An α-myosin mutant and its application

Abstract

The present invention relates to the field of biomedicine technology, and in particular to an α-myosin mutant and its application. Lysine (K) at position 1897 in the wild-type Myh6 protein is mutated to arginine (R). The nucleotide sequence corresponding to the wild-type Myh6 protein is shown in the bases in SEQ ID NO.1. Application of α-myosin mutants as Ang II-induced heart failure pathology models. The present invention obtains α-myosin mutants through the gene editing method of the CRISPR / Cas9 system, confirms that the 1897 site in α-myosin participates in and regulates the degree of interaction between α-MHC and Titin, thereby reflecting the degree of heart failure. The obtained α-myosin mutant is used as a pathology model leading to heart failure, which can lay the foundation for the study of organic heart failure, especially heart failure caused by abnormal myofilament structure.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an α-myosin mutant and applications thereof. Background Art

[0002] Cardiac myocytes are highly differentiated terminal cells whose contractile proteins are primarily α-myosin (α-MHC), responsible for contractile function. Contractile proteins include myosin, actin, globin, and globin. Myosin consists of two heavy chains (MHC) and two light chains (MLC). The heavy chains (MHC) are encoded by two genes: α-myosin heavy chain (α-MHC) and β-myosin heavy chain (β-MHC). These encoded proteins form three isoforms: V1 (α, α homolog), V2 (α, β heterolog), and V3 (β, β homolog). Different isoforms have different ATPase and contractile activities. The ATPase activity of myosin is primarily determined by the number of V1 or V3 isoforms present in the myocyte. α-MHC has higher ATPase activity than β-MHC. Hearts expressing α-MHC protein generate more energy than those expressing β-MHC protein, making α-MHC the primary contractile protein and thus primarily responsible for cardiomyocyte contraction. Both α-MHC and β-MHC are expressed in human and mouse cardiomyocytes. Generally, β-MHC predominates in humans, while α-MHC predominates in mice. However, under certain physiological and pathological conditions, MHC class switching can occur, accompanied by changes in myosin ATPase activity and cardiac contractility. Therefore, switching between MHC classes is an adaptive change in cardiomyocytes, allowing the heart to regulate its load and energy supply. α-MHC is relatively conserved across species and also regulates cardiac growth and gene expression in response to stress and hormonal signals. Therefore, while β-MHC is the predominant isoform in the normal adult heart, subtle changes in α-MHC can lead to significant changes in cardiomyocytes. Summary of the Invention

[0003] The present invention aims to provide an α-myosin mutant and its application.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] An α-myosin mutant, characterized in that the lysine (K) at position 1897 in the wild-type α-MHC protein is mutated to arginine (R).

[0006] The nucleotide sequence corresponding to the wild-type α-MHC protein is shown in the bases in SEQ ID NO.1.

[0007] An application of the α-myosin mutant is the application of the α-myosin mutant as a heart failure model induced by Ang II.

[0008] A primer for detecting α-myosin mutants, wherein the detection primer is

[0009] Forward primer: ‘5-TGTGCAGAAAACCCCCAAGTCTG-3’;

[0010] Back primer: '5-GAGGAACTCACCTTGGCACC-3'.

[0011] A kit for detecting α-myosin mutants contains the primers.

[0012] Specifically:

[0013] Exons 38 and 39, containing a point mutation and a pGK-Neomycin-polyA cassette, were targeted for mutation, and a c.5690A>G (K1897R) mutation vector was designed targeting the α-MHC gene. The targeting vector was electroporated into C57BL / 6J embryonic stem (ES) cells, and G418-resistant colonies were selected according to conventional protocols. Homologous recombinant ES cell clones were identified by PCR and Southern blot. Tail DNA was genotyped using a primer set specific for the α-MHC-(K1897R) mouse mutation region. Highly chimeric mice were crossed with Flp mice, and their genotypes were confirmed by PCR and primer pair sequencing to confirm the successful mutation.

[0014] The advantages of the present invention are:

[0015] The present invention obtains α-myosin mutants through the gene editing method of the CRISPR / Cas9 system, confirming that the 1897 site in α-myosin is involved in and regulates the degree of interaction between α-MHC and titin, thereby reflecting the degree of heart failure. The obtained α-myosin mutant is used as a pathological model leading to heart failure, which can lay the foundation for the study of organic heart failure, especially heart failure caused by abnormal myofilament structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram of the construction of α-MHC-K1897R transgenic mice provided in an embodiment of the present invention; wherein, a is a schematic diagram of the construction of α-MHC-K1897R transgenic mice; b is a display of sequencing results of the α-MHC-WT mutation-related region; c is a display of sequencing results of the α-MHC-K1897R mutation-related region.

[0017] Figure 2Figure 1 shows that the α-MHC K1897R mutation reduces the binding to titin provided in the embodiments of the present invention, wherein: a) a schematic diagram of mouse heart titin; b) a schematic diagram of the structure of titin; c) an IP experiment using Myc antibody after exogenous transfection of 293T cells; d) a schematic diagram showing that the α-MHC-K1897 site mutation results in reduced binding to titin; (eh) HEK293T cells were transfected with plasmids expressing Myc-α-MHC-WT, Myc-α-MHC-K1249R, Myc-α-MHC-K1533R, and Myc-α-MHC-K1897R; and (e) HEK293T cells were transfected with plasmids expressing titin's Fn3 fragments His-I106-108 (e), His-A77-78 (f), and His-A HEK293T cells were transfected with plasmids expressing Myc-α-MHC-WT, Myc-α-MHC-K1249R, Myc-α-MHC-K1533R, and Myc-α-MHC-K1897R; HEK293T cells without or with Ang II induction were transfected with plasmids expressing the Fn3 fragment of Titin His-I106-108(i), His-A77-78(j), His-A80-82(k), and His-A84-86(l).

[0018] Figure 3The α-MHC-K1897R mutation provided in the embodiments of the present invention reduces the binding of α-MHC to titin and aggravates heart failure in mice; wherein, a. Schematic diagram of the α-MHC-K1897R mutation in an Ang II-induced mouse model; b, c Myocardial tissue of WT and α-MHC-K1897R mice after 2 weeks of NaCl or Ang II treatment, immunoprecipitation with anti-Titin antibody, and detection of α-MHC; d M-mode echocardiogram of WT and α-MHC-K1897R mice after 2 weeks of NaCl or Ang II treatment, scale = 100 ms; e Fractional shortening (FS%) was used to evaluate cardiac function (n = 10 per group); f Ejection fraction (EF%) was used to evaluate cardiac function (n = 10 per group); g Masson staining was used to detect myocardial fibrosis and quantify the degree of fibrosis, scale = 20 μm; h Quantification of fibrosis area (n = 10 per group); i Transmission electron microscopy images of cardiac ultrastructure in WT and α-MHC-K1897R mice after 2 weeks of NaCl or Ang treatment. Scale bars: 2 μm, 10,000× (upper panel); 1 μm, 20,000× (lower panel). j Horizontal sections of the heart stained with H&E (upper panel) and WGA (lower panel, 10 μg / ml) to detect cardiomyocyte hypertrophy. Scale bars: 1 mm for H&E staining and 40 μm for WGA staining. k Relative quantitative analysis of cardiomyocyte cross-sectional area (n = 10 per group). l, m Representative immunoblot analysis and quantification of cardiac tissue fibrosis markers (α-SMA, Col-1), using tubulin as an internal control (n = 3 per group); n, o Representative immunoblot analysis and quantification of cardiac tissue injury markers (cleaved-caspase 3 and cleaved-PARP1), using tubulin as an internal control (n = 3 per group). (c, e, f, h, k, m, o) Data are expressed as mean ± SD. Two-way ANOVA with Bonferroni's multiple comparison test was used (P values ​​were adjusted for six comparisons; ns p > 0.05 indicates no statistical significance; *P < 0.05; **P < 0.01; ***P < 0.001). DETAILED DESCRIPTION

[0019] The specific embodiments of the present invention are further described below with reference to examples. It should be noted that the specific embodiments described here are only for illustrating and explaining the present invention, and are not intended to limit the present invention.

[0020] Example 1

[0021] Construct mutants:

[0022] 1) To evaluate the role of the α-MHC-K1897 site in Ang II-induced heart failure,

[0023] Ang II was used to induce heart failure in α-MHC-WT and α-MHC-K1897R mice for 2 weeks. Figure 3 a) We observed that α-MHC-Titin interaction was significantly reduced in α-MHC-K1897R mutant mice compared with α-MHC-WT mice under both physiological and pathological conditions ( Figure 3 b, 3c). Next, we evaluated the physiological effects of the α-MHC-K1897R mutation. Echocardiography showed that under physiological conditions, the ejection fraction (EF%) and fractional shortening (FS%) of α-MHC-K1897R mutant mice were slightly reduced compared with α-MHC-WT mice. Under Ang II-induced heart failure conditions, EF% and FS% of α-MHC-K1897R mutant mice were significantly impaired compared with α-MHC-WT mice ( Figure 3 df). Masson staining showed that compared with α-MHC-WT mice, myocardial fibrosis was mild in α-MHC-K1897R mutant mice under physiological conditions, while Ang II-induced myocardial fibrosis was significantly aggravated in α-MHC-K1897R mutant mice ( Figure 3 g, 3h). Electron microscopy showed that compared with α-MHC-WT mice, the myofibrils of α-MHC-K1897R mutant mice were slightly disordered and swollen. Ang II stimulation significantly aggravated these changes ( Figure 3 i). We used H&E staining and WGA staining to examine cardiac hypertrophy and observed that Ang II caused significant hypertrophy in the hearts of α-MHC-WT mice, while α-MHC-K1897R mutant mice showed only mild hypertrophy ( Figure 3 j, 3k). At the molecular level, we evaluated fibrosis markers (α-SMA, Col-1), as well as myocardial apoptosis-related damage (cleaved-PARP1, cleaved-caspase3). In α-MHC-K1897R mutant mice, all of these markers were slightly upregulated at baseline and significantly upregulated upon Ang II-induced heart failure ( Figure 3 lo). In summary, we speculate that the α-MHC-K1897R mutation may directly cause heart failure without obvious myocardial hypertrophy.

[0024] 2) Mutant construction:

[0025] Specifically, the base AAG encoding lysine at the α-MHC-K1897 site was mutated to AGG. The specific operation is as follows:

[0026] A mutant sequence and vector compatible with the CRISPR / Cas9 system were designed based on the sequence at the mutation site. After assembly, transgenic mice were constructed using gene editing technology. The constructed α-MHC-K1897R mutant was subsequently validated, and mutant mice were obtained using the mutant. The α-MHC-K1897R mutant mice are derived from C57BL / 6 mice with the c.5690A>G (K1897R) mutation in the α-MHC gene. The experimental animals were designed and produced by the Shanghai Model Organisms Research Center (Shanghai, China).

[0027] like Figure 1 As shown in a, the targeting construct for the α-MHC-K1897R mutant mouse was designed in exons 38 and 39 with a point mutation and a pGK-Neomycin-polyA cassette. The targeting construct was electroporated into C57BL / 6J embryonic stem (ES) cells, and colonies were selected for G418 resistance according to conventional protocols. Homologous recombinant ES cell clones were identified by PCR and Southern blot. Tail DNA was genotyped using a primer set specific for the α-MHC-(K1897R) mouse mutant region. Highly chimeric mice were crossed with Flp mice, and their genotypes were confirmed by PCR and sequencing using the following primer pair:

[0028] Forward primer: ‘5-TGTGCAGAAAACCCCCAAGTCTG-3’;

[0029] Back primer: '5-GAGGAACTCACCTTGGCACC-3'.

[0030] After the construction is completed, the mice are fed to the appropriate age, 2mm tissue from the tail tip of the mouse is cut, and lysed at 55°C for 5 hours using a lysis solution containing proteinase K. Saturated saline is added and fully shaken to remove impurities. After centrifugation, the supernatant is obtained and anhydrous ethanol is added to separate the DNA. After centrifugation again, the supernatant is discarded, and the white precipitate is retained and dried naturally, and an appropriate amount of ddH2O is added to fully dissolve it for use. Subsequently, a PCR reaction system is configured, which contains 1μl DNA, 3.2μld ddH2O, 0.4μl of each of the front and back primers, and 5μl of 2xPreMix enzyme. A standard PCR program is used (98°C 15s-98°C 30s-60°C 30s-72°C 2min-72°C 10min-4°C 10min, where steps 3 to 5 are repeated 35 cycles), and the obtained PCR products are sequenced to confirm that the mutation is successful ( Figure 1 b,1c).

[0031] Example 2

[0032] Our previous results demonstrated that the interaction between α-MHC and titin is reduced in heart failure. To investigate whether the α-MHC K1897 site plays a role in promoting titin binding, we evaluated the effects of K1897, K1533, and K1249 on the α-MHC-titin interaction under physiological and Ang II-treated conditions. We used PCR to create the corresponding K1897R, K1533R, and K1249R mutations in the Myc-α-MHC-WT plasmid. The PCR products were digested with Dpn1 and amplified. DNA was extracted and sequenced, and plasmids with successful mutations were selected for transfection. We transfected cells with His-Titin-I106-108, His-Titin-A77-78, His-Titin-A80-82, His-Titin-A84-86, or Myc-α-MHC-K1897R, Myc-α-MHC-K1533R, or Myc-α-MHC-K1249R plasmids and treated with Ang II or a control. Cells were harvested and lysed using lysis buffer (137 mM NaCl, 10 mM NaF, 50 mM Tris-HCl [pH 7.6], 1 mM EDTA, 0.1 mM sodium orthovanadate, 10% glycerol, 1% Nonidet P-40 [NP-40], and 1 mM protease inhibitors). To obtain a protein solution, 30 μL of prewashed magnetic beads were added per 1 mg of protein lysate and incubated with rotation at room temperature for 20 minutes. Subsequently, the beads were separated from the lysate using a magnetic separation rack, and the pre-cleared lysate was transferred to a clean tube. The beads were then coupled with 30 μl of anti-Myc magnetic beads (Cat# B23202, Biotool, USA) at 4°C for 12 hours. The complexes were washed with cold lysis buffer and eluted with sodium dodecyl sulfate (SDS) loading buffer. The complexes were separated by SDS-polyacrylamide gel electrophoresis and then electrotransferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with 5% bovine serum albumin at room temperature for 1 hour, followed by incubation with the primary antibody (4°C, overnight) and the secondary antibody (room temperature, 1 hour). Protein expression levels were quantified using Image J v1.46 (National Institutes of Health, US).

[0033] Our results showed that four fragments of titin (I106-108, A77-78, A80-82, and A84-86) interacted with α-MHC-WT under physiological conditions ( Figure 2 ac), which is consistent with previous research results. The binding of α-MHC-K1897R to the four fragments of titin was significantly weakened ( Figure 2ch), while the binding of α-MHC-K1249R and α-MHC-K1533R to the four fragments of titin was slightly weakened ( Figure 2 ch), indicating that the K1897 site may be a key component of the interaction between α-MHC and titin. After Ang II treatment, the interaction between the four titin fragments and α-MHC-WT was greatly weakened. The interaction between the four titin fragments and α-MHC-K1533R and α-MHC-K1249R was also weakened, but the degree of weakening was slightly less than that of α-MHC-WT. In addition, the interaction between the four titin fragments and α-MHC-K1897R remained at a low level and did not decrease significantly after Ang II treatment ( Figure 2 These results indicate that the α-MHC K1897 site, rather than K1533 and K1249, is the major site affected by Ang II-induced attenuation of the interaction between α-MHC and titin, confirming our decision to focus our studies on the α-MHC K1897 site.

[0034] Example 3

[0035] A heart failure model was further established using the mutants constructed above. In this embodiment, mice were used as an example. Specifically:

[0036] We established a control group of α-MHC-WT mice, an Ang II-treated group of α-MHC-WT mice, a control group of α-MHC-K1897R mice, and an Ang II-treated group of α-MHC-K1897R mice. Ang II treatment was administered via subcutaneous micropump for 14 days. After modeling, thoracic and ventral cardiac ultrasound was performed under anesthesia, and cardiac function-related indices were calculated using M-mode ultrasound. After ultrasound examination, cardiac tissue was obtained. A portion of the cardiac tissue was fixed with 2.5% glutaraldehyde, treated with 2% osmium tetroxide, and then embedded in resin and sectioned. Transmission electron microscopy was used to observe cardiac tissue ultrastructure. A portion of the tissue was fixed with 4% formalin for 48 hours, embedded in paraffin, and sectioned (5 μm thickness). After treatment with xylene and ethanol, the tissues were stained with H&E staining kit (G1120, Solarbio, China), Masson staining kit (G1340; Solarbio, China), and WGA staining reagent (L-1021, fluorescein F) for observation. The remaining tissues were lysed with lysis buffer for immunoprecipitation experiments and protein blotting experiments.

[0037] We used Ang II to induce heart failure in α-MHC-WT and α-MHC-K1897R mice for 2 weeks. Figure 3a) We observed that α-MHC-Titin interaction was significantly reduced in α-MHC-K1897R mutant mice compared with α-MHC-WT mice under both physiological and pathological conditions ( Figure 3 b, 3c). Next, we evaluated the physiological effects of the α-MHC-K1897R mutation. Echocardiography showed that under physiological conditions, the ejection fraction (EF%) and fractional shortening (FS%) of α-MHC-K1897R mutant mice were slightly reduced compared with α-MHC-WT mice. Under Ang II-induced heart failure conditions, EF% and FS% of α-MHC-K1897R mutant mice were significantly impaired compared with α-MHC-WT mice ( Figure 3 d, 3e, 3f). Masson staining showed that compared with α-MHC-WT mice, myocardial fibrosis was mild in α-MHC-K1897R mutant mice under physiological conditions, while Ang II-induced myocardial fibrosis was significantly aggravated in α-MHC-K1897R mutant mice ( Figure 3 g, 3h). Electron microscopy showed that compared with α-MHC-WT mice, the myofibrils of α-MHC-K1897R mutant mice were slightly disordered and swollen. Ang II stimulation significantly aggravated these changes ( Figure 3 i). We used H&E staining and WGA staining to examine cardiac hypertrophy and observed that Ang II caused significant hypertrophy in the hearts of α-MHC-WT mice, while α-MHC-K1897R mutant mice showed only mild hypertrophy ( Figure 3 j, 3k). At the molecular level, we evaluated fibrosis markers (α-SMA, Col-1), as well as myocardial apoptosis-related damage (cleaved-PARP1, cleaved-caspase3). In α-MHC-K1897R mutant mice, all of these markers were slightly upregulated at baseline and significantly upregulated upon Ang II-induced heart failure ( Figure 3 lo). In summary, we speculate that the α-MHC-K1897R mutation may directly cause heart failure without obvious myocardial hypertrophy.

[0038] SEQ ID NO.1 (nucleotide sequence corresponding to the wild-type α-MHC protein, where the bold position is the mutation site)

[0039]

[0040]

[0041]

Claims

1. An α-myosin mutant, characterized in that: Lysine (K) at position 1897 in the wild-type α-MHC protein is mutated to arginine (R); The nucleotide sequence of the nucleic acid encoding the wild-type α-MHC protein is shown in SEQ ID NO.

1.

2. A use of the α-myosin mutant according to claim 1, characterized in that: Application of α-myosin mutants in constructing Ang II-induced heart failure model.

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