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Cardiomyocytes
(Cardiac Muscle Cells)

Structure, Function and Histology


Definition:  What are cardiomyocytes?


Also known as myocardiocytes, cardiomyocytes are cells that make up the heart muscle/cardiac muscle.

As the chief cell type of the heart, cardiac cells are primarily involved in the contractile function of the heart that enables the pumping of blood around the body. In human beings, as well as many other animals, cardiomyocytes are the first cells to terminally differentiate thus making the heart one of the first organs to form in a developing fetus.

In the embryo of a mouse, for instance, precursor cells of the cardiac muscles have been shown to start developing about 6 days after fertilization. Although cardiomyocytes contain many of the organelles found in other animal cells, they also contain others (e.g. myofibrils) that allow them to effectively perform their function.


Some of the main characteristics include:

 

  • Are elongated cylindrical cells and striated
  • A majority of cardiomyocytes have a single nucleus
  • Have contractile proteins
  • Cardiomyocytes are attached to each other through intercalated discs

 


Ultrastructure of Cardiomyocytes


While cardiomyocytes are muscle cells, they are different from other muscle cells in a number of ways. Unlike other muscle cells in the body, cardiomyocytes are highly resistant to fatigue and therefore always contracting and relaxing to ensure proper circulation of blood around the body.


This is made possible by the structural components of the cell that consists of:



The Basement Membrane


The basement membrane of myocytes is the boundary that separates the intracellular part of the cell from the extracellular environment. It is composed of glycoproteins laminin and fibronectin, type IV collagen as well as proteoglycans that contribute to its overall width of about 50nm.

As such, the membrane is composed of two main layers that include the lamina densa and the lamina lucida. By providing an interface for continuity with the extracellular environment, the basement membrane helps trap such ions as calcium as well as acting as the barrier through which various macromolecules are exchanged.



Sarcolemma


The sarcolemma is a specialized structure that also serves as an outer covering of the cell. The sarcolemma is composed of collagen, glycocalyx (which contracts the basement membrane) and the plasmalemma.

Because it is composed of the lipid bilayer, the sarcolemma also controls the type of molecules that enter the cell. For instance, due to the hydrophobic core of the lipid bilayer, the sarcolemma is impermeable to some molecules. 

The sarcolemma is also part of the intercalated disks as well as the transverse tubular system of the cardiac muscle. It serves as the mechanical linkage between the cardiac cells (cardiomyocytes) through the specialized intercalated disks.

In addition, it contributes to the excitation and contraction coupling through the transverse tubules (invaginations of the sarcolemma into the cytoplasm of cardiac cells). Transverse tubules (T-tubules) also organize cells of the cardiac muscle into pairs thus creating striated muscle strands.


Desmosomes


Gap junctions, which are part of the sarcolemma, are channels between adjacent fibers of the cardiac muscle. These structures allow the depolarizing current to flow through the cardiac muscle cells from one to another and thus contribute to the contraction and relaxation of the cells.

Unlike gap junctions, desmosomes, also part of the sarcolemma, serve to anchor ends of cardiac muscle fibers together. This prevents the cells of the cardiac muscles from pulling apart during contraction. Desmosomes are able to withstand mechanical stress which allows them to hold cells together.

* Desmosomes have been shown to be able to resist mechanical stress because of the fact that they are hyper-adhesive. As such, desmosomes are resistant to chelating agents.

* The presence of the lipid bilayer in sarcolemma allows it to act as the barrier for diffusion

* Membrane proteins on the sarcolemma act as pumps, receptors, and channels that regulate the movement of ions. And so, the sarcolemma is actively involved in the contractile process of the cell.

* A number of receptors are also found on the membrane of cardiomyocytes. These include α, muscarinic and the endothelin receptor systems.


Sarcomeres
(Contractile Proteins and Cytokeletal Proteins)


Essentially, sarcomeres are the functional units that line the myofibrils.


Sarcomeres are divided into two important components that include:

·  Contractile proteins - (acting and myosin) involved in the contraction of myofilament.


· Cytoskeletal proteins - Proteins that help maintain the shape of the cell, stabilize proteins of the sarcomere and maintain mechanical integrity as well as resistance.


Myofilament


Myofilaments are contractile proteins that consist of myosin (the thick filament about 15nm in diameter) and actin proteins (thin filaments about 7nm in diameter).

In the cell, myosin makes up an important group of motor proteins that produce muscular contraction. In cardiomyocytes, myosin II is responsible for the contraction of muscle that allows blood to be pumped around the body.

This type of myosin is composed of two heavy (with motorheads) and light chains. With the energy obtained from ATP, it is the head portion of myosin that binds to actin resulting in muscle contraction.

Actin, on the other hand, is composed of single units of actin known as globular actin (G-actin). The filament is also bound to regulatory proteins that include troponin-T, troponin- C, troponin- I and tropomyosin.

Whereas troponin lies in the grooves between the actin filaments, tropomyosin covers the sites on which actin binds to myosin. Their respective actions, therefore, control the binding of myosin to actin and consequently in the contraction and relaxation of cardiac muscles. 



Mitochondria


Like other body cells, cardiomyocytes are densely packed with different types of organelles that keep the cell alive and contribute to its function.

Unlike other cells, however, cardiomyocytes contain high numbers of mitochondria (occupies about 40 percent of the cell) that maintain high levels of ATP required by the cells.

As previously mentioned, cardiac muscles are constantly contracting and relaxing as the blood is pumped around the body. This requires high levels of energy since these muscles do not rest as is the case with other types of muscles. Here, then, high amounts of mitochondria ensure that the cells get the sufficient energy needed to sustain cardiac contraction.


 

Some of the other important organelles found in cardiomyocytes include:


See more information on Organelles here.


Function of Cardiomyocytes (Mechanism)



Cardiomyocytes go through a contraction-relaxation cycle that enables cardiac muscles to pump blood throughout the body. This is achieved through a process known as excitation-contraction coupling that converts action potential (an electric stimulus) into muscle contraction.


Mechanism of Contraction


During an action potential, membrane depolarization results in an influx of calcium ions into the cell. As the calcium binds to receptors inside the cell, this results in the release of even more calcium into the cell (through calcium channels in the T-tubules). In turn, this results in the shortening of actin-myosin fibrils in the cell and consequently in the overall contraction of the cell.


This process may be represented through the following steps:

·      An action potential is induced by cells of the pacemaker (in the sinoatrial and atrioventricular nodes) and is first conducted to the cardiomyocytes through the gap junctions (intercalated discs)

· Calcium channels in the T-tubules are activated by the action potential as it passes between the sarcomeres of the myofibril to release calcium ions into the cell


·      In the cytoplasm of cardiomyocytes, calcium binds to cardiac troponin-C, which in turn moves the troponin complex from the actin binding site. As a result, actin is free to bind myosin thus initiating contraction


·      As the myosin binds to an ATO molecule, actin filaments are pulled to the central part of the sarcomere which causes the muscle to contract


·      In the repolarization phase, calcium is removed from the cytoplasm of the cell (from the cytosol into the sarcoplasmic reticulum or extracellular fluid). This allows the troponin complex to return to its original position which in turn ends the contraction 


Cardiomyocytes Renewal


Although the regeneration of cardiac muscle cells was thought to be absent, studies have shown that these cells renew at a significantly low rate throughout the life of an individual. For instance, for younger people, about 25 years of age, the annual turnover of cardiomyocytes is said to be about 1 percent. This, however, decreases to about 0.45 percent for older individuals (75 and above).

Over the lifespan of an individual, less than 50 percent of these cells are renewed which shows that compared to many of the other cells, cardiomyocytes have a very long lifespan.

* In the event of injuries or myocardial infarction, monocytes are recruited to remove any damaged/necrotic cardiomyocytes. Phagocytosis by these cells has been suggested to be one of the prerequisites to cardiac repair.



Cardiomyocytes Regeneration


Unlike some animals like Zebrafish, injured heart muscles of human beings do not regenerate sufficiently to allow the heart to heal itself. For this reason, a majority of people who experience various injuries to the heart as well as heart attacks (which affects cardiomyocytes) consequently develop heart failure which is likely to cause death.

This ability, according to research studies, is lost after about a week of life which means that it is impossible for cardiac regeneration in human beings. Because of the inability of the heart muscle to regenerate, implantation of mechanical ventricular devices and heart transplantation has been the only solution for the most part.

Although cardiac cells are unable to regenerate (fast enough to repair damage/ injuries), studies have shown that progenitor cells in adults are capable of producing new cells. These cells, known as cardiac stem cells, reside in the heart and efforts have been directed towards their isolation.

Currently, a number of methods have been suggested for regenerating cardiomyocytes.


These include:


Implanting iPSC-derived cardiomyocytes

Using induced pluripotent stem cell (iPSC) technology, researchers have been able to obtain function in cardiomyocytes thus eliminating the need to use human embryos for this purpose.  The transplantation of cardiomyocytes obtained through this method (iPSC) into damaged hearts has proved successful allowing cardiac muscles to function normally.

Direct reprogramming of fibroblasts

Cardiac fibroblasts make up about 50 percent of the total cardiac cells. Because of their ability to survive very well and couple with other neighboring cells, fibroblasts have been shown to be particularly ideal for direct reprogramming to convert them into cells that resemble cardiomyocytes.

Over the past decade, a number of studies have been successfully conducted, reprogramming fibroblasts into cardiomyocyte-like cells. For instance, in a study conducted in 2012 by Olson and his colleagues, the reprogramming proved to be a success with the cells not only exhibiting improved performance, but also showing reduced formation of scars following myocardial infarction.

Using pathways that promote the division of cardiac cells - According to recent studies, taking advantage of certain pathways (e.g. in Hippo-YAP signaling) that promote division can also promote regeneration.


Microscopy (Histology)


In order to observe cardiomyocytes under the microscope, it is necessary to fix and attach the cells on a microscope. Once the cells have been fixed and permeabilized on the slide, then they are ready for staining and viewing.

Requirements

  • A sample of cardiomyocytes (obtained from such an animal as rodents)
  • Paraformaldehyde
  • Phosphate buffered saline (pH 7.4)
  • Tissue adhesive
  • 0.1M sodium bicarbonate (pH 8.0)
  • Nutator
  • A centrifuge (controlled temperature)
  • A cover glass (chambered)
  • Microscope slide
  • Culture medium (consisting of 5 percent fetal bovine serum, 47.5 percent MEM, 10 mM pyruvic acid, Tyrode’s solution, 6.1 mM glucose and 4.0 mM HEPES)
  • Blocking solution consisting of 0.01 percent BSA in PBS

Stains

  • MitoTracker Deep Red 633
  • Alexa Fluor 568 phalloidin
  • SYTO 11 Green-Fluorescent Nucleic Acid
  • Bovine Serum Albumin

Sample preparation

· Suspend the cells in the medium at 5 percent carbon dioxide and 30 degrees Celsius


· Label the sample with MitoTracker Deep Red 633 and incubate for about 30 minutes in the CO2 incubator - This step is aimed at staining the mitochondria


· Wash the sample using PBS two times in the dark - This may be achieved by using aluminum foil to cover the tube containing the sample


· Using low g force at about 30 degrees Celsius, pellet isolated cells for a minute then suspend the cells in 4 percent paraformaldehyde in PBS to (at 30 degrees Celsius) - Mix the contents using the nutating mixer for about 30 minutes to fix


· Pellet the cells again for about a minute at low g force and resuspend the cells in PBS


· Layer the cardiomyocytes over the chambered cover glass with the tissue adhesive and Cell-Tak Cell


· Allow the chamber to stand for about 2 hours at room temperature - The chamber should not be disturbed


· Wash the cells using PBS - on the glass surface


· Using 0.1 percent Triton X-100 in PBS, permeabilize the cells at room temperature for about 3 minutes


· Wash the cells using PBS two times for about 2 minutes


· Treat the cells using the blocking solution for about 30 minutes at room temperature

· Label the cells for 30 minutes in the dark at room temperature using:

  • Alexa Fluor 568 phalloidin - to stain actin
  • SYTO 11 Green-Fluorescent Nucleic Acid - to stain the nucleus


· Wash the cells using PBS three times (three minutes for each wash)

· Maintain the cells in PBS containing antibiotics

· View the slide under the microscope (under oil immersion objective of 60X)

Observation

When viewed under the microscope, mitochondria will appear blue in color; actin will appear red while the nucleus will take up the green color of the Green-Fluorescent Nucleic Acid stain.




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References

C. Allyson Walker, and Francis G. Spinale. (1999). The Structure and Function of the Cardiac Myocyte: A Review of Fundamental Concepts. Basic Science Review. 

Jeremy Pinnell, Simon Turner, and Simon Howell. (2007). Cardiac muscle physiology. Continuing Education in Anaesthesia Critical Care & Pain, Volume 7, Issue 3, June 2007, Pages 85–88. 

Shirley Dehn and Edward Benjamin Thorp. (2018). Myeloid receptor CD36 is required for early phagocytosis of myocardial infarcts and induction of Nr4a1-dependent mechanisms of cardiac repair. 

Woodcock EA and Matkovich SJ. (2005). Cardiomyocytes structure, function and associated pathologies.. 

Yueqiu Chen, Ziying Yang, Zhen-Ao Zhao and Zhenya Shen. (2017). Direct reprogramming of fibroblasts into cardiomyocytes. Chen et al. Stem Cell Research & Therapy. 

Links


https://www.bcm.edu/news/heart/promote-adult-heart-tissue-regeneration


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