Consequences of Cardiac Pathologies and Cardiac Cycle and Potential Therapy (Dr Murdoch)

Question 1

How was E-C coupling in heart failure (SR calcium release) tested in rats

Answer 1

1) Rats with hypertrophy and hypertension
2) Cardiac myocytes loaded with a calcium sensitive dye
3) Sparks are individual calcium release from the sarcoplasmic reticulum and through ryanodine receptors
4) The sparks sum to make a calcium transient
5) The sparks are indistinguishable between healthy and hypertrophied cells
6) This means there is normal SR calcium content and normal ryanodine receptors in hypertrophic myocytes
7) What is lower however is the FREQUENCY of sparks (as shown below)

Question 2

Why do hypertrophied hearts show normal LTCC currents, normal spark magnitude but lower spark frequency?

Answer 2

Functional uncoupling between LTCC and RyR. This leads to the intracellular calcium transient being prolinged and therefore the action potential being prolonged

Question 3

Give 2 reasons for action potential duration prolongation

Answer 3

1) Decreased repolarising potassium currents (transient outward current, inward rectifier and delayed rectifier)
2) Increased depolarising inward current (background sodium current, NCX and T-type calcium current

Question 4

What blocks SERCA?

Answer 4

CPA (Cyclopiazonic acid)

Question 5

What is the proof that failing cells are more dependent on calcium extrusion mechanisms

Answer 5

CPA reduced the AP in control and failing but there is less of a differences between pre-CPA and CPA in the failing heart as shown below

Question 6

What are failing cells dependent on for calcium extrusion

Answer 6

Na/Ca exchange (this can be arrhythmogenic

Question 7

What are the levels of SERCA and NCX in failing cells

Answer 7

Less SERCA2a and more NCX

Question 8

Why are failing cells less responsive to iontropies?

Answer 8

Failing cells cannot shorten the contractile cycle

Question 9

Describe the negative force-frequency relationship

Answer 9

Because SERCA cannot load the SR with Ca & an up regulation of Na/Ca exchange
elevation of intracellular Na
more Ca extrusion between beats and less Ca cycling through the SR
Failing hearts don’t respond to exercise/beta stimulation

Question 10

Summary of E-C coupling in heart failure

Answer 10

Key changes:

action potential prolonged

SR Ca uptake compromised (SERCA)

SR Ca load decreased /Ca release compromised

Intracellular Na increased

Incomplete relaxation

Force-frequency relationship becomes negative

-receptor down-regulation

Compensatory changes:

Na/Ca exchange increased (NCX)

Myofilament sensitivity increased?

Question 11

What is the job of the extracellular matrix

Answer 11

Support myocytes/framework against which to contract: Collagen 1>3

Question 12

What produces the extracellular matrix

Answer 12

Fibroblasts

Question 13

Extracellular matrix adaptions in heart failure

Answer 13

• Disproportionate fibroblast proliferation
• Excess collagen production, 3>1 (aldosterone, AngII, ET-1, TGF-beta, FGF, stretch)
• Reduced collagen breakdown
• Replacement fibrosis: Necrotic cells die and are replaced by collagen
• Reactive fibrosis: Around vessels, interstitium; May improve force generation initially

Question 14

Neurohumoral compensatory mechanisms in heart failure

Answer 14

1) LV failure
2) CO falls
3) BP falls
4) Baroreceptor reflex sympathetic stimulation
5) Increased HR and contractility
6) CO returns to normal
7) BP returns to normal

Question 15

Global adaptions in heart failure

Answer 15

1) Collagen deposition in the extracellular. This makes the muscle stiffer and impairs relaxation in diastole
2) Baroreceptor reflex: Increased sympathetic drive to the heart (Alleviated by beta-blockers)
3) Activation of RAAS, sodium and water retention and increase in BP (Alleviated by ACEi and diuretics)
4) Beta blockers, diuretics and ACEi are first line in HF

Question 16

Describe the action of beta-blockers

Answer 16

• Reduce sympathetic drive to the heart (oxygen sparing)
• Reduces remodelling (fetal genes)
• Paradoxically restore beta-receptor density and signalling
• Reduce blood pressure via reduced renin secretion (afterload)

Question 17

Describe the action of ACEi

Answer 17

• Reduce Ang II production meaning they reduce blood glucose

- Reduce aldosterone secretion meaning they reduce sodium and water retention (preload)

Question 18

Describe the action of diuretics

Answer 18

-Oppose sodium and water retention (preload)

Question 19

How often is the entire ATP content turned over

Answer 19

Every 4-5 seconds

Question 20

Can the heart generate new myocytes? If so how did they prove this?

Answer 20

• There was a huge spike of carbon-14 (radioactive isotope of carbon) due to nuclear testing in the cold war
• Radiocarbon dating can be used to determine the cellular age
• The amount of carbon 14 in a cell’s DNA report on the age of that cell
• If cardiac myocytes are renewed postnatal cell turnover will change the amount of carbon-14 in the DNA compared to DOB
• This showed that the DNA of ventricular myocytes were younger than the patient (suggesting regeneration of myocytes)

Question 21

Early abnormalities in ischaemia

Answer 21

• Functional impairment is almost immediate
• Switch to anaerobic ATP production (i.e. glycolysis) within a minute of perfusion impairment)
• Metabolite accumulation: Protons, phosphate, potassium, free radicals, oxidised lipids.

Question 22

The impaired pumping function of the heart activates the baroreceptor reflex. What impact does this have?

Answer 22

1) Bad: Sympathetic stimulation of the heart is increased. Attempts to normalised CO by increasing contractility and increased oxygen demand
2) Good: Accelerates glycolysis, increase glucose uptake (briefly) , inhibits glycogen synthetase (glucose storage) and activates phosphofructokinase reaction (rate limiting step in glycolysis)

Question 23

Describe the pH effect of calcium sensitivity

Answer 23

• Reduced calcium affinity, max tension and contractility
• Acidosis occurs rapidly following ischaemia: This is one of the main reasons for impaired contractility and precedes the depletion of high energy phosphates
• Increased pH of myocytes increases sodium hydrogen exchanger activity
• During ischaemia there is acidification extracellularly, sodium overload, inhibition of sodium pump, calcium overload via reverse mode of NCX and calcium rise buffered by mitochondria

Question 24

Describe ATP depletion and mitochondria

Answer 24

• Mitochondrial membrane potential (MMP) must be maintained
• In the absence of electron transport MMP starts to dissipate
• The ATP synthesis counters this by working in reverse:

1) ATP hydrolysis: H+ pumped out and MMP restored
2) ATP depletion: Maintains mitochondrial integrity and allows mitochondria to buffer calcium

Question 25

What protein occurs, relating to ROS accumulation, after ischaemia. How was this shown?

Answer 25

HNE (4-hydroxy-2-noneal). Western blot showed after ischaemia this occurs

Question 26

What does HNE inhibit

Answer 26

Inhibits various enymes: Particularly the sodium pump and glyceraldehyde-3-phosphate dehydrogenase (glycolysis)

Question 27

Describe the garden hose effect

Answer 27

- Perfusion of the intracoronary vessels has a distending effect on the cardiac muscle - Distension of the cardiac muscle is positively ionotropic (due to frank starling mechanism) - Occlusion of a coronary artery reduces the perfusion of the cardiac muscle - Hence the contractility is reduced: Less stretch, less positive ionotropy by the frank-starling mechanism - Onset is immediate upon vessel blockage

Question 28

Describe the differences of diastolic dysfunction (i.e. impaired relaxation) in early and late ischaemia

Answer 28

- In early ischaemia: - Impaired cytosolic calcium efflux: - Sodium accumulation and acidosis inhibits calcium efflux (NCX) - SERCA and PMCA compromised - Mitochondria take up calcium but in order to do so must hydrolyse ATP to maintain membrane potential - In late ischaemia: - Contracture develops - ATP is required to dissociate myosin from actin in the absence of ATP rigor bonds form (rigor mortis)

Question 29

Describe the ischaemia progression

Answer 29

1) Garden hose effect (early) 2) Acidosis reduces myofilament calcium sensitivity 3) Extracellular potassium depolarises 4) Cellular ATP depleted 5) ROS damage contractile machinery 6) Rigor bonds form 7) Cell death (late)

Question 30

Describe the ischaemic AP

Answer 30

- Delayed activation - Slower to rise (fewer sodium channels) - Slower to trigger voltage gated calcium channels - Reaches threshold to activate adjacent tissue more slowly - Propagates more slowly and acidosis impairs gap junctions - ‘Substrate’ for arrhythmias

Question 31

Describe SERCA gene therapy

Answer 31

Restoration of contractile function in isolated cardiomyocytes from failing human hearts by gene transfer. This improves survival, cardiac metabolism and LV function. It is done through adenoviral gene transfer

Question 32

Describe stem cell therapy for M.I.

Answer 32

- Cardiomyocyte precursors to graft replacement tissue onto scars caused by MI - Inject bone marrow cells post MI in mice - The results showed the injected cells forming viable myocardium after 1-2 weeks - The injected cells form gap junctions in the regenerating tissue

Question 33

Describe experiment: Induce differentiation of human embryonic stem cells into cardiac myocytes in culture

Answer 33

- Induce MI in mice - Inject ES-derived cardiomyocytes into infarcted region - Injected cells constitutively express green fluorescent protein to allow cell lineage tracking

Question 34

Describe methods of myocardial targeted targeted gene delivery

Answer 34

1) Antegrade intracoronary injection 2) Retrograde injection through the coronary sinus with simultaneous blockage of the antegrade flow 3) Direct myocardial injections through the left ventricle using A) Catheter based and B) Surgical approach 4) Intrapericardial injection