Method for measuring energy efficiency in muscle cell contraction

Abstract

The present disclosure provides a method for measuring energy efficiency in muscle cell contraction by measuring ATP, which is the energy source used by muscle cells during contraction, over time.　The method includes: applying an electric stimulus to a muscle cell that expresses a fusion protein comprising, in this order from the amino terminus, a first fluorescent protein, an ATP binding part, and a second fluorescent protein, and then measuring a change in cell size of the muscle cell and a change in a fluorescent signal associated with the contraction or relaxation of the muscle cell; and calculating the energy efficiency in the contraction of the muscle cell from the change in cell size and the change in the fluorescence signal. The calculation of the energy efficiency includes calculating: the amount of change in the cell size; and the amount of change in the fluorescence signal on the basis of a fluorescence signal emitted from the fusion protein as the result of fluorescence resonance energy transfer ("FRET") caused between the first fluorescent protein and the second fluorescent protein.

Description

A method for measuring the energy efficiency of muscle cell contraction

[0001] The present invention relates to the field of cell biology, and more particularly to a method for measuring the energy efficiency of muscle cell contraction.

[0002] Heart disease is one of the leading causes of death. Adult cardiac muscle cells have lost their ability to proliferate. Therefore, once damaged, cardiac muscle cannot be repaired, resulting in permanent dysfunction and heart failure. With the aging population and Westernized lifestyles, the number of patients with heart failure is increasing.

[0003] The heart is an organ that beats continuously from birth to death, circulating blood throughout the body. The heart beats when the cardiac muscle contracts. The contraction of cardiac muscle requires adenosine triphosphate (ATP) as an energy source.

[0004] The method of supplying ATP to the myocardium differs between adults and fetuses. In adult myocardium, glucose taken in from the blood is broken down in the mitochondria to synthesize ATP. Oxygen is required to maintain efficient ATP production by mitochondria. Oxygen is supplied to fetuses via the maternal placenta. The amount of oxygen shared in the fetal bloodstream is lower than in adults. In fetal myocardium, ATP is synthesized through the glycolytic pathway, which breaks down glucose obtained from glycogen without using oxygen. Therefore, even if the supply of oxygen decreases, fetal myocardium maintains a certain level of ATP and continues contracting. Thus, even within the cardiomyocytes of the same mammalian species, the mechanism of ATP production, which is related to cellular energy, can differ depending on the developmental state of the animal.

[0005] A mouse with cardiac muscle that constitutively expresses a fluorescent probe (ATeam) that visualizes ATP concentration using the function of fluorescence resonance energy transfer (FRET) has been developed. Research using this mouse has visualized the fluctuations in ATP levels when the heart beats (Patent Document 1).

[0006] International Publication No. WO2015 / 108102

[0007] Conventional cardiac disease diagnosis involves detecting and analyzing changes over time in the entire heart at low resolution. However, no method has been developed to detect increases or decreases in energy sources as a function of individual cells that make up the heart.

[0008] The present disclosure provides a method for measuring the energy efficiency of muscle cell contraction by measuring ATP, the energy source used by muscle cells during contraction, over time. The method can identify whether a test substance alters the energy efficiency of muscle cell contraction.

[0009] The present disclosure provides the following inventions: [Item 1-1] A method for measuring the energy efficiency of muscle cell contraction, comprising: applying a stimulus to a muscle cell expressing a fusion protein comprising, from the amino terminus, a first fluorescent protein, an ATP-binding moiety, and a second fluorescent protein, in this order, to measure changes in the cell size and a fluorescent signal of the muscle cell that accompany the contraction or relaxation of the muscle cell; and calculating the energy efficiency of the muscle cell contraction from the changes in cell size and the fluorescent signal, wherein calculating the energy efficiency comprises calculating the amount of change in the fluorescent signal based on the amount of change in cell size and the amount of change in the fluorescent signal emitted from the fusion protein by fluorescence resonance energy transfer ("FRET") that occurs between the first fluorescent protein and the second fluorescent protein. [Item 1-2] A method for measuring the energy efficiency of muscle cell contraction, comprising: applying a stimulus to a muscle cell expressing a fusion protein comprising, from the amino terminus, a first fluorescent protein, an ATP-binding moiety, and a second fluorescent protein, in this order, to measure a change in cell size and a change in a fluorescent signal of the muscle cell that accompanies the contraction or relaxation of the muscle cell; and calculating the energy efficiency of the muscle cell contraction from the change in cell size and the change in the fluorescent signal, wherein calculating the energy efficiency comprises calculating an amount of change in cell size based on the change in cell size, and calculating an amount of change in the fluorescent signal based on a change in the fluorescent signal emitted from the fusion protein due to fluorescence resonance energy transfer ("FRET") that occurs between the first fluorescent protein and the second fluorescent protein.[Item 2-1] A method for identifying a substance that changes the energy efficiency of muscle cell contraction, the method comprising: contacting a test substance with muscle cells expressing a fusion protein comprising, from the amino terminus, a first fluorescent protein, an ATP-binding moiety, and a second fluorescent protein, in that order; applying a stimulus to the muscle cells to measure changes in the cell size and fluorescent signal of the muscle cells that accompany the contraction or relaxation of the muscle cells; calculating the energy efficiency of the muscle cell contraction from the changes in cell size and the change in the fluorescent signal; and comparing the energy efficiency with a threshold value to identify whether the test substance changes the energy efficiency of muscle cell contraction, wherein calculating the energy efficiency comprises calculating a change in the fluorescent signal based on a fluorescent signal emitted from the fusion protein by FRET occurring between the first fluorescent protein and the second fluorescent protein. [Item 2-2] The method of Item 1, further comprising contacting the muscle cells with the test substance; and comparing the energy efficiency with a threshold value to identify whether the test substance changes the energy efficiency of muscle cell contraction. [Item 3] The method of Item 1 or 2, wherein the ATP-binding moiety is the CBS domain of inosine monophosphate dehydrogenase 2 (IMPDH2) or a fragment or variant thereof, GlnK1 or a fragment or variant thereof, or the epsilon subunit of ATP synthase or a fragment or variant thereof. [Item 4] The method of any of Items 1 to 3, wherein the muscle cells are cardiomyocytes, smooth muscle cells, or skeletal muscle cells derived from a non-human transgenic animal that expresses the fusion protein; or cardiomyocytes, smooth muscle cells, or skeletal muscle cells that contain transfected DNA encoding the fusion protein and express the fusion protein derived from the DNA.[Item 5] The method according to any one of Items 1 to 4, wherein measuring the change in the fluorescent signal comprises: 1) before applying the stimulus, irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein; detecting a first fluorescent signal emitted from the excited fluorescent protein; and detecting a second fluorescent signal emitted from the fusion protein by FRET; and 2) after applying the stimulus, irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein; detecting a third fluorescent signal emitted from the excited fluorescent protein; and detecting a fourth fluorescent signal emitted from the fusion protein by FRET; and calculating the amount of change in the fluorescent signal comprises calculating a first fluorescent intensity ratio from the first fluorescent signal and the second fluorescent signal; calculating a second fluorescent intensity ratio from the third fluorescent signal and the fourth fluorescent signal; and calculating from the first fluorescent intensity ratio and the second fluorescent intensity ratio. [Item 6] The method of any one of Items 1 to 4, wherein measuring the change in the fluorescent signal comprises: 1) irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein before applying the stimulus; measuring a first fluorescent signal emitted from the excited fluorescent protein and detecting a first fluorescent lifetime; and 2) irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein after applying the stimulus; measuring a second fluorescent signal emitted from the excited fluorescent protein and detecting a second fluorescent lifetime, and calculating the amount of change in the fluorescent signal comprises calculating it from the first fluorescent lifetime and the second fluorescent lifetime. [Item 7] A kit for measuring energy efficiency in muscle cell contraction, comprising: muscle cells expressing a fusion protein comprising a first fluorescent protein, an ATP-binding moiety, and a second fluorescent protein, in this order from the amino terminus; and a cell-holding chamber equipped with a cell-holding compartment.[Item 8] The kit according to Item 7, wherein the muscle cells are cardiomyocytes, smooth muscle cells, or skeletal muscle cells derived from a non-human transgenic animal that express the fusion protein; or cardiomyocytes, smooth muscle cells, or skeletal muscle cells that contain transfected DNA encoding the fusion protein and express the fusion protein derived from the DNA.

[0010] Figure 1 is a scatter plot of ATP concentration in ATP-visualized fertilized eggs over time after ATP production was suppressed. The symbol ○ indicates the ATP concentration estimated based on the FRET ratio (FRET / GFP) obtained from fluorescence measurement. The symbol ◇ indicates the ATP concentration estimated based on the luminescence intensity obtained from luminescence measurement. Figure 2a is a graph showing the change in the FRET ratio in cardiomyocytes over time. Figure 2b is a graph showing the change in cardiomyocyte area over time. Figure 3 is a scatter plot showing the energy efficiency during contraction-relaxation of cardiomyocytes derived from young ATP-visualized mice. The horizontal axis represents the change in FRET efficiency corresponding to the change in ATP, and the vertical axis represents the rate of change in area. Figure 4A is a scatter plot showing the energy efficiency during contraction-relaxation of cardiomyocytes from young ATP-visualized mice treated with a selective cardiac myosin activator (omecamtib mecarbim). FIG. 4B is a scatter plot showing the energy efficiency during contraction-relaxation of cardiomyocytes from young ATP-visualized mice treated with digoxin. In FIGS. 4A and 4B, the horizontal axis represents the change in FRET efficiency, and the vertical axis represents the percentage change in area. FIG. 5A is a scatter plot showing the energy efficiency during contraction-relaxation of cardiomyocytes from old ATP-visualized mice. FIG. 5B is a scatter plot showing the energy efficiency during contraction-relaxation of cardiomyocytes from old ATP-visualized mice treated with omecamtib mecarbim. In FIGS. 5A and 5B, the horizontal axis represents the change in FRET efficiency, and the vertical axis represents the percentage change in area. FIG. 6A is a scatter plot showing the energy efficiency during contraction-relaxation of cardiomyocytes from ATP-visualized mice derived from a hereditary cardiomyopathy model ATP-visualized mouse. FIG. 6B is a scatter plot showing the energy efficiency during contraction-relaxation of the cardiomyocytes from the ATP-visualized mice treated with (□) or not treated with (●) omecamtib mecarbim. In Figures 6A and 6B, the horizontal axis represents the change in FRET efficiency. The vertical axis in Figure 6A represents the rate of change in area, and the vertical axis in Figure 6B represents the change in area. Figure 7A is a scatter plot showing the energy efficiency during contraction-relaxation of young ATP-visualized cardiomyocytes derived from an ATP-visualized mouse model of drug-induced heart failure. Figure 7B is a scatter plot showing the energy efficiency during contraction-relaxation of ATP-visualized cardiomyocytes derived from an ATP-visualized mouse model of drug-induced heart failure and treated with omecamtib mecarbim.7A and 7B, the horizontal axis represents the amount of change in FRET efficiency, and the vertical axis represents the rate of change in area.

[0011] The term "fusion protein" refers to a protein containing two or more functional domains. Fusion proteins according to the present disclosure contain a first fluorescent protein, an ATP-binding moiety, and a second fluorescent protein, in this order from the amino terminus. The first fluorescent protein and the second fluorescent protein can undergo fluorescence resonance energy transfer (FRET) between the two proteins. In one example, the first fluorescent protein functions as a FRET donor, and the second fluorescent protein functions as an acceptor. The fusion protein can be prepared, for example, by genetic engineering techniques using DNA encoding a gene for a known ATP-binding moiety, DNA encoding a fluorescent protein as the FRET donor, and DNA encoding a fluorescent protein as the acceptor. The fusion protein can be prepared, for example, according to the method described in Nakano, M., ACS Chem Biol, 2011; 6, 709-715.

[0012] The fusion protein may further include, for example, a linker sequence and a cleavage sequence. The first fluorescent protein and the second fluorescent protein are arranged, for example, so that FRET occurs or FRET is abolished when ATP binds to the ATP-binding portion of the fusion protein. The first fluorescent protein and the second fluorescent protein are preferably arranged so that FRET occurs when ATP binds to the ATP-binding portion of the fusion protein.

[0013] The term "fluorescence resonance energy transfer (FRET)" refers to a phenomenon in which, when a fluorescent protein serving as a donor and a fluorescent protein serving as an acceptor come close to each other within a certain distance, light energy absorbed by the fluorescent protein serving as a donor is transferred to the fluorescent protein serving as an acceptor. FRET is measured, for example, by irradiating the fluorescent protein serving as a donor with light of an excitation wavelength and measuring either or both of a first fluorescent signal derived from the fluorescent protein and a second fluorescent signal derived from the fluorescent protein serving as an acceptor. FRET is preferably measured by measuring the fluorescent signals derived from the first and second fluorescent proteins, respectively.

[0014] When measuring the fluorescence intensity as a fluorescent signal emitted from a fusion protein according to the present disclosure, preferably, the first fluorescent protein serving as a donor is irradiated with an excitation wavelength, and both the first fluorescent signal emitted from the first fluorescent protein and the second fluorescent signal emitted from the second fluorescent protein serving as an acceptor are measured. When measuring the fluorescence intensity as a fluorescent signal, for example, the fluorescence intensity from the first fluorescent protein serving as a donor is small when the two fluorescent proteins (the first fluorescent protein and the second fluorescent protein) are closer than a certain distance. When the distance is greater than a certain distance, the fluorescence intensity from the first fluorescent protein is large. In this example, the fluorescence intensity from the second fluorescent protein serving as an acceptor is large when the two fluorescent proteins are closer than a certain distance. When the distance is greater than a certain distance, the fluorescence intensity from the second fluorescent protein is small.

[0015] When measuring the fluorescence lifetime as a fluorescent signal emitted from a fusion protein according to the present disclosure, preferably, the fluorescent protein serving as a donor is irradiated with light of an excitation wavelength, and the fluorescent signal emitted from the fluorescent protein is measured. When measuring the fluorescence lifetime as a fluorescent signal, for example, when the two fluorescent proteins (the first fluorescent protein and the second fluorescent protein) are separated by a certain distance, the first fluorescent protein serving as a donor exhibits a fluorescence lifetime intrinsic to the first fluorescent protein. In this example, when the distance is closer than a certain distance, the first fluorescent protein exhibits a fluorescence lifetime shorter than its intrinsic fluorescence lifetime.

[0016] The "energy efficiency" of contraction of muscle cells expressing a fusion protein according to the present disclosure is calculated from the change in cell size and the change in fluorescent signal of the muscle cells accompanying the contraction or relaxation. The change in cell size or fluorescent signal of the muscle cells may be, for example, the difference between the cell size or fluorescent signal of the muscle cells before application of a stimulus and the cell size or fluorescent signal of the muscle cells after application of a stimulus and the muscle cells contract. The change in cell size or fluorescent signal of the muscle cells may be, for example, the difference between the cell size or fluorescent signal of the muscle cells after application of a stimulus and the muscle cells contract and the cell size or fluorescent signal of the muscle cells after removal of the stimulus and the muscle cells relax. Calculating the energy efficiency includes calculating the amount of change in cell size based on the change in cell size and the amount of change in fluorescent signal based on the change in fluorescent signal.

[0017] "Cell size" refers to, for example, the volume, area, or length of a cell. Cell size can be measured, for example, using a microscope (e.g., bright field). When the cell membrane of a cell is fluorescently stained, cell size can be measured, for example, using a fluorescence microscope. In one example, cell size can be measured by acquiring an image of a cell using a microscope, specifying a region of interest (ROI) in the image, and counting the number of pixels in the ROI.

[0018] Measuring the "change in cell size" can be done, for example, by measuring the cell size before deforming the outer shape of the cell (t０ ) observing the cells under a microscope to obtain a first image, and after transformation (t １ ) and observing the cells under a microscope to obtain a second image. While the above-described measuring of the change in cell size involves observing the cells at two time points, before and after the change in cell shape, the time points for measurement are not limited to these timings. In one example, measuring the change in cell size includes continuously observing and recording the morphology of at least one cell (e.g., multiple cells) for a predetermined period of time. In one example, the timing for measuring the change in cell size may be after the change in cell shape and after the cell has returned to its original shape.

[0019] The "amount of change in cell size" can be calculated based on, for example, the change in cell size. １ ) and the second image (t ２ ) and measuring the first cell size and the second cell size of the cell, respectively, and dividing the difference between the second cell size and the first cell size by the first cell size (= [(second cell size) - (first cell size)] / (first cell size); if the cell size is the volume or area of ​​the cell, this may also be referred to as the "rate of change in cell volume" or "rate of change in cell area"). In one example, １ ) may be an image recording the smallest cell size (also referred to as the "base size of the cell") exhibited by a single cell within a predetermined time period during which the cell size is observed and recorded. In one example, the base size of the cell may be the average value of the smallest cell sizes among the cell sizes that change within a predetermined time period. In one example, the second image (t ２ ) may be an image recording the largest cell size exhibited by a single cell within the predetermined time. In one example, the amount of change in cell size may be determined by creating a line graph showing the change in size exhibited by a single cell within the predetermined time, with the peak at which the cell size is largest being the second cell size and the valleys before and after the peak being the first cell size.

[0020] Calculating the amount of change in cell size includes, for example, observing the muscle cell under a microscope before deforming the outer shape of the muscle cell to obtain a first image; observing the muscle cell under a microscope after deformation to obtain a second image; designating an ROI along the periphery of the cell in the first image and counting a first number of pixels; designating an ROI along the periphery of the cell in the second image and counting a second number of pixels; and dividing the difference between the first and second pixel counts by the first pixel count (= [(second pixel count) - (first pixel count)] / (first pixel count)).

[0021] The amount of change in cell size can be expressed as, for example, t １ First cell size and t ２ (If the cell size is the volume or area of ​​the cell, this may also be referred to as the "change in cell volume" or "change in cell area"). In this example, the change in cell size is ２ Second cell size and t １ is the difference between the first cell size at (= (t ２ Second cell size at t １ The amount of change in cell size may be, for example, the amount of change per unit time from the first cell size to the second cell size (=[(t ２ Second cell size at t １ (first cell size at t ２ -t １ ): also referred to as "rate of cell size change").

[0022] The "fluorescence signal" is, for example, fluorescence intensity, fluorescence intensity ratio, or fluorescence lifetime. The fluorescence signal can be measured by a known method. For example, the fluorescence signal can be measured using a commercially available device such as a spectrophotometer or a fluorescence lifetime measurement device. For example, the fluorescence signal is obtained by measuring the fluorescence signal emitted from the entire muscle cell for a predetermined time to obtain a fluorescence image, and then specifying an ROI in the fluorescence image, and the fluorescence signal is obtained from the ROI.

[0023] Measuring a "change in fluorescent signal" includes, for example, measuring a first fluorescent signal from a cell expressing a fusion protein according to the present disclosure before changing the ATP concentration in the cell, and measuring a second fluorescent signal from the cell after changing the ATP concentration. The ATP concentration in the cell is changed, for example, by a chemical or electrical stimulus that promotes cell movement (e.g., contraction or relaxation). Although the above-described measurement of the change in fluorescent signal is performed at two time points, before and after changing the ATP concentration in the cell, the time points of measurement are not limited to these timings. In one example, the change in the ATP concentration in the cell may be measured immediately after changing the ATP concentration in the cell and after ATP recovery in the cell.

[0024] The "amount of change in the fluorescent signal" can be calculated, for example, based on the change in the fluorescent signal. The amount of change in the fluorescent signal may be, for example, a value obtained by measuring the first fluorescent intensity and the second fluorescent intensity from the first fluorescent signal and the second fluorescent signal, and dividing the second fluorescent intensity by the first fluorescent intensity (= (second fluorescent intensity) / (first fluorescent intensity): also referred to as the "FRET ratio"). In one example, it may be the ratio of the fluorescent intensity derived from the fluorescent protein serving as the acceptor ("acceptor fluorescent intensity") to the fluorescent intensity derived from the fluorescent protein serving as the donor ("donor fluorescent intensity") (= [acceptor fluorescent intensity] / [donor fluorescent intensity]). The FRET ratio can be used as an index of FRET efficiency.

[0025] Although the above-described change in the fluorescent signal has been described in terms of fluorescent intensity, the change in the fluorescent signal is not limited to this. In one example, the change in the fluorescent signal may be a change in the fluorescent lifetime. When the fluorescent lifetime is used as the fluorescent signal, the microscope system includes, for example, a pulsed laser as a light source that excites the fluorescent protein on the donor side, and a device (fluorescence lifetime measurement device) that detects the fluorescent signal from the fluorescent protein. Pulse lasers and fluorescence lifetime measurement devices are commercially available.

[0026] In one example, when 50% of a fusion protein population according to the present disclosure is bound to ATP and the remaining 50% is not bound to ATP, the fluorescence lifetime measured from the fluorescent signal emitted from the fusion protein population reflects the ratio (50%:50%) of the first fluorescence lifetime (first fluorescence decay curve) measured from the fluorescent signal emitted from the fusion protein when ATP is bound to the second fluorescence lifetime (second fluorescence decay curve) measured from the fluorescent signal emitted from the fusion protein when ATP is not bound. From the third fluorescence decay curve, the ratio of the ATP-bound fusion protein exhibiting the first fluorescence lifetime to the ATP-unbound fusion protein exhibiting the second fluorescence lifetime can be calculated by fitting. The fluorescence lifetime obtained from the fluorescent signal emitted from the fusion protein population in this manner can be used as an indicator of FRET efficiency.

[0027] When the fluorescence intensity obtained from the fusion protein of the present disclosure is used to measure the change in the fluorescence signal, the first fluorescent protein and the second fluorescent protein are preferably two different fluorescent proteins. In FRET, the donor fluorescent protein is preferably excited by light with a shorter wavelength than the acceptor fluorescent protein and has an emission spectrum that overlaps with the absorption spectrum of the fluorescence intensity of the acceptor. The fusion protein, for example, comprises a donor fluorescent protein, an ATP-binding moiety, and an acceptor fluorescent protein in this order from the amino terminus. The fusion protein, for example, comprises an acceptor fluorescent protein, an ATP-binding moiety, and a donor fluorescent protein in this order from the amino terminus. Combinations of the first and second fluorescent proteins include, for example, CFP and RFP, YFP and RFP, BFP and RFP, GFP and RFP, CFP and OFP, BFP and OFP, YFP and OFP, or GFP and OFP.

[0028] When the fluorescence lifetime obtained from the fusion protein according to the present disclosure is used to measure the change in the fluorescence signal, the first and second fluorescent proteins may be, for example, two different fluorescent proteins or the same fluorescent protein. The first and second fluorescent proteins are preferably the same fluorescent protein. Examples of combinations of the first and second fluorescent proteins include CFP and CFP, YFP and YFP, BFP and BFP, GFP and GFP, RFP and RFP, or OFP and OFP.

[0029] The change in fluorescent signal, FRET efficiency, or FRET ratio according to the present disclosure can be converted into ATP concentration using, for example, a calibration curve, which can be obtained by maintaining cells expressing a fusion protein according to the present disclosure in a culture medium or buffer, permeabilizing the cell membrane of the cells, adding a series of known concentrations of ATP to the culture medium or buffer, calculating the FRET efficiency, and creating a graph plotting the ATP concentrations used and the corresponding FRET efficiency.

[0030] The term "ATP-binding moiety" refers to a peptide whose conformation undergoes a structural change upon binding to or dissociation from adenosine triphosphate (ATP). The ATP-binding moiety exhibits, for example, adenosine diphosphate (ADP), adenosine monophosphate (AMP), or adenosine acid that is 10 times or more, 100 times or more, or 1000 times or more weaker than its binding to ATP. The ATP-binding moiety may be, for example, the CBS domain of inosine monophosphate dehydrogenase 2 (IMPDH2). The ATP-binding moiety may be, for example, GlnK1 or a fragment or variant thereof. GlnK1 is a trimeric PII-like signaling protein known to be involved in the regulation of ammonium assimilation and nitrogen fixation in bacteria. The ATP-binding moiety may be, for example, ATP synthase F. ０ F １The ATP-binding moiety may be the ε subunit of ATPase, or a fragment or variant thereof. The ε subunit may be derived from, for example, any biological species. The ATP-binding moiety may be, for example, an ε subunit derived from a microorganism (e.g., the ε subunit derived from Bacillus sp. PS3 (GenBank accession No. AB044942) or Bacillus subtilis (GenBank accession No. Z28592)).

[0031] The ε subunit contains two domains: an amino-terminal domain and a C-terminal domain. The amino-terminal domain is composed of approximately 85 amino acids from the amino terminus of the ε subunit and consists of approximately 10 β-strands. The C-terminal domain is composed of approximately 45 amino acids from the C-terminus of the ε subunit and consists of two α-helices. The ε subunit may be, for example, a full-length subunit, a fragment thereof, or a variant thereof. The fragment is a peptide that carries at least a site in the ε protein that can undergo an ATP-specific structural change. The variant has, for example, one or several (e.g., 2 to 5) amino acids substituted, deleted, inserted, or added to the amino acid sequence of the native ε subunit or a fragment thereof. The responsiveness of the fusion protein to ATP concentrations can be modified by introducing a mutation into the ATP-binding moiety described above.

[0032] Examples of "fluorescent proteins" include cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), green fluorescent protein (GFP), red fluorescent protein (RFP), and orange fluorescent protein (OFP). Fluorescent proteins are described, for example, in Table 1 of Nature Methods, vol. 9, No. 10, 1005-1012 (2012). Fluorescent proteins can be prepared by known methods or are commercially available. For example, CFP is commercially available from Invitrogen. YFP is commercially available from Invitrogen, or Phi-Yellow is commercially available from Evrogen. GFP is commercially available from Clontech, or Tag-GFP is commercially available from Evrogen. As RFP, DsRed2-monomer from Clontech or HcRed-Tandem from Evrogen are commercially available.

[0033] The term "non-human transgenic animal expressing a fusion protein" refers to an animal other than a human that has been engineered to express the fusion protein according to the present disclosure. The non-human transgenic animal can be obtained, for example, by incorporating DNA encoding the fusion protein into the animal's chromosome. The DNA encoding the fusion protein is expressed under the control of a promoter so that it functions in the cells of the non-human transgenic animal. Examples of the promoter include a β-actin promoter, a CMV promoter, and a CAG (CAGGS) promoter. From the viewpoint of expression efficiency, the promoter is, for example, a CAG (CAGGS) promoter. The promoter is, for example, a stage-specific promoter or a tissue-specific promoter.

[0034] When DNA encoding a fusion protein replaces an endogenous gene on a chromosome by homologous recombination, the DNA contains, for example, a sequence homologous to a target site on the chromosome. In one example, the target site on the chromosome is the Rosa26 locus. Examples of the Rosa26 locus include, but are not limited to, the human Rosa26 locus (Irion S. et al., Nature Biotechnology, volume 25, pages 1477-1482 (2007)), the mouse Rosa26 locus, and corresponding loci in other animals. The mouse ROSA26 locus was discovered in 1991 by Friedrich and Soriano through a gene trap experiment using embryonic stem (ES) cells infected with a retrovirus (Friedrich, G. and P. Soriano, Genes & Development, 1991, 5, 1513-1523).

[0035] The DNA encoding the fusion protein may contain a recognition sequence, such as a lox-based sequence, recognized by a recombinant protein such as Cre recombinase or Flp recombinase. In one example, a stop codon and / or a drug resistance gene may be placed between the promoter and the DNA encoding the fusion protein, so that upon Cre recombinase expression, the promoter and the DNA encoding the fusion protein are directly linked, initiating transcription by the promoter. Examples of lox-based sequences include loxP, lox71, lox66, lox511, lox2272, Vlox (VCre), and Slox (SCre). In another example, an FRT sequence recognized by FLP may be used as the recognition sequence.

[0036] The non-human transgenic animal can be produced according to the methods described in, for example, International Publication WO2015 / 108102, Hogan et al., "Manipulating the Mouse Embryo: A Laboratory Manual," Cold Spring Harbor Laboratory (1986), and U.S. Patent Nos. 5,616,491 and 5,750,826. The non-human transgenic animal may be a disease model non-human transgenic animal produced according to a known method.

[0037] The DNA encoding the fusion protein may include, for example, a selection marker (e.g., a drug resistance gene), a promoter, an enhancer, a start codon, and a stop codon. The term "selection marker" refers to a genetic element that provides a selectable phenotype to a cell into which the selection marker has been introduced. A selection marker is, for example, a gene encoding a protein that confers resistance to a drug that inhibits cell growth or kills the cell. A selection marker may be, for example, the Neo gene, Hyg gene, hisD gene, Gpt gene, or Ble gene.

[0038] A "non-human transgenic animal" or a "non-human mammal" may be, for example, a rodent, such as a mouse, rat, guinea pig, or hamster; a non-human primate, such as a chimpanzee; an even-toed ungulate, such as a cow, goat, or sheep; a perissodactyl, such as a horse; or a companion animal, such as a rabbit, dog, or cat. A non-human mammal is, for example, a rodent or a non-human primate. A "mammal" includes humans and non-human mammals.

[0039] The term "muscle cell" refers to a cell that can contract or relax in response to a stimulus. Examples of muscle cells include mammalian muscle cells. Examples of muscle cells include cardiac muscle cells, skeletal muscle cells, and smooth muscle cells. Examples of muscle cells include mammalian cardiac muscle cells, skeletal muscle cells, or smooth muscle cells. Examples of muscle cells include muscle cells (e.g., cardiomyocytes) derived from a non-human transgenic animal expressing a fusion protein according to the present disclosure. Muscle cells derived from a non-human transgenic animal can be obtained, for example, by harvesting a tissue or tissue fragment containing the muscle cells (e.g., the heart in the case of cardiac muscle cells) from the transgenic animal and isolating the cells constituting the tissue or tissue fragment into individual cells. Obtaining the muscle cells includes, for example, selecting desired muscle cells from an isolated cell population. Isolating a cell population includes, for example, treating a tissue or tissue fragment containing the cell population with a cell-dissociating enzyme such as collagenase, trypsin, or TrypLE™.

[0040] The muscle cells are, for example, muscle cells (e.g., cardiomyocytes) that have been transfected with DNA encoding the fusion protein and that express the fusion protein. The muscle cells are, for example, muscle cells (e.g., cardiomyocytes) that have been induced to differentiate from stem cells transfected with DNA encoding the fusion protein and that constitutively express the fusion protein. The muscle cells are, for example, muscle cells (e.g., cardiomyocytes) that have been transfected with DNA encoding the fusion protein and that transiently express the fusion protein. Transfection of DNA into muscle cells can be carried out according to known methods such as calcium chloride precipitation and electroporation.

[0041] "Stem cells" may be, for example, embryonic stem cells and induced pluripotent stem cells. Embryonic stem cells are, for example, publicly available or commercially available. Induced pluripotent stem cells are, for example, commercially available or can be prepared by introducing reprogramming factors into somatic cells.

[0042] "Contraction" of a muscle cell includes a decrease in cell volume or area. When a muscle cell expresses a fusion protein according to the present disclosure, contraction of the muscle cell includes an increase in apparent fluorescence intensity per unit area. Contraction of a muscle cell can be measured, for example, based on a change in cell size using a microscope. Contraction of a muscle cell can be measured, for example, by detecting a fluorescent signal. Contraction of a muscle cell can be induced, for example, by applying an extracellular stimulus (e.g., a compound or an electrical stimulus) to the muscle cell.

[0043] "Relaxation" of a muscle cell includes an increase in the volume or area of ​​the muscle cell. When a muscle cell expresses a fusion protein according to the present disclosure, relaxation of the cell includes a decrease in apparent fluorescence intensity per unit area. Relaxation of a muscle cell can be measured, for example, based on a change in cell size using a microscope. Relaxation of a muscle cell can be measured, for example, by detecting a fluorescent signal. Relaxation of a muscle cell can be induced, for example, by removing an extracellular stimulus (e.g., a stimulus with a compound or an electrical stimulus) from the muscle cell.

[0044] "Applying a stimulus" to muscle cells includes, for example, bringing a compound into coexistence with muscle cells, applying an electrical stimulus, or changing the pH of the environment in which muscle cells are maintained. Applying a stimulus includes, for example, removing the stimulus. "Removing a stimulus" to muscle cells includes, for example, removing a compound that was in coexistence with muscle cells, ceasing the application of an electrical stimulus, or changing the pH of the environment in which muscle cells are maintained. The stimulus is preferably an electrical stimulus.

[0045] "Stimulation with a compound" includes, for example, causing a compound capable of causing contraction or relaxation of muscle cells to coexist with muscle cells. The coexistence of the compound with muscle cells includes, for example, introducing (e.g., adding) the compound to the environment (e.g., culture medium) in which the muscle cells are maintained. Stimulation with a compound includes, for example, removing a compound that was coexisting with muscle cells. Removing a coexisting compound includes, for example, changing the environment (e.g., culture medium) in which the muscle cells are maintained (replace with a medium that does not contain the compound). A compound that can cause contraction or relaxation of muscle cells may be, for example, a cardiac inotropic agent (e.g., catecholamine).

[0046] The "electrical stimulation" is appropriately set by, for example, adjusting the pulse voltage, pulse frequency, and pulse duration. The electrical stimulation can be generated, for example, by a commercially available electrical stimulation device. The pulse voltage is, for example, 10 to 100 V, 30 to 100 V, 40 to 100 V, 50 to 100 V, 10 to 80 V, 30 to 80 V, 40 to 80 V, 50 to 80 V, 10 to 70 V, 30 to 70 V, 40 to 70 V, 50 to 70 V, 10 to 60 V, 30 to 60 V, 40 to 60 V, 50 to 60 V, or 50 V. The pulse frequency is set, for example, so that the contraction of muscle cells due to the nth electrical stimulation can be relaxed to the original cell size by the time of the n+1th electrical stimulation. The pulse frequency is, for example, 0.1 to 10 Hz, 0.3 to 10 Hz, 0.5 to 10 Hz, 0.7 to 10 Hz, 1 to 10 Hz, 3 to 10 Hz, 0.1 to 7 Hz, 0.3 to 7 Hz, 0.5 to 7 Hz, 0.7 to 7 Hz, 1 to 7 Hz, 3 to 7 Hz, 0.1 to 5 Hz, 0.3 to 5 Hz, 0.5 to 5 Hz, 0.7 to 5 Hz, 1 to 5 Hz, 3 to 5 Hz, 0.1 to 3 Hz, 0.3 to 3 Hz, 0.5 to 3 Hz, 0.7 to 3 Hz, 1 to 3 Hz, or 1 Hz. The duration of the pulse can be, for example, 1 to 100 seconds, 3 to 100 seconds, 5 to 100 seconds, 7 to 100 seconds, 10 to 100 seconds, 20 to 100 seconds, 1 to 70 seconds, 3 to 70 seconds, 5 to 70 seconds, 7 to 70 seconds, 10 to 70 seconds, 20 to 70 seconds, 1 to 50 seconds, 3 to 50 seconds, 5 to 50 seconds, 7 to 50 seconds, 10 to 50 seconds, 20 to 50 seconds, 1 to 30 seconds, 3 to 30 seconds, 5 to 30 seconds, 7 to 30 seconds, 10 to 30 seconds, 20 to 30 seconds, 1 to 20 seconds, 3 to 20 seconds, 5 to 20 seconds, 7 to 20 seconds, 10 to 20 seconds, or 10 seconds.

[0047] "pH change" or "altering the pH" includes, for example, adding a pH-altering reagent (e.g., a phosphate buffer of a predetermined pH) to the environment (e.g., culture medium) in which the muscle cells are maintained.

[0048] The "test substance" may be, for example, a small molecule compound, a protein (e.g., an antibody), DNA, RNA, small interfering RNA, or an antisense oligonucleotide. The test substance may be, for example, a drug for treating heart disease, or a candidate substance thereof. The test substance may be, for example, one type, or a mixture of two or more types. The test substance is preferably one type of substance.

[0049] The term "cardiac disease" refers to a disease of the heart, including, for example, heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated phase hypertrophic cardiomyopathy, and dilated cardiomyopathy, as well as diseases associated with these diseases or disorders.

[0050] <Method for Measuring Energy Efficiency in Muscle Cell Contraction> One aspect of the present disclosure provides a method for measuring the energy efficiency of muscle cell contraction. The measurement method includes (step 1) applying a stimulus to a muscle cell expressing a fusion protein according to the present disclosure and measuring changes in the cell size and fluorescent signal of the muscle cell that accompany the contraction or relaxation of the muscle cell; and (step 2) calculating the energy efficiency of the muscle cell contraction from the changes in the cell size and the fluorescent signal. Calculating the energy efficiency includes calculating the amount of change in cell size and the amount of change in the fluorescent signal based on a fluorescent signal emitted from the fusion protein by FRET occurring between the first fluorescent protein and the second fluorescent protein.

[0051] Step 1 includes applying a stimulus to the muscle cells to contract or relax the muscle cells (step 1-1), and measuring changes in cell size and fluorescent signals accompanying the contraction or relaxation (step 1-2). Step 1-1 is performed, for example, before step 1-2. Step 1-2 includes, for example, continuously measuring the cell size and fluorescent signals for a predetermined period of time. Step 1-2 may be performed, for example, at a time including before, simultaneously with, and after step 1-1. In this example, step 1-1 is performed substantially simultaneously with step 1-2. In one example, step 1 includes applying a stimulus to the muscle cells (step 1-1) while measuring the cell size and fluorescent signals (step 1-2).

[0052] Step 1-1 includes, for example, introducing at least one muscle cell into a chamber equipped with a cell-holding compartment and electrodes. The introduction of at least one muscle cell or an isolated muscle cell population into the chamber can be performed using, for example, a pipette or other dispenser. The introduction of the muscle cells into the chamber further includes, for example, leaving the introduced muscle cells to settle on the wall or bottom of the cell-holding compartment. The electrodes included in the chamber are positioned so that electrical stimulation can be applied to the cell-holding compartment. The chamber includes, for example, a means for supplying a culture medium to the cell-holding compartment (e.g., a perfusion tube). Such a chamber is commercially available, for example, from Warner Instruments (RC-27NE2: Narrow Bath Chamber with Field Stimulation).

[0053] The "electrode" includes, for example, conductive carbon, gold, platinum, palladium, stainless steel, tin, tungsten, titanium, or a combination thereof. The electrode includes, for example, a first electrode and a second electrode separated by a gap in the cell-holding compartment. The gap is appropriately set, for example, in the range of 1 mm to 1 cm. The first electrode is arranged parallel or substantially parallel to the second electrode. The first electrode and the second electrode can be electrically connected, for example, to an electrode pair of an electrical stimulation device. The electrodes are embedded in the bottom or wall of the well. The bottom is made of, for example, a transparent material (e.g., resin or glass). The wall is made of, for example, a transparent material.

[0054] Step 1-1 further includes, for example, installing the chamber in a fluorescence microscope system so that fluorescence observation of the muscle cells introduced therein is possible, and electrically connecting electrodes of the chamber to an electrical stimulation device. The installation in the microscope system includes, for example, mounting the chamber on a stage of the microscope system. When the chamber includes the culture medium supply means, step 1-1 further includes, for example, supplying a culture medium to the muscle cells in the cell holding compartment through the culture medium supply means.

[0055] Step 1-1 further includes, for example, generating a predetermined electrical stimulus from the electrical stimulator. The electrical stimulus emitted from the electrical stimulator electrically stimulates the muscle cells introduced into the cell-holding compartment through electrically connected electrodes, thereby causing the muscle cells to contract. The electrical stimulus to the muscle cells causes a change in their external shape. The change in external shape includes, for example, a reduction in cell area or a reduction in cell volume. The change in external shape is preferably a reduction in cell area. The muscle cells that have contracted after the electrical stimulation preferably relax to the cell size of the muscle cells before the electrical stimulation.

[0056] Although the above describes an embodiment in which muscle cells are contracted or relaxed by electrical stimulation, step 1-1 is not limited to this embodiment. Step 1-1 also includes an embodiment in which muscle cells are contracted or relaxed by stimulation with a compound. When muscle cells are contracted or relaxed by stimulation with a compound, the chamber for observing the contraction or relaxation of muscle cells includes a cell-holding compartment, but does not necessarily need to include electrodes. The chamber includes a means (e.g., a perfusion tube) for supplying a culture medium to the cell-holding compartment that holds the muscle cells, for example, to facilitate replacement of the culture medium with one containing or not containing a compound that can cause muscle cell contraction or relaxation.

[0057] Step 1-2 includes measuring a change in cell size accompanying contraction or relaxation of the muscle cells (step 1-2a) and measuring a change in a fluorescent signal (step 1-2b). Step 1-2a may be performed before, simultaneously with, or after step 1-2b. Step 1-2a is preferably performed simultaneously with step 1-2b.

[0058] Step 1-2a includes, for example, observing the external shape of the muscle cells using a microscope system (for example, bright-field observation). The microscope system includes, for example, a device (observation device) capable of observing the external shape of the muscle cells. The observation device is, for example, a CCD camera. The external shape of the muscle cells is observed, for example, from before to after at least one stimulation.

[0059] Step 1-2b includes, for example, detecting a fluorescent signal emitted by the fusion protein according to the present disclosure expressed in the muscle cells using a microscope system. The microscope system includes, for example, a light source capable of exciting the donor fluorescent protein in the fusion protein. The light source may be a device capable of emitting laser light of a specific wavelength or a lamp that emits white light. When a lamp that emits white light is included, the microscope system includes an optical filter that passes light from the white light in a wavelength range that can excite the donor fluorescent protein.

[0060] When fluorescence intensity is used as the fluorescence signal, the microscope system is equipped with, for example, a device (fluorescence detection device) that detects the fluorescence intensity derived from the fluorescent protein on the donor side ("donor side fluorescence intensity") and the fluorescence intensity derived from the fluorescent protein on the acceptor side ("acceptor side fluorescence intensity"). The fluorescence detection device is, for example, a CCD camera. The image acquisition by the CCD camera is performed with a sufficiently large number of frames to capture, for example, the contraction and relaxation of the cell area. The fluorescence intensity on the donor side and the fluorescence intensity on the acceptor side may be the fluorescence intensity obtained from the entire cell, or may be the fluorescence intensity obtained from an ROI.

[0061] The image acquisition rate with the CCD camera is, for example, 10 frames / second or more, 20 frames / second or more, 30 frames / second or more, 40 frames / second or more, or 50 frames / second or more. The image acquisition rate with the CCD camera is, for example, a number of frames per second that can provide a sufficient exposure time to capture changes in the fluorescent signal (ATP concentration). The image acquisition rate with the CCD camera is, for example, 100 frames / second or less, 80 frames / second or less, 70 frames / second or less, 60 frames / second or less, or 50 frames / second or less. Image acquisition with a CCD camera is performed, for example, at 10 to 100 frames / second, 20 to 100 frames / second, 30 to 100 frames / second, 40 to 100 frames / second, 50 to 100 frames / second, 10 to 80 frames / second, 20 to 80 frames / second, 30 to 80 frames / second, 40 to 80 frames / second, 50 to 80 frames / second, 10 to 70 frames / second, 20 to 70 frames / second, 30 to 70 frames / second, 40 to 70 frames / second, 50 to 70 frames / second, 10 to 60 frames / second, 20 to 60 frames / second, 30 to 60 frames / second, 40 to 60 frames / second, 50 to 60 frames / second, or 50 frames / second.

[0062] The conditions for applying the stimulus and observing the cells (observation of the cell's external shape and fluorescence observation including detection of a fluorescent signal) in step 1 can be appropriately set by a person skilled in the art within a range that does not impair the contraction or relaxation function of the muscle cells, for example.

[0063] Step 2 includes calculating the amount of change in cell size from the change in cell size of muscle cells accompanying contraction or relaxation (step 2-1), and calculating the amount of change in fluorescent signal from the change in fluorescent signal emitted from the muscle cells accompanying the contraction or relaxation (step 2-2). Step 2 further includes calculating the energy efficiency of muscle cell contraction from the amount of change in cell size and the amount of change in fluorescent signal (step 2-3). Step 2-1 may be performed before, simultaneously with, or after step 2-2. Step 2-1 is preferably performed simultaneously with step 2-2.

[0064] Step 2-1 is, for example, the difference between the cell area or volume before stimulating a muscle cell and the cell area or volume before and after stimulation. Step 2-1 is, for example, the difference between the cell area or volume after contraction due to application of a stimulus and the cell area or volume of the muscle cell after relaxation due to removal of the stimulus. The change in cell size includes, for example, the change in cell area due to contraction and relaxation of the muscle cell caused by stimulation. When the cell size is cell area, the change in cell area can be calculated by, for example, specifying the cell size before stimulation as base value b, specifying the cell size at which the cell area is smallest as minimum value m, and dividing the difference (absolute value) between base value b and minimum value m by base value b ([Δ Area] = [base value b - minimum value m] / base value b).

[0065] The base value is preferably the cell area before a stimulus that induces contraction is applied. The base value may be, for example, the cell area after the muscle cell has fully relaxed after the stimulus. A fully relaxed cell area is, for example, about 80% or more (e.g., about 85% or more, 90% or more, 95% or more) of the cell area 3 minutes after a single stimulus. When the stimulus is an electrical stimulus and the electrical stimulus is repeatedly applied to the muscle cell, the base value is the cell area when the muscle cell is fully relaxed after the electrical stimulus. When the electrical stimulus is repeatedly applied to the muscle cell, the base value is set appropriately depending on, for example, the interval between electrical stimuli. For example, when the electrical stimulus is applied once per second, the base value may be the cell area 0.5 seconds after the electrical stimulus.

[0066] The above description is for the case where cell size is defined as cell area, but the present invention is not limited to the case where cell size is defined as cell area. The present invention also includes the case where cell size is defined as cell volume. The above description of cell area is appropriately applied to the case where cell size is defined as cell volume.

[0067] Step 2-2 includes, for example, calculating a change in a first fluorescent signal from the ratio of the acceptor-side fluorescence intensity to the donor-side fluorescence intensity before contraction of a muscle cell (also referred to as a "first fluorescent intensity ratio") (= [acceptor-side fluorescence intensity] / [donor-side fluorescence intensity]), and calculating a change in a second fluorescent signal after contraction of the muscle cell (also referred to as a "second fluorescent intensity ratio"). Step 2-2 includes, for example, calculating a change in a first fluorescent signal from the acceptor-side fluorescence intensity to the donor-side fluorescence intensity after contraction due to application of a stimulus, and calculating a change in a second fluorescent signal of the muscle cell after relaxation due to removal of the stimulus.

[0068] In one example, in the measurement method according to the present disclosure, measuring a change in a fluorescent signal (step 1-2b) comprises: before applying the stimulus, irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein; detecting a first fluorescent signal emitted from the excited fluorescent protein; and detecting a second fluorescent signal emitted from the fusion protein by FRET; and after applying the stimulus, irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein; detecting a third fluorescent signal emitted from the excited fluorescent protein; and detecting a fourth fluorescent signal emitted from the fusion protein by FRET; and calculating an amount of change in a fluorescent signal (step 2-2) comprises calculating a first fluorescent intensity ratio from the first fluorescent signal and the second fluorescent signal; calculating a second fluorescent intensity ratio from the third fluorescent signal and the fourth fluorescent signal; and calculating from the first fluorescent intensity ratio and the second fluorescent intensity ratio.

[0069] Step 2-2 includes, for example, calculating a first fluorescence lifetime on the donor side of a muscle cell before contraction and calculating a second fluorescence lifetime on the donor side of a muscle cell after contraction of the muscle cell. Step 2-2 includes, for example, calculating a first fluorescence lifetime on the donor side of a muscle cell after contraction due to application of a stimulus and calculating a second fluorescence lifetime on the donor side of the muscle cell after relaxation due to removal of the stimulus.

[0070] In one example, in the measurement method according to the present disclosure, measuring a change in a fluorescent signal (step 1-2b) comprises: before applying the stimulus, irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein; and detecting a first fluorescent signal emitted from the excited fluorescent protein and measuring a first fluorescent lifetime; after applying the stimulus, irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein; and detecting a second fluorescent signal emitted from the excited fluorescent protein and measuring a second fluorescent lifetime; and calculating an amount of change in the fluorescent signal (step 2-2) comprises calculating the amount of change in the fluorescent signal from the first fluorescent lifetime and the second fluorescent lifetime.

[0071] Step 2-3 involves calculating the energy efficiency of cardiomyocyte contraction by dividing the change in cell size (e.g., the rate of change in cell area) by the change in fluorescent signal (e.g., the FRET efficiency or FRET ratio) (=[change in cell size] / [change in fluorescent signal]).

[0072] The measurement method according to this embodiment can be used to identify substances that change the energy efficiency in muscle cell contraction, as described below.

[0073] One aspect of the present disclosure provides a method for identifying a substance that alters the energy efficiency of muscle cell contraction. The method includes: (1) contacting a test substance with muscle cells expressing a fusion protein including, from the amino terminus, a first fluorescent protein, an ATP-binding moiety, and a second fluorescent protein; (2) applying a stimulus to the muscle cells and measuring changes in cell size and fluorescent signal accompanying contraction or relaxation of the muscle cells; (3) calculating the energy efficiency from the changes in cell size and the changes in the fluorescent signal; and (4) comparing the energy efficiency with a threshold value to identify whether the test substance alters the energy efficiency of muscle cell contraction, wherein the change in the fluorescent signal is measured based on a fluorescent signal emitted from the fusion protein by FRET occurring between the first fluorescent protein and the second fluorescent protein.

[0074] Step 1, which involves contacting muscle cells with a test substance, includes, for example, allowing the muscle cells and the test substance to be in contact with each other. Step 1 includes, for example, adding the test substance to a culture medium for maintaining the muscle cells. Step 1 can be performed before, simultaneously with, or after step 2. Step 1 is preferably performed before step 2. When step 1 is performed simultaneously with step 2, the test substance can be present, for example, in a perfusion fluid supplied to the muscle cells. When step 1 is performed after step 2a, it is preferable to further perform step 2b after performing step 1. In this case, energy efficiency can be calculated from the change in cell size and the change in fluorescent signal obtained in step 2a, and used as a threshold in step 3a and step 4. Energy efficiency can be calculated from the change in cell size and the change in fluorescent signal in step 2b, and used as the energy efficiency to be compared with the threshold in step 4.

[0075] The explanations for steps 1 and 2 in the measurement method according to the above-mentioned embodiment apply, as appropriate, to steps 2 and 3 in the identification method according to this embodiment. In step 4, when the energy efficiency obtained from muscle cells not contacted with the test substance is used as the threshold, if the energy efficiency is higher than the threshold, the test substance can be identified as changing (more specifically, increasing) the energy efficiency. In the above case, if the energy efficiency is lower than the threshold, the test substance can be identified as changing (more specifically, decreasing) the energy efficiency. In the above case, if the energy efficiency is unchanged from the threshold, the test substance can be identified as not changing the energy efficiency.

[0076] The muscle cells are preferably derived from a non-human transgenic animal that expresses a fusion protein according to the present disclosure, since the muscle cells can reflect the condition of the animal (e.g., age, disease). The muscle cells are preferably differentiated from stem cells transfected with DNA encoding the fusion protein, since muscle cells that stably express the fusion protein according to the present disclosure can be prepared. The muscle cells are preferably transfected with DNA encoding the fusion protein, since muscle cells that transiently express the fusion protein according to the present disclosure can be easily prepared.

[0077] A test substance identified as increasing energy efficiency by the identification method according to this embodiment may be, for example, a candidate substance capable of treating heart disease. "Treatment" includes the maintenance, reduction, or elimination of the symptoms or pathology.

[0078] One aspect of the present disclosure provides a kit for measuring the energy efficiency of muscle cell contraction, comprising: (1) muscle cells expressing a fusion protein comprising, from the amino terminus, a first fluorescent protein, an ATP-binding moiety, and a second fluorescent protein, in that order; and (2) a cell-holding chamber equipped with a cell-holding compartment (also referred to as a "well").

[0079] The kit may include instructions (3) for guidance in carrying out the method for measuring the energy efficiency of muscle cell contraction according to the present disclosure. The kit may include a control substance (4) known to increase or decrease the energy efficiency of muscle cell contraction. The instructions (3) may, for example, state that the method for measuring the energy efficiency of muscle cell contraction according to the present disclosure can be used to identify substances that alter the energy efficiency.

[0080] The muscle cells (1) may be stored in a solution containing, for example, a pH adjuster, a buffer, or a cryoprotectant (e.g., glycerol). When the chamber (2) is equipped with electrodes, the electrodes are arranged so as to apply electrical stimulation to the cell-holding compartment. The electrodes include, for example, a first electrode and a second electrode separated by a gap in the cell-holding compartment. The cell-holding compartment (well) includes, for example, an internal space defined by a bottom and a wall. A liquid containing cells is introduced into the internal space using a dispenser such as a pipette. The chamber (2) includes, for example, at least one well (e.g., 2-well, 4-well, 6-well, 8-well, 12-well, 24-well, 96-well, or 384-well). The chamber (2) includes, for example, a means for supplying a culture medium to the cell-holding compartment (e.g., a perfusion tube).

[0081] In this specification, ordinal numbers such as first and second are used for the purpose of conveniently distinguishing between substances and spaces having the same or similar structures or properties. The ordinal numbers do not in any way limit the structures or properties.

[0082] The term "comprising" means that the recited elements and / or steps are present, and that other elements and / or steps may be added. The term "consisting of" means that the recited elements and / or steps are present, and that other elements and / or steps are excluded. The term "consisting essentially of" means that the recited elements and / or steps are present, and that other elements and / or steps may be present to the extent that they do not adversely affect the technical features of the present disclosure. As used herein, the term "substantially free" does not exclude "completely free."

[0083] The terms and descriptions referred to in this disclosure for a particular aspect or embodiment also apply to other aspects or embodiments as appropriate, unless expressly stated otherwise.

[0084] Specific test examples and examples are described below, but they are intended to illustrate preferred embodiments of the present invention and are not intended to limit the invention described in the appended claims in any way.

[0085] Test Example 1 ATP Concentration-Dependent Fluorescence Measurement in ATP-Visualized Primary Cardiomyocytes (ATP-Visualized Mice) ATP-visualized mice were produced according to the method described in International Publication WO 2015 / 108102. The cells of the produced ATP-visualized mice constitutively express a FRET probe containing, from the amino terminus, green fluorescent protein (GFP), the ε protein subunit of ATP synthase, and orange fluorescent protein (OFP), in this order.

[0086] (Obtaining ATP-Visualized Primary Cardiomyocytes) The heart was removed from the mature ATP-visualized mouse and treated with collagenase to isolate mature cardiomyocytes. The mature cardiomyocytes were treated with a Plasma Membrane Permeabilizer (Agilent Seahorse XF). Treating the treated mature cardiomyocytes with a CIB solution containing ATP allowed ATP to flow into the cytoplasm of the mature cardiomyocytes. The amount of ATP in the buffer was varied from 0.1 mM to 20 mM by changing the MgATP concentration.

[0087] (Fluorescence measurement based on changes in intracellular ATP concentration) When ATP entered the cytoplasm, changes in the fluorescence emitted by the cells were measured. The FRET probe in the mature cardiomyocytes was excited with 488 nm light. The fluorescence emitted from the fibroblasts was passed through DualView (manufactured by Nippon Roper Co., Ltd.) and spectrally analyzed using DM540. The fluorescence was then passed through absorption filters of 515 / 30 nm and 575 / 40 nm, and the GFP-derived fluorescence (G) and RFP-derived fluorescence (O) were photographed using a CCD camera ORCA-Flash 4.0 (Hamamatsu Photonics). The image acquisition rate was 30 to 100 frames per second. The acquired images were separated into two images (GFP fluorescence image and RFP fluorescence image) using the image analysis software metamorph.

[0088] From the fluorescence images of isolated cardiomyocytes, the fluorescence intensity ratio of the OFP-derived fluorescence intensity (O) to the GFP-derived fluorescence intensity (G) (FRET ratio = [fluorescence intensity (O)] / [fluorescence intensity (G)]) was calculated. A scatter plot was created in which the calculated FRET ratios were plotted against the ATP concentration. The FRET ratio changed linearly from 0.4 to 2.0 with ATP concentrations of 0.1 to 6 mM. The error range was approximately 4.5% or less. This result demonstrates that changes in intracellular ATP concentration (0.1 to 6 mM) can be measured as changes in the FRET ratio. Test Example 1 demonstrates that the obtained relationship between ATP concentration and FRET ratio (calibration curve) can be used to calculate changes in ATP in the cells based on the FRET ratios measured using other cells.

[0089] Test Example 2 (ATP Concentration Changes Based on Fluorescence Measurement in ATP-Visualized Fertilized Eggs) ATP-visualized mice were mated, and two days after mating, multiple 2-cell stage fertilized eggs were obtained from the oviducts of female mice. The fertilized eggs were flashed out using M16 culture medium and cultured in phenol red-free M2 culture medium. During culture, 2-deoxyglucose and oligomycin, inhibitors of the glycolysis and electron transport chain, were added to the culture medium to suppress ATP production in the cytoplasm of the fertilized eggs. The process of the decrease in ATP concentration was photographed using a fluorescence microscope. Fluorescence emitted by the fertilized eggs under a fluorescence microscope was measured in substantially the same manner as in Test Example 1. The FRET ratio was obtained from the measured fluorescence intensity, and the ATP concentration was calculated from the obtained FRET ratio and the calibration curve obtained in Test Example 1. The ATP concentration was plotted against the time course after ATP production was inhibited to create a graph (derived from fluorescence) showing the change in ATP concentration (FIG. 1, ○: FRET / GFP).

[0090] (ATP Concentration Changes Based on Luminescence Measurement from ATP-Visualized Fertilized Eggs) The ATP concentration in the cytoplasm of fertilized eggs was measured using the luciferase method, a well-known ATP measurement method. Fertilized eggs were prepared in which ATP production in the cytoplasm was inhibited using 2-deoxyglucose and oligomycin, as described above. Every 3 minutes after inhibition of ATP production, the ATP concentration in the cytoplasm of the fertilized eggs was measured using the luciferase method (Toyo B-Net Co., Ltd., Intracellular ATP Measurement Kit). The ATP concentration was plotted against the time course after inhibition of ATP production to create a graph showing the change in ATP concentration (derived from luminescence) (Figure 1, ◇: Luciferase).

[0091] The graph showing the change in ATP concentration (derived from fluorescence) was compared with the graph showing the change in ATP concentration (derived from luminescence). The ATP concentration changes over time shown in the two graphs showed similar rates of change (slope). This result indicates that ATP concentration can be quantitatively measured by measuring fluorescence. Furthermore, unlike the luciferase method, which requires cell disruption, fluorescence measurement allows for non-invasive measurement. This makes it possible to measure ATP concentration over time in the same cells.

[0092] Example 1 Energy Efficiency in Contraction-Relaxation in Cardiomyocytes Derived from Juvenile ATP-Visualized Mice After intraperitoneal injection of heparin, an anticoagulant, into 8-week-old adult ATP-visualized mice, the hearts were removed from the mice. The hearts were perfused with modified Tyrode's solution containing collagenase for 20 minutes using a Langendorff perfusion system (Radnoti, USA). The hearts were cut into small pieces with scissors and filtered through a 100-μm mesh. The cell population in the resulting filtrate was subjected to static culture. The culture medium was replaced with a culture medium containing a series of calcium concentrations ranging from 0.25 mM to 1 mM, and the cells were cultured in the presence of gradually increasing calcium concentrations. This static culture procedure removed damaged cells, yielding isolated, undamaged cardiomyocytes. The isolated cardiomyocytes were perfused with 10 mg of BSA and 1 M CaCl . ２ The solution was stored in 5 ml of cell isolation buffer (CIB) to which 2.5 μl had been added.

[0093] The isolated cardiomyocytes were electrically stimulated under a microscope to observe their contraction and relaxation. At the same time, fluorescence from the cardiomyocytes was measured. Electrical stimulation of the cardiomyocytes was performed using a chamber equipped with silver wire electrodes on a glass-bottom dish. The isolated cardiomyocytes were introduced into the chamber. The silver wire electrodes were connected to an electrical stimulator (SD9 square pulse stimulator). The electrical stimulator applied electrical stimulation to the cardiomyocytes for 10 seconds at a frequency of once per second. Fluorescence measurement was performed under substantially the same conditions as in Test Example 1, except that the image acquisition rate was 30 to 100 frames per second. During observation, the cardiomyocytes were maintained in Tyrode's solution.

[0094] The acquired images were separated into two images (GFP fluorescence image and OFP fluorescence image) using the image analysis software Metamorph and converted into IMD image ratio images. Each image was binarized using the image analysis software Imaged. A region of interest (ROI) was set to correspond to the periphery of the cardiomyocyte, and the area of ​​the cardiomyocyte was quantified and plotted (Figure 2b). One peak in Figure 2b corresponds to the contraction-relaxation of the cardiomyocyte in response to one electrical stimulus. Figure 2b shows the dynamics of cardiomyocytes, in which they repeatedly contract (cell area becomes smaller) and relax (cell area becomes larger).

[0095] The mean fluorescence intensity within the ROI in each of the two images was quantified to calculate the FRET ratio. The FRET ratio over time was plotted (Figure 2a). A single peak in Figure 2a corresponds to the contraction-relaxation of cardiomyocytes in response to a single electrical stimulus. Figure 2a shows that the FRET ratio, i.e., the ATP concentration, fluctuates as cardiomyocytes contract. The smallest FRET ratio value (a1 in Figure 2) in the 0.5 seconds preceding a peak in Figure 2a was defined as the base value of that peak, and the apex of that peak (a2 in Figure 2) was defined as the maximum value. The range of change in the FRET ratio due to cardiomyocyte contraction was determined by subtracting the base value a1 from the maximum value a2. The range of change in the FRET ratio is an indicator of ATP consumption due to cardiomyocyte contraction and is also referred to as the "ATP amplitude."

[0096] The peaks in Figure 2b, which occur during the time span in which a certain peak in Figure 2a is observed, represent the change in area of ​​the cardiomyocyte corresponding to the contraction-relaxation of the cardiomyocyte that resulted in the peak in Figure 2a. The rate of change in cell area during contraction-relaxation of cardiomyocytes in response to electrical stimulation was calculated by subtracting the maximum cell area (b1) from the base value of the peak (0.5 seconds before the peak), and dividing the difference (absolute value) obtained by subtracting the base value from the maximum value by the base value ([area change rate] = [(maximum value b1 - base value b2) / base value b2]). The energy efficiency of a single contraction of cardiomyocytes (= [area change rate] / [ATP amplitude]) was calculated by dividing the area change rate at one peak by the ATP amplitude (an index of ATP consumption).

[0097] The same test as above was performed using multiple ATP-visualized cardiomyocytes, and the area change rate (ΔArea ratio) was plotted against the ATP amplitude (ΔFRET ratio) ( FIG. 3 ). FIG. 3 shows that there is a positive correlation between the rate of increase in ATP amplitude and the increase in the area change rate, with a slope of approximately 3.3 as determined by the least squares method. The slope indicated by the least squares method in the scatter plot can be an indicator of the energy efficiency of cardiomyocyte contraction under specific conditions. Example 1 suggests that the value obtained by dividing the area change rate (ΔArea ratio) by the ATP amplitude (ΔFRET ratio) may be used as an indicator of the energy efficiency of cardiomyocyte contraction-relaxation.

[0098] Example 2 (Changes in energy efficiency during contraction-relaxation in cardiomyocytes due to drug addition) Energy efficiency was calculated in the same manner as in Example 1, except that the selective cardiac myosin activators omecamtib mecarbim (Fig. 4A) and digoxin (Fig. 4B) were added at predetermined concentrations to the culture medium of cardiomyocytes 60 minutes before measuring energy efficiency.

[0099] In heart failure, the contractile force of the myocardium is often weakened. This results in a lack of powerful heartbeat, leading to congestive heart failure. A selective cardiac myosin activator (omecamtib mecarbim) has been developed as a therapeutic agent with the potential to improve symptoms in subjects with heart failure due to reduced contractility. Figure 4A shows that the slope, which is an index of energy efficiency during contraction-relaxation, is approximately 3.7. In Example 2, the addition of omecamtib mecarbim increased the energy efficiency compared to the slope of approximately 3.3 shown in Example 1 without omecamtib mecarbim.

[0100] Digoxin has a history of over 200 years since it was first reported as a therapeutic agent for heart failure. The pharmacological effects of digoxin include the inotropic effect caused by the accumulation of calcium ions in cardiomyocytes, which enhances the contraction of cardiomyocytes. Figure 4B shows that the slope, which is an index of the energy efficiency, is approximately 3.5. In Example 2, the addition of digoxin increased the energy efficiency compared to the slope of approximately 3.3 shown in Example 1 without digoxin.

[0101] These results indicate that omecamtib mecarbim or digoxin, which are used as therapeutic agents for heart failure, can exert their effects by improving energy efficiency at the cardiomyocyte level. Example 2 also supports the idea that [Area change rate (ΔArea ratio)] / [ATP amplitude (ΔFRET ratio)] can be an index of energy efficiency during contraction-relaxation of cardiomyocytes.

[0102] Example 3 (Energy Efficiency During Contraction-Relaxation in Cardiomyocytes Derived from Aged ATP-Visualized Mice) The energy efficiency during contraction-relaxation of aged ATP-visualized cardiomyocytes was obtained using essentially the same method as in Example 1, except that cardiomyocytes obtained from 1.5-year-old adult ATP-visualized mice were used ( FIG. 5A ). The resulting energy efficiency during contraction-relaxation (slope) was approximately 3.4. This value was almost the same as the energy efficiency during contraction-relaxation (approximately 3.3) obtained using cardiomyocytes obtained from 8-week-old young ATP-visualized mice. It is known that the heart undergoes structural changes with aging, such as an increase in size, thickening of the cardiac chamber walls, and hardening of the cardiac valves. Surprisingly, Example 3 demonstrated no significant difference in energy efficiency at the cardiomyocyte level between young and old mice.

[0103] The energy efficiency during contraction-relaxation of aged ATP-visualized cardiomyocytes was obtained in substantially the same manner as in Example 1, except that the cardiomyocytes were treated with omecamtib mecarbim (FIG. 5B). The obtained energy efficiency (slope) during contraction-relaxation was approximately 3.3. This value was smaller than the energy efficiency index of approximately 3.7 obtained for young ATP-visualized cardiomyocytes treated with omecamtib mecarbim (Example 2, FIG. 4A).

[0104] These results surprisingly indicate that even in cardiomyocytes in a state where energy efficiency remains unchanged, the effects of drugs (e.g., myosin activators) may vary. Examples 1 to 3 demonstrate that, at least, the effects of omecamtib mecarbim, a drug used to treat heart failure, differ between cardiomyocytes from young and old mice, despite the cardiomyocytes having nearly the same energy efficiency. Such differences can be revealed by the method of the present disclosure, which enables measurement of energy efficiency at the cellular level.

[0105] Example 4 Energy Efficiency During Contraction-Relaxation in Cardiomyocytes Derived from a Mouse Model of Hereditary Cardiomyopathy The energy efficiency during contraction-relaxation of ATP-visualized cardiomyocytes was obtained using substantially the same method as in Example 1, except that cardiomyocytes obtained from a mouse model of hereditary dilated cardiomyopathy (DCM) related to the MYBPC3 gene were used ( FIG. 6A ). DCM is known to cause hypertrophy and fibrosis of cardiomyocytes, leading to progressive myocardial dysfunction and cardiac contractile dysfunction. The obtained energy efficiency during contraction-relaxation (slope) was approximately 2.9, which was smaller than the value (approximately 3.3) of cardiomyocytes from young mice obtained in Example 1. This result indicates that DCM is associated with a decrease in energy efficiency at the cardiomyocyte level.

[0106] Using ATP-visualized cardiomyocytes from a genetic DCM model mouse, contraction-relaxation energetic efficiency was obtained in substantially the same manner as in Example 1, except for the use of omecamtiv mecarbim ( FIG. 6B ). When omecamtiv mecarbim was not used, the contraction-relaxation energetic efficiency (slope) was approximately 931, whereas when omecamtiv mecarbim was used, the slope was a larger value of approximately 1076. These results suggest that omecamtiv mecarbim is effective in treating DCM, which is accompanied by a decrease in energetic efficiency.

[0107] However, a study was conducted to evaluate the efficacy of omecamtib mecarbim (a selective cardiac myosin activator) in patients with reduced systolic function (HFrEF) with a left ventricular ejection fraction (LVEF) of 35% or less. This study failed to demonstrate the efficacy of omecamtib mecarbim, which may increase energy efficiency (The New England Journal of Medicine, Vol. 384, No. 2, pp. 105-116, 2021). Example 3 demonstrated that selective cardiac myosin activators may or may not be effective depending on age. While it is unclear from the study whether HFrEF patients were grouped by age, the results of Example 3 suggest that failure to distinguish between elderly patients may have contributed to the inability to confirm the efficacy of omecamtib mecarbim.

[0108] Examples 3 and 4 suggest that, in determining whether a particular drug is effective for heart failure, it is useful to preliminarily classify patients to be treated as responsive to a drug that alters energy efficiency in terms of their condition, such as age. Testing compounds that can alter energy efficiency may be effective for patients who are responsive to a drug that alters energy efficiency.

[0109] Example 5 Energy Efficiency During Contraction-Relaxation of ATP-Visualized Cardiomyocytes Derived from an ATP-Visualized Mouse Model of Drug-Induced Heart Failure Energy efficiency was calculated in substantially the same manner as in Example 1, except that cardiomyocytes were isolated from a mouse model of heart failure created by administering isoprotelenol to an ATP-visualized mouse in vivo ( FIG. 7A ). The resulting energy efficiency (slope) during contraction-relaxation was approximately 3.4, which was not significantly different from the energy efficiency during contraction-relaxation of ATP-visualized cardiomyocytes from young mice in Example 1 (approximately 3.3).

[0110] When omecamtib mecarbim was added to ATP-visualized cardiomyocytes treated with the heart failure model inducer, the energy efficiency during contraction-relaxation was approximately 3.4 (Figure 7B). This result suggests that it is useful to preclassify patients who will respond to drugs that alter energy efficiency based not only on the condition of the patient being treated but also on the type of heart failure being treated. It may be effective to test compounds that alter energy efficiency for heart failures that respond to drugs that alter energy efficiency.

[0111] Example 6 (Energy efficiency during contraction-relaxation in skeletal muscle cells) Except for isolating skeletal muscle cells from young ATP-visualized mice, energy efficiency was calculated using essentially the same method as described in Example 1. As a result, as with cardiac muscle cells, energy efficiency could also be measured in skeletal muscle cells from fluctuations in ATP concentration and the area of ​​skeletal muscle cells between sarcomeres.

Claims

1. A method for measuring the energy efficiency in the contraction of muscle cells, comprising: stimulating muscle cells that express a fusion protein containing a first fluorescent protein, an ATP-binding moiety, and a second fluorescent protein in this order from the amino terminus, and measuring changes in the cell size and fluorescence signal of the muscle cells accompanying the contraction or relaxation of the muscle cells; and calculating the energy efficiency in the contraction of the muscle cells from the changes in the cell size and the fluorescence signal, wherein calculating the energy efficiency includes calculating the amount of change in the fluorescence signal based on the amount of change in the cell size and the fluorescence signal emitted from the fusion protein by fluorescence resonance energy transfer ("FRET") that occurs between the first fluorescent protein and the second fluorescent protein.

2. A method for identifying a substance that changes the energy efficiency in the contraction of muscle cells, comprising: contacting muscle cells that express a fusion protein containing a first fluorescent protein, an ATP-binding moiety, and a second fluorescent protein in this order from the amino terminus with a test substance; stimulating the muscle cells and measuring changes in the cell size and fluorescence signal of the muscle cells accompanying the contraction or relaxation of the muscle cells; calculating the energy efficiency in the contraction of the muscle cells from the changes in the cell size and the fluorescence signal; and comparing the energy efficiency with a threshold value to identify whether the test substance changes the energy efficiency in the contraction of muscle cells, wherein calculating the energy efficiency includes calculating the amount of change in the fluorescence signal based on the amount of change in the cell size and the fluorescence signal emitted from the fusion protein by FRET that occurs between the first fluorescent protein and the second fluorescent protein.

3. The method according to claim 1 or 2, wherein the ATP-binding moiety is the CBS domain of inosine monophosphate dehydrogenase 2 (IMPDH2) or a fragment or a variant thereof, GlnK1 or a fragment or a variant thereof, or the ε subunit of ATP synthase or a fragment or a variant thereof.

4. The method according to any one of claims 1 to 3, wherein the muscle cells are cardiomyocytes, smooth muscle cells or skeletal muscle cells derived from a non-human transgenic animal expressing the fusion protein; or cardiomyocytes, smooth muscle cells or skeletal muscle cells containing the DNA encoding the transfected fusion protein and expressing the fusion protein derived from the DNA.

5. Measuring the change in the fluorescence signal includes, before applying the stimulus, irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein; detecting a first fluorescence signal emitted from the excited fluorescent protein; detecting a second fluorescence signal emitted from the fusion protein by the FRET; and after applying the stimulus, irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein; detecting a third fluorescence signal emitted from the excited fluorescent protein; detecting a fourth fluorescence signal emitted from the fusion protein by the FRET. Calculating the amount of change in the fluorescence signal includes calculating a first fluorescence intensity ratio from the first fluorescence signal and the second fluorescence signal; calculating a second fluorescence intensity ratio from the third fluorescence signal and the fourth fluorescence signal; and calculating from the first fluorescence intensity ratio and the second fluorescence intensity ratio. The method according to any one of claims 1 to 4.

6. Measuring the change in the fluorescence signal includes, before applying the stimulus, irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein; detecting a first fluorescence signal emitted from the excited fluorescent protein and measuring a first fluorescence lifetime; and after applying the stimulus, irradiating the fusion protein with excitation light for the first fluorescent protein or the second fluorescent protein; detecting a second fluorescence signal emitted from the excited fluorescent protein and measuring a second fluorescence lifetime. Calculating the amount of change in the fluorescence signal includes calculating from the first fluorescence lifetime and the second fluorescence lifetime. The method according to any one of claims 1 to 4.

7. A kit for measuring the energy efficiency in the contraction of muscle cells, comprising: muscle cells expressing a fusion protein containing, in this order from the amino terminus, a first fluorescent protein, an ATP-binding moiety, and a second fluorescent protein; and a chamber for cell retention having a cell retention compartment.

8. The kit according to claim 7, wherein the muscle cells are cardiomyocytes, smooth muscle cells or skeletal muscle cells derived from a non-human transgenic animal expressing the fusion protein; or cardiomyocytes, smooth muscle cells or skeletal muscle cells containing the DNA encoding the transfected fusion protein and expressing the fusion protein derived from the DNA.

Images