Cardiac myocyte

Myocardial fibers belong to involuntary muscles with transverse stripes

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Myocardial cells, also known as myocardial fibers, have striations Vegetative nerve Domination belongs to the involuntary muscle with transverse stripes and has the ability to excite contraction. It is short cylindrical with branches, and its nucleus is located in the center of the cell, usually only one. The ends of each myocardial fiber branch can be connected to form a muscle fiber network. Broadly speaking, myocardial cells include those that form the sinoatrial node, intra atrial bundle Atrioventricular junction Ministry Atrioventricular fasciculus (i.e. Hiss beam) and Purkinje fiber The specially differentiated cardiomyocytes, as well as the general working cells of atrial and ventricular muscles.

Chinese name: Cardiac myocyte
Alias: Myocardial fiber
Composition: Sinus node, intra atrial bundle, atrioventricular junction,

Innervating nerve: Vegetative nerve
Type: Striated muscle,
structure characteristics: Short columnar

catalog

five Electrophysiology
six cell culture

classification

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According to their Histology The differences in characteristics, electrophysiological characteristics and functions can be roughly divided into two types. The two types of myocardial cells perform certain functions respectively, cooperate with each other, and complete the overall activities of the heart^[1] 。

Working cell

Cardiac myocyte

One is ordinary myocardial cells, including atrial and ventricular muscles, which are rich in Myofibril It performs contraction function, so it is also called working cell. Working cells cannot be generated automatically Rhythmic excitation That is, there is no automatic rhythm; But it has excitability, which can produce excitability under the action of external stimuli; It also has the ability to transmit excitations, but it is different from the corresponding special Conduction tissue By comparison, the conductivity is low.

Autonomic cell

The other is some special differentiation The myocardial cells of heart Special conduction system; It mainly includes P cells and Purkinje cell In addition to excitability and conductivity, they also have the ability to automatically generate rhythmicity and excitement, so they are called autonomic cells. They contain very small myofibrils or are completely lacking, so their contractile function has basically lost. Another kind of cell is located in a special Conduction system The area of the node has neither contractive function nor self-discipline. Only a very low conductivity is retained. It is a non autonomic cell in the conduction system. The special conduction system is the tissue that generates and spreads excitement in the heart, and plays a role in controlling the rhythmic activity of the heart.

structure characteristics

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Membrane potential diagram

1. Myocardial cells are short columnar and generally have only one nucleus, while Skeletal muscle fibers yes Multinucleated cell 。 There are Intercalary disk Structure. The cell membrane here is concavoconvex, and specially differentiated Desmosome , each other Tight connection But there is no Protoplasm Continuity of. Myocardial tissue It used to be mistaken for Syncytium The electron microscope study found that there was an obvious membrane between myocardial cells, which was corrected. The intercalated disk of myocardium is conducive to the excitation transmission between cells. On the one hand, due to the low impedance of the structure to the current, the excited wave is easy to pass; On the other hand, there are 15~20 angstroms of hydrophilic tubules in the gap connection, which can allow calcium ion plasma to penetrate and transport. Therefore, although normal atrial or ventricular myocytes are separated from each other, they are excited almost at the same time and contract synchronously, which greatly improves the efficiency of myocardial contraction and functionally reflects the characteristics of syncytium, so they are often called "functional syncytium".

2. The nucleus of myocardial cell is mostly located in the middle of the cell, which is oval or rectangular in shape, and its long axis is consistent with the direction of myofibril. Myofibrils go around the nucleus, and both ends of the nucleus are rich Myoplasm , which contains rich Glycogen Particles and mitochondrion In order to meet the needs of continuous rhythmic contraction of myocardium. In cross section, the diameter ratio of myocardial cells skeletal muscle Small, the former is about 15 microns, while the latter is about 100 microns. From the perspective of profile Sarcomere The length is also shorter than the sarcomere of skeletal muscle.

3. On electron microscope The myofibrils Transverse tubule 、 Sarcoplasmic reticulum , mitochondria, glycogen, fat and other ultrastructure. But myocardial cells are different from skeletal muscle; Myofibril thickness of myocardial cells varies greatly, ranging from 0.2 to 2.3 microns; At the same time, the thick myofibrils and the thin myofibrils can migrate to each other, and the adjacent ones are close to each other so that the boundary is not clear. The transverse tubules of cardiac myocytes are located at the Z line level, and many mammals have longitudinal axis stretching out, with a diameter of about 0.2 μ m. The transverse tubule of skeletal muscle is located at the junction of A-I band, without longitudinal extension, and its diameter is about 0.4 μ m. The sarcoplasmic reticulum of myocardial cells is clustered in the middle Terminal pool Not many, not widely connected with transverse tubules^[2] 。

Bioelectricity

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Cardiac myocyte Bioelectricity Basis of production: myocardial cells Transmembrane potential Depends on the transmembrane electrochemical gradient of ions and the selective permeability of the membrane to ions^[3] 。

Transmembrane potential

Ventricular myocyte Transmembrane potential and its mechanism

1. Resting potential: When ventricular myocytes are at rest, the cell membrane is in a positive external and negative internal polarization state, which is mainly formed by K+outflow.

2. Action potential: the whole process of ventricular muscle action potential includes phase 0 and repolarization The first, second, third and fourth periods of the process.

Cardiac myocyte

Phase 0: When ventricular myocytes are excited, the intramembrane potential rises from - 90mV at rest to about+30mV, forming the ascending branch of action potential, which is called depolarization process (phase 0). It is mainly formed by Na+internal flow.

Phase 1: at repolarization At the initial stage, the intracellular potential of ventricular myocytes rapidly decreased from+30 mV to about 0 mV, which was mainly formed by K+outflow.

Phase 2: Phase 1 repolarization to about 0 mV, at this time, the membrane potential drops very slowly, which is mainly formed by the internal flow of Ca2+and the external flow of K+.

Phase 3: In this phase, the membrane repolarization speed of ventricular myocytes is accelerated, and the membrane potential drops rapidly from about 0 mV to - 90 mV, lasting about 100~150 ms. It is mainly formed by the outward ion current of K+(Ik1 and Ik, Ik is also called Ix).

Phase 4: Phase 4 is the completion of phase 3 repolarization, Membrane potential Basically stable at the resting potential level, myocardial cells have been in a resting state, so it is also called Rest period 。 The transport of Na+, Ca2+and K+is mainly related to the activities of Na+-- K+pump and Ca2+pump. About Ca2+ Active transshipment At present, most scholars believe that the outward transport of Ca2+in the reverse concentration gradient is coupled with the inward flow of Na+in the parallel concentration, forming Na+- Ca2+exchange.

purkinje cell Transmembrane potential and its mechanism

purkinje cell Action potential and its generation mechanism Ventricular myocyte Basically similar, but it has 4 stages of automatic depolarization. Phase 4 automatic depolarization is the result of the progressive increase of the membrane's Na+permeability over time (If inward current). The main differences between If channel and fast Na+channel are as follows: ① The selectivity of If channel to ions is not strong. Although Na+is mainly selected, K+is also involved. The fast Na+channel has strong selectivity, mainly allowing Na+to penetrate. ② If channel is activated when repolarization reaches about - 60mV, while fast Na+channel is activated when electric depolarization reaches about - 70mV in the membrane. ③ If channel can be blocked by cesium (Cs), while fast Na+channel can be blocked by Puffer fish Toxic blockade.

Sinus node P cells Transmembrane potential and its mechanism

1. The main characteristics of action potential of P cells The membrane potential in phase 4 is unstable and can occur automatically Depolarization This is the most significant feature of action potential of autonomic cells.

In addition:

Cardiac myocyte

1) The peak value of depolarization phase 0 is small, and the depolarization speed is slow, about 10V/s. The depolarization phase 0 is only about 0mV.

2) The repolarization was completed in three phases, with almost no one phase and two phases.

3) After the completion of repolarization phase 3, enter phase 4. The maximum membrane potential that can be reached at this time is called the maximum diastolic potential (or the maximum repolarization potential), which is about - 70mV.

Cardiac myocyte

2. P cell action potential formation and ion current activity

(1) Formation of phase 0 depolarization: the inward current of phase 0 depolarization is mainly loaded by calcium ion.

(2) The formation of phase 3 repolarization: after phase 0 depolarization, the slow calcium channel gradually became inactive. The third stage is the result of the combined action of calcium ion inflow and potassium ion outflow.

(3) Formation of phase 4 automatic depolarization: the current research is related to three ion currents.

A: Progressive attenuation of potassium ion outflow;

B: The internal flow of sodium ion is gradually enhanced;

C: Electrogenic Na+-- Ca2+ion exchange.

Electrophysiology of cardiac myocytes

Bioelectric classification

Electrophysiological classification of myocardial cells

In addition to anatomical and physiological characteristics, cardiac myocytes can be divided into working cells (non self regulating cells) and self regulating cells. According to the electrophysiological characteristics of action potential of cardiac myocytes (especially 0 in addition to the extreme rate), the action potential generated by cardiac myocytes can be divided into two categories: fast response potential and slow response potential. Cells with these two different potentials are called Fast reaction cell and Slow reaction cell ：

1. Fast reaction cells include: atrial muscle, ventricular muscle and Purkinje cell, and their action potential characteristics are: extremely fast, large amplitude and long duration.

2. Slow reaction cells include sinoatrial node cells and atrioventricular junction cells. Their action potential characteristics are: very slow, small amplitude, short duration.

The classification of myocardial cells is as follows:

Autonomic cell

fast reactive autorhythmic cell : e.g Purkinje cell

Slow reaction autonomic cells: Sinoatrial node Cells in the atrioventricular junction area (atrial node area, nodal area)

Non self regulating cells Fast reaction Non self regulating cells: Atrial muscle , Ventricular myocytes

Slow reaction non autonomic cells: node area cells

Cardiac myocyte

American scientists Nature The research report published in the journal Nature pointed out that a group of stem cells 。 The scientist leading this research is William Pu, a Chinese. Researchers from Massachusetts Boston Children's Hospital said that the newly discovered stem cells are located in the outermost layer of the heart epicardium , or it can repair the damaged heart tissue. William Pu said: "When patients have heart problems, they will lose drive heartbeat Of myocardial cells. The remedy is to make more of these cells. " It is reported that researchers discovered new stem cells by accident. They were studying epicardium So we need to label specific cells with the red fluorescent protein complex on the embryos of live mice. To their surprise, they witnessed the transformation of epicardial cells into cardiomyocytes. William Pu's research results show that gene Stem cells numbered "Wt1" can produce cardiomyocytes Synoviocyte and endothelial cells 。

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cardiomyocyte hypertrophy

Myocardial tissue includes myocardial cells and Stroma Two parts, of which myocardial cells account for 75% of the total volume of the heart; Interstitium accounts for 25%. Cardiomyocyte hypertrophy refers to the increase in the size, diameter, or length of cardiac myocytes and Sarcomere The number of cardiomyocytes increases, and cardiomyocytes proliferate when the myocardium is excessively hypertrophic.

Fundamentals of Cytology

Cardiac myocyte

Myocardial cell is a highly differentiated terminal cell. Its contractile protein is mainly α - myosin (α - MHC), which is in charge of contractile function. Contractile proteins include myosin, Actin Myosin and myosin. among Myosin Including 2 heavy chains (MHC) and 2 light chains (MLC), the heart has only two kinds of MHC gene expression That is, α - MHC and β - MHC form α - α, β - homodimer and α - β heterodimer, forming isoenzymes V1, V2 and V3 respectively. Normally, embryo Atrium and adult atrium α - MHC (V1 isoenzyme) are dominant, while left and right ventricles from embryo to adult β - MHC are always maintained at 80%~90%, and V3 isoenzyme is dominant. Myocardial cells generally cannot proliferate, but only have hypertrophy of cell volume and are in contraction state. Embryonic cardiomyocytes are derived from muscle stem cells Myoblast Gradually differentiate into mature cardiomyocytes, and their contractile proteins are dominated by β - MHC, which is in a "synthetic state", Cardiac hypertrophy It is a phenomenon that myocardial cells transform from mature "contraction state" to "embryonic synthesis state".

When cardiac myocytes become hypertrophic, their phenotype changes, their volume increases, and the type of contractile protein in cardiac myocytes changes Interstitial cell Proliferation. The growth of cardiomyocytes and interstitial cells have their own regulatory mechanisms. Cardiomyocyte hypertrophy may or may not be accompanied by the proliferation of interstitial cells.

Cause and mechanism

In 1958, Teare Hypertrophic cardiomyopathy Has been described, since then, people have been myocardial hypertrophy The research shows that myocardial hypertrophy is a complex dynamic process involving multiple factors. The biochemical basis of cardiac myocyte hypertrophy is the increase of cardiac protein synthesis, which leads to the increase of cell volume. All kinds of mechanical stimulation and chemical factors can lead to cardiac hypertrophy.

1. The direct effect of mechanical stimulation is long-term pressure and/or volume overload. send Ventricular wall Stress increases, leading to cardiac hypertrophy. The overall experiment shows that when the heart is stimulated by load Cardiac hypertrophy 。 Mechanical stimulation can be achieved by promoting protein synthesis Increase or/and promote Protein degradation Decrease and lead to cardiac hypertrophy. The mechanism is that (1) intracellular CAMP increases. When the aortic pressure of a beating or stopped heart rises from 7.98kPa (60mmHg) to 15.96kPa (118mmHg), its protein synthesis increases, Nucleoprotein The increased formation, CAMP content and CAMP dependent protein kinase activity suggest that increased arterial pressure can accelerate protein synthesis through CAMP dependent mechanism. (2) Intracellular muscle Alkophosphate Add reports such as Portzer, Aortic stenosis When left ventricular hypertrophy was caused, the activity of cytoplasmic protein kinase (PKC) in hypertrophic myocardium increased by 15% compared with the control, and the activity of cell membrane PKC increased by 40%. explain myocardium The increase of inositol phosphonate content in myocardium due to stretch may be due to phospholipase Activation of C. (3) Increased expression of proto oncogenes due to pressure overload Cardiac hypertrophy In the early stage, the increase of proto oncogene expression can be observed. (4) The gene expression of β - MHC and α - actin increased when cultured cardiomyocytes were continuously stimulated by stretch. (5) Other myocardial cells calcium ion channels , sodium ion influx and intracellular environment alkalinity ratio, etc myocardial hypertrophy Medium importance Regulatory action 。

2. Chemical stimulating humoral factors can also promote cell hypertrophy or proliferation 。 (1) Go Methylepinephrine (NE), animal experiments have proved that long-term injection of NE at a dose of sub hypertension can induce cardiac hypertrophy, which may mainly act through α 1-R. Some scholars also found that the transcription of myc gene increased by 5 to 10 times when NE was added to the myocardial cell culture medium, and promoted cardiac hypertrophy. This reaction can be blocked by specific α 1 receptor antagonists Protein kinase C 、 Activator PNA is strengthened and NE is enhanced through α - receptor, activation Phosphatidylinositol Protein kinase C system, which plays a role in activating oncogene expression. (2) androgen Cabral et al found in the neurogenic hypertension induced by denervation of the baroreceptor nerve in rats, male The left ventricular weight/body weight ratio of rats was significantly higher than that of females testosterone Can cause the left ventricle of male neurogenic hypertensive rats myocardial hypertrophy However, estradiol can inhibit the increase of left ventricular weight, and its mechanism is unclear, which may be related to oncogenes. (3) Angiotensin II (Ang Ⅱ) Ang Ⅱ receptors are divided into AT1 and AT2 subtypes. AT1 receptors are related to the positive inotropic and chronotropic effects of myocardium and the growth and hypertrophy of cardiomyocytes. When Ang Ⅱ is added to the culture medium of cardiomyocytes, the expression of c-fos, c-jun, c-mye and other proto oncogenes is rapidly enhanced, and the protein synthesis is increased and induced Cardiac hypertrophy 。 Ang Ⅱ can also cause angiotensinogen gene and transforming growth factor β 1 gene is up-regulated, thus promoting cardiomyocyte hypertrophy. This reaction can be blocked by Ang Ⅱ AT1 receptor blocker, and can also be enhanced by PKC, suggesting that Ang Ⅱ activates inositol phosphate through AT1 receptor - protein kinase C system, which activates the expression of proto oncogenes. (4) Endothelin (ET) In 1988, Yangisawa et al. isolated a vasoconstrictor polypeptide from porcine aortic endothelial cells. ET is achieved by Target cell Upper ET receptor binding, functioning, regulating cell proliferation Of. ET receptors are divided into ETA, ETB and ETC. Cardiovascular myocytes are rich in ETA. The cardiac hypertrophy caused by ETA may be caused by increasing myosin light bond 2, α - actin troponin And α, β Myosin heavy chain MRNA expression. It is reported that ETA may also be caused by NE in the body Cardiac hypertrophy Play a role in. (5) growth hormone (GH) and insulin-like growth factor (TGF) Antonio et al. observed the effects of GH and TGF1 on the cardiovascular system of rats, and found that the myocardium is the target organ of GH and IGF-1. Administration of exogenous GH and IGF-1 to normal adult rats can cause cardiac hypertrophy. The increase of volume and pressure load can increase the expression of IGF-1 gene in the heart. GH and IGF-1 may also be indirectly metabolized through insulin or adrenaline The system works. (6) Interleukin 6 (IL-6) and cell hypertrophy factor (CT-1) keiko et al. found that myocardial cells can secrete a large amount of IL-6 in hypoxia, reperfusion and stimulation by other factors, and Cardiac hypertrophy of As a ligand, IL-6 binds to the IL-6 receptor to form a homodimer with GP130 connected to it Tyrosine kinase When activated, a series of reactions such as Ras Paf map kinase occur, promoting the increase of cell gene transcription activity. CT-1 is derived from mice embryonic stem cell A 21.5KDa protein was isolated from the supernatant during differentiation induction. It has been reported that myocardial cells also produce CT-1, which also affects the intracellular transmission of information through GP130. CT-1 can also increase the expression of c-fos, c-jun, ANP and mRNA in cardiomyocytes, indicating that CT-1 regulates the level of gene activation and transcription.

Electrophysiology

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Cardiac myocyte

The ion channel and ion current of cardiac myocytes are the main content of electrophysiology of cardiac myocytes. Its research progress is very rapid, especially the combination of molecular biology research, which can be said to be a rapid progress. Today, it is difficult to understand modern cardiac physiology, pharmacology, and the pathogenesis and treatment of some heart diseases without understanding the electrophysiology of myocardial cells, especially their ion channels and ion currents. Many chapters of cardiac pharmacology cannot become modern pharmacology without these contents. This shows the importance of this field.

In foreign countries, there are more and more works on electrophysiology of myocardial cells, while in China, there are few works, especially those read by clinicians.

cell culture

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Culture and isolation of rat cardiomyocytes

objective

To establish the culture model of myocardial cells for the experiment of myocardial cells in vitro.

method

Using trypsin digestion and neonatal rat cardiomyocytes and non cardiomyocytes such as Fibroblast 、 blood corpuscle Myocardial cells were isolated and purified by the method of equal time difference attachment, and the myocardial culture model was established.

result

myocardium cell culture The longest survival time is up to 3 days. Myocardial cells have different morphological manifestations under the influence of digestion time.

conclusion

Myocardial cells of newborn rats can be isolated by differential adhesion method and cultured in vitro. Pancreatin digestion in low concentration and short time is preferred.

Material Science

1. Digestion of myocardial cells

Take a beaker with a cap and put one in it for use Ether Saturated gauze, put the young rats aged 0 to 3 days into the beaker for anesthesia, sterilize the chest and abdomen skin with 2% iodine and 75% alcohol, open the chest and take out the heart under sterile conditions, immediately put them into 4 º CD Hanks solution (mmol/L: Nacl137, Kcl5.4, Na2HPO40.37, K2HPO40.44, NaHCO34.2) for cutting ventricle Muscle, wash the residual blood, cut it into a tissue block of about 1mm ³, discard D-Hanks solution, add 10~15ml of 0.08% trypsin solution, stand at 37 ° C for 5min, suck out the upper suspension, and add the same amount of serum containing medium. After digestion was terminated, the supernatant was centrifuged (1800r/min) and added with culture medium containing serum. Blow away the precipitated cells, centrifugate them under the same conditions, make cell suspension with 10% serum culture medium, and place it at 37 ° C, containing 5% CO2 incubator Medium.

Myocardial cell isolation

Myocardial fibroblasts and cardiomyocytes were obtained by 2-hour differential attachment according to the different attachment time of myocardial cells and non myocardial cells. Myocardial cells are seeded in 50ml according to 1 * 106 cells/ml Culture medium In the first 2 days of culture, 5-bromodeoxylation was added to the myocardial cell culture medium Pyrimidine nucleoside 0.1 mmol/l to inhibit the proliferation of non cardiac myocytes. All cultured cardiac myocytes were changed every 2 days.

1.3 Serum free culture of myocardial cells

When the myocardial cells are cultured for 24 hours, change the serum free culture medium (containing DMEM culture medium, insulin 10 μ g/ml, ferritin 10 μ g/ml, vitamin C100 μ g/L, vitamin B121.5µ mol/L) to continue to culture for 48 hours, change the medium every 8 hours, try to keep the concentration of each added ingredient unchanged, and finally collect the cells for determination.

2. Experimental results

In this experiment, the longest survival time of neonatal rat cardiomyocytes was 3 days. The method of trypsin digestion with low concentration and short time was adopted in the experiment. Different digestion time leads to different morphological changes of the cells obtained: after digestion for 3-5 minutes, most of the myocardial cells were crescent shaped under high power microscope, and the cells were connected head to tail in the culture medium, showing dynamic changes. However, the myocardial cells that were digested for 5-10 minutes were round, with low density and poor activity. After about 8 hours of culture, the cells gathered in one place and hooked up with each other to show high-density areas, which may be related to the formation of connections between cells. Due to some unknown reason, unknown microorganisms appeared in the central muscle cells during multiple cultures, resulting in the basic death of the cells 3 days later.

3. Experimental discussion

There are many literature reports that the culture time of neonatal rat cardiomyocytes is 10-12 days, and the culture time of this experiment is 3 days, which is shorter than the survival time. However, this experiment can show that the digestion time effect of low concentration trypsin digestion solution (0.08%) is better than that of ordinary concentration (0.125%), Digestion time can reduce cell mortality within 3~5 minutes. The phenomenon of cell aggregation and visible continuous morphological changes during the experiment can indicate that it is possible for cultured myocardial cells in vitro to reconnect into larger unit cell clusters, and they may hook up with each other and form a network through morphological changes.

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