BOD L5 Ischaemic preconditioning and cardioprotection

Question 1

What is ischaemia?

Answer 1

Decreased blood flow to tissue leading to hypoxia

Question 2

What happens when cells receive less O₂?

Answer 2

Decrease in ATP production.

KEY CELL SENSOR!!!

Question 3

What are some physicological consequences of ischaemia on ion homeostasis?

Answer 3

Glycolysis rate (anaerobic respiration) increased following hypoxia.

Increase in acidity of cell. Activates Na⁺/H⁺ exchanger NHE (secondary active transporter, uses sodium gradient to pump H⁺ against its gradient).

Rise in intracellular Na⁺ from this.

This activates Na⁺/Ca²⁺ exchanger. Same principle, uses calcium gradient to pump sodium out.

Huge influx of Ca²⁺. If persists, leads to calcium ion overload of cell. Very toxic to cell. Triggers pathways which cause cell to spiral out of control, protease activity (calpain) which leads to cell damage and apoptosis.

Question 4

The sodium potassion ion pump is vital to cell homeostasis and survival. How does it function?

Answer 4

Active transport, uses ATP.

Question 5

In apoptosis, what is the common link-up between extrinsic (type I) and intrinsic (type II) pathways?

Answer 5

Caspase activation. Both activate caspase 9. Extrinsic activates it via death receptor activation by e.g. FAS ligand and downstream. Intrinsic activates it via mitochondrial activation of cytochrome C, which acts upon it.

Question 6

What are caspases?

Answer 6

Family of proteases linked to apoptotic cell death.

Target and cleave large variety of proteins in the cell, structural proteins (fodrin and lamin) DNA repair enzymes and protein kinases.

Question 7

What is reperfusion injury?

Answer 7

Myocardial cell death once blood flow is restored (reperfusion) to heart tissues.

Question 8

What is ischaema/reperfusion injury associated with that is response for this cell death?

Answer 8

Generation of reactive oxygen species.

Three main are:

Hydrogen peroxide, (H₂O₂₎

Superoxide (O2^-)

Hydroxyl radical (OH•)

Question 9

How are ROS generated during reperfursion injury?

Answer 9

Electron leakage during electron transport chain.

Question 10

How are ROS usually deat with by the body?

Answer 10

Removed by antioxidants! Many pathways exist within a cell.

Question 11

Briefly what is ischaemic preconditioning?

Answer 11

Phenomenom where brief episodes of ischaemia can protect against subsequent prolonged ischaemia.

Four sequential 5 min periods of isc. each followed by 5 mins of reperfusion before 40 min of sustained isc. Infarct size damage reduced to 23%! (7% from 30%).

Question 12

How long does Ischaemic preconditioning (IPC) last?

Answer 12

Only lasts 2/3 hours, however there is a second window of protection that appears 24/72 hours later.

Question 13

What is the rationale between the two phases of IPC?

Answer 13

Initial/classical IPC result of modulating pre-existing proteins/pathways.

Delayed IPC result of changes in gene expression.

Question 14

What ischaemic postconditioning?

Answer 14

Short periods of re-perfusion before a longer period of reprefusion protects tissue much more.

Much more cninically applicable.

Question 15

Why is studying cardiac issues often multifactoral and not as simple as standard models suggest?

Answer 15

Cardiac problems often associated with other diseases/conditions, diabetes/obesity/age.

In clinic, factors from these other conditions may effect treatments.

Question 16

Many end points can be used when studying IPC. What are the three main ways and briefly what would they involve?

Answer 16

Mechanical performace of heart, (left ventricular developed pressure LVDP, end diastolic pressure EDP, Blood flow).

Infarct size, heart slices stained with Tetrazolium red (viable tissue red, infarcted tissue is pale pink.)

Biochemical analysis, ATP levels, protein release, necrosis/apoptosis.

Question 17

Out of the three end points used when studying IPC, which is the gold standard?

Answer 17

Infarct size is gold standard. Measuring heart death itself, using Tetrazolium Red staining which stains living tissue.

Question 18

What cell lines / in vitro / vivo models can be used to study IPC and what specialist incubator is used?

Answer 18

Cell lines such as human atrial cardiocytes, primary cells from cardiomyocytes (chick/rat). Isolated heart preparations can be used (Langendorff system).

In vivo studies use anaethetized animals.

Hypoxic incubators are used, that low oxygen in time intervals with absence of glucose in media.

Question 19

What role do GPCR’s play in IPC?

Answer 19

In ischaemia, ATP levels go down, adeosine levels rise, pumped out of cell. They activate GPCRs.

Question 20

how can the role of specific GPCRs be studied in cardioprotection?

Answer 20

Instead of preconditioning, treating with small molecule drugs / agonists that may mimic IPC.

Can also confirm study with antagonist.

Studies may confirm that agonists to GPCRs are produced during IPC

Question 21

How was it investigated and confirmed whether ROS generation played a role in IPC?

Answer 21

Treatment with ROS scavengers (anti-oxidants) abolished protection seen in IPC.

Suggests that ROS plays crucial protective role in IPC.

Other studies confirmed that IPC can be generated by artificial ROS generation.

Question 22

What are the two experimental evidences that ROS plays a role in IPC?

Answer 22

N-2-mercaptopropionyl glycine: cell permeable ROS scavenger abolished protection induced by IPC.

Infusion of hypoxanthine & xanthine oxidase (ROS generating system) mimicked IPC

Question 23

What GPCR and G-protein action was found to be involved in IPC?

What does this protein do and what is its downstream target?

Answer 23

Phospholipase C (PLC).

PLC hydrolyses membrane phospholipid PIP2.

Downstream of this, Protein Kinase C.

Question 24

What is protein kinase C?

Answer 24

Family of serine/threonine protein kinase enzymes. Control function of other proteins via phosphorylation of Ser/Thr residues.

15 isozymes split into three subfamilies.

Conventional, novel and atypical activation different.

Question 25

What is the role of PKC in IPC?

Answer 25

PKC has protective role in ischaemia.

Investigated using inhibitors (that block IPC).

Activators trigger protection (IPC).

IPC induces selective translocation of PKC-epsilon and eta.

Question 26

What are the downstream targets of PKC?

Answer 26

sarcolemma K_ATP channel (sarcK_ATP).

mitoK_ATP channel.
mitochondrial permeability transition pore (MPTP)
BAD (pro-apoptotic protein)
Bcl-2 (anti-apoptotic protein)
these four associated with outer mitochondrial membrane.

Question 27

How does the sarcK_ATP channel function?

Answer 27

Inhibited by ATP and activated by ADP.

When active/open, potassium ions leave the cell, hyperpolarising cell membrane.

In abnormal ATP production and ADP buildup, channel opens and causes hyperpolarisation.

Question 28

What are two clinically used sarcK_ATP modulators?

Answer 28

Glibenclamide - channel blocker used to treat diabetes mellitus, promotes insulin release from beta cells.

Diazoxide - channel opener used to treat hypertension. Promotes smooth muscle relaxation.

Question 29

What happens to sarcK_ATP channel in cardiomyocyte during ischaemia?

Answer 29

Contractions reduced to conserve ATP and enhance cells survival chance. Calcium channels inhibited in this way.

sarcK_ATP opening, hyperpolarisation, causes reduction in Ca²⁺ influx. Reduces calcium ion overload.

Question 30

How can pharmacological preconditioning be performed.

Answer 30

Agonists for these various receptors involved.

Generation of ROS.

PKC activators

sarcK_ATP (poteassium channel ATP channels) openers.

All elicit pharma PC.