RU2802995C1 - Method of control of heart rhythm and reduction of individual cardiomyocytes using thermogenetics - Google Patents

Abstract

FIELD: medicine; biotechnology.

SUBSTANCE: invention can be used to control the contraction of cardiomyocytes using thermogenetics. A genetic construct containing a cardiospecific promoter and a human thermally activated TRPV1 channel gene is delivered to cardiomyocytes, followed by heating with laser or ultrasonic radiation pulses with a frequency of 2 to 7 Hz.

EFFECT: method provides the possibility of non-invasive control of the heart rhythm and contractions of individual cardiomyocytes by imposing a rhythm with a frequency of 2 to 7 Hz.

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Description

The present invention relates to the field of medicine and biotechnology, namely to technologies for controlling the activity of excitable tissues, and can be used to control the heart rhythm in vivo, as well as to control the contractions of cardiomyocytes in vitro.

Cardiac rhythm and conduction disorders are a significant medical and social problem. For example, with sick sinus syndrome, the sinoatrial pacemaker does not generate impulses with sufficient frequency, which leads to a decrease in heart rate and pauses in the heart. These pauses can lead to fainting and can be potentially life-threatening. Other rhythm disturbances can cause sudden ventricular fibrillation and death within minutes (sudden cardiac death). The most common practice of such rhythm disturbances is the installation of a pacemaker or an automatic cardioverter-defibrillator with the implantation of one or two electrodes in the heart cavity. This approach is quite effective, however, there is a risk of perforation of the heart wall when installing electrodes, the likelihood of endocarditis, thrombosis, dislocation of electrodes and other complications both in the early postoperative and long-term periods. Automatic cardioverter-defibrillators, in addition, when triggered, generate extremely painful discharges for the patient, which leads to a significant decrease in the quality of life.

Technical solutions are known from the prior art that make it possible to stimulate cardiac tissue using light.

There is a known method for optical depolarization of heart tissue using the expression of channelrhodopsins in cardiomyocytes - protein ion channels that open under the influence of light (patent US9138596B2). The channelrhodopsin gene can be delivered to cardiomyocytes using a viral vector. An implantable device with a light source is used for depolarization. When irradiating the heart with a series of flashes, you can set the heart rate to match the frequency of the flashes. In addition, Bruegmann et al. used transgenic mice carrying the channelrhodopsin-2 gene and stimulation of the heart with laser light (Bruegmann, T., Malan, D., Hesse, M. et al. Optogenetic control of heart muscle in vitro and in vivo. Nat Methods 7, 897–900 (2010).https://doi.org/10.1038/nmeth.1512). A method for optical monitoring of cardiac function is known from the prior art, which avoids genetic modification of patient tissue (patent WO2012054484A1). It involves the use of a biological device consisting of non-excitable cells carrying channelrhodopsins, depolarized by light and forming gap junctions with cardiac cells.

The disadvantage of the above methods is the use of channelrhodopsins, which are not found in mammals and cause an immune reaction. The immune reaction in a relatively short time leads to the death of cells expressing channelrhodopsins, be they the cells of the heart itself or a biological device.

Technical solutions are known from the prior art that make it possible to control cells using temperature-sensitive ion channels. However, these solutions are not used to control heart rhythm.

In particular, methods are known to stimulate immune cells by expressing mechano- and temperature-sensitive ion channels in them, followed by exposure to ultrasound (patent WO2018098315A1). Regulation of cellular activity using thermally activated channels of the TRP (Transient Receptor Potential) superfamily is a promising approach since these channels are present in the human body and thus their expression in cardiac tissue will not trigger an immune response.

The closest to the claimed technical solution is a method for non-invasive neuromodulation of the deep parts of the brain (Yang Y, Pacia CP, Ye D, Zhu L, Baek H, Yue Y, Yuan J, Miller MJ, Cui J, Culver JP, Bruchas MR, Chen H. Sonothermogenetics for noninvasive and cell-type specific deep brain neuromodulation. Brain Stimul. 2021 Jul-Aug;14(4):790-800. doi: 10.1016/j.brs.2021.04.021). This method uses thermosensitive ion channels TRPV1 and heats brain cells using high-intensity focused ultrasound. However, this method did not implement rhythmic stimulation with sufficient frequency and was not used to control the heart rhythm.

Disclosure of the Invention

Technical problem,solved by the present invention is the impossibility of non-invasive (without the use of devices implanted directly into the myocardium) heart rate control.

By solving the stated technical problem, the following technical results are achieved: the ability to non-invasively control the heart rhythm, as well as the ability to control the contractions of individual cardiomyocytes using heating.

To solve the stated technical problem and achieve the stated technical result, a method is proposed for controlling the heart rhythm and contraction of individual cardiomyocytes using thermogenetics, which includes the following actions:

- creation of genetic constructs containing a cardiac-specific promoter and genes for thermally activated channels of the TRP superfamily;

- delivery of these constructs to cardiomyocytes for subsequent expression of the corresponding channel;

- heating the heart or individual cardiomyocytes using heat pulses of a given frequency or in a constant mode.

Brief description of the figures

Fig. 1. Map of a genetic construct based on the AAV plasmid containing the cardiac-specific promoter of the chicken troponin T (cTnT) gene, as well as the sequence of the human thermally activated TRPV1 channel with a FLAG-tag signal peptide sequence stitched to it.

Fig. 2. Results of heating the heart of a mouse that was injected with viral particles containing the AAV-cTnT-hTRPV1-FLAG construct. Heating is carried out at a frequency of 5 Hz using an IR laser. The lower graph on a large scale shows the moment of synchronization of heating and heart rate (from the 5th to the 7th second of recording).

Fig. 3. Map of a genetic construct based on the AAV plasmid containing the sequence of the human thermally activated channel TRPV1 (truncated for better packaging into viral particles without loss of functionality), the self-cleaving peptide P2A, and the fluorescent protein mRuby.

Fig. 4. Recording the membrane potential of single cardiomyocytes during their heating using an IR laser. The upper graph shows a recording of a cardiomyocyte containing the AAV-cTnT-hTRPV1(-110AA)-P2A-mRuby construct, the lower graph shows a recording of a control cardiomyocyte without the construct. Cardiomyocytes with the construct, in contrast to controls, demonstrate synchronization of action potentials with heating.

Carrying out the invention

In general, the method of controlling heart rhythm and contraction of individual cardiomyocytes using thermogenetics includes the following steps:

- creation of a genetic construct by cloning into a commercially available vector a cardiac-specific promoter and a thermally activated channel gene, as well as a reporter, allowing, if necessary, to visualize the expression of a thermally activated channel in cardiomyocytes;

- development of the resulting genetic construct using the transformation of E. coli bacteria with their subsequent expansion and isolation of plasmid DNA;

- generating a viral vector suitable for delivering the above-mentioned genetic construct into cardiomyocytes;

- delivery of the genetic construct through intravenous injection of viral particles obtained at the previous stage, or transduction of cardiomyocytes with viral particles in vitro;

- non-contact thermal effects on the heart or individual cardiomyocytes using an infrared (IR) laser or high-intensity focused ultrasound with simultaneous recording of electrical activity.

Examples

Example 1. Controlling mouse heart rate using thermogenetics

Creation and development of a genetic construct

To create genetic constructs, we used the AQUA cloning method (Beyer et al., 2015). The AAV plasmid was taken as the basis for cloning, into which the cardiac-specific promoter of the chicken troponin T (cTnT) gene was cloned, as well as the sequence of the human thermally activated TRPV1 channel with the FLAG signal peptide sequence stitched to it. This signal peptide is required for subsequent detection of TRPV1 in cardiac tissue using immunohistochemistry. A map of the resulting construct (SEQ ID NO:1) AAV-cTnT-hTRPV1-FLAG is shown in Fig. 1

Generation of a viral vector and delivery of a genetic construct

Adeno-associated viruses carrying the AAV-cTnT-hTRPV1-FLAG construct were generated using a commercially available service. Transduction of myocardial tissue for subsequent control of heart rhythm was carried out by intravenous injection 28 days before the experiment according to the following protocol:

1. Anesthetize the animal with isoflurane (5% isoflurane for induction of anesthesia, 1.5% for surgery).

2. Locally anesthetize the skin and subcutaneous tissue with 0.25% bupivacaine.

3. Under an operating microscope, make a skin incision in the area of the animal’s neck to gain access to the jugular vein.

4. Using an insulin syringe with a sterile 30G needle, draw up a viral suspension containing AAV particles AAV-cTnT-hTRPV1-FLAG (100 µl, concentration of at least 10 ¹² genomes per ml).

5. Inject the suspension into the jugular vein, remove the needle, press the injection site with a sterile cotton swab for 2 minutes.

6. Apply sutures to the skin, bring the animal out of anesthesia

Non-contact thermal effects on the heart using an IR laser

To determine the possibility of controlling the heart rhythm using thermogenetics, a software and hardware complex was used, including an electrocardiographic signal amplifier, a pulse generator, an IR laser radiation source with a cooling unit, a visible laser radiation source, an optical system with an output optical fiber attached to a stereotactic system, and a thermocouple with an amplifier, an analog-to-digital converter (ADC) and a computer with software that provides signal recording from the ADC. The electrocardiographic signal amplifier and thermocouple amplifier are connected directly to the ADC, providing electrocardiogram (ECG) and myocardial temperature recordings, respectively. The pulse generator has an output, the control signal from which is simultaneously supplied to the laser radiation source and the ADC. Thus, synchronous recording of the laser operating mode, temperature in the heating area and ECG is ensured. The optical system mixes IR radiation and visible light in the output optical fiber. Moving this optical fiber using a stereotactic system allows for targeted heating of the target area of the myocardium and visualization of the area of heating.

1. For anesthesia, intraperitoneally inject tiletamine hydrochloride/zolazepam hydrochloride (Zoletil, Virbac Sante Animale, France) - 40 mg/kg and xylazine hydrochloride (Rometar, Bioveta, Czech Republic) - 10 mg/kg.

2. Using depilatory cream, remove hair from the animal’s limbs.

3. Apply gel to the animal’s limbs to record an ECG.

4. Fix the animal’s limbs to the heated table using disposable self-adhesive electrodes for ECG recording.

5. Connect the wires of the ECG signal amplifier to the disposable electrodes.

6. Intubate the animal

7. Connect a fan for small animals. Use the following parameters of artificial lung ventilation: frequency - 90 min ^-1 , tidal volume - 0.25 ml, inspiratory to expiratory time ratio - 50%, positive end expiratory pressure (PEEP) - 5 cm of water column.

8. Locally anesthetize the skin and subcutaneous tissue with 0.25% bupivacaine.

9. Open the chest by cutting the sternum along the midline.

10. Implant a thermocouple at the heating site

11. Turn on the visible light source and bring the optical fiber to the heating site

12. Using a pulse generator, set the desired frequency and intensity of heating.

13. Start recording ADC signals, then use a pulse generator to start the IR radiation source.

In the experiment, it was shown that pulsed heating of the apex of the mouse heart using an IR laser allows one to impose a rhythm with a frequency of up to 7 Hz (420 beats per minute). An example of imposing a rhythm with a frequency of 5 Hz is shown in Fig. 2.

Example 2. Controlling the contraction of individual cardiomyocytes in vitro using thermogenetics

Creation and development of a genetic construct, generation of a viral vector

To create genetic constructs, we used the AQUA cloning method (Beyer et al., 2015). The AAV plasmid was taken as the basis for cloning, into which the cardiac-specific promoter of the chicken troponin T (cTnT) gene, the sequence of the human thermally activated TRPV1 channel (shortened by 110 amino acids for better packaging into viral particles without loss of functionality), the self-cleaving peptide P2A, as well as the fluorescent protein mRuby. This design allows visualization of transduced cells using a fluorescence microscope. A map of the resulting construct (SEQ ID NO:2) AAV-cTnT-hTRPV1(-110AA)-P2A-mRuby is shown in FIG. 3. Adeno-associated viruses carrying the AAV-cTnT-hTRPV1(-110AA)-P2A-mRuby construct were generated using a commercially available service.

Isolation and transduction of neonatal cardiomyocytes

To isolate a primary culture of neonatal cardiomyocytes, mice aged 1 to 3 days were used using a cardiac tissue dissociation kit (Miltenyi biotec) and erythrocyte lysis buffer (Miltenyi biotec). Preparation for isolation was carried out according to the following protocol:

1. Decapitate the animal with scissors, fix the limbs on the support with needles.

2. Make an incision along the chest. Use tweezers to remove the heart and place it in a Petri dish with PBS on ice. Squeeze out any remaining blood using tweezers. Repeat steps 1-2 to get the required number of hearts. The total cell yield from one heart is about 700,000.

3. Clear the hearts of connective tissue. If there is any remaining blood, squeeze it out using tweezers.

The procedure was then carried out according to the protocol of the manufacturer of the reagent kit. Cells were seeded onto gelatin-coated glasses at a rate of 10,000 cells per ^cm2 . The day after isolation, transduction was carried out by introducing AAV viral particles carrying the AAV-cTnT-hTRPV1(-110AA)-P2A-mRuby construct into the culture medium at a rate of 7.5 thousand particles per cell. A day later, the culture medium was replaced, and after another three days, experiments were carried out to control the contractions of individual cardiomyocytes.

Non-contact thermal effect on cardiomyocytes using an IR laser

To control the contractions of individual cardiomyocytes, a setup similar to that described in example 1 was used, but instead of an ECG, the membrane potential of cardiomyocytes was recorded using electrophysiological setups known from the prior art. An optical fiber with a diameter of 100 microns was used, which made it possible to heat single cells. To correlate the intensity of laser radiation with the temperature in the heating spot, calibration was previously performed.

In the experiment, it was shown that when individual cardiomyocytes are pulsed using an IR laser, action potentials arise, which makes it possible to cause contractions with a frequency of up to 7 Hz. An example of the occurrence of action potentials during stimulation with a frequency of 2 Hz is shown in Fig. 4.

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LIST OF SEQUENCES

<110> Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of

the Russian Academy of Sciences

<120> METHOD OF CONTROLLING HEART RHYTHM AND CONTRACTION OF INDIVIDUAL

CARDIOMYOCYTES USING THERMOGENETICS

<130> 0

<160> 2

<170> Patent In version 3.5

<210> 1

<211> 6898

<212> DNA

<213> Artificial Sequence

<220>

<223> Plasmid for adeno-associated viruses generation

<220>

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cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg

4281

agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg

4341

tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc

4401

gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc

4461

gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct

4521

ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa

4581

aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc

4641

cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca

4701

ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat

4761

tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg

4821

tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag

4881

ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc

4941

gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca

5001

tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc

5061

atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc

5121

cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc

5181

tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc

5241

gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg

5301

gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat

5361

ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc

5421

acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa

5481

ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa

5541

aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt

5601

gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct

5661

tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat

5721

gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg

5781

cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg

5841

atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt

5901

attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg

5961

ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg

6021

gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg

6081

tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa

6141

aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt

6201

tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt

6261

tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt

6321

ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag

6381

ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta

6441

gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat

6501

aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg

6561

ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg

6621

agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac

6681

aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga

6741

aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt

6801

ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta

6861

cggttcctgg ccttttgctg gccttttgct cacatgt

6898

<210> 2

<211> 7285

<212> DNA

<213> Artificial Sequence

<220>

<223> Plasmid for adeno-associated viruses generation

<220>

<221>exon

<222> (1045)..(3234)

<220>

<221>exon

<222> (3310)..(4020)

<400> 2

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca

120

actccatcac taggggttcc tgcggccgca cgcgtggagc tagttattaa tagtaatcaa

180

ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa

240

atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg

300

ttcccatagt aacgtcaata gggactttcc attgacgtca atgggtggag tatttacggt

360

aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg

420

tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc

480

ctacttggca gtacatctac gtattagtca tcgctattac catggttcgc ccttacgggc

540

cccccctcga ggtcgggata aaagcagtct gggctttcac atgacagcat ctggggctgc

600

ggcagaggt cgggtccgaa gcgctgcctt atcagcgtcc ccagccctgg gaggtgacag

660

ctggctggct tgtgtcagcc cctcgggcac tcacgtatct ccgtccgacg ggtttaaaat

720

agcaaaactc tgaggccaca caatagcttg ggcttatatg ggctcctgtg ggggaagggg

780

gagcacggag ggggccgggg ccgctgctgc caaaatagca gctcacaagt gttgcattcc

840

tctctgggcg ccgggcacat tcctgctggc tctgcccgcc ccggggtggg cgccgggggg

900

accttaaagc ctctgccccc caaggagccc ttcccagaca gccgccggca cccaccgctc

960

cgtgggacga tccccgaagc tctagagctt tattgcggta gtttatcaca gttaaattgc

1020

taacgcagtc aggagctcgc cacc atg agg ctc tat gat cgc agg agt atc

1071

Met Arg Leu Tyr Asp Arg Arg Ser Ile

15

ttt gaa gcc gtt gct cag aat aac tgc cag gat ctg gag agc ctg ctg

1119

Phe Glu Ala Val Ala Gln Asn Asn Cys Gln Asp Leu Glu Ser Leu Leu

10 15 20 25

ctc ttc ctg cag aag agc aag aag cac ctc aca gac aac gag ttc aaa

1167

Leu Phe Leu Gln Lys Ser Lys Lys His Leu Thr Asp Asn Glu Phe Lys

30 35 40

gac cct gag aca ggg aag acc tgt ctg ctg aaa gcc atg ctc aac ctg

1215

Asp Pro Glu Thr Gly Lys Thr Cys Leu Leu Lys Ala Met Leu Asn Leu

45 50 55

cat gac gga cag aac acc acc atc ccc ctg ctc ctg gag atc gcg cgg

1263

His Asp Gly Gln Asn Thr Thr Ile Pro Leu Leu Leu Glu Ile Ala Arg

60 65 70

caa acg gac agc ctg aag gag ctt gtc aac gcc agc tac acg gac agc

1311

Gln Thr Asp Ser Leu Lys Glu Leu Val Asn Ala Ser Tyr Thr Asp Ser

75 80 85

tac tac aag ggc cag aca gca ctg cac atc gcc atc gag aga cgc aac

1359

Tyr Tyr Lys Gly Gln Thr Ala Leu His Ile Ala Ile Glu Arg Arg Asn

90 95 100 105

atg gcc ctg gtg acc ctc ctg gtg gag aac gga gca gac gtc cag gct

1407

Met Ala Leu Val Thr Leu Leu Val Glu Asn Gly Ala Asp Val Gln Ala

110 115 120

gcg gcc cat ggg gac ttc ttt aag aaa acc aaa ggg cgg cct gga ttc

1455

Ala Ala His Gly Asp Phe Phe Lys Lys Thr Lys Gly Arg Pro Gly Phe

125 130 135

tac ttc ggt gaa ctg ccc ctg tcc ctg gcc gcg tgc acc aac cag ctg

1503

Tyr Phe Gly Glu Leu Pro Leu Ser Leu Ala Ala Cys Thr Asn Gln Leu

140 145 150

ggc atc gtg aag ttc ctg ctg cag aac tcc tgg cag acg gcc gac atc

1551

Gly Ile Val Lys Phe Leu Leu Gln Asn Ser Trp Gln Thr Ala Asp Ile

155 160 165

agc gcc agg gac tcg gtg ggc aac acg gtg ctg cac gcc ctg gtg gag

1599

Ser Ala Arg Asp Ser Val Gly Asn Thr Val Leu His Ala Leu Val Glu

170 175 180 185

gtg gcc gac aac acg gcc gac aac acg aag ttt gtg acg agc atg tac

1647

Val Ala Asp Asn Thr Ala Asp Asn Thr Lys Phe Val Thr Ser Met Tyr

190 195 200

aat gag att ctg atc ctg ggg gcc aaa ctg cac ccg acg ctg aag ctg

1695

Asn Glu Ile Leu Ile Leu Gly Ala Lys Leu His Pro Thr Leu Lys Leu

205 210 215

gag gag ctc acc aac aag aag gga atg acg ccg ctg gct ctg gca gct

1743

Glu Glu Leu Thr Asn Lys Lys Gly Met Thr Pro Leu Ala Leu Ala Ala

220 225 230

ggg acc ggg aag atc ggg gtc ttg gcc tat att ctc cag cgg gag atc

1791

Gly Thr Gly Lys Ile Gly Val Leu Ala Tyr Ile Leu Gln Arg Glu Ile

235 240 245

cag gag ccc gag tgc agg cac ctg tcc agg aag ttc acc gag tgg gcc

1839

Gln Glu Pro Glu Cys Arg His Leu Ser Arg Lys Phe Thr Glu Trp Ala

250 255 260 265

tac ggg ccc gtg cac tcc tcg ctg tac gac ctg tcc tgc atc gac acc

1887

Tyr Gly Pro Val His Ser Ser Leu Tyr Asp Leu Ser Cys Ile Asp Thr

270 275 280

tgc gag aag aac tcg gtg ctg gag gtg atc gcc tac agc agc agc gag

1935

Cys Glu Lys Asn Ser Val Leu Glu Val Ile Ala Tyr Ser Ser Ser Glu

285 290 295

acc cct aat cgc cac gac atg ctc ttg gtg gag ccg ctg aac cga ctc

1983

Thr Pro Asn Arg His Asp Met Leu Leu Val Glu Pro Leu Asn Arg Leu

300 305 310

ctg cag gac aag tgg gac aga ttc gtc aag cgc atc ttc tac ttc aac

2031

Leu Gln Asp Lys Trp Asp Arg Phe Val Lys Arg Ile Phe Tyr Phe Asn

315 320 325

ttc ctg gtc tac tgc ctg tac atg atc atc ttc acc atg gct gcc tac

2079

Phe Leu Val Tyr Cys Leu Tyr Met Ile Ile Phe Thr Met Ala Ala Tyr

330 335 340 345

tac agg ccc gtg gat ggc ttg cct ccc ttt aag atg gaa aaa att gga

2127

Tyr Arg Pro Val Asp Gly Leu Pro Pro Phe Lys Met Glu Lys Ile Gly

350 355 360

gac tat ttc cga gtt act gga gag atc ctg tct gtg tta gga gga gtc

2175

Asp Tyr Phe Arg Val Thr Gly Glu Ile Leu Ser Val Leu Gly Gly Val

365 370 375

tac ttc ttt ttc cga ggg att cag tat ttc ctg cag agg cgg ccg tcg

2223

Tyr Phe Phe Phe Arg Gly Ile Gln Tyr Phe Leu Gln Arg Arg Pro Ser

380 385 390

atg aag acc ctg ttt gtg gac agc tac agt gag atg ctt ttc ttt ctg

2271

Met Lys Thr Leu Phe Val Asp Ser Tyr Ser Glu Met Leu Phe Phe Leu

395 400 405

cag tca ctg ttc atg ctg gcc acc gtg gtg ctg tac ttc agc cac ctc

2319

Gln Ser Leu Phe Met Leu Ala Thr Val Val Leu Tyr Phe Ser His Leu

410 415 420 425

aag gag tat gtg gct tcc atg gta ttc tcc ctg gcc ttg ggc tgg acc

2367

Lys Glu Tyr Val Ala Ser Met Val Phe Ser Leu Ala Leu Gly Trp Thr

430 435 440

aac atg ctc tac tac acc cgc ggt ttc cag cag atg ggc atc tat gcc

2415

Asn Met Leu Tyr Tyr Thr Arg Gly Phe Gln Gln Met Gly Ile Tyr Ala

445 450 455

gtc atg ata gag aag atg atc ctg aga gac ctg tgc cgt ttc atg ttt

2463

Val Met Ile Glu Lys Met Ile Leu Arg Asp Leu Cys Arg Phe Met Phe

460 465 470

gtc tac atc gtc ttc ttg ttc ggg ttt tcc aca gcg gtg gtg acg ctg

2511

Val Tyr Ile Val Phe Leu Phe Gly Phe Ser Thr Ala Val Val Thr Leu

475 480 485

att gaa gac ggg aag aat gac tcc ctg ccg tct gag tcc acg tcg cac

2559

Ile Glu Asp Gly Lys Asn Asp Ser Leu Pro Ser Glu Ser Thr Ser His

490 495 500 505

agg tgg cgg ggg cct gcc tgc agg ccc ccc gat agc tcc tac aac agc

2607

Arg Trp Arg Gly Pro Ala Cys Arg Pro Pro Asp Ser Ser Tyr Asn Ser

510 515 520

ctg tac tcc acc tgc ctg gag ctg ttc aag ttc acc atc ggc atg ggc

2655

Leu Tyr Ser Thr Cys Leu Glu Leu Phe Lys Phe Thr Ile Gly Met Gly

525 530 535

gac ctg gag ttc act gag aac tat gac ttc aag gct gtc ttc atc atc

2703

Asp Leu Glu Phe Thr Glu Asn Tyr Asp Phe Lys Ala Val Phe Ile Ile

540 545 550

ctg ctg ctg gcc tat gta att ctc acc tac atc ctc ctg ctc aac atg

2751

Leu Leu Leu Ala Tyr Val Ile Leu Thr Tyr Ile Leu Leu Leu Asn Met

555 560 565

ctc atc gcc ctc atg ggt gag act gtc aac aag atc gca cag gag agc

2799

Leu Ile Ala Leu Met Gly Glu Thr Val Asn Lys Ile Ala Gln Glu Ser

570 575 580 585

aag aac atc tgg aag ctg cag aga gcc atc acc atc ctg gac acg gag

2847

Lys Asn Ile Trp Lys Leu Gln Arg Ala Ile Thr Ile Leu Asp Thr Glu

590 595 600

aag agc ttc ctt aag tgc atg agg aag gcc ttc cgc tca ggc aag ctg

2895

Lys Ser Phe Leu Lys Cys Met Arg Lys Ala Phe Arg Ser Gly Lys Leu

605 610 615

ctg cag gtg ggg tac aca cct gat ggc aag gac gac tac cgg tgg tgc

2943

Leu Gln Val Gly Tyr Thr Pro Asp Gly Lys Asp Asp Tyr Arg Trp Cys

620 625 630

ttc agg gtg gac gag gtg aac tgg acc acc tgg aac acc aac gtg ggc

2991

Phe Arg Val Asp Glu Val Asn Trp Thr Thr Trp Asn Thr Asn Val Gly

635 640 645

atc atc aac gaa gac ccg ggc aac tgt gag ggc gtc aag cgc acc ctg

3039

Ile Ile Asn Glu Asp Pro Gly Asn Cys Glu Gly Val Lys Arg Thr Leu

650 655 660 665

agc ttc tcc ctg cgg tca agc aga gtt tca ggc aga cac tgg aag aac

3087

Ser Phe Ser Leu Arg Ser Ser Arg Val Ser Gly Arg His Trp Lys Asn

670 675 680

ttt gcc ctg gtc ccc ctt tta aga gag gca agt gct cga gat agg cag

3135

Phe Ala Leu Val Pro Leu Leu Arg Glu Ala Ser Ala Arg Asp Arg Gln

685 690 695

tct gct cag ccc gag gaa gtt tat ctg cga cag ttt tca ggg tct ctg

3183

Ser Ala Gln Pro Glu Glu Val Tyr Leu Arg Gln Phe Ser Gly Ser Leu

700 705 710

aag cca gag gac gct gag gtc ttc aag agt cct gcc gct tcc ggg gag

3231

Lys Pro Glu Asp Ala Glu Val Phe Lys Ser Pro Ala Ala Ser Gly Glu

715 720 725

aag gcgggaagcg gagctactaa cttcagcctg ctgaagcagg ctggagacgt

3284

Lys

730

ggaggagaac cctggaccta gatct atg gtg tct aag ggc gaa gag ctg atc

3336

Met Val Ser Lys Gly Glu Glu Leu Ile

735

aag gaa aat atg cgt atg aag gtg gtc atg gaa ggt tcg gtc aac ggc

3384

Lys Glu Asn Met Arg Met Lys Val Val Met Glu Gly Ser Val Asn Gly

740 745 750 755

cac caa ttc aaa tgc aca ggt gaa gga gaa ggc aat ccg tac atg gga

3432

His Gln Phe Lys Cys Thr Gly Glu Gly Glu Gly Asn Pro Tyr Met Gly

760 765 770

act caa acc atg agg atc aaa gtc atc gag gga gga ccc ctg cca ttt

3480

Thr Gln Thr Met Arg Ile Lys Val Ile Glu Gly Gly Pro Leu Pro Phe

775 780 785

gcc ttt gac att ctt gcc acg tcg ttc atg tat ggc agc cgt act ttt

3528

Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr Gly Ser Arg Thr Phe

790 795 800

atc aag tac ccg aaa ggc att cct gat ttc ttt aaa cag tcc ttt cct

3576

Ile Lys Tyr Pro Lys Gly Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro

805 810 815

gag ggt ttt act tgg gaa aga gtt acg aga tac gaa gat ggt gga gtc

3624

Glu Gly Phe Thr Trp Glu Arg Val Thr Arg Tyr Glu Asp Gly Gly Val

820 825 830 835

gtc acc gtc atg cag gac acc agc ctt gag gat ggc tgt ctc gtt tac

3672

Val Thr Val Met Gln Asp Thr Ser Leu Glu Asp Gly Cys Leu Val Tyr

840 845 850

cac gtc caa gtc aga ggg gta aac ttt ccc tcc aat ggt ccc gtg atg

3720

His Val Gln Val Arg Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met

855 860 865

cag aag aag acc aag ggt tgg gag cct aat aca gag atg atg tat cca

3768

Gln Lys Lys Thr Lys Gly Trp Glu Pro Asn Thr Glu Met Met Tyr Pro

870 875 880

gca gat ggt ggt ctg agg gga tac act cat atg gca ctg aaa gtt gat

3816

Ala Asp Gly Gly Leu Arg Gly Tyr Thr His Met Ala Leu Lys Val Asp

885 890 895

ggt ggt ggc cat ctg tct tgc tct ttc gta aca act tac agg tca aaa

3864

Gly Gly Gly His Leu Ser Cys Ser Phe Val Thr Thr Tyr Arg Ser Lys

900 905 910 915

aag acc gtc ggg aac atc aag atg ccc ggt atc cat gcc gtt gat cac

3912

Lys Thr Val Gly Asn Ile Lys Met Pro Gly Ile His Ala Val Asp His

920 925 930

cgc ctg gaa agg tta gag gaa agt gac aat gaa atg ttc gta gta caa

3960

Arg Leu Glu Arg Leu Glu Glu Ser Asp Asn Glu Met Phe Val Val Gln

935 940 945

cgc gaa cac gca gtt gcc aag ttc gcc ggg ctt ggt ggt ggg atg gac

4008

Arg Glu His Ala Val Ala Lys Phe Ala Gly Leu Gly Gly Gly Met Asp

950 955 960

gag ctg tac aag taaagatcta cgggtggcat ccctgtgacc cctccccagt

4060

Glu Leu Tyr Lys

965

gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt

4120

aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt

4180

ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga

4240

accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca

4300

agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct

4360

cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc

4420

caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc

4480

gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt gcggaccgag

4540

cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc

4600

actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg

4660

agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat

4720

ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg

4780

cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc

4840

tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc

4900

gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg

4960

accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg

5020

tttttcgccc tttgacgttg gagtccaacgt tctttaatag tggactcttg ttccaaactg

5080

gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt

5140

cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa

5200

tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt

5260

taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc

5320

cggcatccgc ttacagacaa gctgtgaccg tctccggggag ctgcatgtgt cagaggtttt

5380

caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg

5440

ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc

5500

gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac

5560

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt

5620

tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag

5680

aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg

5740

aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa

5800

tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc

5860

aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag

5920

tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa

5980

ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc

6040

taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg

6100

agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa

6160

caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa

6220

tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg

6280

gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag

6340

cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg

6400

caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt

6460

ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt

6520

aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac

6580

gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag

6640

atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg

6700

tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca

6760

gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga

6820

actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca

6880

gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc

6940

agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca

7000

ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa

7060

aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc

7120

cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc

7180

gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg

7240

cctttttacg gttcctggcc ttttgctggc cttttgctca catgt

7285

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Claims (6)

1. A method for controlling the contraction of cardiomyocytes using thermogenetics, characterized by the fact that a genetic construct containing a cardiac-specific promoter and the gene for the human thermally activated channel TRPV1 is delivered to the cardiomyocytes, followed by heating of the cardiomyocytes using pulses of laser or ultrasound radiation with a frequency of 2 to 7 Hz. 2. The method according to claim 1, characterized by the use of vectors based on adeno-associated viruses as a method for delivering genetic constructs. 3. The method according to claim 1, characterized in that a genetic construct containing a cardiac-specific promoter and the human thermally activated TRPV1 channel gene is delivered to individual cardiomyocytes in vitro through transduction, followed by heating of individual cardiomyocytes. 4. The method according to claim 1, characterized in that a genetic construct containing a cardiac-specific promoter and the gene for the human thermally activated channel TRPV1 is delivered to the cardiomyocytes located in the myocardium in vivo by intravenous injection, followed by heating of the myocardium. 5. The method according to claim 1, characterized by the use of an infrared laser for heating. 6. The method according to claim 1, characterized by the use of high-intensity focused ultrasound to heat the emitter.