Macrophage – the new miracle cell? How the phagocytic cell helps maintaining a healthy heart beat

For some people, when they think of a macrophage, they will imagine a real life microscopic version of Pac-Man running around their body ingesting any microbial intruder lying in its way. While this has been the central dogma surrounding this immune cell for the last 100 years - it is becoming increasingly more obvious that this isn’t the entire truth. Within the last 10 years, numerous papers have been published identifying new functions and processes carried out by the humble macrophage. More importantly, we have shown that tissue resident macrophages carry out organ-specific functions. For instance, macrophages have been shown to contribute to iron recycling in the spleen and liver (Theurl et al. 2016), thermogenesis regulation in adipose tissue (Nguyen et al. 2011) and more recently, electrical conduction in the heart (Hulsmans et al. 2017). 

Are you trying to tell me that these tiny white blood cells are better at multitasking than I am? Well….Yes. That is exactly what I am telling you.

A study published in Cell by a team of researchers from Harvard Medical School, revealed that cardiac-resident macrophages play an important role in facilitating electrical conduction in the steady state heart.

By coupling cardiac research techniques such as surface and telemetric ECG monitoring with immunological techniques like flow cytometry and the use of transgenic mice, they uncovered the secret life of a cardiac macrophage.

Cardiac macrophages form Cx43-containing gap junctions with neighbouring cardiomyocytes

Cardiomyocytes, or heart cells, communicate with one another via gap junctions. Gap junctions allow for the transmission of small molecules and ions between neighbouring cells, which in the case of the heart, facilitates the synchronous contraction of the tissue and subsequent generation of a heartbeat. Connexin (Cx) proteins are the nuts and bolts of these intercellular channels and can come in various isoforms. The particular isoform Cx43, has been shown to connect cardiomyocytes with non-cardiomyocytes.

Using Flow cytometry and RNA sequencing, the team were able to show that AV node resident macrophages express Cx43. To prove that the macrophages were using the Cx43 connexin proteins to form gap junctions with neighbouring cardiac cells, they took a closer look at the cells using electron microscopy. This allowed the team to visualize direct membrane-membrane contact between the two cell types, indicating possible cell-cell communication.

Macrophages influence AV node conduction

To investigate whether cell-cell communication was happening through the Cx43-junctions, the team used a loss-of-function experiment where they deleted Cx43 in AV node resident macrophages. To do this, they used a tamoxifen inducible system, where by injecting the transgenic mice with tamoxifen, they could specifically delete Cx43 in the cardiac macrophages. To measure the effects of removing the Cx43 junctions on AV node conduction, they performed an in vivo electrophysiology (EP) study using a Millar Mikro-Tip octapolar catheter (EPR-800) inserted into the right atrium and ventricle in conjunction with surface ECG/EKG recordings.

Surface ECG recordings we measured using subcutaneous electrodes connected to an Animal Bio amplifier and PowerLab. Data was later analyzed using the ECG module in LabChart Pro. This enabled the team to stimulate the heart in different ways and measure the subsequent effects on the electrical activity of the heart. Comparing results from the control and transgenic mice, they found that in the absence of Cx43, there was impaired AV node conduction, indicating that cardiac macrophages can influence the electrical conduction of the heart.

ECG telemetry reveals mice lacking macrophages have irregular heart beats 

Next the team wanted to investigate if depleting the resident macrophage population would have a similar effect on AV-node conduction. They did this by genetically modifying mice to expressess a diphtheria toxin (DT) inducible system, that upon administration of DT, would result in the depletion of macrophages (and other myeloid cells). To monitor the subsequent effects on the heart, they used an implantable ECG telemetry device (DSI) placed in a lead II position within the abdomen. This enabled ECG data to be recorded continuously over 8 days. The data was later analyzed using LabChart software. Interestingly, within 24 hours of a single dose of DT, all mice had developed a first degree AV block, that overtime progressed to a third degree block. An AV-block happens when the conduction between the atria and ventricles is impaired. Clinically, AV-blocks can worsen pre-existing conditions such as heart failure.

The unique integration of cardiac and immunological research techniques used in this study, has uncovered the unlikely role that macrophages play in facilitating electrical conduction in the heart and their potential involvement in heart disease. For years we have compartmentalized our bodies into distinct physiological systems, made up of different cell types that have distinct functions. However, it is through such studies like this that we are beginning to understand the amazing adaptability and cooperativity of these so called ‘distinct’ cell types, and discover how intertwined these systems really are.

Courtesy: Adinstruments

Types of Monocytes

Monocytes are a group of immune cells that originate in bone marrow and are released into peripheral blood, where they circulate for several days. They belong to the mononuclear-phagocyte system, which also include macrophages, dendritic cells, and their bone-marrow precursors. Monocytes represent 5–10% of peripheral leucocytes .

Monocytes have been divided into three subtypes based on relative surface expression of LPS co-receptor CD14 and FCγIII receptor CD16. The most predominant of the three, termed “classical monocytes”, express high levels of CD14 on their surface, are devoid of surface CD16, and account for approximately 80% of the total monocyte population. The remaining 20% express CD16 and have been further classified into two subtypes. The more abundant “nonclassical monocytes”, are characterized by very low expression of surface CD14 and high levels of CD16, whereas the third monocyte subtype, called “intermediate monocytes”, express high levels of both the receptors.

Protein targeting

Different proteins need to be sent to different parts of a eukaryotic cell, or, in some cases, exported out of the cell and into the extracellular space. How do the right proteins get to the right places?

Cells have various shipping systems, kind of like molecular versions of the postal service, to make sure that proteins arrive at their correct destinations. In these systems, molecular labels (often, amino acid sequences) are used to "address" proteins for delivery to specific locations. Let’s take a look at how these shipping systems work.

Overview of cellular shipping routes

Translation of all proteins in a eukaryotic cell begins in the cytosol (except for a few proteins made in mitochondria and chloroplasts). As a protein is made, it passes step by step through a shipping "decision tree." At each stage, the protein is checked for molecular tags to see if it needs to be re-routed to a different pathway or destination.

Diagram based on similar diagram in Alberts et al. $^1$

The first major branch point comes shortly after translation starts. At this point, the protein will either remain in the cytosol for the rest of translation, or be fed into the endoplasmic reticulum (ER) as it is translated$^2$.

• Proteins are fed into the ER during translation if they have an amino sequence called a signal peptide. In general, proteins bound for organelles in the endomembrane system (such as the ER, Golgi apparatus, and lysosome) or for the exterior of the cell must enter the ER at this stage.
• Proteins that do not have a signal peptide stay in the cytosol for the rest of translation. If they lack other "address labels," they'll stay in the cytosol permanently. However, if they have the right labels, they can be sent to the mitochondria, chloroplasts, peroxisomes, or nucleus after translation. 

The endomembrane system and secretory pathway

Proteins destined for any part of the endomembrane system (or the outside of the cell) are brought to the ER during translation and fed in as they're made.

Signal peptides

The signal peptide that sends a protein into the endoplasmic reticulum during translation is a series of hydrophobic (“water-fearing”) amino acids, usually found near the beginning (N-terminus) of the protein. When this sequence sticks out of the ribosome, it’s recognized by a protein complex called the signal-recognition particle (SRP), which takes the ribosome to the ER. There, the ribosome feeds its amino acid chain into the ER lumen (interior) as it's made.

In some cases, the signal peptide is snipped off during translation and the finished protein is released into the interior of the ER (as shown above). In other cases, the signal peptide or another stretch of hydrophobic amino acids gets embedded in the ER membrane. This creates a transmembrane (membrane-crossing) segment that anchors the protein to the membrane.

Transport through the endomembrane system

In the ER, proteins fold into their correct shapes, and may also get sugar groups attached to them. Most proteins are then transported to the Golgi apparatus in membrane vesicles. Some proteins, however, need to stay in the ER and do their jobs there. These proteins have amino acid tags that ensure they are shipped back to the ER if they "escape" into the Golgi.

"The endomembrane system and proteins: Figure 1," by OpenStax College, Biology (CC BY 3.0).

In the Golgi apparatus, proteins may undergo more modifications (such as addition of sugar groups) and before going on to their final destinations. These destinations include lysosomes, the plasma membrane, and the cell exterior. Some proteins need to do their jobs in the Golgi (are "Golgi-resident), and a variety of molecular signals, including amino acid tags and structural features, are used to keep them there or bring them back.

If they don't have any specific tags, proteins are sent from the Golgi to the cell surface, where they’re secreted to the cell exterior (if they’re free-floating) or delivered to the plasma membrane (if they’re membrane-embedded). This default pathway is shown in the diagram above for a membrane protein, colored in green, that bears sugar groups, colored in purple.

Proteins are shipped to other destinations if they contain the right molecular labels. For example, proteins destined for the lysosome have a molecular tag consisting of a sugar with a phosphate group attached. In the Golgi apparatus, proteins with this tag are sorted into vesicles bound for the lysosome.

Targeting to non-endomembrane organelles

Proteins that are made in the cytosol (don't enter ER during translation) may stay permanently in the cytosol. However, they may also be shipped to other, non-endomembrane destinations in the cell. For instance, proteins bound for the mitochondria, chloroplasts, peroxisomes, and nucleus are usually made in the cytosol and delivered after translation is complete.

To be delivered to one of these organelles after translation, a protein must contain a specific amino acid "address label." The label is recognized by other proteins in the cell, which help transport the protein to the right destination.

As an example, let's consider delivery to the peroxisome, an organelle involved in detoxification. Proteins needed in the peroxisome have a specific sequence of amino acids called a peroxisomal targeting signal. The classic signal consists of just three amino acids, serine-lysine-leucine, found at the very end (C-terminus) of a protein. This pattern of amino acids is recognized by a helper protein in the cytosol, which brings the protein to the peroxisome. 

Mitochondrial, chloroplast, and nuclear targeting are generally similar to peroxisomal targeting. That is, a certain amino acid sequence sends the protein to its target organelle (or a compartment inside that organelle). However, the nature of the "address labels" is different in each case.

[Credit: Khan Academy]