Method for up-regulating L-type calcium channel of myocardial cell

Abstract

The invention discloses a method for up-regulating L-type calcium channels of myocardial cells, and belongs to the technical field of molecular biology. In order to solve the problem that a method for up-regulating the L-type calcium channel of the myocardial cell with high safety and high efficiency is lacked in the prior art, the invention provides a method for up-regulating the L-type calcium channel of the myocardial cell by combining in-vivo intravenous injection or in-vitro direct incubation of a sound-sensitive agent with low-frequency and low-intensity ultrasonic irradiation treatment. According to the method for up-regulating the L-type calcium channel of the myocardial cell, the L-type calcium channel of the myocardial cell can be up-regulated, the percentage and frequency of spontaneous atrial fibrillation of a rabbit with atrial fibrillation can be reduced, the atrial fibrillation induction rate can be reduced, the effective atrial refractory period can be prolonged, the action potential time history of acute separation rabbit atrial muscle cells can be prolonged, and the current density of the L-type calcium channel of the atrial muscle cells can be increased; and mRNA and protein expression of the L-type calcium channel pore forming subunit CACNA1C are increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a method for upregulating L-type calcium channels in myocardial cells. Background Art

[0002] L-type calcium channels are a type of voltage-gated calcium ion channel, mainly distributed in cardiomyocytes and cardiovascular smooth muscle cells. They are the main channel for calcium ion influx when myocardial cells are excited. The inward current they mediate is the initiating condition for myocardial excitation-contraction coupling, and they are also the main inward current ion channel during the plateau phase of the action potential of myocardial cells.

[0003] The CACNA1C gene encodes the α1C subunit (CaV1.2), the primary component of the voltage-dependent L-type calcium channel. This pore-forming subunit is central to its structure and function. Patients with atrial fibrillation (AF) often experience a shortened atrial effective refractory period (AERP), primarily due to a decrease in L-type calcium channel current density during phase 2 of the action potential. Studies have reported that this decrease in ion channel current is due to a reduction in channel number rather than a change in channel function. Compared with the sinus rhythm group, patients with chronic AF showed significantly decreased mRNA and protein levels of CACNA1C, a pore-forming subunit of the atrial L-type calcium channel, with protein expression decreasing by approximately 40%.

[0004] L-type calcium channels are core components of myocardial electrical activity and contraction, and their dysfunction is closely related to the occurrence and development of various cardiovascular diseases. In the existing technology, research and application of L-type calcium channel regulation are mostly focused on calcium channel blockers, which are the most commonly used antihypertensive drugs, such as verapamil, diltiazem and nifedipine. However, in the existing technology, there are few studies on methods that can upregulate the expression and function of L-type calcium channels. Although gene editing technology, nanocarrier targeted myocardial technology, etc. can achieve certain regulatory purposes, they are all in the early research stage and their safety needs to be further verified.

[0005] Therefore, those skilled in the art are eager to develop a method for upregulating L-type calcium channels in cardiomyocytes with high safety and high efficiency. Summary of the Invention

[0006] The invention provides a method for upregulating L-type calcium channels in myocardial cells in order to solve the problem of lack of a highly safe and efficient method for upregulating L-type calcium channels in myocardial cells in the prior art.

[0007] One of the purposes of the present invention is to provide a method for upregulating L-type calcium channels in myocardial cells, the method comprising the following steps: administering a sonosensitizer to myocardial cells for absorption and conversion reaction, and after the reaction is completed, irradiating the cells with low-frequency, low-intensity ultrasound.

[0008] In a preferred embodiment of the present application, the administration mode of the sonosensitizer is intravenous injection or in-vitro incubation.

[0009] In a preferred embodiment of the present application, the sonosensitizer is a drug or a prodrug with sonosensitivity; including but not limited to sodium porfimer, 5-aminolevulinic acid or protoporphyrin IX.

[0010] In a preferred embodiment of the present application, the concentration of the sonosensitizer in the myocardial cells is .

[0011] In a preferred embodiment of the present application, the time of the absorption conversion reaction is 4-12 hours.

[0012] In a preferred embodiment of the present application, the condition of the irradiation treatment is: the ultrasonic frequency is 0.5 MHz-1.0 MHz, the ultrasonic intensity is 0.1 W / cm 2 -0.5 W / cm 2 , and the treatment time is 5-15 minutes.

[0013] Compared with the prior art, the present application has the beneficial effects: the present application provides a method for up-regulating L-type calcium channels of myocardial cells, which realizes the purpose of up-regulating L-type calcium channels of myocardial cells by intravenous injection or in-vitro incubation of sonosensitizer for absorption conversion reaction, and combined with low-frequency and low-intensity ultrasonic irradiation treatment.

[0014] The present application establishes a rabbit model of atrial fibrillation by implanting a pacemaker for rapid pacing, and uses the combination of sonosensitizer and low-frequency and low-intensity ultrasonic irradiation treatment. Through Holter monitoring and electrical stimulation to induce atrial fibrillation, it is proved that the combination of sonosensitizer and low-frequency and low-intensity ultrasonic irradiation treatment up-regulates L-type calcium channels of myocardial cells, reduces the percentage and frequency of spontaneous atrial fibrillation of the rabbit, reduces the atrial fibrillation induction rate, prolongs the atrial effective refractory period (AERP), prolongs the action potential duration (APD) of acute isolated rabbit atrial myocardial cells, and increases the L-type calcium channel current density of atrial myocardial cells.

[0015] The present application simulates the inflammatory environment during atrial fibrillation by stimulating HL-1 atrial myocardial cells with lipopolysaccharide (LPS), and uses the combination of sonosensitizer and low-frequency and low-intensity ultrasonic irradiation treatment for intervention. Through real-time fluorescent quantitative PCR and immunofluorescence methods, it is proved that the method for up-regulating L-type calcium channels of myocardial cells provided by the present application has the ability to increase the mRNA and protein expression of the L-type calcium channel pore-forming subunit CACNA1C, and reverses the effect of LPS; it is shown that the combination of sonosensitizer and low-frequency and low-intensity ultrasonic irradiation treatment in the present application synergistically increases the expression of the L-type calcium channel pore-forming subunit, thereby increasing the L-type calcium channel current density. It can be seen that the present application provides a more controllable, effective and safer method for up-regulating L-type calcium channels of myocardial cells BRIEF DESCRIPTION OF DRAWINGS Figure 1 Figure 2 is a graph showing the results of spontaneous atrial fibrillation detection in Example 1; A is a graph of Holter recording of 24 h spontaneous atrial fibrillation of different treatment groups of surface electrocardiogram; B is a graph of the number of rabbits with spontaneous atrial fibrillation in different treatment groups, **** represent P <0.0001; C is the frequency of spontaneous atrial fibrillation (times / 24 h) in different treatment groups; wherein, Dynamic ECG is dynamic electrocardiogram, Atrial premature beats is atrial premature beats, Atrial arrhythmia is atrial fibrillation, sinus rhythm is sinus rhythm, Spontanouos atrial arrhythmal is spontaneous atrial arrhythmia, and AF frequency is AF frequency; Figure 2 Figure 3 is a graph showing the results of atrial fibrillation induction rate detection in Example 1; A is a graph of the induction rate of atrial fibrillation in each group at an electric stimulation frequency S1=20, 40, 60 ms; B is a graph of AERP in each group at an electric stimulation frequency S1=150 ms and 200 ms, ** represent P <0.01, *** represent P <0.001, **** represent P <0.0001; AF episodes / rabbit is the average number of AF episodes per rabbit; Figure 3 Figure 4 is a graph showing the detection of action potential duration of atrial muscle cells in Example 2; A is a graph of action potential duration of atrial muscle cells; B is a graph of APD50 of atrial muscle cells; C is a graph of APD90 of atrial muscle cells. Compared with the Control group, * represent P <0.05; compared with the Paced group, # represent P <0.05; Figure 4 Figure 5 is a graph showing the detection of L-type calcium channel current density in Example 2; compared with the Paced group, * represent P <0.05, ** represent P <0.01, *** represent P <0.001; Figure 5 Figure 6 is a graph showing the detection of toxicity of DVDMS on inflammatory HL-1 atrial muscle cells in Example 3; n=6, compared with the 0 μmol / L group, *** represent P<0.001; Figure 6 Figure 3 is a graph showing the results of detecting the inflammatory HL-1 atrial myocytes in Example 3 after incubation with DVDMS for different times; A is a fluorescence intensity graph, n = 6, compared with the 0 h group, * represent P <0.05, ** represent P <0.01, *** represent P <0.001; B is a fluorescence graph of inflammatory HL-1 atrial myocytes incubated with DVDMS for 4 hours using a laser confocal microscope, the scale is 50 μm; Figure 7 Figure 4 is a graph showing the results of mRNA and protein expression of CACNA1C of HL-1 atrial myocytes in Example 3 in different treatment groups; A is a real-time quantitative PCR result graph of CACNA1C of HL-1 atrial myocytes, n = 3, compared with the LPS group, ** represent P <0.01, ns represents P >0.05; compared with the Control group, ## represent P <0.01; B is an immunofluorescence photograph of CACNA1C of HL-1 atrial myocytes, the scale is 200 μm. DETAILED DESCRIPTION

[0016] Those skilled in the art can improve the process parameters according to the content herein. In particular, it should be pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present application, to realize and apply the present application technology.

[0017] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0018] The experimental animals used in the following examples are 4-5 month old male New Zealand white rabbits (body weight 2.5-3.0 kg), purchased from the First Affiliated Hospital of Harbin Medical University Animal Experimental Center; the HL-1 atrial myocyte line used in the following examples is purchased from Hunan Fenghui Biological Technology Co., Ltd.

[0019] Example 1: (1) Preparation of atrial fibrillation rabbit model and detection of spontaneous atrial fibrillation In this example, 21 New Zealand white rabbits were housed individually in standard laboratory conditions (23°C, 12 h light intensity). Preoperatively, 3% sodium amobarbital (30 mg / kg) was injected via the ear vein for anesthesia. 5 mL of 2% lidocaine was injected intramuscularly. Endotracheal intubation and connection to a ventilator (oxygen flow rate of 1.5 L / min) were performed. A thoracotomy was performed at the right 3rd-4th intercostal space to expose the heart. A pericardial window was created, and a pacing electrode was fixed to the right atrial appendage. The pacemaker was activated by a magnet and then closed. A chest drainage tube was placed, and the chest wall was sutured layer by layer to drain all pleural fluid and air. A subcutaneous pouch was created to secure the pacemaker battery. The rabbits were then rinsed with 80,000 U of gentamicin, then disinfected and bandaged. Postoperatively, 800,000 U of penicillin were injected intramuscularly for 5 consecutive days. One week later, the pacemaker was activated and pacing was continued in AOO mode at 600 beats / min for 4 weeks to establish a rabbit model of atrial fibrillation.

[0020] The atrial fibrillation rabbit models obtained above were divided into a sham operation group (Sham group), an atrial fibrillation model group (Paced group), and a sonosensitizer + low-frequency, low-intensity ultrasound irradiation group (Paced+SDT group, abbreviated as +SDT group), with 7 rabbits in each group; rapid pacing with a pacemaker can increase spontaneous atrial fibrillation and atrial fibrillation susceptibility in rabbits. After 4 weeks of pacing, the rabbits were treated with sonosensitizer + low-frequency, low-intensity ultrasound irradiation in the atrial region. Two weeks later, 24-hour Holter monitoring was performed.

[0021] The specific steps of sonosensitizer + low-frequency, low-intensity ultrasound irradiation treatment are as follows: 1 μM sonosensitizer (the sonosensitizer is 1 μM sodium chloroporphyrin, 5-aminolevulinic acid or protoporphyrin IX) is administered to the myocardial cells by intravenous injection for absorption and conversion reaction. The reaction time is 5 hours. After the reaction is completed, low-frequency, low-intensity ultrasound is used for irradiation treatment. The conditions of the irradiation treatment are: ultrasound frequency of 1.0 MHz and ultrasound intensity of 0.5 W / cm 2 , processing time is 15 minutes.

[0022] like Figure 1 As shown in A, the surface electrocardiogram of rabbits in the Sham group showed no spontaneous atrial fibrillation, while the surface electrocardiogram of rabbits in the Paced group showed atrial fibrillation. Figure 1 In the middle part A, the black box of the Paced group is the atrial fibrillation area; the surface electrocardiogram of the rabbits in the +SDT group showed no atrial fibrillation. Figure 1 The black boxes in the middle A part +SDT group are atrial premature beats.

[0023] like Figure 1 As shown in B and C, compared with the Sham group, the percentage and frequency of spontaneous atrial fibrillation in rabbits treated with the method for upregulating the L-type calcium channel in myocardial cells provided by the present invention (+SDT group) were significantly reduced.

[0024] (2) Detection of the rate of atrial fibrillation induction The rabbits in the sham group, the paced group and the +SDT group obtained in (1) were subjected to transvenous catheterization via the internal jugular vein for intraluminal electrical stimulation and intraluminal electrocardiogram detection (IEGM), and at the same time, surface electrocardiogram detection (ECG) was performed.

[0025] The specific method is as follows: the rabbit to be detected was anesthetized with 3% sodium amobarbital (30 mg / kg) at room temperature, and the neck was shaved with a depilatory cream. The skin of the neck was incised and the subcutaneous tissue was bluntly dissected to expose the jugular vein. The distal end of the jugular vein was ligated, a micro-incision was made on the surface of the vein, and after heparin was given, the catheter was gently pushed into the right atrium. The position of the catheter was adjusted, and the induction of atrial fibrillation of the heart was recorded.

[0026] ① The atrial fibrillation induction program was: S1S1 (20 ms, 40 ms, 60 ms) promoting stimulation, each lasting 10 seconds, with an interval of 60 seconds, repeated 4 times, 12 times for each rabbit, 84 times for 7 rabbits, and the number of successful atrial fibrillation induction for each rabbit was recorded. Atrial fibrillation or atrial tachycardia is defined as a duration of more than 1000 milliseconds, and if the duration of atrial fibrillation reaches 30 minutes and still does not recover to sinus rhythm, synchronous direct current cardioversion is required to terminate atrial fibrillation.

[0027] ② The atrial effective refractory period (AERP) detection program was: S1S2 program stimulation (decrement method), with AERP 150 ms and AERP 200 ms as the basis for program stimulation, 8 times of basic S1 stimulation, S2 premature stimulation at atrial diastole, starting from 150 ms, decreasing by 5 ms each time, to AERP, the decrement interval was adjusted to 2 ms, and the definition of AERP was the longest S1S2 interval that failed to induce atrial activation by S2 premature stimulation. The AERP value was repeatedly tested three times under the basis cycle length of AERP 150 ms and AERP 200 ms, and the average value of AERP was finally taken.

[0028] As shown in Figure 2 In the experiment of electrical stimulation-induced atrial fibrillation, the rabbits in the +SDT group significantly reduced the rate of atrial fibrillation induction when the program stimulation S1=20 ms or 40 ms; the rabbits in the +SDT group had a tendency to inhibit the rate of atrial fibrillation induction when S1=60 ms, but there was no statistical difference; at the same time, the rabbits in the +SDT group could significantly prolong the atrial effective refractory period (AERP) of the atrial fibrillation rabbit when the program stimulation S1=150 ms or 200 ms.

[0029] Example 2: (1) Detection of atrial myocyte action potential duration The action potential duration of atrial myocytes in the rabbits of the sham operation group (Sham group), atrial fibrillation model group (Paced group) and sonosensitizer + low-frequency, low-intensity ultrasound irradiation group (Paced+SDT group, abbreviated as +SDT group) obtained in Example 1 was detected by patch clamp.

[0030] The specific method is as follows: acutely isolated rabbit atrial myocytes to be tested are dropped into a bath, the liquid is aspirated, and extracellular fluid for recording action potential duration is added. The cells are observed under an inverted microscope, and single cells that are adherent to the wall and have clear transverse striations and good refractive index are selected for operation. After attaching the electrode to the cell membrane, negative pressure is applied to quickly seal it, and negative pressure is applied instantaneously to rupture the membrane. The whole-cell mode is constructed, and fast and slow capacitance compensation is performed after high-resistance sealing. The action potentials are recorded in current clamp mode, and the collected currents are measured and analyzed under Clamfit 10.0.

[0031] The results of action potential duration (APD) test on rabbit atrial myocytes are as follows Figure 3 As shown, the APD of rabbit atrial myocytes in the sham group was significantly shortened. 50 and APD 90 , and the rabbit atrial myocytes APD after being treated with the method of upregulating the L-type calcium channel of myocardial cells provided by the present invention (+SDT group) 50 and APD 90 Significantly extended.

[0032] (2) Detection of L-type calcium channel current density The L-type calcium channel current density of atrial myocytes of rabbits in the sham operation group (Sham group), atrial fibrillation model group (Paced group) and sonosensitizer + low-frequency, low-intensity ultrasound irradiation group (Paced+SDT group, abbreviated as +SDT group) obtained in Example 1 was detected by patch clamp.

[0033] The specific method is as follows: drop the acutely isolated rabbit atrial myocytes to be tested into the bath, aspirate the liquid, add extracellular solution for recording L-type calcium current, observe under an inverted microscope, select a single cell that is adherent to the wall with clear transverse striations and good refractive index for operation, attach the electrode to the cell membrane and apply negative pressure to quickly seal it, apply negative pressure instantaneously to break the membrane, establish the whole-cell mode, perform fast and slow capacitance compensation after high-resistance sealing, record calcium current in voltage clamp mode, and measure and analyze the collected current under Clamfit 10.0.

[0034] like Figure 4 As shown, the L-type calcium channel current density of the rabbit atrial myocytes in the Paced group was significantly reduced, and the L-type calcium channel current density of the rabbit atrial myocytes treated with the method for upregulating the L-type calcium channel in myocardial cells provided by the present invention (+SDT group) was significantly increased.

[0035] Example 3: (1) Optimization of the concentration of DVDMS for inflammatory HL-1 atrial myocytes In order to avoid the death of inflammatory HL-1 atrial myocytes caused directly by drug toxicity due to too high concentration of DVDMS, and at the same time allow the cells to uptake as much photosensitizer DVDMS as possible, inflammatory HL-1 atrial myocytes were incubated with 0, 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3 μmol / L DVDMS for 24 hours, and the absorbance value at 450 nm was detected by CCK-8 method.

[0036] The results are shown in Figure 5 Compared with the 0, 0.2, 0.4 μmol / L treatment groups, the cell survival rate of the 0.6, 0.8, 1, 1.5, 2, 2.5, 3 μmol / L treatment groups was significantly decreased; therefore, 0.4 μmol / L was selected as the DVDMS drug concentration for inflammatory HL-1 atrial myocytes.

[0037] (2) Optimization of the incubation time of DVDMS for inflammatory HL-1 atrial myocytes In this example, 0.4 μmol / L DVDMS was used to incubate inflammatory HL-1 atrial myocytes for 1, 2, 3, 4, 5, 6, 7, 12 hours, and it was found by fluorescence microplate reader detection that the more DVDMS was taken up by inflammatory HL-1 atrial myocytes as the incubation time was prolonged, and when the incubation time was ≥3 hours, the cell fluorescence intensity was significantly increased (as shown in Figure 6 A of FIG. 6); in order to keep consistent with the DVDMS drug incubation time in the previous animal experiment, the DVDMS incubation time for inflammatory HL-1 atrial myocytes was determined to be 4 hours.

[0038] After inflammatory HL-1 atrial myocytes were incubated with DVDMS for 4 hours, Hoechst was used to label the nuclei of living cells, and under confocal microscopy, bright DVDMS red fluorescence was observed in the cytoplasm of inflammatory HL-1 atrial myocytes around the blue nucleus, indicating that the cytoplasm was rich in DVDMS (as shown in Figure 6 B of FIG. 6).

[0039] (3) Detection of mRNA and protein expression of L-type calcium channel pore-forming subunit CACNA1C of inflammatory HL-1 atrial myocytes In this example, HL-1 atrial myocytes were divided into a control group (Control group), a lipopolysaccharide stimulation group (LPS group), a photosensitizer + low-frequency low-intensity ultrasound irradiation group (LPS+SDT group), a photosensitizer group (LPS+DVDMS group), and a low-frequency low-intensity ultrasound irradiation group (LPS+Ultrasound group) for real-time fluorescent quantitative PCR detection of CACNA1C.

[0040] The specific method is as follows: the culture dish containing the cells to be detected is taken out and placed on ice, the cell supernatant is discarded, the Trizol method is used to extract cell RNA, the RNA concentration of each sample is measured in turn, the RNA concentration is adjusted to 2 μg / 20 μL, the TOYOBO reverse transcription kit is used to reverse transcribe the RNA into cDNA, the CACNA1C amplification primer is used for the first time, centrifugation is carried out at 12000 rpm for 2-3 minutes, DEPC water is added to prepare a 100 μM storage solution, and the primer is diluted 10 times when used; 10 μL of SYBR Green, 6 μL of DEPC water, 1 μL of upstream and downstream primers, and 2 μL of cDNA are added to the reaction well for amplification, and PCR amplification reaction is carried out; the reaction program is: pre-denaturation at 95℃ for 1 minute, 40 cycles; 95℃ for 15 seconds; 60℃ for 15 seconds; 72℃ for 45 seconds; the melting curve is: 95℃ for 15 seconds; 60℃ for 60 seconds; 95℃ for 30 seconds; 60℃ for 15 seconds; after the PCR amplification reaction is completed, the data is collected.

[0041] The CACNA1C amplification primer sequence is: SEQ ID NO. 1 upstream primer-CTACAGAAACCCATGTGAGCAT; SEQ ID NO. 2 downstream primer-CAGCCACGTTGTCAGTGTTG.

[0042] The real-time fluorescent quantitative PCR result is shown in Figure 7 As shown in part A of the figure, compared with the Control group, the mRNA level of the L-type calcium channel subunit CACNA1C of the inflammatory HL-1 atrial muscle cells in the LPS group decreased significantly, the mRNA transcription level of CACNA1C in the inflammatory HL-1 atrial muscle cells in the LPS+SDT group increased significantly, and the mRNA transcription level of CACNA1C in the inflammatory HL-1 atrial muscle cells in the LPS+Ultrasound group and the LPS+DVDMS group had no significant change. It can be seen that the method for up-regulating the L-type calcium channel of the myocardial cells (LPS+SDT group) provided by the application can increase the mRNA transcription level of CACNA1C in the inflammatory HL-1 atrial muscle cells.

[0043] In this embodiment, the cell immunofluorescence method is used to detect the CACNA1C protein expression of the HL-1 atrial muscle cells, and the specific method is as follows: After washing the to-be-tested inflammatory HL-1 atrial muscle cells 3 times with PBS buffer, the cells were fixed with 4% paraformaldehyde for 10 minutes, then washed 2 times with PBS buffer, permeabilized with 0.1% Triton for 2 minutes, washed 2 times with PBS buffer, blocked with room-temperature goat serum blocking solution for 30 minutes, incubated with an anti-L-type calcium channel antibody prepared by dissolving 1% BSA in PBS buffer at 4°C overnight, washed 3 times with PBS buffer the next day, incubated with a fluorescent secondary antibody at room temperature for 90 minutes, washed with PBS buffer, stained with DAPI for 10 minutes, added an anti-fluorescence quencher to the cell slide, inverted the slide on a glass slide, and collected images under a fluorescence microscope.

[0044] Results are shown in Part B of Figure 7 As shown in Part B of the results, bright red fluorescence of CACNA1C protein was observed in the Control group, and the red fluorescence was significantly weakened in the LPS group; after the method for up-regulating L-type calcium channels of myocardial cells provided by the application (LPS+SDT group) was used for 24 hours, the red fluorescence of CACNA1C protein was significantly enhanced in the immunofluorescence image. Therefore, the method for up-regulating L-type calcium channels of myocardial cells provided by the application (LPS+SDT group) increases the protein expression of CACNA1C.

[0045] The specific embodiments of the application disclosed above are only used to help explain the application. The specific embodiments do not describe all the details, nor limit the application to the specific embodiments. According to the content of the specification, many modifications and variations can be made. The specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the application, so that those skilled in the art can well understand and utilize the application.

Claims

1. A method for upregulating L-type calcium channels in myocardial cells, characterized in that: The method comprises the following steps: administering a sonosensitizer to myocardial cells for absorption and conversion reaction, and after the reaction is completed, performing irradiation treatment using low-frequency and low-intensity ultrasound.

2. The method according to claim 1, characterized in that The sonosensitizer is administered by intravenous injection or in vitro incubation.

3. The method according to claim 1, characterized in that The sonosensitizer is a sonosensitizing drug or prodrug, including but not limited to sodium thiocyanate, 5-aminolevulinic acid or protoporphyrin IX.

4. The method according to claim 1, wherein The concentration of the sonosensitizer in the myocardial cells is .

5. The method according to claim 1, wherein The absorption conversion reaction time is 4-12 hours.

6. The method according to claim 1, wherein The irradiation treatment conditions are: ultrasonic frequency of 0.5 MHz-1.0 MHz, ultrasonic intensity of 0.1 W / cm 2 -0.5 W / cm 2 , processing time is 5-15 minutes.

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