Application of Rab26 protein in preparing drugs for preventing or treating myocardial hypertrophy

Abstract

The present invention relates to the field of biomedical technologies, and specifically relates to the use of Rab26 protein in the preparation of a drug for preventing or treating myocardial hypertrophy. When the expression level of the Rab26 gene is low (less Rab26 protein is produced), the degree of myocardial cell hypertrophy increases significantly; when the Rab26 gene is overexpressed (more Rab26 protein is produced), the degree of myocardial cell hypertrophy decreases significantly. Therefore, the present invention uses Rab26 protein in the preparation of a drug for preventing or treating myocardial hypertrophy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of Rab26 protein in the preparation of drugs for preventing or treating myocardial hypertrophy. Background Art

[0002] Myocardial hypertrophy usually serves as the pre-stage of heart failure, which will lead to further deterioration of cardiac function over time and eventually develop into end-stage heart failure.

[0003] Pathological myocardial hypertrophy is a phenomenon of myocardial cell hypertrophy and hyperplasia caused by the heart's long-term exposure to high load, ischemia or other pathological stimuli. This process is not only accompanied by changes in myocardial structure and function, but also involves a series of complex molecular mechanisms, including the activation of intracellular signal transduction pathways, inflammatory responses, oxidative stress responses, etc. Although there are certain treatment methods currently, the effective means to prevent pathological myocardial hypertrophy from progressing to heart failure are still very limited, and there is an urgent need to develop new treatment strategies. Summary of the Invention

[0004] Based on this, the present invention provides the application of Rab26 protein in the preparation of drugs for preventing or treating myocardial hypertrophy, which solves at least one problem in the prior art.

[0005] In the first aspect, the present invention provides the application of Rab26 protein in the preparation of drugs for preventing or treating myocardial hypertrophy.

[0006] Rab26 protein is mainly expressed in myocardial cells. When the expression level of the Rab26 gene is low (less Rab26 protein is produced), the degree of myocardial cell hypertrophy increases significantly; when the Rab26 gene is overexpressed (more Rab26 protein is produced), the degree of myocardial cell hypertrophy decreases significantly. Therefore, Rab26 protein can be used to prepare drugs for preventing or treating myocardial hypertrophy. Rab26 protein can promote the degradation of eEF1α1 protein, thereby slowing down the occurrence and development of myocardial hypertrophy.

[0007] In some alternative embodiments, the myocardial hypertrophy is pathological myocardial hypertrophy.

[0008] In some alternative embodiments, Rab26 protein relieves myocardial hypertrophy by regulating the degradation of eEF1α1.

[0009] In the second aspect, the present invention provides a drug for preventing or treating myocardial hypertrophy, which comprises Rab26 protein.

[0010] In some alternative embodiments, the myocardial hypertrophy is pathological myocardial hypertrophy.

[0011] In some alternative embodiments, the drug for preventing or treating myocardial hypertrophy further comprises pharmaceutically acceptable excipients.

[0012] In some alternative embodiments, the pharmaceutically acceptable excipient is at least one of a solvent and a diluent.

[0013] In some alternative embodiments, the pharmaceutically acceptable excipient is water or physiological saline.

[0014] In some alternative embodiments, the dosage form of the drug for preventing or treating myocardial hypertrophy is an injection.

[0015] Due to the adoption of the above technical solutions, the embodiments of the present invention have at least the following beneficial effects:

[0016] (1) By constructing a Rab26 gene-specific knockout and overexpression mouse model, the potential of Rab26 protein in improving cardiac function was verified; overexpression of the Rab26 gene can significantly improve cardiac ejection fraction, single-cell contraction function, and reduce cardiomyocyte hypertrophy, providing a new idea for the clinical treatment of pathological myocardial hypertrophy;

[0017] (2) It was found that Rab26 protein regulates the degradation of eEF1α1 through the autophagy pathway. This discovery provides new insights into the molecular mechanism of heart failure, provides new molecular targets for drug research and development and clinical treatment, and promotes the development of heart failure treatment research; through precise targeting of Rab26 and eEF1α1 regulation, customized treatment options can be provided for patients with different types of pathological myocardial hypertrophy, improving the treatment effect. Description of the Drawings

[0018] Figure 1 It is the Western blot detection results and statistical data graphs of each group of mice or rats in Example 1.

[0019] Figure 2 It is the immunofluorescence staining results of the Sham group and the TAC group in Example 1.

[0020] Figure 3 It is the immunofluorescence co-localization analysis results of the Sham group and the TAC group in Example 1.

[0021] Figure 4 It is the immunofluorescence staining results of the Control group and the PE group of adult mouse cardiomyocytes and HL-1 cardiomyocytes in Example 1.

[0022] Figure 5 It is the immunofluorescence staining analysis results of the Control group and the PE group of adult mouse cardiomyocytes and HL-1 cardiomyocytes in Example 1.

[0023] Figure 6Western blot detection results and statistical data graphs of mice in each group in Example 2.

[0024] Figure 7 Cardiac ultrasound detection results of mice in each group in Example 2.

[0025] Figure 8 Cardiac function detection results of mice in each group in Example 2.

[0026] Figure 9 Ratio of heart weight to body weight and ratio of heart weight to tibia length of mice in each group in Example 2.

[0027] Figure 10 Atrial natriuretic peptide and brain natriuretic peptide values of mice in each group in Example 2.

[0028] Figure 11 Pathological staining of myocardial cell size of mice in each group in Example 2.

[0029] Figure 12 Statistical data graph of cross-sectional area size of pathological staining myocardial cells of mice in each group in Example 2.

[0030] Figure 13 Single myocardial cell function detection results of mice in each group in Example 3.

[0031] Figure 14 Myocardial cell mitochondrial oxygen consumption of mice in each group in Example 3.

[0032] Figure 15 Seahorse detection results of mice in each group in Example 3.

[0033] Figure 16 Western blot detection results and statistical data graphs of mice in each group in Example 4.

[0034] Figure 17 Cardiac ultrasound detection results of mice in each group in Example 4.

[0035] Figure 18 Cardiac function detection results of mice in each group in Example 4.

[0036] Figure 19 Ratio of heart weight to body weight and ratio of heart weight to tibia length of mice in each group in Example 4.

[0037] Figure 20 Atrial natriuretic peptide and brain natriuretic peptide values of mice in each group in Example 4.

[0038] Figure 21 Pathological staining of myocardial cell size of mice in each group in Example 4.

[0039] Figure 22 Statistical data graph of the cross-sectional area of cardiomyocytes in pathological staining of each group of mice in Example 4.

[0040] Figure 23 Results of single cardiomyocyte function detection in each group of mice in Example 5.

[0041] Figure 24 Mitochondrial oxygen consumption of cardiomyocytes in each group of mice in Example 5.

[0042] Figure 25 Results of Seahorse detection in each group of mice in Example 5.

[0043] Figure 26 Results of co-immunoprecipitation of Rab26 and eEF1A proteins in Example 6.

[0044] Figure 27 Results of co-localization of Rab26 and eEF1A proteins by immunofluorescence in Example 6.

[0045] Figure 28 Results of analysis of co-localization of Rab26 and eEF1A proteins by immunofluorescence in Example 6.

[0046] Figure 29 Results of Doulink-PLA analysis of Rab26 and eEF1A proteins in Example 6. Detailed implementation manners

[0047] The concept of the present invention and the resulting technical effects will be clearly and completely described below to fully elaborate the purpose, solution, and effects of the present invention.

[0048] As a member of the Rab protein family, Rab26 protein has been found to be involved in various cellular processes, such as endocytosis, cell migration, and secretion. However, the function of Rab26 protein in heart diseases has not been fully studied, especially its role in myocardial hypertrophy and heart failure remains unclear.

[0049] The inventors found that the expression of Rab26 protein was significantly down-regulated in a mouse model of pathological myocardial hypertrophy, especially in spontaneous hypertensive rats, aortic coarctation mice, and angiotensin II-induced pathological myocardial hypertrophy mouse models. Immunofluorescence staining results showed that Rab26 protein was mainly expressed in cardiomyocytes, suggesting that it may play a key role in myocardial function and pathological changes. In a mouse model with specific knockout of the myocardial Rab26 gene, the deletion of the Rab26 gene aggravated the deterioration of cardiac function, including a decrease in cardiac ejection fraction, a weakening of single-cell contractile function, and a significant increase in cardiomyocyte hypertrophy.

[0050] Through further research, the inventors found that Rab26 protein may alleviate the process of pathological myocardial hypertrophy by regulating the degradation of eEF1α1 protein. The eEF1α1 gene is an important factor involved in protein synthesis. Rab26 protein can bind to eEF1α1 protein, and there is a negative correlation between the two, suggesting that Rab26 protein may regulate the degradation of eEF1α1 protein through the autophagy pathway, thereby slowing down the occurrence and development of myocardial hypertrophy.

[0051] Accordingly, the present invention provides the use of Rab26 protein in the preparation of drugs for preventing or treating myocardial hypertrophy, and provides drugs for preventing or treating myocardial hypertrophy, providing a new targeted treatment strategy for treating pathological myocardial hypertrophy. The technical solutions and technical effects of the present invention can be reflected by the following examples.

[0052] Example 1: Determine the change trend of Rab26 protein in a mouse model of pathological myocardial hypertrophy

[0053] Animal experiments were conducted, and the following experimental groups were set up:

[0054] Spontaneously hypertensive rats (SHR group): Spontaneously hypertensive rats were purchased from Vital River Laboratories and normally raised for 16 weeks.

[0055] Spontaneously hypertensive rat control group (WKY group): Spontaneously hypertensive control (Wistar-Kyoto) rats were purchased from Vital River Laboratories and normally raised for 16 weeks.

[0056] Angiotensin II-induced group (Ang-II group): C57 mice were purchased from Vital River Laboratories and treated with angiotensin II injection (1000 ng / kg·min) through a microinfusion pump for four weeks.

[0057] Angiotensin II-induced control group (Control group): C57 mice were purchased from Vital River Laboratories and treated with PBS injection through a microinfusion pump for four weeks.

[0058] Transverse aortic constriction surgery group (TAC group): Male C57BL / 6J mice at 8 - 9 weeks of age were selected. The hair on the front of the mice's necks was fully exposed, and a depilatory cream was used to depilate the necks of the mice. Then, the depilated mice were placed in an anesthesia box with 1% - 2% isoflurane gas for anesthesia. When the mice were in an anesthetized state, their limbs and teeth were fixed on a mouse board, and 1% isoflurane was used to maintain the anesthetic state. The skin of the mice's necks was thoroughly disinfected with medical iodophor, and then the skin of the mice was cut along the anterior median margin of the neck. Forceps were used for blunt dissection of the anterior cervical thyroid tissue and muscle layer, and the thyroid tissue was gently pushed to both sides (as much as possible without affecting the blood supply of the thyroid tissue). After the anterior cervical tissue was separated cleanly, the sternum of the mice was cut open to the second rib with an ophthalmic scissors. A retractor was used to hook the neck muscles of the mice upward for fixation, fully exposing the trachea below. The muscle tissue in front of the trachea was bluntly dissected to fully expose the right common carotid artery. The branch vessels of the aortic arch were searched for, and a 6 - 0 silk thread was delivered through the aortic arch between the first and second branches of the aortic arch with a pre - threaded needle. After passing around the aortic arch, the silk thread was pulled out, and the thread end was clamped with forceps for surgical knotting. A 27G needle was first fixed and placed inside the knotted thread, and then the needle was carefully pulled out after tightening the knot. In this way, the circumferential constriction of the ascending aorta could be made of the same size, and the degree of aortic arch constriction was 50% - 60%. Then, the sternum and skin were sutured layer by layer, and the local area was disinfected again.

[0059] Sham operation group (Sham group): Male C57BL / 6J mice at 8 - 9 weeks of age were selected. The handling operations of the mice were basically the same as those in the TAC group, but only the thread was passed through and the aortic arch was not ligated.

[0060] Mice or rats in each group were fed and raised with regular diet, drinking water, and lighting for 4 weeks after the operation, and then cardiac function detection, Western blot detection, and immunofluorescence staining were performed. The results are as Figures 1 - 3 shown. The expression of Rab26 protein was down - regulated in various types of myocardial hypertrophy models. Among them, as Figure 1 shown in A in Figure 1 and B in Figure 1 , compared with the Sham group, the ratio of the content of Rab26 protein to β - Tubulin protein (Rab26 / β - Tubulin) in the cardiac tissue of mice in the TAC group decreased by about 50%; as Figure 1 shown in C in Figure 1 and D in Figure 1 , compared with the WKY group, the Rab26 / β - Tubulin in the cardiac tissue of rats in the SHR group decreased by about 20%; as Figure 2 and Figure 3As shown, compared with the Sham group, the expression of Rab26 protein in cardiomyocytes of mice in the TAC group was downregulated.

[0061] In addition, cell experiments were conducted, and the following experimental groups were set up:

[0062] Phenylephrine stimulation group (PE group): HL-1 cardiomyocytes and adult mouse cardiomyocytes were cultured in vitro, and HL-1 cardiomyocytes and adult mouse cardiomyocytes were stimulated with phenylephrine (50 μmol / L, 48 hours) to construct a cardiomyocyte hypertrophy model.

[0063] Control group (Control group): HL-1 cardiomyocytes and adult mouse cardiomyocytes were treated with PBS.

[0064] After 48 hours of modeling, Western blot detection and immunofluorescence staining were performed. The results are as Figure 4 and Figure 5 shown, and the expression of Rab26 protein in HL-1 cardiomyocytes and adult mouse cardiomyocytes in the PE group was significantly downregulated.

[0065] Example 2: Overexpression of Rab26 gene in cardiomyocytes improves pathological myocardial hypertrophy

[0066] Animal experiments were conducted, and the following experimental groups were set up:

[0067] Vector group: 4-6-week-old C57 mice were selected, and each mouse was injected with Vector (2×10^11 vg). After 4 weeks, the model was established.

[0068] Rab26 group: 4-6-week-old C57 mice were selected, and each mouse was injected with adeno-associated virus type 2 (2×10^11 vg) to overexpress the Rab26 gene through adeno-associated virus type 2. After 4 weeks, the model was established.

[0069] Vector + TAC group: On the basis of the Vector group, aortic arch constriction surgery was performed, and the operation of aortic arch constriction surgery was the same as that in Example 1.

[0070] Vector + Sham group: On the basis of the Vector group, sham surgery was performed, and the operation of sham surgery was the same as that in Example 1.

[0071] Rab26 + TAC group: On the basis of the Rab26 group, aortic arch constriction surgery was performed, and the operation of aortic arch constriction surgery was the same as that in Example 1.

[0072] Rab26 + Sham group: On the basis of the Rab26 group, sham surgery was performed, and the operation of sham surgery was the same as that in Example 1.

[0073] Mice in each group were fed a regular diet, given water, and kept under normal lighting conditions for 4 weeks after the operation, and then underwent cardiac function tests, morphological tests, plasma ANP and BNP tests, Western blot tests, and immunofluorescence staining. As Figure 6 shown, the construction of the mouse myocardial cell Rab26 gene-specific overexpression model was successful. As Figure 7 and Figure 8 shown, compared with mice in other groups, the mice with overexpression of the Rab26 gene had good cardiac function, indicating that overexpression of Rab26 could significantly improve the cardiac function of heart failure mice. As Figure 9 shown, the HW / BW and HW / TL ratios of the mice with overexpression of the Rab26 gene were smaller, indicating that overexpression of the Rab26 gene could improve the heart weight of heart failure mice. As Figure 10 shown, the levels of ANP and BNP in the mice with overexpression of the Rab26 gene were lower, indicating that overexpression of the Rab26 gene could significantly improve the levels of heart failure markers. As Figure 11 and Figure 12 shown, the results of H&E and WGA staining showed that overexpression of the Rab26 gene improved myocardial hypertrophy.

[0074] Example 3: Overexpression of the Rab26 gene in myocardial cells improves the contractile function and mitochondrial metabolism of myocardial cells in pressure overload heart failure mice

[0075] An animal experiment was conducted, and the following experimental groups were set up:

[0076] Vector group: C57 mice aged 4-6 weeks were selected, and each mouse was injected with Vector (2×10^11 vg). After 4 weeks, the model was established.

[0077] Rab26 group: C57 mice aged 4-6 weeks were selected, and each mouse was injected with adeno-associated virus type 2 (2×10^11 vg) to overexpress the Rab26 gene through adeno-associated virus type 2. After 4 weeks, the model was established.

[0078] Vector+TAC group: On the basis of the Vector group, aortic arch constriction surgery was performed, and the operation of aortic arch constriction surgery was the same as that in Example 1.

[0079] Vector+Sham group: On the basis of the Vector group, sham surgery was performed, and the operation of sham surgery was the same as that in Example 1.

[0080] Rab26+TAC group: On the basis of the Rab26 group, aortic arch constriction surgery was performed, and the operation of aortic arch constriction surgery was the same as that in Example 1.

[0081] Rab26+Sham group: On the basis of the Rab26 group, sham surgery was performed, and the operation of sham surgery was the same as that in Example 1.

[0082] Each group of mice was fed with regular diet, water, and lighting for 4 weeks after the operation, and single cardiomyocyte isolation, function detection, and Seahorse detection were performed. As Figure 13 shown, overexpression of the Rab26 gene can improve the contractile function of cardiomyocytes, and indexes such as PS, ±dL / dt, and TR90 are restored to a certain extent compared with other groups. As Figure 14 and Figure 15 shown, after TAC treatment, various indexes of primary cardiomyocytes in mice decreased. After overexpression of the Rab26 gene, mitochondrial respiration indexes such as basal respiration value, ATP production, and maximum oxygen consumption were improved. The above results indicate that overexpression of the Rab26 gene can improve the contractile function and mitochondrial energy metabolism of mouse cardiomyocytes.

[0083] Example 4: Knockout of the Rab26 gene in cardiomyocytes exacerbates pathological myocardial hypertrophy

[0084] An animal experiment was conducted, and the following experimental groups were set up:

[0085] WT group: Rab26 flox / flox mice were selected as control mice, and tamoxifen (40 mg / kg) was intraperitoneally injected continuously for three days.

[0086] CKO group: Cre-Rab26 flox / flox mice were selected as Rab26 conditional knockout mice, and tamoxifen (40 mg / kg) was intraperitoneally injected continuously for three days.

[0087] WT+TAC group: On the basis of the WT group, aortic arch constriction surgery was performed, and the operation of aortic arch constriction surgery was the same as that in Example 1.

[0088] CKO+TAC group: On the basis of the CKO group, aortic arch constriction surgery was performed, and the operation of aortic arch constriction surgery was the same as that in Example 1.

[0089] Each group of mice was fed with regular diet, water, and lighting for 4 weeks after the operation, and cardiac function detection, morphological detection, and H&E and Masson staining were performed. As Figure 16 shown, the mouse model of specific knockout of the Rab26 gene in cardiomyocytes was successfully constructed. As Figures 17 - 18 shown, compared with other groups of mice, Rab26 knockout can significantly exacerbate the deterioration of cardiac function in heart failure mice. As Figure 19 shown, knocking out the Rab26 gene can significantly increase the heart weight of heart failure mice. As Figure 20 shown, knocking out the Rab26 gene can significantly increase the level of heart failure markers. As Figures 21 - 22 shown, knocking out the Rab26 gene can significantly exacerbate myocardial cell hypertrophy in heart failure mice.

[0090] Example 5: Myocardial cell Rab26 gene knockout exacerbates myocardial cell contractile function and mitochondrial metabolism in mice with pressure overload heart failure

[0091] Animal experiments were conducted, and the following experimental groups were set up:

[0092] WT group: Rab26 flox / flox mice were selected as control mice, and tamoxifen (40 mg / kg) was intraperitoneally injected continuously for three days.

[0093] CKO group: Cre-Rab26 flox / flox mice were selected as Rab26 conditional knockout mice, and tamoxifen (40 mg / kg) was intraperitoneally injected continuously for three days.

[0094] WT+TAC group: On the basis of the WT group, aortic arch constriction surgery was performed, and the operation of aortic arch constriction surgery was the same as that in Example 1.

[0095] CKO+TAC group: On the basis of the CKO group, aortic arch constriction surgery was performed, and the operation of aortic arch constriction surgery was the same as that in Example 1.

[0096] Mice in each group were fed with regular diet, water and lighting for 4 weeks after the operation, and single myocardial cell isolation, function detection and Seahorse detection were carried out. As Figure 23 shown, myocardial-specific knockout of Rab26 exacerbates myocardial cell contractile function. As Figure 24 and Figure 25 shown, after TAC treatment, various indexes of primary myocardial cells in mice decreased, and myocardial-specific knockout of Rab26 exacerbated mitochondrial respiration indexes such as basal respiration value, ATP production, and maximum oxygen consumption. The above results indicate that specific knockout of Rab26 gene exacerbates myocardial cell contractile function and mitochondrial energy metabolism in mice.

[0097] Example 6: Interaction between Rab26 protein and eEF1α1 protein

[0098] Proteins that interact with Rab26 protein were analyzed by immunoprecipitation mass spectrometry. The mass spectrometry results showed that Rab26 protein could interact with eEF1α1 protein, and the binding between the two was further verified by immunoprecipitation, immunofluorescence and PLA. The results were as Figures 26 - 29 shown, Rab26 could significantly bind to eEF1α1.

[0099] In the above examples, the methods for detecting mice (or rats) are as follows:

[0100] ① Cardiac function detection:

[0101] The M-mode ultrasound examination was performed on mice using a Visual Sonics Vevo 2100 small animal ultrasound detection system, and the heart rate was recorded. During the examination, the measurement frequency was 15 MHz, the depth was 3 cm, and the speed was 200 mm / s. To obtain a relatively clear parasternal long-axis image of the left ventricle, the direction and position of the ultrasound probe were appropriately adjusted, and the corresponding M-mode ultrasound image was measured and recorded at the level of the left ventricular papillary muscle. Indexes such as ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at end-systolic (LVID;s), and left ventricular internal diameter at end-diastolic (LVID;d) were recorded.

[0102] ② Hemodynamic pressure detection:

[0103] Measure hemodynamic indices using a Millar catheter of model 1.4F, including aortic systolic pressure (SP) and diastolic pressure (DP). The detection steps are as follows: fully expose the hair on the front of the mouse's neck, use depilatory cream to depilate the mouse's neck, and then place the depilated mouse in an anesthesia box with 1%-2% isoflurane gas for anesthesia; when the mouse is in an anesthetized state, fix the mouse's limbs and teeth on the mouse board, and maintain the anesthetized state with 1% isoflurane; cut the mouse's skin along the anterior median margin of the neck, use forceps for blunt dissection of the anterior cervical thyroid tissue and muscle layer, gently push the thyroid tissue to both sides (as little as possible affecting the blood supply of the thyroid tissue), after the anterior cervical tissue is separated cleanly, use an ophthalmic scissors to cut the mouse's sternum to the second rib, use a retractor to hook the mouse's neck muscles upward for fixation, fully expose the trachea below, bluntly dissect the muscle tissue in front of the trachea, and fully expose the right common carotid artery; strip the tissues around the common carotid artery and the carotid sheath membrane, and carefully separate the accompanying vagus nerve; after separating the tissues around the carotid artery cleanly, tie a slipknot with 6-0 silk thread at the proximal end of the artery and a dead knot with 6-0 silk thread at the distal end, and thread a silk thread under the right common carotid artery in advance for standby; use a vascular micro-scissors to cut a "V"-shaped small opening (the opening is preferably upward) at the distal end of the right common carotid artery, fix the blood vessel with micro-forceps and slightly expand the "V"-shaped incision, slowly insert the Millar catheter from the opening of the right common carotid artery, tie the prepared silk thread, slowly loosen the slipknot at the proximal end of the right common carotid artery, push the Millar catheter forward to the aorta, stay for 2 minutes, and start recording data after the signal is stable; then continue to push the catheter forward into the left ventricle, stay for 2 minutes and record the signal again after the signal is stable. Use the instrument software paired with the Millar catheter for data analysis.

[0104] ③Morphological detection:

[0105] After anesthetizing and sacrificing the mouse, record the mouse's body weight, quickly cut out the heart and lungs and record the heart and lung weights. Embed a part of the myocardial tissue with OCT and perform frozen section, and then perform WGA staining, Tunel staining, etc.; fix another part of the myocardial tissue with 4% paraformaldehyde and perform paraffin section, and then perform H&E staining, Masson staining and other operations. Calculate the HW / BW and HW / TL ratios according to the data of the mouse's heart weight (Heart weight, HW), body weight (Body weight, BW) and tibial length (Tibial length, TL).

[0106] ④Plasma ANP and BNP detection:

[0107] The mice were anesthetized and sacrificed, and their plasma was collected. Using Nanjing Jiancheng ANP (H180-1-2) and BNP (H166-1-2) kits, the content of atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP) in the samples was detected by the competitive method. Samples were added to the enzyme-labeled wells pre-coated with antibodies, and then biotin-labeled antigen-recognizing agents were added. They were incubated at 37 °C for 30 min. The two competed with the solid-phase antibody for binding to form an immune complex. Unbound biotin antigen was removed by washing with PBST. Then avidin (HRP) was added and incubated at 37 °C for 30 min. The avidin bound to the biotin antigen. After washing, the bound HRP catalyzed tetramethylbenzidine (TMB) to turn blue, and then it was converted to yellow under the action of acid. There was an absorption peak at a wavelength of 450 nm, and the absorbance value was negatively correlated with the concentration of the antigen in the sample.

[0108] ⑤ Isolation and functional detection of single cardiomyocytes:

[0109] After anesthetizing and disinfecting the mice, the hearts were quickly removed and continuously perfused with KHB buffer for 15 min. Collagenase D (0.05 mg / ml) was added and digestion was terminated after 15 - 20 min. Then the left ventricular part was minced, and long strip-shaped cardiomyocytes were observed under the microscope. The isolated cardiomyocytes were placed on a transparent glass slide, covered with contraction buffer, and suprathreshold stimuli were given at a frequency of 0.5 Hz and an interval of 3 ms. A differential interference contrast microscope was connected to a computer monitor, and the IonOptix soft-edge software was used to record the peak shortening (PS), ± maximum shortening / re-elongation rate (± dL / dt), and shortening and re-elongation duration (TPS, TR90) values.

[0110] ⑥ Apoptosis staining of cardiomyocytes:

[0111] Using an immunohistochemical pen, marks were drawn around the OCT-embedded heart tissue sections. The tissue sections were washed 3 times with PBS for 5 minutes each time. After discarding the PBS, the tissue sections were fixed with 4% paraformaldehyde at room temperature for 15 minutes. Then the tissue sections were washed 3 times with PBS for 5 minutes each time. PBS containing 0.5% Triton X-100 was added to the surface of the sections and incubated at room temperature for 5 minutes, and then the tissue sections were washed 3 times with PBS for 5 minutes each time. The tissue sections were blocked with goat serum blocking solution at room temperature for 1 hour. The goat serum blocking solution was aspirated, and the pre-prepared Tunel detection solution was added and incubated at 37 °C in the dark for 60 minutes. After discarding the Tunel detection solution, the tissue sections were washed 3 times with PBS for 5 minutes each time. Finally, a little anti-fluorescence quenching mounting medium was added to the surface of the heart tissue for mounting, and laser confocal imaging detection and photography were performed as soon as possible.

[0112] ⑦ Western blot detection:

[0113] Use Western blot to detect the expression of proteins such as ANP, BNP, Rab26, eEF1α1 and their active components in myocardial tissues or cardiomyocytes.

[0114] ⑧ Detection of cardiomyocyte hypertrophy:

[0115] Use an immunohistochemical pen to draw marks around the OCT-embedded heart tissue sections. Wash the tissue sections with PBS 3 times, 5 minutes each time. After discarding the PBS, fix the tissue sections with 4% paraformaldehyde at room temperature for 15 minutes. Wash the tissue sections with PBS 3 times, 5 minutes each time. Block the tissue sections with goat serum blocking solution at room temperature for 1 hour. Aspirate the goat serum blocking solution, add the pre-prepared WGA staining solution, place it in a wet box and incubate at 37°C for 30 minutes. After discarding the WGA staining solution, wash the tissue sections with PBS 3 times, 5 minutes each time. Finally, add a little anti-fluorescence quenching mounting medium on the surface of the heart tissue, mount the slides, and perform laser confocal imaging detection and photography as soon as possible.

[0116] ⑨ Seahorse detection:

[0117] Use a Seahorse energy metabolism analyzer to detect the mitochondrial oxygen consumption rate (OCR) of mouse cardiomyocytes, and quantitatively analyze various indicators of mitochondrial respiratory function, including basal respiration, ATP production, maximal respiration, and spare respiration capacity.

[0118] As mentioned above, these are only the preferred embodiments of the present invention. The present invention is not limited to the above-mentioned implementation manners. As long as the same or equivalent means are used to achieve the technical effects of the present invention, they should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and changes can be made to its technical solutions and / or implementation manners.

Claims

1. Application of Rab26 protein in the preparation of drugs for preventing or treating myocardial hypertrophy.

2. The use according to claim 1, characterized in that: Myocardial hypertrophy is pathological myocardial hypertrophy.

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