), Gibco, catalog number 10091148; Penicillin and streptomycin, Gibco, catalog number 15140122.

[0044] 2.2 Experimental Methods (1) The AC16 cells were seeded into a conventional cell culture dish; (2) Use DMEM / F-12 medium supplemented with 10% FBS and 1% penicillin and streptomycin; (3) Incubate in a 37°C, 5% CO2 humidifier; (4) After the cells reach a suitable degree of confluence, they are used for lentiviral infection and subsequent hypertrophic phenotype analysis. The above culture conditions have been clearly described in the method.

[0045] 2.3 Experimental Conclusions AC16 cells have well-defined culture conditions and stable proliferation, making them a suitable supplementary human cardiomyocyte model for the present invention, used for parallel validation and rapid screening of ESRRA-induced hypertrophy.

[0046] 3. Construction of ESRRA overexpression vector 3.1 Experimental Reagents Human ESRRA coding sequence, NM_004451.4; Lentiviral expression vector pCDH-CMV-MCS-3xFlag-EF1-GFP+Puro, System Biosciences; 3.2 Experimental Methods (1) Obtain the human ESRRA encoded sequence; (2) Using the standard restriction endonuclease cloning method, ESRRA was inserted into the pCDH-CMV-MCS-3xFlag-EF1-GFP+Puro vector; (3) In the constructed vector, the CMV promoter drives the expression of the ESRRA coding sequence with the N-terminal 3×Flag tag, and the EF1α promoter drives the GFP reporter gene and the puromycin gene; (4) After lentivirus transduction, puromycin was added for resistance screening. Cells that were not successfully transduced were eliminated, while cells that were successfully transduced and carried the resistance gene were retained.

[0047] (5) After the recombinant vector is constructed, the inserted sequence is sequenced for verification; (6) After verification, it is used for lentivirus packaging.

[0048] The specific details of the above-mentioned carrier construction information are as follows: Figure 1As shown, the design advantages of the plasmid vector provided by the present invention are: it can achieve stable expression of ESRRA, facilitate subsequent observation of infection efficiency through GFP, and obtain a more uniform positive cell population through puromycin.

[0049] 4. Lentiviral packaging and cardiomyocyte infection 4.1 Experimental Materials HEK293T cells; ESRRA expression plasmid; psPAX2 packaging plasmid, Addgene, catalog number 12260; pMD2.G envelope plasmid, Addgene, catalog number 12259; jetPRIME® transfection reagent, Polyplus-transfection, catalog number 101000046; Opti-MEM, Gibco, part number 31985070; 0.45μm PES filter membrane, Millipore, catalog number SLHP033RB; Polybrene, Sigma-Aldrich, catalog number TR-1003-G; Puromycin, Gibco, catalog number A1113803.

[0050] 4.2 Experimental Methods (1) Seed HEK293T cells in a 10 cm culture dish; (2) When the fusion rate reaches 70%-80%, co-transfect 10 μg of ESRRA expression plasmid, 7.5 μg of psPAX2 and 2.5 μg of pMD2.G; (3) Use jetPRIME® as the transfection reagent to prepare the transfection system in Opti-MEM; (4) Viral supernatant was collected at 48 h and 72 h after transfection; (5) The lentivirus solution was obtained by filtration through a 0.45 μm PES filter membrane; (6) The obtained viral titer is approximately 1×10⁻⁶. 8 TU mL - ¹; (7) Add the filtered viral supernatant to the hiPSC-CM or AC16 cell culture system, and add 8 μg mL of the supernatant. - ¹polybrene, overnight incubation; (8) Replace with fresh culture medium the next day; (9) GFP signal was detected by fluorescence microscopy 48 h post-infection to assess infection efficiency; (10) Using 1 μg mL - ¹Puromycin was screened for 48-72 h before being used in subsequent experiments.

[0051] Figure 2 This is a graph verifying lentiviral transfection efficiency using the GFP reporter gene. "hiPSC Esrra-OE" refers to the hiPSC experimental group transduced with the ESRRA overexpression vector, and "hiPSC Vector" refers to the hiPSC control group transduced with the empty vector. "Field 1, Field 2, and Field 3" represent three different microscopic fields selected from each group. The results show that GFP-positive signals were visible in both groups of cells under different fields of view, indicating that the vector was successfully introduced into hiPSC cells. The GFP-positive cells in the Esrra-OE group indicate successful construction of the ESRRA overexpression vector and successful cell transduction.

[0052] Using the lentivirus system described above, a stable ESRRA overexpression system can be established in hiPSC-CM and AC16, providing a reliable model basis for subsequent morphological phenotypic analysis.

[0053] Test Example 1: Molecular Validation of ESRRA Overexpression Model Molecular-level validation was performed on the post-infection hiPSC-CM to confirm the successful construction of the ESRRA model.

[0054] 1-1 Experimental Methods (1) Total RNA was extracted, reverse transcribed, and then qRT-PCR was performed to detect ESRRA transcription levels; (2) Total protein was extracted and subjected to Western blot to detect the expression level of ERRα protein; (3) The empty vector group was used as a negative control; (4) After infection with hiPSC-CM, cells were cultured for 5 days, and then ESRRA group and control group cells were collected for analysis. This time window was used for phenotypic and molecular verification in the experiment.

[0055] 2-2 Experimental Results (1) mRNA level: Total RNA was extracted, reverse transcribed to generate cDNA, and the expression level of ESRRAmRNA was detected by real-time quantitative PCR. The specific results are as follows: Figure 3 As shown; from Figure 3 As can be seen from the data, in both hiPSC-CM and AC16 human cardiomyocytes, compared with the empty vector control group, the ESRRA mRNA level in the ESRRA overexpression group was significantly increased, and the difference was statistically significant. (P<0.001), indicating that the constructed ESRRA overexpression system can effectively upregulate ESRRA expression at the transcriptional level.

[0056] (2) Protein level: Total protein was extracted, and SDS-PAGE electrophoresis, membrane transfer, and Western blotting were performed to detect the expression level of ERRα protein. The specific results are as follows: Figure 4 As shown; from Figure 4 As can be seen, in hiPSC-CM and AC16 cells, the ERRα protein band in the ESRRA overexpression group was significantly stronger than that in the corresponding empty vector control group, while the GAPDH band was basically the same. This indicates that under the condition of internal control standardization, the ERRα protein expression level in the ESRRA overexpression group was significantly increased, indicating that the constructed overexpression system can stably enhance ESRRA expression at the protein level.

[0057] (3) The empty vector group was used as a negative control, and the internal reference gene or internal reference protein was used for standardized analysis.

[0058] (4) Only when ESRRA is significantly elevated at both the mRNA and protein levels can it proceed to the subsequent functional evaluation.

[0059] Test Example 2: Immunofluorescence analysis of hypertrophic phenotype in human cardiomyocytes Morphological analysis was performed on the ESRRA overexpression group and the control group hiPSC-CM to evaluate whether cardiomyocytes underwent hypertrophic growth.

[0060] 2-1 Experimental Materials (1) F-actin staining reagent, used to mark the cytoskeleton boundary; (2) DAPI or similar nuclear dyes are used for counterstaining cell nuclei; (3) ImageJ image analysis software.

[0061] 2-2 Experimental Methods (1) Collect hiPSC-CM^ESRRA, hiPSC-CM^Ctrl, AC16^ESRRA and AC16^Ctrl cells respectively; (2) Fix cells, permeate and seal them; (3) Perform F-actin staining to reveal cell outline boundaries; (4) Perform nuclear staining; (5) Single-cell images were acquired using a fluorescence microscope; (6) Use ImageJ software to draw the boundaries of each single cell and calculate the cross-sectional area; (7) Divide the cell area into four intervals: <800μm², 800-1600μm², 1600-2400μm² and >2400μm², and perform morphological stratification statistics.

[0062] 2-3 Experimental Results Figure 5 The size of hiPSC-CMs overexpressing ESRRA was observed using immunofluorescence staining. Figure 6 The image shows the size of AC16 cardiomyocytes overexpressing ESRRA observed by immunofluorescence staining. As can be seen from the figure, in hiPSC-CM, the cell area of ​​the control group was mainly distributed in the 800-1600 μm² range, while after ESRRA upregulation, the proportion of cells in this range decreased, and the proportion of large cells >2400 μm² increased significantly, proving that ESRRA can promote hypertrophy of human cardiomyocytes. A consistent hypertrophic response was also observed in AC16 cells, indicating that this effect is not limited to a single cell system.

[0063] The results above demonstrate that the human cardiomyocyte hypertrophy cell model provided by this invention directly uses ESRRA as the core driving factor, resulting in a clearer technical route and more defined intervention points. Furthermore, it can be stably produced with good reproducibility. Using human cardiomyocytes as the target avoids the species difference issues associated with relying solely on animal-derived cardiomyocytes, making it more conducive to humanized research and broadening its application scenarios.

[0064] Furthermore, this invention uses F-actin immunofluorescence combined with ImageJ single-cell area analysis as the primary determination method, which is clear in operation, intuitive in results, and easy to standardize. It clearly demonstrates that ESRRA overexpression can stably increase the proportion of large areas of cardiomyocytes.

[0065] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A cell model of human cardiomyocyte hypertrophy, characterized in that, This cell model of human cardiomyocyte hypertrophy was obtained through ESRRA induction.

2. A method for constructing a cell model of human cardiomyocyte hypertrophy, characterized in that, The method includes: introducing a viral vector containing a human ESRRA coding sequence into human cardiomyocytes, where ESRRA is stably and highly expressed in human cardiomyocytes, thereby inducing a cell model of human cardiomyocyte hypertrophy.

3. The method according to claim 2, characterized in that, The viral vector is selected from at least one of lentivirus, adenovirus, and adeno-associated virus; And / or, the human cardiomyocytes are selected from at least one of human induced pluripotent stem cell-derived cardiomyocytes, human cardiomyocyte-like cell lines, primary human cardiomyocytes, or human heart organoid-derived cardiomyocytes.

4. The method according to claim 2 or 3, characterized in that, The specific operations of the method include: (1) The human ESRRA coding sequence was inserted into the plasmid vector using the standard restriction endonuclease cloning method to obtain the ESRRA expression plasmid; (2) co-transfect the packaging plasmid, the envelope plasmid and the ESRRA expression plasmid into the packaging cell in the presence of a transfection reagent, collect the virus supernatant at 45-50 h and 70-75 h after transfection respectively to obtain a virus liquid; the titer of the virus liquid is 0.8 x 10 8 -1.2 x 10 8 TU / mL - ¹; (3) The viral solution was added to a human cardiomyocyte culture system containing polybrene and incubated for 12-16 h to obtain a cell model of human cardiomyocyte hypertrophy.

5. The method according to claim 4, characterized in that, The method of step (3) further comprises: screening the incubated cells with puromycin for 48-72 h to obtain the hypertrophic cell model of the human myocardial cells; and the screening concentration of the puromycin is 1-2 μg mL - ¹.

6. A method for constructing a cell model of human cardiomyocyte hypertrophy, characterized in that, This method includes: introducing the human ESRRA coding sequence into human cardiomyocytes by plasmid transfection or mRNA transfection, inducing stable high expression of ESRRA in human cardiomyocytes, and obtaining a cell model of human cardiomyocyte hypertrophy.

7. A method for constructing a cell model of human cardiomyocyte hypertrophy, characterized in that, This method includes: introducing the human ESRRA coding sequence into human cardiomyocytes using CRISPR activation technology; ESRRA is stably and highly expressed in human cardiomyocytes; and a cell model of human cardiomyocyte hypertrophy is induced.

8. A cell model of human cardiomyocyte hypertrophy prepared by the method of any one of claims 2-5, the method of claim 6, or the method of claim 7.

9. The application of the human cardiomyocyte hypertrophy cell model as described in claim 1 or claim 8 in engineered exosomes.

10. The use of the human cardiomyocyte hypertrophy cell model of claim 1 or claim 8 in screening candidate molecules, drugs, small interfering RNAs, nucleic acid preparations or protein factors that regulate cardiomyocyte hypertrophy.

Images