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FHS - Cardiovascular Science > Theme 4: Cardiovascular Biology During Stress and Disease > Flashcards

Theme 4: Cardiovascular Biology During Stress and Disease Flashcards

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1
Q

What are the main categories of birth defects?

A

Structural:

  • Abnormalities in the development of body parts
  • Not amenable to treatment, but may be correctable by surgery

Functional:

  • Abnormalities in the systems that run the body
  • Vary in severity from inconsequential (hammer toe) to life-threatening (heart defects)
  • Includes metabolic disorders (phenylketonuria) and degenerative diseases (muscular dystrophy)
  • May be treatable in some cases
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2
Q

Give some statistics about congenital heart defects.

A
  • Heart defects are the most common class of structural birth defects
  • CHD affects ~1% of all live births, on average 13 per day in the UK
  • Despite modern surgery, still the biggest cause of infant mortality and morbidity
  • May affect up to 10% of still births and spontaneous terminations
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3
Q

How are congenital heart defects detected?

A
  • Phenotyping is a full investigation of the nature and severity of defects
  • This is done via 2D and 3D ultrasound from 20 weeks of pregnancy
  • After birth, phenotyping may continue using Magnetic Resonance Imaging (MRI) or X-Ray/CT scan
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4
Q

What are the most common types of congenital heart defect?

A

The most common is a bicuspid aortic valve, which is not usually a problem until later life when there is a higher risk of calcification.

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5
Q

What percentage of congenital heart defects have a simple genetic cause?

A

About 30%

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6
Q

What are the main types of inherited congenital heart defect?

A
  • Chromosomal abnormalities
  • Copy number variation (CNV)
  • Small nucleotide polymorphisms (SNP)
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7
Q

Describe what chromosomal abnormalities are and how they can be diagnosed.

A
  • These can involve duplication or absence of an entire chromosome
  • Alternatively they can be insertion, deletion, duplication, translocation or inversion of a large portion of a chromosome (many Mb)
  • The best known example is Down syndrome (trisomy 21) where 40% of patients have CHD
  • They can be diagnosed by isolating cells from the embryo or placenta and looking at the karyotype, fluorescence in situ hybridization (FISH) or PCR -> The latest techniques enable embryonic cells to be isolated from a sample of the mother’s blood from 9-12 weeks
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8
Q

Describe what copy number variations (CNVs) are and how they can be diagnosed.

A
  • These are duplications or deletions of small regions of chromosome, typically 1-18 kb
  • These can be detected by fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) or next generation sequencing
  • Typically cause deletion or duplication of one entire gene
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9
Q

How does searching for SNPs that cause congenital heart defects work?

A
  • The human genome has ~3 billion base pairs
  • The reference genome is derived from the DNA of 13 anonymous donors (USA)
  • Any particular individual has ~3 million SNPs that vary from the reference genome
  • Exome sequencing is often used:
    • Exome sequencing requires exon isolation (hybridisation) followed by next generation sequencing
    • This is rapid but only covers <90% of the exome, and misses non-coding variation
    • An exome dataset has ~60k variants, with ~100 “private” or unique to that individual/family
    • It then remains a task to identify which of these variants is causative of the disease (see table)
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10
Q

How do we distinguish between natural variation (benign) and disease-causing variation (pathogenic)?

A
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11
Q

What standards are used for classifying how pathogenic a variant is pathogenic?

A
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12
Q

What are the main limitations of exome sequencing and what is the alternative?

A
  1. It doesn’t cover ~10% of exons
  2. It misses variation in gene regulatory regions that might be causing defects

Whole genome sequencing is the alternative. It is now cheap enough to use clinically (around £1000 per sample), but it generates huge amounts of data, which become difficult to store.

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13
Q

What are some environmental risk factors for congenital heart defects?

A
  • Infectious diseases (e.g. Rubella and Zika viruses)
  • Environmental teratogens (e.g. hypoxia, hyperthermia)
  • Maternal nutritional deficiencies (e.g. Vitamin B3, iron)
  • Maternal non-communicable diseases (e.g. pre-gestational diabetes)
  • Maternal genetic conditions (e.g. phenylketonuria)
  • Teratogenic therapeutic drugs (e.g. Thalidomide, Roaccutane)
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14
Q

What animal models can be used for studying congenital heart defects?

A
  • Mouse embryos
  • However, they do not always respond in the same was as humans (e.g. they do not respond to thalidomide)
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15
Q

What type of epidemiological study is usually used to study congenital heart defects?

A

Retrospective observational case-control studies

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16
Q

What are the limits of epidemiological study of congenital heart defects?

A
  • All the key events of human embryonic heart development occur before week 8 of gestation. Therefore, the mother may not accurately remember her exposure to risk factors when the birth defect is diagnosed.
  • Likewise, clinical workup of maternal physiological parameters when the birth defect is first detected will not necessarily reflect her conditions during weeks 1-8.
  • Comprehensive maternal histories often not recorded
  • Can take decades to satisfactorily test if the association is real (e.g. folic acid)
  • Can take years before risk factor and/or corrective therapy becomes widely known (Valproate for epilepsy)
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17
Q

Explain the importance of gene-environment interaction (GxE) in congenital heart defects.

A
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18
Q

What is the difference between myocardial ischaemia and infarction?

A

Infarction is when the cells die.

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19
Q

Define ischaemia.

A
  • Ischaemia occurs when blood supply to a tissue is inadequate to meet the tissue’s demand.
  • There are 3 main components:
    • Hypoxia
    • Insufficiency of metabolic substrates
    • Accumulation of metabolic waste
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20
Q

Name some ways in which supply and demand of the heart may become unbalanced, leading to ischaemia.

A
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21
Q

Compare angina and myocardial infarction.

A
  • Angina (stable, unstable)
    • Chest pain caused by myocardial ischaemia
    • Usually due to atherosclerosis
  • Myocardial infarction
    • Complete occlusion of coronary flow
    • Plaque rupture with occlusive thrombus
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22
Q

How is a myocardial infarction diagnosed?

A
  • The gold-standard marker of myocardial injury is blood test (ELISA) for cardiac troponin (cTn) I or T.
  • This is because troponin is usually found inside cardiac myocytes.
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23
Q

What is an increased blood cTnT (cardiac troponin T) associated with? Give experimental evidence.

A
  • It is not only an indicator of myocardial infarction, but it also appears to be associated with all-cause mortality
  • (Chesnaye, 2022):
    • It has previous been shown that cardiac troponin T (cTnT) is associated with mortality in chronic kidney disease (CKD).
    • Studied 176 patients with stage 4-5 CKD aged ≥65 years and not on dialysis
    • Took cTnT measurements over a median follow-up of 2.4 years
    • Found that 3 factors were associated with all-cause mortality@
      • Longitudinally measured cTnT
      • Slope of the cTnT trajectory
      • Area under the cTnT trajectory
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24
Q

How does extreme exercise influence cardiac troponin T (cTnT)?

A
  • Extreme exercise has been shown to lead to increases in troponin T
  • 2/3rds of individuals after a marathon gave a positive blood troponin test
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25
Q

Describe how myocardial ischaemia can be treated.

A
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26
Q

Describe the consequences of myocardial ischaemia.

A
  1. Impairment of contraction
  2. Ischaemia-induced arrhythmias
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27
Q

Give a summary of the metabolic response to myocardial ischaemia.

A
  • There is a shift from oxidation of fatty acids (aerobic) to glycolysis (anaerobic)
  • This leads to increased lactate levels and impaired contractility and ion fluxes
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28
Q

State the two main metabolites that change during myocardial ischaemia.

A
  • Inorganic phosphate levels increase
  • Lactate levels increase -> This leads to reduced pH
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29
Q

Describe how phosphates change during myocardial ischaemia.

A
  • Phosphocreatine (PCr) is rapidly depleted and ATP levels decline upon ischaemia
  • (Cao, 2018):
    • Used NMR spectroscopy in a Langendorff-perfused heart
    • Studied PCr and ATP levels (as well as contractile function) over time before, during and after a period of ischaemia
    • Found that PCr, ATP and contractile function fell during the ischaemia
    • Both the PCr and ATP levels at least partially recovered after the ischaemia, but the contractile function only recovered briefly
  • As a result, inorganic phosphate begins to accumulate
  • (Wu, 2008):
    • Used in vivo canine NMR spectroscopy and modelling
    • Found that inorganic phosphate concentration ([Pi]) rises during ischaemia
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30
Q

Why is there accumulation of lactate in acute myocardial ischaemia?

A
  1. Increased anaerobic glycolysis and lactate production
  2. Decreased lactate clearance
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31
Q

Why is accumulation of lactate in myocardial ischaemia a problem?

A

(Mohabir, 1991):

  • Measured intracellular pH change over time during myocardial ischaemia
  • Found that intracellular pH fell with time
  • Hence, increased lactate leads to falls in intracellular pH
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32
Q

What are the acid extruders responsible for correcting intracellular acidosis? Why do these not correct reduced intracellular pH during ischaemia?

A
  • NHE1 and NBC
  • These do not correct pH during ischaemia because:
    • There is a local fall in extracellular pH due to buffering of intracellular H+ -> This reduces the extruder function
    • NHE is inactivated as ischaemia progresses
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33
Q

Why does contractility decrease during myocardial ischaemia?

A
  • Reduced intracellular pH -> H+ ions bind to cTnI and reduce cTnC affinity for Ca2+
  • Increased intracellular Pi -> Slows the removal of Pi from myosin head after power stroke
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34
Q

How do calcium transients change during myocardial ischaemia? Give experimental evidence.

A
  • (Mohabir, 1991):
    • Studied global ischaemia in isolated rabbit heart perfused with Indo-1 (Ca2+ indicator) with electrical pacing.
    • Found that calcium transients INCREASED during ischaemia.
    • The decline in contraction despite this inrease can be explained by H+ outcompeting Ca2+ for calcium binding sites on cTnC.
  • The mechanism for this is:
    • Na+/K+-ATPase activity falls as ATP is depleted (Fuller, 2002)
    • This leads to sodium retention in the cell (Williams, 2007)
    • This reduces the activity of the NCX, so intracellular calcium increases
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35
Q

Describe how ion channel activity is affected by myocardial ischaemia.

A
  • Na+/K+-ATPase activity falls as ATP is depleted (Fuller, 2002)
    • This leads to sodium retention in the cell (Williams, 2007)
      • This reduces the activity of the NCX, so intracellular calcium increases
      • However, it has no effect because the increased intracellular H+ during ischaemia outcompetes the calcium for binding to TnI
    • It also leads to increased extracellular potassium (Gleber, 1983)
      • This depolarises the resting membrane potential
      • So the cell becomes more prone to EADs and arrythmias
  • KATP channels open when ATP is depleted
    • This raises [K]o, leading to hyperpolarisation (by a different mechanism), early repolarisation and shortening of the action potential
    • (Noma, 1983):
      • Plotted action potential duration against time during a period of ischaemia
      • The action potential duration gradually decreased
      • Addition of ATP briefly restored action potential duration
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36
Q

What does continued opening of KATP channels in cardiac myocytes lead to?

A
  • The gradual shortening of the action potential eventually leads to failure of the action potential
  • This is reversible and serves a protective role, which is reduced workload
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37
Q

What are two main determinants of infarct size (in myocardial ischaemia)?

A
  • Residual blood flow
  • Ischaemia duration
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38
Q

How does ischaemia/reperfusion injury happen? Give a summary.

A
  • Reperfusion leads to:
    • Calcium overload
      • During reperfusion, the extracellular pH (that has become acidic during ischaemia) returns to normal
      • This leads to activation of H+-efflux transporters (NHE and NBC)
      • This leads to Na+ overload in the cell, which activates NCX, leading to increased intracellular calcium
      • The SERCA pump is activated to store this calcium in the SR, leading to calcium overload
    • ROS generation
      • Succinate accumulates during ischaemia in the heart
      • Upon reperfusion, it is re-oxidised by succinate dehydrogenase (SDH) to generate ROS
  • Calcium overload and ROS lead to opening of mPTP channels -> This causes caspase activation, leading to cell death
  • Calcium overload also leads to calcium waves -> This causes hypercontraction, whcih drives sarcolemmal rupture and cell death
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39
Q

How does calcium overload happen in ischaemia/reperfusion injury? Give experimental evidence.

A
  • During reperfusion, the extracellular pH (that has become acidic during ischaemia) returns to normal
  • This leads to activation of H+-efflux transporters (NHE and NBC)
    • (Park, 1999) -> Found that MIA (NHE inhibitor) partly inhibits the return of intracellular pH to normal after ischaemia
  • This leads to Na+ overload in the cell, which activates NCX, leading to increased intracellular calcium
  • The SERCA pump is activated to store this calcium in the SR, leading to calcium overload
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40
Q

Give experimental evidence for the idea that NHE activation during ischaemia/reperfusion leads to spontaneous calcium waves.

A

(Ford, 2017):

  • Measured intracellular pH, calcium wave frequency and intracellular sodium concentration during an acid insult (acetate in this case)
  • Found that intracellular pH dropped, calcium wave frequency increased and intracellular sodium concentration increased
  • When dimethyl amiloride (DMA) was used to inhibit NHE, the pH still fell, but calcium wave frequency and intracellular sodium concentration fell
  • This is evidence for the importance of NHE in calcium overload and spontaneous calcium waves during I/R injury
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41
Q

What are the problems with SR calcium overload during ischaemia/reperfusion injury?

A
  • Leads to calcium waves -> These can trigger hypercontraction, which leads to sarcolemmal rupture and then cell death
  • Leads to DADs -> Stimulation of NCX increases net inward current, triggering premature APs and delayed after-depolarisations (DADs)
  • Leads to opening of mPTP channels
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42
Q

Give experimental evidence for how calcium overload triggers DADs.

A

(Boyman, 2011):

  • Showed that intracellular acidification led to increased NCX currents

(Wit, CHECK YEAR):

  • Showed that DADs are triggered during reperfusion
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43
Q

Describe the involvement of ROS in cardiac pathology.

A
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44
Q

How do ROS lead to calcium overload in ischaemia/reperfusion?

A

They lead to activation of NHE1.

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45
Q

How do calcium overload and ROS evoke ischaemia/reperfusion injury?

A
  • Primarily, they lead to opening of mPTP channels on mitochondria
  • This collapses the mitochondrial membrane potential, which uncouples oxidative phosphorylation
  • Hence, it results in ATP depletion and cell death
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46
Q

How are ROS generated in ischaemia/reperfusion injury? Give experimental evidence.

A

(Chouchani, 2014):

  • Studied succinate abundance with time -> It increased during ischaemia and fell upon reperfusion
  • Also used dimethyl succinate as an analogue of succinate in adult primary cardiomyocytes:
    • DHE (marker of ROS) oxidation increased with time relative to reperfusion
    • When rotenone (an ETC inhibitor) was added, this increase was blocked -> This shows the importance of the ETC in generation of ROS in I/R injury
  • Dimethyl malonate (a succinate dehydrogenase inhibitor) reduces the infarct size

Hence, overall it appears that succinate accumulates during ischaemia, after which succinate dehydrogenase produces ROS during reperfusion.

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47
Q

How does mPTP channel opening in ischaemia/reperfusion lead to cell death?

A
  • Opening of mPTP releases cytochrome C
  • This activates caspases
  • (Yang, 2003):
    • Used MX1013 (a novel caspase inhibitor) in an acute myocardial infarction and reperfusion model in rats.
    • Ischemia was effected by occlusion of the left anterior descending artery of rat hearts for 1 h, followed by reperfusion for 23 h.
    • At the end of the reperfusion, the hearts were sectioned, stained, and analyzed for infarct volume.
    • The infarct size was much smaller in the treated animals compared to the controls.
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48
Q

Describe some cardioprotective measures to reduce ischaemia/reperfusion injury.

A

There are many cardioprotection targets under active research, including:

  • Ischaemic ‘pre-conditioning’ -> Subjecting the heart to brief bouts of ischaemia to pre-condition it for myocardial ischaemia
  • mPTP inhibitors
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49
Q

Draw and describe the renal function curve.

A
  • The renal sodium excretion increases as blood pressure increases
  • There is an equilibrium point at which the sodium excretion is exactly equal to sodium intake -> This is the point at which the blood pressure settles
  • The mechanism of correction is called renal pressure-natriuresis.

(Guyton, 1990)

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50
Q

Give an experiment that demonstrates renal pressure-natriuresis.

A

(Dobbs, 1970):

  • Increased blood volume was induced in dogs in which nervous pressure control mechanisms had been blocked.
  • The blood pressure returned to normal over a period of about 2 hours.
  • This showed the speed at which RPN works.
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51
Q

Describe the arrangement used to determine the acute renal function curve.

A
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52
Q

Describe the two ways in which long-term changes in blood pressure (e.g. in hypertension) can occur.

A
  1. Change in the kidney’s capacity to excrete Na+ -> Represented by a shift (red trace) or change in slope of the renal function curve
  2. Change in the level of dietary Na+ intake
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53
Q

When there is a change in the kidney’s capacity to excrete Na+, why does the body not correct the blood pressure?

A

High blood pressure is a compensatory mechanism that allows maintenance of sodium balance when RPN is altered, but this exposes organs to the damaging effects of elevated MAP.

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54
Q

Describe long-term adaptation to high salt intake.

A
  • In case of prolonged high Na+ intake, the renal function curve becomes steeper to allow increased Na+ excretion and restoration of normal MAP.
  • The RAAS is involved in this process.
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55
Q

Are changes in total peripheral resistance involved in hypertension? Give experimental evidence.

A

(Guyton, 1980):

  • Studied a number of conditions with altered total peripheral resistance (e.g. hypothyroidism)
  • These conditions do not have abnormal blood pressure, so changes in TPR are not sufficient to drive hypertension

(FIND REFERENCE):

  • Studied volume loading in dogs that had had 70% of their kidneys removed (to induce salt-sensitivity)
  • Volume-loading induced hypertension, but over the first couple of days there was a fall in TPR -> This shows that TPR is not essential for hypertension
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56
Q

Describe how linear approximation of the renal function curve is done.

A
  • MAP and rate of urinary sodium excretion (UNaV) are measured under two different amounts of sodium intake.
  • Linear fit between the resulting two points allows determination of A and B.
  • Physiological significance of A and B:
    • A, x-intercept: level of MAP below which urinary sodium excretion stops.
    • B, slope (reciprocal of sodium sensitivity of blood pressure).
      • Steep –> small changes in MAP result in large changes in sodium excretion. Thus, even if sodium intake is increased the change in MAP is minimal.
      • Shallow –> MAP is more “sodium-sensitive”.
      • B is also a time constant –> shallow slope means longer time needed to achieve sodium balance.
  • At the equilibrium level of MAP (MAPeq) sodium intake (QNa) and output (UNaV) are equal:
    • QNa = UNaV
    • QNa = B(MAPeq-A)
    • Thus, MAPeq = A + (1/B)QNa = A + CQNa where C=1/B is a constant that reflects the sensitivity of MAP to sodium.
  • Thus, steady state blood pressure can be viewed as the sum of two factors:
    • A (x-intercept of the linear renal function curve) -> Non sodium-sensitive component.
    • CQNa (product of the reciprocal of the slope and the sodium intake) -> Sodium-sensitive component.
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57
Q

Compare sodium-sensitive and non-sodium-sensitive changes in arterial blood pressure in hypertension.

A

Reno-vascular HT (non sodium-sensitive) -> Increased resistance in afferent arterioles:

  • x-intercept A is rightward shifted.
  • Increased renal vascular resistance (caused by stenotic vascular lesion), which initially reduces GFR.
  • MAP raises high enough to maintain adequate filtration.

Primary aldosteronism (sodium sensitive) -> Increased tubular reabsorption:

  • Slope B is shallower – sodium sensitivity is elevated.
  • Increased Na+ reabsorption by renal tubules.
  • This in turn requires elevated glomerular capillary hydrostatic pressure and elevated GFR to overcome the enhanced Na+ reabsorption.
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58
Q

Draw the water tank model of body fluid volume and MAP regulation.

A
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59
Q

Draw the water tank model for salt-sensitive and non-salt-sensitive changes in MAP.

A
60
Q

Name the main types of evidence for the importance of the kidneys in hypertension.

A
  1. Conceptual framework – Guyton’s model.
  2. Renal transplantation studies – hypertension “follows the kidneys”.
  3. In experimental models of hormone-induced HT (e.g. due to infusion of angiotensin II, aldosterone, vasopressin or noradrenaline), shielding the kidneys from the increased MAP exacerbates HT and leads to fluid retention.
  4. Genetic forms of HT due to mutations in genes coding for tubular solute transport proteins.
  5. Vascular conditions (including congenital) that affect renal blood flow and produce HT.
61
Q

Give some experimental evidence involving kidney transplantation for the importance of the kidneys in hypertension.

A
  • (Guidi, 1996):
    • Looked at the long-term historical prospective follow-up of recipients grafted with kidneys coming from donors with or without hypertension in their families
    • Found that hypertension may be transplanted with the kidney in humans
  • (Bianchi, 1974):
    • From a single strain of Wistar rats developed two lines of normotensive rats (NR) and two lines of hypertensive rats (SHR)
    • Carried out cross-transplantation of kidneys from hypertensive to normotensive rats and vice versa. Also transplanted hypertensive to hypertensive and normotensive to normotensive.
    • Normotensive recipients of kidneys from hypertensive rats developed hypertension.
    • Transplantation of normotensive kidneys reduced the blood pressure of hypertensive recipients.
  • (Crowley, 2006):
    • Looked at 4 types of mice: wild-type, kidney-only angiotensin receptor KO, systemic-only angiotensin receptor KO and total angiotensin receptor KO
    • Angiotensin only elevated the blood pressure in the wild-type and systemic-only KO
    • The mice that developed hypertension had lower rates of sodium excretion and retained more water
62
Q

What is servo-control of renal perfusion?

A

When a cuff is placed around the renal artery, allowing the renal arterial pressure to be controlled.

63
Q

Give some experimental evidence involving servo-control of the kidney for the role of the kidney in hypertension.

A

(Mizelle, 1993):

  • Studied dogs with servo-controlled renal perfusion pressure of one kidney.
  • Urinary bladder was split for determination of water and sodium excretion from each kidney individually.
  • Found that sodium and water excretion were reduced in the kidney exposed to low pressure and significantly increased in the collateral kidney.

(Hall, 1988):

  • Studied control dogs and servo-controlled dogs.
  • When exposed to noradrenaline, the control dogs’ blood pressure rose and remained high. In response, there was a transient natriuresis/diuresis followed by normal urinary sodium excretion at elevated MAP.
  • In the servo-controlled dogs (where the cuff leads to lower renal arterial pressure), the blood pressure continued to rise during the infusion. The renal pressure did not rise. There was no transient natriuresis/diuresis, showing that the kidney needs to detect the hypertension in order to trigger these processes.
64
Q

Give an example of a genetic condition that involves hypertension.

A

Liddle’s syndrome (Pseudo-hyperaldosteronism) (Liddle, 1963)

  • This involves a gain-of-function mutation in the ENaC channel, leading to sodium retention
  • Index patient G.S. was treated with low Na+ diet & triamterene (ENaC inhibitor) -> This led to normalization of MAP.
  • G.S. eventually developed end stage renal disease (from presumed long-standing hypertension) and underwent kidney transplant -> This resulted in good kidney function and normotension
65
Q

Describe congenital coarctation of the aorta.

A
  • Congenital coarctation of the aorta is when there is a narrowing in the aorta
  • This reduces the perfusion pressure of the kidneys, so there is increased RAA axis activity
  • This leads to very high blood pressure in the area above the point of narrowing
66
Q

What are some potential misinterpretations of Guyton’s model of long-term control of MAP?

A

Points that Guyton’s paradigm does not imply:

  1. Renal dysfunction is the direct cause of all forms of HT.
  2. Changes in MAP are inevitably associated to changes in blood volume.
  3. The Guyton’s concept does not take into account the role of CNS in the long-term control of MAP.
67
Q

Explain why this myth is wrong: Renal dysfunction is the direct cause of all forms of HT.

A
  • Long-term alterations in MAP require a shift in the renal function curve.
  • However, this does not need to be the initial step in the series of events leading to HT.
  • Examples of HT with extra-renal origin: mineralocorticoid-producing tumor, coarctation of the aorta, sympathetic over-reactivity.

In all cases, there is a modification of the capacity of the kidneys to excrete salt and water for a given level of BP.

68
Q

Explain why this myth is wrong: Changes in MAP are inevitably associated to changes in blood volume.

A

MAP does not depend on blood volume as such, but on the “volume in excess” in the arterial tree in relation to the resting size of this vasculature.

69
Q

Explain why this myth is wrong: The Guyton’s concept does not take into account the role of CNS in the long-term control of MAP.

A

(Symplicity HTN-2 Investigators, 2010):

  • Carried out a multicentre, prospective, randomized trial of patients with treatment-resistant hypertension.
  • Utilised radiofrequency-mediated catheter ablation of renal nerves -> This led to a large and sustained decrease in MAP after six months.
  • A possible underlying mechanism: leftward shift of RFC and enhanced natriuresis.
  • Consistent with this proposition, bilateral renal nerve ablation provokes a pronounced decrease of (i) NA spillover from the sympathetic nerve terminals in kidneys and (ii) renin activity (factors that shift the RFC to the right).
70
Q

Define heart failure.

A

A complex clinical syndrome that can result from any structural or functional cardiac disorder that impairs the ability of the ventricle to fill with or eject blood.

71
Q

What are some different methods of assessment of cardiac metabolism?

A
  • Measurement of plasma concentrations of different metabolites (e.g. glucose, lactate, NEFA, BHB)
  • Enzyme activity assays and measurement of protein levels (Western blotting)
  • Measurement of respiratory quotient (RQ)
  • PET scans
  • Langendorff perfused heart
  • Radiolabelled uptake experiments (using either 14C or 3H)
  • Cardiac MRI + MR Spectroscopy
72
Q

What does an RQ of 1 indicate?

A

Carbohydrate metabolism

73
Q

How are PET scans used to assess cardiac metabolism?

A
  • Uses radiolabeled compounds (e.g. 18FDG) to image
    metabolism
  • FDG provides a measure of glucose uptake
  • Other compounds can monitor other pathways

But:

  • It provides a radiation dose
  • Can’t distinguish the tracer from downstream products
74
Q

What is a type of spetrosocopy that is useful in studying cardiac metabolism?

A

Phosphorous Spectroscopy:

  • Involves high energy phosphates: Phosphocreatine (PCr) and Adenosine-Triphosphate (ATP).
  • Ratio of PCr/ATP peaks gives an indication of the energetic state of the heart.
  • It can be used in a Langendorff perfused heart to give live indication of the energy state of the heart.

Carbon Spectroscopy (13C):

  • 13C has low natural abundance, so enrichment is usually done (e.g. via infusion)
  • Can provide information on metabolism via studies of
    glycolysis & TCA cycle
  • There are more advanced techniques that allow us to determine where in a molecule it is incorporated.
75
Q

What are the advnatages and disadvantages of 13C MRI?

A

Advantages:

  • Wide range of metabolites can be detected
  • Can only see the carbon we put in and so there is lack of background signal

Limitations:

  • Very low concentration of carbon in the body
  • Inherently low signal-to-noise ratio
  • Very few studies of non-steady state rates of metabolism
  • Even fewer in vivo studies possible
76
Q

Describe the impact of heart failure. Give stats.

A
77
Q

Describe how heart failure can be classified.

A
  • New York Heart Association (NYHA) classification is based on symptoms (shortness of breath / angina) during physical activity.
  • Heart failure can also be considered as:
    • Systolic Heart Failure (Heart Failure with Reduced Ejection Fraction – HFREF)
    • Diastolic Heart Failure (Heart Failure with Preserved Ejection Fraction – HFPEF)
78
Q

Describe some causes of heart failure.

A
  • Pressure Overload
    • Aortic Stenosis
    • Hypertension
  • Ischemic
    • Coronary Heart Disease
  • Atrial Fibrillation
  • Dilated Cardiomyopathy
    • Idiopathic
    • Chemotherapeutics
  • Various Other Cardiomyopathies
    • HCM
    • Diabetic
    • Genetic
79
Q

Describe some animal models of heart failure.

A
  • Hypertension/Hypertrophy
    • TAC – Transverse Aortic Constriction
    • AAB - Abdominal Aortic Banding
  • Ischemic Heart Failure
    • Ischemia/Reperfusion – LAD Ligation
    • Cryoinjury
    • Isoproterenol
    • Intracoronary Micro-embolism
  • Dilated Cardiomyopathy
    • Pacing Induced DCM -> Making the heart beat faster
    • Genetic Models
80
Q

What is abdominal aortic banding and what is it used to study?

A
81
Q

How can myocardial infarction be studied in a model?

A
82
Q

What is the heart in heart failure considered to be like “an engine out of fuel”? Give experimental evidence.

A

(Neubauer, 1995):

  • Plotted a graph of PCr/ATP ratio against ejection fraction
  • Found that lower ejection fractions were associated with lower PCr/ATP ratios, since PCr is running low
  • However, this is a bit of a chicken and the egg situation
  • The ejection fraction was also a predictor of mortality:
    • PCr/ATP ratio less than 1.6 resulted in higher mortality than a ratio above 1.6
83
Q

Are energy-costly or energy-sparing treatments for heart failure better? Give some examples.

A

Energy-sparing

84
Q

Draw a diagram to show the way in which different metabolites change during heart failure.

A

Note how everything is changed at the end, but in moderate heart failure some things are different.

85
Q

What happens to fatty acid oxidation during heart failure? Give experimental evidence.

A

It decreases in various types of heart failure.

In human models:

  • (Davila-Roman, 2012) -> Idiopathic dilated cardiomyopathy
  • (de las Fuentas, 2013) -> Hypertensive heart disease
  • (Stride, 2013) -> Left ventricular systolic dysfunction

In animal models:

  • (Heather, 2006) -> Myocardial infarction
  • (Allard, 1994) -> Pressure overload
  • (el Alaoui-Talibi, 1992) -> Volume-overload hypertrophy
  • (Heather, 2009) -> Isoproterenol-induced hypertrophy
86
Q

Why is fatty acid oxidation decreased in heart failure?

A

Fatty acid oxidation is affected at many points:

  • (Sack, 1996) -> β-oxidation enzymes
  • (Heather, 2006) -> Sarcolemmal fatty acid transporters
  • (Heather, 2011) -> Krebs cycle and OXPHOS enzymes
87
Q

How does fatty acid oxidation change with heart failure progression? Give experimental evidence.

A
  • A fall in fatty acid oxidation is seen as the disease progresses.
  • (Heather, 2011):
    • Looked at patients with aortic stenosis
    • Plotted FAT/CD36 and Complex I against heart size (relative to body size)
  • (Heather, 2006):
    • Look at a rat model of myocardial infarction
    • Found that palmitate oxidation decreased as ejection fraction decreased
88
Q

How does glucose oxidation change with heart failure progression? Give experimental evidence.

A

It is gradually upregulated (and then it falls during severe disease):

  • Human models
    • (Davila-Roman, 2002) + (Neglia, 2007) -> Idiopathic dilated cardiomyopathy
    • (Heather, 2011) -> Aortic stenosis
  • Animal models
    • (Allard, 1994) + (Lydell, 2002) + (Nascimben, 2004) + (Wambolt, 2000)

Overall, the results show that glycolysis is increased, but the products do not enter the mitochondria. They are instead shuttle away as lactate.

89
Q

Give some experimental evidence for the uncoupling of glycolysis from glucose oxidation in heart failure.

A

(Giles, 2015):

  • Abdominal aortic banding in rats was used to induce hypertrophy
  • Based on pyruvate dehydrogenase flux and lactate production, the hypertrophied hearts showed uncoupling of glycolysis from glucose oxidation (compared to controls)

There is also some evidence that the uncoupling may be downstream of PDH:

(Schroeder, 2014):

  • Used 13C magnetic resonance to track pyruvate metabolism in a pacing-induced model of hypertrophy
  • Found that glutamate (a product of the TCA cycle) fell in early heart failure
  • PDH flux remained constant much longer

(Dodd, 2014):

  • Observed similar results
90
Q

Give some experimental evidence for how energy transfer is changed during heart failure.

A

(Weiss, 2005):

  • Found that creatine kinase activity was reduced in heart failure
  • This suggests that it’s not just mitochondrial activity that may be affected, but also the transfer of energy away from the mitochondria
91
Q

Describe some therapeutic targets for treating heart failure, given the metabolic changes that occur in heart failure.

A

(Liao, 2002):

  • Increasing glucose uptake in the heart could help with heart failure
  • Transgenic mice that overexpress GLUT1 have increased ability to uptake glucose and show decreased mortality compared to wild-type animals in response to pressure overload (induced by ascending aortic constriction
  • The PCr/ATP ratio fell more in the wild-type mice than in the transgenic mice

(Nikolaidis, 2004):

  • Glucagon-like peptide 1 works to increase insulin release and suppress glucagon in a glucose concentration-dependent way
  • Infusion of GLP-1 for 48 hrs in a rapid pacing model of HF led to improvements in stroke volume, ejection fraction and cardiac output

(Lee, 2005):

  • Blocking mitochondrial FFA uptake with Perhexiline improved VO2max and ejection fraction in CHF patients
92
Q

When in life does atherosclerosis start developing? Give experimental evidence.

A

(Napoli, 1997):

  • Found a correlation between maternal lipid levels and fatty streaks are found in foetus (mean age 6 months)
  • This suggests that atherosclerosis starts developing before birth
93
Q

Where does atherosclerosis tend to occur? Why? Give experimental evidence.

A
  • It tends to be at bifurcation points
  • This is where the endothelial cells experience more turbulkent flow and greater sheer forces, which triggers atherosclerosis
  • This is because endothelial cells convert mechanical stimuli into biochemical signals via mechanotransduction
  • (Suo, 2006):
    • Looked at flow in the aortic arch of mice
    • Found that VCAM-1 expression was increased in areas of turbulent flow
  • (Jongstra-Bilen, 2006):
    • Expression of a reporter gene under the control of eNOS promoter is reduced in plaque prone areas
  • Overall this suggests that endothelial cells determine where atherosclerosis can occur due to changes in NO production
94
Q

How is NO involved in atherosclerosis? Give experimental evidence.

A

NO has a well-defined protective role in vascular function and disease:

  • Potent vasodilator
  • Inhibits leukocyte adhesion
    • (Lefer, 1999):
      • Used intravascular microscopy to study leukocyte adhesion
      • Found that adhesion was increased in eNOS knockout mice
  • Inhibits vascular smooth muscle cell proliferation and migration
    • (Ignarro, 2001):
      • Used 4 different NO donors
      • Each one led to a decrease in cell proliferation
  • Inhibits platelet aggregation
    • (Kirby, 2013):
      • Used a thrombin assay to study platelet aggregation
      • Increasing the concentration of NO inhibited the aggregation
      • Blockade of P2Y receptors increased this blockade
  • Inhibits coagulation

As a result, eNOS knockout leads to atherosclerosis formation:

  • (Ponnuswamy 2012):
    • apoE and eNOS knockout mice show higher levels of leukocyte rolling, leukocyte adhesion and increased lesion area compared to just apoE knockout mice
    • This shows the importance of NO in suppressing atherosclerosis
95
Q

Give experimental evidence for the importance of the endothelial cell layer in predicting atherosclerosis in the long-term.

A

(Schächinger, 2000):

  • Divided individuals into those with healthy and damaged endothelium based on two criteria:
    • Reactivity to ACh (a vasoconstrictor response indicates damaged endothelium)
    • Flow-dependent vasodilation (reduced vasodilation indicates damaged endothelium)
  • 10 year follow-up indicates that damaged endothelium was a predictor of cardiovascular events
96
Q

Is endothelial damage sufficient for formation of atherosclerosis?

A
  • No, mice with endothelial damage alone do not show signs of atherosclerotic plaques, since they carry all their TAG in HDL
  • It’s only when we cross these mice onto a hyperlipademic background (e.g. ApoE-/- or LDLR-/-) that atherosclerosis begins
97
Q

Give experimental evidence for the importance of LDL in atherosclerosis.

A

(Ference, 2017):

  • Found a linear association between achieved low-density lipoprotein cholesterol (LDL-C) level and absolute coronary heart disease (CHD) event rate

(Cohen, 2006):

  • PCSK9 controls the expression of the LDL receptor on the cell surface
  • Increased PCSK9 leads to decreases in LDLR expression, increased plasma levels and increased plaque
  • The study compared individuals with the PCSK946L allele (which leads to a 15% reduction in LDL) and compared them to controls
  • Both populations were disease free at the start of the study and followed for 15 years
  • The PCSK946L allele led to a 50% decreased risk of coronary heart disease risk
98
Q

Describe how endothelial cell damage and LDL interact to lead to atherosclerosis.

A
  • In areas of endothelial cell damage, LDL can enter via the paracellular route or active transcytosis via multiple mechanisms such as caveolae, scavenger receptor B1 and the LDL receptor
  • This leads to pro-inflammatory accumulation of LDL within the subendothelial space
  • Any factor which increases endothelial cell damage such as smoking or diabetes will increase endothelial cell damage and lead to more LDL deposition
99
Q

Should we treat atherosclerosis in at risk young adults?

A

(Luirink, 2019):

  • Compared children with familial hypertension with their parents
  • The children were given life-long statin treatment, resulting in 32% decrease in LDL
  • 20 year follow up showed that cumulative incidence of cardiovascular events and of death from cardiovascular causes at 39 years of age was lower among the patients with FH treated with statin from childhood vs their affected parents who were not treated in childhood
100
Q

Can atherosclerotic plaques regress? Give experimental evidence.

A

(Reis, 2001):

  • Transplanted an atherosclerotic arch from an ApoE-/- mouse into a wild-type or another ApoE-/- recipient
  • Transplantation into the wild-type mouse led to a significant reduction in plaque area

Clinically the ability to detect regression was limited due to imaging resolution of angiography, but intravascular ultrasonography enabled measurement of wall thickness, composition and lumen diameter.

(Nicholls, 2016):

  • PCSK9 antibodies which lower circulating LDL-C were shown to induce regression (1%) of atherosclerosis
  • However, clinically changes in plaque volume have been very small, further research is needed to improve regression

(Ference, 2017):

  • Found a linear association between plasma LDL cholesterol levels and percent atheroma volume change
101
Q

Why are lipids in the sub-endothelial cell layer so dangerous?

A
  • LDL binds to proteoglycans and are modified by ROS to form oxidised LDL
  • This then causes further inflammation and recruitment of monocytes to the endothelial cell surface
  • Monocytes convert to macrophages and engulf the LDL
  • These foam cells are mostly trapped in the vessel wall
102
Q

Are antioxidants rational therapeutic strategies for atherosclerosis?

A

Since LDL oxidation to OxLDL is a pro-inflammatory stimulus that contributes to atherosclerosis initiation and progression, antioxidants in theory make sense as a therapeautic strategy.

(Malekmohammad, 2019):

  • This is a review article that found that antioxidants show limited success in clinical trials

(Stephens, 1996):

  • Ran a RCT of vitamin E in patients with coronary disease
  • Vitamin E substantially reduces the rate of non-fatal MI, with beneficial effects apparent after 1 year of treatment.
  • The effect of vitamin E treatment on cardiovascular deaths requires further study.

(Gale, 1995):

  • Carried out a 20 year follow up study of a cohort of randomly selected elderly people
  • Mortality from stroke was highest in those with the lowest vitamin C status. Those in the highest third of the distribution of vitamin C intake had a relative risk of 0.5 compared with those in the lowest third, after adjustment for age, sex, and established cardiovascular risk factors.
  • The relation between vitamin C intake and stroke was independent of social class and other dietary variables.
  • No association was found between vitamin C status and risk of death from coronary heart disease.
103
Q

Can inhibiting OxLDL uptake by macrophages reduces atherosclerosis? Give experimental evidence.

A

(Que, 2018):

  • Authors created a mouse which overexpresses a fragments of a natural antibody which recognises oxidised but not non-oxidised phospholipids
  • This transgene was under the control of a liver promotor which drives expression in the liver
  • The antibody inhibited the update of OxLDL by macrophages and reduced atherosclerosis
  • Hence, inhibition of OxLDL uptake could be used to reduce atherosclerosis
104
Q

What role does inflammation play in atherosclerosis?

A

Uptake of OxLDL by macrophages leads to a number of downstream effects:

  • Release of pro-inflammatory cytokines
  • Release of chemokines
  • Release of proteases
  • Release of ROS
105
Q

Can we target chemokines to reduced atherosclerosis?

A
  • Monocytes rely on CCR2 to migrate to inflammatory sites
  • CCR2 expression is 3 fold higher on monocytes from patients with FH compared with healthy volunteers and was positively associated with intracellular lipid accumulation
  • (Boring, 1998):
    • Produced CCR2 knockout mice (along with ApoE knockout to induce hypercholesterolaemia)
    • These mice showed reduced lesion size compared to control mice, which persisted to 13 weeks
106
Q

Can we target inflammation to treat atherosclerosis? Give experimental evidence.

A
  • Troponin is the current gold standard biomarker of MI -> Levels are proportion to myocardial damage
  • (Kaura, 2022):
    • C-reactive protein (CRP) is a protein produced by the liver and released into the bloodstream in response to inflammation
    • Showed that CRP could be a used additional marker for myocardial infarction
    • This revealed an at-risk population that would usually not be identified based on just troponin
  • (Bouabdallaoui, 2020):
    • Colchicine decreases CRP levels and inflammasome activation
    • Found that early initiation of low-dose colchicine after MI led to a 48% reduction in the risk of ischaemic CV events (compared to placebo)
107
Q

Can we target macrophage related cytokines to treat atherosclerosis? Give experimental evidence.

A
  • CANTOS trail:
    • This was a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction
    • IL-1beta is produced in response to an activated NLRP3 inflammasome
    • Canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering.
    • HOWEVER, since it led to an increase in fatal infections, the all-cause mortality was not signficantly different between the control and placebo groups
  • (Ridker, 2018):
    • Performed an analysis of the CANTOS trial data
    • Found that the beneficial cardiovascular outcomes of canakinumab were mediated through interleukin-6 (IL-6)
    • CANTOS participants receiving canakinumab who achieved on-treatment IL-6 levels below the study median value of 1.65 ng/L experienced a 32% reduction in major adverse cardiovascular events

There are a number of limitations to this approach, however:

  • Monoclonal antibodies are expensive
  • Short lived effect
  • Increased risk of infection

A vaccine would potentially be better.

108
Q

What is the evidence for an autoimmune response in atherosclerosis?

A
  • Sparked by the presence of T cells and B cells in plaques and confirmed in human and mouse by single cell RNA seq, flow cytometry and immunohistochemistry
  • CD4+ T cells in murine lesions recognize apoB100
  • (Sjorgen, 2018):
    • Immunoglobulin G (IgG) antibodies to apoB100 are negatively correlated with cardiovascular disease in humans
    • This suggest that LDL is a relevant self-antigen that drives an autoimmune response against self-proteins in the plaque
109
Q

Give some experimental evidence for the involvement of T cells in atherosclerosis.

A

(Emersone, 1996):

  • Mice were fed a high fat diet for 20 weeks
  • Intra peritoneal injection of antibodies against CD4 or CD8 were given weekly
  • There was a significant reduction in plaque in mice given CD4 but not CD8 antibodies -> This shows the importance of T cells in atherosclerosis
  • Further studies have shown a small effect of CD8 deletion on atherosclerosis -> It appears that B cells can be either protective or deleterious, depending on the stage of atherosclerosis

TH cells

(Fernandez, 2019):

  • TH1 cells are enriched in plaques of patients with recent history of stroke compared with those with asymptomatic atherosclerosis

TH1 cells are considered proinflammatory through their affiliation with M1 macrophages and secretion of IFNγ. IFNγ protein and mRNA are detected in plaques from mice and humans.

(Whitman, 2000):

  • Administered either recombinant IFN-γ or PBS daily for 30 days to atherosclerosis-susceptible apoE knockout mice
  • The lesion size, T cell mean number and MHC class II cell number were all increased in the IFN-γ group
  • This suggests that TH1 cells are likely to play a role in atherosclerosis development, since they produce IFN-γ, but the activation of other cells is not necessarily specific to the action of the TH1

Treg cells

Treg cells are negative regulators of the immune system and have been shown to play an atheroprotective role. Treg cell numbers and IL-10, a cytokine secreted by Tregs are lower in patients with myocardial infarction than in patients with stable angina or individuals without coronary artery disease.

(Klingenberg, 2013):

  • Produced DEREG (depletion of regulatory T cells) mice.
  • These mice express a diphtheria toxin (DT) receptor under the control of the Foxp3 gene locus, allowing selective and efficient depletion of Foxp3+ T reg cells by DT injection (this allows the mice to develop as normal)
  • The lesion size was smaller in the DEREG mice than PBS (phosphate-buffered saline) controls

(Sharma, 2020):

  • Treg cells are increased in plaques which are undergoing atherosclerosis regression
  • Depletion of Treg by treating with antibodies to CD25 cause a reduction in atherosclerosis regression
  • This indicates that Treg cells are required for plaque regression

The role of other T cell subtypes in atherosclerosis is controversial with inconsistent findings across studies. Driven in part by the models used but also the lack of technical ability to accurately differentiate sub-types.

110
Q

Name some advances in experimental techniques that enable a more detailed analysis of cell subtypes in atherosclerosis.

A
  • Single cell sequencing
    • Limitations include: Need to digest tissue, Limited detection for low abundance transcripts, Expensive so small sample numbers
  • Abseq -> Enables the detection and quantification of proteins in single cells at throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest. Antibodies are labeled with sequence tags. Increases detection of low abundance proteins.
    • Limitations include: Need good antibodies, Need to digest tissue
111
Q

Are EMT important in atherosclerosis? Give experimental evidence.

A
  • In response chronic inflammation and pathological flow endothelial cells undergo endothelial-to-mesenchymal transition.
  • Endothelial cells gain mesenchymal cell characteristics such as extracellular matrix production and contractile function.
  • (Chen, 2019):
    • Knockout of TGF-beta was used to inhibit EMT
    • This led to a significant reduction in atherosclerosis
112
Q

Are vascular smooth muscle cells important in atherosclerosis?

A
  • Classically VSMC were thought to just be involved in the formation of the fibrous cap
  • Later realised that VSMC can undergo transdifferentiaton into a macrophage like state upon cholesterol loading
  • Lineage tracing studies have enable us to track VSMC even after they have taken on characteristic markers of other cell types
  • Studies using these models have shown that >80% of VSMC-derived cells in mouse plaques are negative for the contractile protein ACTA2
113
Q

Can we use genetics to find novel treatment strategies for coronary artery disease?

A
  • Atherosclerosis is a complex disease not explained by a single gene – GWAS enables a noncandidate driven approach so has the potential to identify novel mechanism of disease
  • People with atherosclerosis and controls are genotyped – common single nucleotide changes (SNPs) are used to mark a region of the genome that may influence disease
  • Most SNPS are not in the protein coding region but are in the non coding region e.g on enhancers
  • Limitations:
    • Need well defined case and controls
    • Need large sample sizes to account for multiple testing and small effect size
    • Over 50% of implicated genes have uncertain biological function
114
Q

Give an example of a novel coronary artery disease gene identified by GWAS.

A

JCAD

(Xu, 2019):

  • CAD-associated risk variants at JCAD locus are associated with increased JCAD gene expression in human aortic artery tissues.
  • This suggested that this SNP is driving the increased expression of JCAD.

(Douglas, 2020):

  • Created JCAD and ApoE knockout mice
  • The JCAD knockout led to decreased atherosclerosis on the aortic arch
115
Q

Are epigenetic modifications important in atherosclerosis?

A
  • Studies have shown significant epigenetic modification in atherosclerosis
  • Extensive epigenetic changes have been shown on endothelial cells, macrophages and VSMC
  • (Edgar, 2021):
    • Transplanted bone marrow from normoglycemic control or streptozotocin-induced diabetic donor mice into atherosclerosis-prone (normoglycemic) Ldlr−/− recipient mice
    • Mice that had received diabetic BM showed markedly increased atherosclerosis, with a majority having more than the greatest atherosclerosis burden in the nondiabetic recipients
116
Q

Describe how systolic, mean and diastolic blood pressure change during dynamic exercise.

A
117
Q

Describe how systolic, mean and diastolic blood pressure change during static exercise.

A
118
Q

Describe the neural control of the circulation during exercise.

A

Afferents:

  • Central command
  • Arterial baroreflex (must be suppressed during exercise or heart rate won’t rise)
  • Muscle pressor reflex

Efferents:

  • Sympathetic efferents
  • Parasympathetic efferents
119
Q

What contributes more to increases in CO during exercise: stroke volume or heart rate?

A

(Donald, 1964):

  • Found that, in dogs, heart rate increased much more than stroke volume during exercise
  • However, when the dogs undergo cardiac transplantation, the heart is denervated and most of the CO increase is due to stroke volume. The heart rate can only increase due to circulating catecholamines.
120
Q

What element of neural control of heart rate must be removed during exercise?

A
  • Vagal activity
  • In essence, the baroreflex is reset to a higher operating pressure, so that for any given pressure, the vagal activity is reduced and heart rate is higher
121
Q

How is the baroreflex inhibited in exercise?

A
  • When blood pressure increases, the baroreflex decreases heart rate
  • This would be a problem during exercise, because it would prevent the heart rate increasing, so the baroreflex must be inhibited during exercise
  • Imaging of the brain shows increased activity in the left insular cortex during imagined exercise
  • The left insular cortex causes GABAergic inhibition of the nucleus ambiguus, which is one of the main vagal output tracts -> Thus, this inhibition leads to decreased vagal control of the heart
122
Q

Give some experimental evidence for the role of central command in the cardio-respiratory response to exercise.

A

(Krogh and Lindhard, 1913):

  • Proposed the concept of “cortical irradiation” to account for anticipatory hyperventilation, HR & ABP responses prior to exercise
  • Found that alterations in perceived load transiently affected ventilatory responses

(Eldridge, 1981):

  • Identified the sub-thalamic nucleus as a key sub-cortical site for cardio-respiratory control, independent of afferent feedback from muscles
  • Stimulated the sub-thalamic nucleus in decorticate cats, which produced locomotion and increases in ABP, ventricular pressure, heart rate and phrenic nerve activity (controls the diaphragm)
  • When the cat was paralysed and the experiment was repeated, the cat did not move but there was an increase in blood pressure and phrenic nerve activity (controls the diaphragm)
  • This is therefore evidence for sub-thalamic control of the cardio-respiratory system independent of afferent feedback -> This is technically not central command since it is not cortical
  • A problem with this study was that the sub-thalamic nucleus is tiny in cats and so the electrode tip that the scientists used may have been larger and there could’ve been accidental stimulation of the surrounding areas too

(Gandevia, 1993):

  • Studied subjects during curare-induced whole body paralysis
  • Told the subjects to attempt to contract muscles
  • This led to increases in heart rate and arterial blood pressure
  • However, the resting heart rate was very high (120bpm), which is because the patients were artificially ventilated, so the results could be altered due to this

(Krough, 2013):

  • Studied the concept of anticipatory hyperventilation, heart rate and blood pressure -> These variables start to increase before exercise does
  • Found that the ventilation, heart rate and ABP increase more if the participants are told that the exercise is more vigorous
  • Regardless of the intensity they are anticipating, the exercise is the same intensity and the variables return to the same point
123
Q

Give experimental evidence for the role of the sub-thalamic nucleus in cardio-respiratory control during exercise.

A

(Note how the STN is not considered part of central control technically, because it is a sub-cortical site)

  • (Eldridge, 1981):
    • Identified the sub-thalamic nucleus as a key sub-cortical site for cardio-respiratory control, independent of afferent feedback from muscles
    • Stimulated the sub-thalamic nucleus in decorticate cats, which produced locomotion and increases in ABP, ventricular pressure, heart rate and phrenic nerve activity (controls the diaphragm)
    • When the cat was paralysed and the experiment was repeated, the cat did not move but there was an increase in blood pressure and phrenic nerve activity (controls the diaphragm)
    • This is therefore evidence for sub-thalamic control of the cardio-respiratory system independent of afferent feedback
    • A problem with this study was that the sub-thalamic nucleus is tiny in cats and so the electrode tip that the scientists used may have been larger and there could’ve been accidental stimulation of the surrounding areas too
  • (Thornton, 2002):
    • Electrically stimulated the STN in awake men
    • This led to an increase in heart rate
  • (Basnayake, 2012):
    • Repeated the classic experiment of (Goodwin, 1972), which involved measuring blood pressure and heart rate changes during bicep contraction and bicep contraction with stimulation of the triceps tendon -> The stimulation of the tricep tendon increases the load on the bicep
    • Inserted an electrode into the sub-thalamic nucleus to study activity
    • As expected, arterial blood pressure was significantly increased in the tricep tendon stimulation group compared to the normal contraction group (although blood pressure was not significantly different)
    • STN activity was highest at rest, lower during bicep contraction and lowest during bicep contraction with tricep tendon stimulation -> This suggests that STN activity is like a handbrake and cardio-respiratory increases occur when the STN activity is lowered

Hence, overall, the results seem to suggets that the STN is like a handbrake on cardio-respiratory drive. When the STN is stimulated, the stimulation acts as a depolarising block, such that the handbrake is released and HR and ABP increase.

124
Q

What is the role of the periaqueductal grey in cardio-respiratory changes during exercise? Give experimental evidence.

A
  • Stimulation of lateral column leads to hypertension
  • Stimulation of ventrolateral column leads to hypotension
  • (Green, 2005):
    • Used electrodes to stimulate the ventral and dorsal sides of the periaqueductal grey
    • Stimulation of the ventral electrodes led to a fall in blood pressure
    • Stimulation of the dorsal electrodes led to a rise in blood pressure
    • This suggests a role of the PAG in reciprocal control of blood pressure
  • (Green, 2007):
    • Used a similar set-up in an exercise setting, except used the electrodes for measurement rather than stimulation
    • Anticipation of exercise led to increased activity in the PAG compared to rest
    • During exercise, the activity was even higher
  • (Basnayake, 2011):
    • Measured blood pressure and PAG activity (via electrodes) changes during rest, exercise, muscle occlusion and recovery
    • Found that PAG activity was increased during exercise and muscle occlusion
    • This suggests that the PAG is involved in the muscle pressor reflex
125
Q

What is the muscle/exercise pressor reflex?

A

When a muscle contracts leading to afferent signals that cause cardiovascular changes, such as an increase in arterial blood pressure.

126
Q

Give some experimental evidence for the role of peripheral feedback in the cardio-respiratory response to exercise.

A

(Alam, 1937):

  • Studied subjects who performed isometric exercise (weightlifting) and occluded the subject’s blood flow in the arm
  • The systolic blood pressure rose and remained high until the occlusion was removed
  • The increase in blood pressure was greater when the weight lifted was higher
  • This suggested some peripheral feedback to the brain due to accumulating metabolites (that activate C fibres in the muscle)

(Rowell, 1981):

  • Performed a very similar study to (Alam, 1937), except this time it was the thigh that was occluded
  • After the exercise stopped, arterial blood pressure remained high until occlusion was stopped, but heart rate and ventilation fell
  • This is arguably an artefact of the fact that exercise was stopped, which led to the decrease in HR and ventilation that would otherwise not happen

(Coote, 1971):

  • Provided direct evidence for the pressor response to muscle stimulation
  • Stimulated a muscle via the cut ventral root, which led to an increase in contraction and also increase in blood pressure
  • When the dorsal root was cut and the ventral root was stimulated again, the muscle still contracted but the blood pressure did not increase
  • This provides evidence for the role of the muscle afferents in blood pressure increases during exercise

(Matsukawa, 1994):

  • Stimulated a triceps muscle via the ventral root and observed how blood pressure, heart rate, cardiac sympathetic nerve activity and tension all increased
  • Cut the L4-S1 ventral and dorsal spinal roots and repeated this
  • Observed that tension was the only one that increased
  • This provides evidence for the role of the muscle afferents in blood pressure and heart rate increases during exercise
127
Q

Give some experimental evidence for how epidural anaesthesia affects cardio-respiratory responses during exercise. What does this tell us?

A

(Fernandes, 1970):

  • Compared subjects undergoing exercise at increasing work intensities until exhaustion
  • During dynamic exercise with epidural anaesthesia, blood pressure was lower than in control experiments, but ventilation and heart rate were not affected.
  • The results indicate that afferent neural activity from the working muscles is important for blood pressure regulation during dynamic exercise (i.e. muscle pressor reflex) but may not be necessary for eliciting the ventilatory and heart rate responses (i.e. muscle heart reflex).
  • However, while the epidural anaesthesia was only supposed to block the afferent sensory fibres from the muscles, it also blocks the efferent sympathetic fibres (since they conduct at similar velocities). This means that the lower blood pressure may not have been due to the blocking of the muscle pressor reflex, but simply due to blocking of the sympathetic outflow.
  • Similarly, they may be problems with lack of muscle heart reflex. This is because of the epidural anaesthesia led to weakening of motor drive slightly, so to match oxygen consumption, the body must work harder, so the sympathetic drive increases. Hence, the heart rate is not allowed to go down. In other words, the lack of change in heart rate upon epidural anaesthesia does not necessarily indicate a lack of muscle heart reflex, but instead could be explained by there being increased sympathetic drive in order to achieve the same oxygen consumption.
128
Q

Draw a summary diagram of cardiovascular control during exercise.

A
  • Afferent and efferent pathways converge on the cardiovascular control centre in the medulla.
  • Cortical areas (central command), the PAG and sub-thalamic nucleus influence the cardiovascular control centre.
  • Afferent fibres from the muscles also influence the cardiovascular system during exercise, which is known as the exercise pressor reflex.
129
Q

Summarise the role of the PAG and sub-thalamic nucleus in cardio-respiratory changes during exercise.

A

The PAG and STN integrate information from central command (in the cortex) and muscles, after which they pass it on to the medulla, which in turn affects cardiovascular and respiratory changes.

130
Q

Why should we target cardiovascular autonomic pathophysiological states?

A

Hypertension, heart failure and post-myocardial infarction are associated with:

  • Lower cardiac vagal function
  • Sympathetic hyperactivity

This change in autonomic balance is a negative prognostic indicator for both morbidity and mortality.

131
Q

Is NO produced by NOS in all parts of the heart to control heart rate?

A

No, neural control of HR by NOS is site-specific.

132
Q

Summarise the roles of NO at sympathetic and parasympathetic sites.

A
  • NO inhibits sympathetic traffic
  • NO facilitates vagal activation
133
Q

Draw the formation of NO and the structure of NOS.

A

The BH4 (tetrahydrobiopterin) is an essential cofactor for the conversion of L-arginine to NO by endothelial nitric oxide synthase. If it becomes uncoupled, this can lead to formation of superoxide.

134
Q

Describe the physiological and pathological roles of NO.

A

Physiological (at low concentrations):

  • Activates transcription factors
  • Binds to heme-containing enzymes
  • Activates guanylate cyclase, which activates cGMP, which:
    • Activates PKA and PKC
    • Activates phosphodiesterases
    • Targets cyclic nucleotide-gated ion channels

Pathological (at high concentrations):

  • DNA deamination
  • Nitration
  • Nitrosylation
135
Q

Draw a diagram to show the different ways in which NO production can be studied.

A
136
Q

Where does NO target the cardiac parasympathetic nervous system in control of cardiac excitability?

A

There are different schools of thought as to the extent that NO plays a role in cardiac excitability:

  • Some say NO is a mediator (i.e. it is obligatory for inducing bradycardia)
  • Some some NO is a modulator (i.e. it plays some role in bradycardia)
  • Some say NO plays no role at all
137
Q

How could NO be a mediator (i.e. play an obligatory role) of bradycardia by acting on the parasympathetic nervous system? Give experimental evidence FOR and AGAINST this.

A
  • NO could be a downstream product of ACh activating muscarinic receptors that activate eNOS. It then would activate guanylate cyclase, leading to the production of cGMP that inhibits cAMP and activates PKG, both of which inhibit LTCCs.
  • (Balligand, 1993):
    • Took spontaneously-beating ventricular myocytes (this is already a limitation of the study)
    • Addition of carbachol (ACh analogue) leads to bradycardia
    • This is blocked by methylene blue (guanylate cyclase inhibitor)
    • It is also blocked by haemoglobin (buffer for NO)
    • This suggests a critical role for NO in enabling the effects of ACh on the heart
  • (Han, 1994):
    • Performed patch-clamping
    • Addition of isoprenaline led to an increase in calcium current that was blocked by carbachol (ACh analogue)
    • When this was repeated in the presence of a NOS inhibitor, the isoprenaline had the same effect, but carbachol did not have an effect
    • This suggests a critical role for NO in enabling the effects of ACh on the heart
  • (Han, 1998):
    • Repeated the experiment from (Han, 1998) with the use of eNOS knockout mice instead of the NOS inhibitor
    • The same results were found
  • However, there was also opposing evidence to this produced:
  • (Vandecasteele, 1999):
    • Carried out a very similar study to (Han, 1998)
    • Found that addition of carbechol (ACh analogue) after adding isoprenaline in eNOS knockout mice DID lead to decreased calcium currents
    • In other words, the effects of ACh were not altered by the lack of NO.
    • Right atrial beating frequency and contraction were not altered either.
    • However, it could be argued that the conditions were not the same in this experiment. For example, the temperature was much lower, which could already have inhibited the calcium current.
  • Some evidence settled somewhere in between
  • (Sears, 1998):
    • Applied carbamylcholine to the heart, which led to bradycardia
    • Inhibition of NOS by a range of inhibitors did not alter this response to carbamylcholine
    • Then looked to see if the NO had any effect on L-type calcium currents
    • nNOS gene transfer into a disease model led to a decreased L-type calcium current in the control experiment and with noradrenaline

Summary: No consistent evidence that NO-cGMP mediates cholinergic inhibition of ICaL (although activation of Gi is crucial for encoding bradycardia)

138
Q

Does NO act only post-synaptically to induce bradycardia? Give experimental evidence.

A

(Herring, 2000):

  • Added graded concentrations of carbamylcholine to the heart, which led to bradycardia
  • When L-VNIO (neural NOS inhibitor) was also added, the degree of bradycardia was unchanged
  • Carried out graded frequency of vagus stimulation, which also led to bradycardia
  • When L-VNIO was added, the degree of bradycardia was reduced, suggesting that NOS may also act pre-synaptically -> L-arginine reversed this

(Dawson, 2008):

  • Carried out NOS-1 gene transfer into the intracardiac ganglia (80% cholinergic)
  • Stimulation of the vagus nerve led to greater bradycardia in the NOS-1 gene transfer heart than in the controls
  • This was reduced by NOS inhibitors or guanylate cyclase inhibitors
  • This suggests that NOS may also act pre-synaptically

(Heaton, 2005):

  • Exposed the cardiac vagus nerve in pigs
  • Gene transferred nNOS into the nerve
  • Upon administration of phenylephrine (vasoconstrictor that increases ABP), this led to a greater baroreflex response than controls
  • This suggests that NOS may also act pre-synaptically

(Herring, 2002):

  • Suggest a model for how NOS may act pre-synaptically (see diagram)
  • Addition of a nitric oxide donor (SNP) led to increased ACh release and bradycardia
139
Q

In what situations might NO control of bradycardia be important? Give experimental evidence.

A

In exercise

(Danson, 2003):

  • Removed the hearts of exercise-trained and not exercise-trained mice
  • The exercise-trained mice showed a greater decrease in heart rate in response to 5Hz vagal nerve stimulation
  • This can be interpreted as improved recovery of heart rate after exercise
  • This difference could be removed by addition of L-VNIO (neural NOS inhibitor) and recovered by addition of L-arginine -> This suggests the importance of NOS in vagal responsiveness
  • However, carbachol (muscarinic agonist, sort of like ACh) did not produce a different in vagal responsiveness, so the NO must be pre-synaptic

(Danson, 2004):

  • Expanded on his previous work by knocking out nNOS in mice
  • When the mice were exercise trained, they did not show increased vagal responsiveness
  • This shows the importance of nNOS in this process

In disease

(Li, 2013):

  • Found that nNOS was impaired in cardiac-neural tissues from pre-hypertensive rats

(Dawson, 2008):

  • Found that ACh release was reduced in areas of myocardial infarction compared to areas of sham intervention
  • Carried out gene transfer of nNOS into the intracardiac ganglia (where the cholinergic neurons are)
  • This led to an increase in ACh release
  • This resulted in greatly improved all-cause mortality in these animals
140
Q

Give a summary of NO in pre-synaptic control of heart rate.

A
  • Pre-synaptically, NO may facilitate ACh release during vagal nerve stimulation to decrease heart rate
  • This occurs naturally as a result of exercise and is decreased in certain diseases
  • Overall, this may provide a therapeutic target
141
Q

Give experimental evidence for how NO affects the sympathetic control of the heart.

A

(FIND REFERENCE):

  • Removed a heart with the sympathetic varicosities still attached
  • Sympathetic nerve stimulation led to an increase in heart rate that was increased by inhibition of NO and decreased by overexpression of NO
142
Q

How is sympathetic control of heart rate different in pre-hypertensive individuals? Give experimental evidence.

A
  • (Larson, 2016):
    • Co-cultured sympathetic neurons with cardiac myocytes
    • Stimulated the neurons using nicotine and measured the resulting increase in cAMP in the myocytes
    • Sympathetic activation caused greater increase in cAMP in myocytes that were taken from pre-hypertensive rats
    • When a healthy neuron was cultured with a diseased myocyte, a healthy phenotype was seen
    • When a diseased neuron was cultured with a healthy myocyte, a diseased phenotype was seen
    • This shows that it is the neuron, not the myocyte, that determines the phenotype
143
Q

Draw how NO could act pre-synaptically in the sympathetic nervous system to affect heart rate.

A

NO drives bradycardia.

144
Q

What is the importance of site-specific gene transfers when studying the effects of NO on the sympathetic nervous system?

A
  • It is important to only express the NOS in the sympathetic neurons, not all cells
  • This can be achieved by using an adenoviral vector with a noradrenergic-specific promoter coupled to nNOS
145
Q

What is CAPON? Give its clinical importance.

A
  • CAPON is a protein that co-localises with NOS in neurons and cardiac myocytes
  • The only SNP found to be associated with long QT syndrome in GWAS was in the CAPON protein
  • CAPON was thought to drive shortening of the action potential in long QT by NO activating the IKr channel -> This makes the person susceptible to sudden cardiac death
  • Sudden cardiac death occurs in moments of stress, etc.
  • Recently, it has been found that CAPON also plays a role in neurons in sudden cardiac death because NO inhibits the release of neurotransmitter, reducing the sympathetic drive and therefore reducing the risk of sudden cardiac death
  • This provides a potential therapeutic target

FHS - Cardiovascular Science (4 decks)

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