The Cardiac Fibroblast and Cardiac Remodelling

Question 1

What cells make up the heart?

Answer 1

• MYOCYTES: 30-40% of cardiac cells yet occupy 2/3 of cellular volume
• NON-MYOCYTES: 60-70%
• Fibroblasts (most prevalent cell type)
• Endothelial cells
• Smooth muscle cells
• Pericytes
• Neuronal cells
• Inflammatory cells
• Progenitor cells

Question 2

How to myocytes and fibroblasts interact in the heart?

Answer 2

Both myocytes and fibroblasts are found in close proximity to one another in the heart
-There is evidence that gap junctions form between the two cell types to allow them to communicate and quickly propagate action potentials (maintains the syncytium)
THIS EVIDENCE has been seen using Rabbit Heart Models and stains for Myocytes, Fibroblasts and Gap Junctions (KOHL, HEART RHYTHM, 2012)

Question 3

How do gap junctions allow communication?

Answer 3

They allow action potential propagation through the movement of ions, but they allow communication between cells via the movement of second messengers

Question 4

How does fibrosis prevent action potential propagation?

Answer 4

Excess fibrosis and the laying down of collage disrupts the gap junctions and prevents ions and second messengers from flowing freely between cells.
-Electrical coupling is therefore disturbed and arrhythmias can occur

Question 5

How are fibroblasts regulated?

Answer 5

They are able to respond to blood cytokines and growth factors that are delivered through capillaries
-They are also able to synthesise and secrete their own bioactive molecules that act in an autocrine or paracrine manner

Question 6

How are fibroblasts associated with the extra cellular matrix?

Answer 6

Fibroblasts are physically associated with the collagen-based ECM through receptors such as DDR2 and Integrins

Question 7

What is the healing process after MI?

Answer 7

1. Cell death due to ischaemia
2. Acute inflammatory response
   - innate immune response is triggered and neutrophils and macrophages are recruited
   - cytokine and chemokine release attracts more cells
3. Formation of GRANULATION TISSUE
   - there is phenotypic differentiation of fibroblasts to myofibroblasts
   - Both myofibroblasts and macrophages accumulate in the damaged tissue
   - Macrophages release MMPs which degrades the ECM
   - Neovascularisation to restore blood flow
4. MATURATION PHASE
   - Myofibroblasts deposit collagen types I and III
   - Myofibroblasts then contract to reduce the area of scar tissue
5. RESOLUTION OF INFLAMMATORY RESPONSE
   - Action of anti-inflammatory cytokines e.g IL-10 and TGF-b
   - Majority of myofibroblasts now undergo apoptosis but a few remain for several years

Question 8

What is the long term outcome of healing post MI?

Answer 8

1. Excessive collagen deposition
2. Fibrosis
3. Adverse remodelling
4. Heart failure

Question 9

Where do cardiac fibroblasts come from?

Answer 9

From Epicardial Epithelial Cells by Epithelial Mesenchymal Transformation
-This is a very different lineage from other cardiac cells and also from other fibroblasts in the body

Question 10

How are fibroblasts identified?

Answer 10

There are no definitive markers for identifying fibroblasts due to the hetergeneous nature of fibroblasts and their similarity to other cell types

Question 11

What are the most commonly used antibody stains for fibroblasts?

Answer 11

1. Vimentin (a filamentous cytoskeletal protein)
2. DDR2 (collagen receptor)
3. FSP (fibroblast specific protein)

Question 12

How can Myofibroblasts be identified?

Answer 12

Myofibroblasts are differentiated forms of fibroblasts and contain contractile units called alpha-Smooth Muscle Actin
-These a-SMCs can be identified using stains and can differentiate them from cardiac myocytes because they do not stain for Smooth-Muscle Myosin Heavy Chain (SM-HC)

Question 13

What roles do cardiac fibroblasts play?

Answer 13

1. Production of cytokines and growth factors
2. Matrix synthesis
3. Matrix degradation by secretion of MMPs and inhibition of TIMPs (tissue inhibitors of Metaloproteases)
4. Myofibroblast differentiation
5. Fibroblast migration
6. Fibroblast proliferation

Question 14

How do fibroblasts behave under normal physiological conditions?

Answer 14

They are QUIESCENT CELLS

Question 15

What happens to cardiac fibroblasts during remodelling?

Answer 15

They become activated due to altered levels of growth factors, cytokines and reactive oxygen species

Question 16

How are fibroblasts different from myocytes?

Answer 16

Fibroblasts once activated can become highly proliferative and migrate to different areas, whereas myocytes are unable to proliferate.
-Fibroblasts tend to migrate to the infarct zone where they undergo phenotypic differentiation into myofibroblasts

Question 17

How are myofibroblasts different from fibroblasts?

Answer 17

They begin to express alpha-smooth muscle actin, focal adhesion proteins and extracellular matrix proteins
-All of these new proteins confer TENSILE properties and so the myofibroblast is able to contract and reduce the size of the infarct area
(focal adhesion proteins are needed to hold on to the ECM and contract it)

Question 18

How do fibroblasts modify the extracellular matrix?

Answer 18

They express matrixmetalloproteases which degrade the ECM, and they can also promote its preservation through expression of TIMPs-tissue inhibitors of metalloproteases

Question 19

What are some pro-inflammatory cytokines that fibroblasts produce?

Answer 19

IL-1
IL-6
IL-8
TNF

Question 20

What are some pro-fibrotic growth factors that fibroblasts produce?

Answer 20

TGF-Beta

GTGF

Question 21

What are some angiogenesis-inducing factors that fibroblasts produce?

Answer 21

PDGF

VEGF

Question 22

What makes cardiac fibroblasts essential?

Answer 22

• They’re needed for maintenance of normal cardiac development and structure
• They support adaptive changes in heart structure both physiologically and pathologically
• They induce repair of the heart in scar formation and healing post-MI
• Prevent cardiac rupture by increasing tensile wall strength

Question 23

Why are fibroblasts detrimental?

Answer 23

• Excessive, sustained fibroblast proliferation and ECM deposition results in Fibrosis and a stiff heart and restricted contraction/relaxation
• ECM deposition disrupts action potential conduction by interferring with Gap junctions and so arrhythmias can result

Question 24

What stimulates production of Myofibroblasts via differentiation of fibroblasts?

Answer 24

1. Mechanical tension (infarct area and hypertrophy)
2. TGF-B
3. Fibronectin (FN-EDA)
4. Collagen VI

Question 25

What do myofibroblasts show increased expression of?

Answer 25

1. Contractile proteins e.g alpha-smooth muscle actin and Vimentin 2. Focal Adhesion Proteins e.g Tensin and Integrins 3. Cell surface receptors e.g Ang I 4. ECM proteins e.g Collagen I and III

Question 26

What type of characteristics do they extra proteins produce in myofibroblasts?

Answer 26

They induce TENSILE CHARACTERISTICS i.e the myofibroblast can contract -This contraction allows ECM contraction and reduction in the size of an infarct or damaged area

Question 27

How do we know about these tensile characteristics?

Answer 27

Nirwenhoven, Matrix Biol,2013 used collagen gel discs to demonstrate the tensile characteristics of myofibroblasts - Myofibroblasts were placed on the gel discs and the surface are of the collagen discs became reduced - Shows that upon contact with collagen i.e ECM, myofibroblasts contract

Question 28

Where do myofibroblasts come from?

Answer 28

It was originally thought that myofibroblasts came solely from differentiation of fibroblasts in the heart. However there is increasing evidence that they also come from elsewhere -CELL LINEAGE EXPERIMENT have now shown that myofibroblasts can be derived from epi- and endothelial cells in a process termed ENDO-/EPITHELIAL TO MESENCHYMAL TRANSFORMATION (as is the case for fibroblasts) -Studies have also shown that myofibroblasts can be derived from progenitor stem cells in the heart or blood (bone marrow dervied fibroblasts are known as Fibrocytes) -AND resident smooth muscle cells may de-differentiate into cardiac myofibroblasts

Question 29

Where do the majority of cardiac myofibroblasts come from?

Answer 29

The relative contributions of different sources of myofibroblast vary greatly in different settings i.e physiological and pathological BUT it still appears that the majority of cardiac myofibroblasts come from the differentiation of resident cardiac fibroblasts

Question 30

In pro-fibrotic environments, what proteins are produced to allow ECM synthesis?

Answer 30

- Collagens I and III - Laminins (structural ECM protein) - Fibronectins (structural ECM protein) - GAGs (glycosaminoglycans) - TIMPS (inhibits breakdown of ECM)

Question 31

In anti-fibrotic environments, what proteins are produced to inhibit ECM synthesis?

Answer 31

- MMPs | - ADAMS and ADAMTS (proteases that breakdown elements of the ECM)

Question 32

What are matrixmetalloproteases?

Answer 32

- A family of >25 zinc-dependent endopeptidases - Can degrade components of the ECM - MMPs are mostly secreted from fibroblasts but can also come from cell membranes called Membrane-Type MMPs - Expression of MMPs is usually low (don't want myocyte slippage in healthy phenotype) but expression can be massively upregulated when needed in response to pro-fibrotic cytokines such as TGF-B - MMPs are secreted as inactive zymogens and require enzymatic activation - MMPs are inhibited by TIMPs 1-4 - Cardiac fibroblasts express MMPs 1-14

Question 33

What stimulates MMP production?

Answer 33

TGF-B and IL-1

Question 34

What inactivates MMPs?

Answer 34

TIMPs and Ang II

Question 35

What is IL-1?

Answer 35

-An inflammatory cytokine that shows elevated levels following MI -Cardiac fibroblasts are particularly sensitive to the effects of IL-1 IL-1 causes fibroblasts to: -Produce more pro-inflammatory molecules -Increases angiogenesis through production of VEGF -Increase cell migration (to move fibroblasts to where they are needed) -Increase ECM degradation (MMP production-to allow for remodelling) -Decrease ECM synthesis (until ready to produce more) -Decrease myofibroblast production (IL-1 is released in the early stages of injury and we don't want infarct contraction until some remodelling has taken place)

Question 36

How might cardiac fibroblasts be used therapeutically?

Answer 36

There is potential that cardiac fibroblasts could be used as a therapeutic target to protect/enhance cardiac repair but inhibit adverse remodelling

Question 37

How might cardiac fibroblasts be targeted?

Answer 37

There are no direct fibroblast therapies yet but direct reprogramming, genetic therapies and microRNAs all hold potential

Question 38

What drugs already have an effect on fibroblasts?

Answer 38

``` it is thought that: 1. ACEis and ARBs 2. Beta Blockers 3. Statins ^all play a role in modulating fibroblasts ```

Question 39

How do ACEis and ARBs affect fibroblasts?

Answer 39

- As well as lowering blood pressure, ACEis and ARBs may reduce adverse myocardial remodelling and reduce heart failure mortality - Angiotensin II is known to have profibrotic effects on the heart - Ang II stimulates: - cardiac fibroblast stimulation - myofibroblast differentiation - ECM synthesis - Inhibits MMPs SO, by inhibiting Ang II we are inhibiting all of these effects thus reducing fibrosis and reducing adverse remodelling

Question 40

How do beta blockers affect cardiac fibroblasts?

Answer 40

(Turner, Cardiovascular Res, 2003) - Block the effects of adrenergic receptors so reduce blood pressure and heart rate - But have also been shown to reduce fibroblast proliferation so reducing fibrosis

Question 41

How do statins affect fibroblasts?

Answer 41

(Porter, 2011) -Statins inhibit the enzyme HMG CoA Reductase needed to synthesise cholesterol -Improves vascular function by raising levels of HDLs whilst lowering LDLs Statins have also been shown to reduce adverse remodelling so appear to have CHOLESTEROL INDEPENDENT PLEIOTROPIC EFFECTS -Lipid intermediates in the cholesterol pathway are needed to synthesise G-proteins Ras and Rho -Statins inhibit this so Ras and Rho signalling cannot occur-it is thought that this cellular signalling is involved in the effects of statins upon remodelling -Simvastatin shown to reduce TNF induced proliferation of fibroblasts and also reduces MMP-9 needed for ECM degradation

Question 42

Summary of Fibroblasts:

Answer 42

1. Essential for normal development and physiological functions of the heart 2. Play a key role in physio- and pathological remodelling 3. Fibroblasts are potential therapeutic targets to aid repair or reduce adverse myocardial remodelling