Theme 1: Signalling in the Cardiac Myocyte Flashcards
Is cardiac muscle striated?
Yes
Describe the nuclei of cardiac muscle.
Each cell only has one nucleus, which is centrally located.
What is unusual about the shape of cardiac myocytes?
They are branched.
What joins cardiac muscle cells?
Intercalated discs
What is the distinguishing characteristic of cardiac muscle in histology?
The presence of dark transverse lines between cells -> These are intercalated discs.
What are intercalated discs?
Structures that interface between adjacent cardiac muscle cells and support synchronised contraction.
What are the 3 components of intercalated discs?
- Desmosomes
- Adherens junctions
- Gap junctions
What is the function of desmosomes in intercalated discs between cardiac myocytes?
Bind cells together.
What is the function of adherens junctions in intercalated discs between cardiac myocytes?
Act as anchoring sites for actin filaments.
What is the function of gap junctions in intercalated discs between cardiac myocytes?
Provide continuity between adjacent cells and allow ions to pass between cells.
What is another name for adherens junctions in intercalated discs?
Fascia adherens (a.k.a. hemi Z-bands)
What is another name for desmosomes in intercalated discs?
Macula adherens
Summarise simply the structure and functioning of cardiac myocytes.
Branching mesh of mononuclear striated cells joined and electrically coupled by intercalated discs (desmosomes and gap junctions: electrically a ‘functional syncytium’).
Draw the structure of a sarcomere. Include the names of the different zones.
- A band (dark) -> All of the length of myosin filaments
- I band (light) -> Just actin filaments
- Z line -> Where actin attaches
- H band -> Just myosin filaments
- M line -> What myosin attaches to
Label this section of cardiac muscle.
- Mt = Mitochondria
- ln D = Border of the myocyte
How is the sarcoplasmic reticulum arranged in cardiac muscle?
The SR run along myofibrils.
What are T tubules and cisternae?
- T tubules -> Invaginations of the plasma membrane into the myocytes (contain LTCCs)
- Cisternae -> Parts of the sarcoplasmic reticulum that are adjacent to the T tubules (contain RyR)
What are dyads?
- The dyad is a structure in the cardiac myocyte composed of a single T-tubule paired with a terminal cisterna of the SR.
- It is like a triad in skeletal muscle.
At what point along the sarcomere in cardiac myocytes are the T-tubules?
At the z-lines (i.e. at the ends of the sarcomere)
Draw the subcellular organisation of cardiac muscle.
Describe excitation-contraction coupling in cardiac muscle.
- Depolarisation of the sarcolemma travels along the membrane and into the T-tubule
- T-tubules form dyads with cisternae of sarcoplasmic reticulum
- Sarcolemma depolarisation causes opening of L-type Ca2+ channels (LTCC) in the sarcolemma
- Calcium entry activates ryanodine receptors (RyR) on the sarcoplasmic reticulum -> This is called calcium-induced calcium release (CICR)
- Calcium exits via the RyR into the cytosol
- Calcium activates troponin C and triggers contraction
Draw the different calcium fluxes that occur within cardiac myocytes.
Give an example of a fluorescent calcium indicator and how it works.
Fura-2 acetoxymethyl ester (Fura-2 AM):
- Extracellularly, it is not fluorescent and is in its ester form
- This enables it to enter cells
- Once it the cell, it is broken down by endogenous methylesterases, removing the acetoxymethyl groups
- This leaves Fura-2, which is the indicator component that binds calcium and then gives off fluorescent light
- The light can be detected by fluorescence microscopy or a fluorometer
What microscopy technique can be used along with calcium-sensitive dyes to visualise calcium release in subcellular compartments?
Confocal fluorescence microscopy -> It essentially removes the out of focus parts of the image, leading to a clearer image
What are ratiometric and non-ratiometric calcium dyes?
Each dye has an excitation spectrum (the wavelengths it is excited at) and an emission spectrum (the wavelengths it emits):
- Non-ratiometric dyes -> Show a shift in only the INTENSITY of the excitation and emission spectra when bound to by calcium
- Ratiometric dyes -> Show a shift in the emission spectrum (or excitation spectrum) when bound to by calcium. This means they have two peak wavelengths that can be measured and ratioed to amplify the signal (i.e. comparing how much of the indicator is bound/not bound to calcium).
Is Fura-2 AM ratiometric or non-ratiometric?
Ratiometric
What are some advantages and disadvantages of ratiometric calcium dyes over non-ratiometric calcium dyes?
Advantages:
- Ratiometric measurements involve ratioing of two wavelength intensities -> This allows amplification of the signal.
- Allows correction of artifacts (e.g. uneven loading of dye)
- Improved visualisation during movement
Disadvantages:
- Makes measurements and data processing more complicated.
- Since you are collecting two different wavelengths, you are at least halving your temporal resolution.
Why do many cardiovascular studies use a single wavelength (non-radiometric) calcium dye?
Non-radiometric dyes have better temporal resolution, which is important in fast events, such as cardiac myocyte contraction.
What is an ex vivo model of the heart that can be used to study it?
Langendorff-perfused heart:
- Heart is removed from the animal’s or human’s body, severing the blood vessels
- It is then perfused in a reverse fashion (retrograde perfusion) via the aorta, usually with a nutrient rich, oxygenated solution
- The backwards pressure causes the aortic valve to shut, forcing the solution into the coronary vessels, which normally supply the heart tissue with blood.
- This feeds nutrients and oxygen to the cardiac muscle, allowing it to continue beating for several hours after its removal from the animal or human.
Describe how the fact that cardiac myocyte depolarisation precedes calcium influx can be demonstrated.
(Lee, 2012):
- Langendorff heart is used and it is contraction-blocked
- A red-shifted calcium dye and voltage-sensitive dye are used, allowing both calcum influx and depolarisation to be viewed live
What are calcium sparks? How can they be represented?
- A calcium spark is the most elemental release of calcium from the sarcoplasmic reticulum
- These sparks are localised and very small -> They appear as small flickers on confocal microscopy
- Calcium sparks can combine to form calcium waves that travel across the myocyte
Describe the different ways in which calcium events (i.e. calcium waves) within a cardiac myocyte can be presented.
- Linescan confocal microscopy -> Confocal microscopy enables a straight line inside a cardiac myocyte to be studied. This enables a plot of length (distance across the line) vs time, as shown in figure c. If two waves travel in opposite directions, they may collide, as shown in figure d.
- Drawing a region of interest for analysis around the cell -> This enables consideration of the cell as a whole. Thus, when a wave starts, there is increased signal, which is maintained until the wave ends. See the blue figure.
Describe a classical experiment showing that depolarisation of cardiac myocytes leads to calcium increases and contraction.
(Allen & Blinks, 1978):
- Depolarisation of cells is followed by intracellular calcium increases (shown by aequorin luminescence)
- This is followed by contraction
- Both calcium increases and contraction are increased in the presence of isoprenaline
The problem with this experiment is a lack of spatial resolution, since it does not show where the calcium is being released from.
Describe how current and voltage clamps can be used to study calcium events in cardiac myocytes.
- In current clamps, a current is injected into the cell, which depolarises it -> This allows us to see the effect that this depolarisation has on calcium events.
- In voltage clamps, a current is injected into the cell constantly via a negative feedback loop in order to maintain the membrane potential at a constant level -> This allows investigation of currents and conductances to calcium at different membrane potentials.
In both cases, when the cell is depolarised, there are calcium influxes. These techniques may be used in patch clamping.
Describe an experiment that used linescan confocal calcium imaging to study intracellular calcium increases in cardiac myocytes.
(Cheng, 1994):
- Linescan confocal imaging showed that intracellular calcium increases following field stimulation
- When this is repeated in the presence of ryanodine (ryanodine receptor blocker) and thapsigargin (SERCA pump inhibitor), the calcium increases are decreased
- This provides information about where calcium is released from in the cell
Describe how calcium-induced calcium release leads to amplification.
- Ca2+ flux through L-type calcium channels is about 0.3pA, while it is around 0.4 - 1.0pA through ryanodine receptors
- Each L-type calcium channel leads to the opening of multiple ryanodine recptors
- Ryanodine receptors open for longer than L-type calcium channels
Localised calcium influx through ryanodine receptors is known as…
Calcium spark
Are calcium sparks evoked or spontaneous?
They can be both:
- They can be evoked by cardiac mycoyte depolarisation
- They can also occur spontaneously when ryanodine receptors open spotaneously
How can calcium sparks occur spontaneously?
- Ryanodine receptors have a small open potential at rest
- This means that some calcium can be spontaneosuly released, even without action potentials
- Each RyR opening leads to the firing of 16-20 ryanodine receptors -> Due to CICR and some mechanical coupling
Action potential-driven calcium waves in the cell are the essentially…
The summation of multiple calcium sparks
What prevents the spread of calcium sparks at rest? How is this different during an action potential?
- Space between dyads (i.e. the T-tubule) is large and thus prevents propagation of SR calcium release.
- When cellular calcium levels are high, the sensitivity of RyR is increased, so that CICR can propagate as a wave along the cell
What is a calcium transient and how is it produced?
- A calcium transient is produced in cardiac myocytes upon an action potential (although it can also happen spontaneously)
- A normal cardiac myocyte calcium transient is the summation of around 10,000 calcium sparks
Are there IP3 receptors in the heart?
Yes, although this is controversial. The evidence for it is quite strong.
Describe some evidence for the existence IP3 receptors in the heart. What types are there?
(Lipp, 2000):
- Gene expression
- In the myocardium as a whole, there are mostly IP3R1 and IP3R2
- Myocytes have mostly IP3R2
- Aortic endothelium has mostly IP3R1
- Protein expression
- IP3R2 is expressed more in the atria than in the ventricles
- Permeabilized cells
- Cells are made to be permeable to IP3
- IP3 administration leads to increases in intracellular calcium, which are evidence for the existence of IP3 receptors
Compare the distribution of IP3R2 and RyR2 in ventricular myocytes. Give some experimental evidence for this.
- IP3R2 are located around the nucleus and at the ends of the myocyte
- RyR2 are located near T-tubules
(Escobar, 2011) used immunolabelling to locate IP3 receptors in cells.
Which IP3R isoforms are seen in the human ventricle?
All 3 isoforms.
Give some experimental evidence for the role of IP3R in the heart.
(Signore, 2013):
- Heart failure may lead to G-protein activation
- This activates IP3Rs, which consequently leads to prolonged action potential duration and increased calcium influx
- This means that IP3R enable an inotropic reserve
Compare the structure of dyads in ventricular and atrial myocytes.
Ventricular myocytes:
- Have T-tubules with LTCC on them
- SR is closely apposed, with RyR on the surface
Atrial myocytes:
- Have Z-tubules (part of the SR) with RyR on the surface
- There are no invaginations of the cell membrane, but LTCC are closely apposed to the Z-tubules
Describe the calcium increases in atrial myocytes upon an action potential. Compare this to ventricular mycoytes.
- Atrial myocytes do not have T-tubules, so all of the LTCC are on the cell surface
- This means that the calcium influx starts on the outside of the cell and progresses inwards as a ring
- On the other hand, in ventricular mycoytes, the calcium increases are approximately simultaneously across the whole cell
(Mackenzie, 2004) showed this as is shown in the diagram. They also showed that if beta-1 stimulation is done simultaneously, then the calcium increases become more widespread within the cell.
Compare the distribution of IP3R2 and RyR2 in atrial myocytes. Give some experimental evidence.
(Lipp, 2000):
- RyR are along Z-tubules, especially near the plasmalemma, since this is where the LTCC are
- IP3R2 are near the plasmalemma, close to the RyR
Give some experimental evidence for IP3 causing calcium sparks in atrial myocytes.
(Lipp, 2000):
- Tagged IP3 with BM (butyryloxymethyl ester) to enable IP3 to enter the cell
- This led to an increase in calcium sparks compared to a control
- The sparks peaked around 5 minutes after administration
What are the two main functional parts of the sinoatrial node cells?
- Primary pacemaker
- Subsidiary pacemaker
What are the functional subunits that enable pacemaking in the primary pacemaker of SAN cells?
Caveolae, which are smallninvaginations of the plasma membrane.
Describe what is found in caveolae of SAN cells.
- HCN4 (hyperpolarisation activated cyclic nucleotide gated) -> Responsible for the funny current upon hyperpolarisation (sodium current)
- Beta-2 receptor
- NCX
- Cav1.2 (L-type calcium channel)
- Cav3 (T-type calcium channel)
The sarcoplasmic reticulum is closely apposed to this, such that most of the RyR are found near the sarcolemma.
Compare the primary pacemaker and subsidiary pacemaker in SAN cells.
- The primary pacemaker features caveolae on the cell surface, with RyR on the SR closely apposed to this.
- The subsidiary pacemaker features axial tubule junctions, where the RyR on the SR are closely apposed to T-tubules
Describe the ionic basis of the SAN action potential in the heart.
- The membrane is gradually depolarised by 3 main inwards currents (phase 4):
- Sodium currents are partly background currents and ‘funny’ currents (If) through non-selective channels that open upon hyperpolarisation, allowing sodium and potassium to flow.
- The second main current in the pacemaker potential is that created by the electrogenic sodium-calcium exchanger, which moves 3 sodium ions in for every calcium ion moving out.
- The third current responsible for the final part of the pacemaker potential is a calcium current that is firstly through transient (T-type) calcium channels, and then also L-type calcium channels at higher membrane potentials.
- Above the threshold, the rapid depolarisation (phase 0) is caused mostly by the opening of L-type calcium channels.
- Repolarisation (phase 3) occur due to efflux of K+ through voltage-gated potassium channels.
Describe the ideas of the membrane and intracellular calcium clock in the SAN cells. How are they related?
(Lakatta, 2010):
Membrane calcium clock:
- The SAN action potential involves calcium via the L-type calcium channels, T-type calcium channels and NCX.
Intracellular calcium clock:
- Calcium released from the SR is then re-uptaken up by the SERCA pump.
Thus, the two clocks are inter-connected. When there is excessive release of calcium from the SR, the NCX is stimulated. Since the NCX has electrogenic activity, this depolarises the cell and can predispose to arrythmias.
Compare L-type and T-type calcium channels.
- The L-type calcium channel is responsible for normal myocardial contractility and for vascular smooth muscle contractility.
- T-type calcium channels are not normally present in the adult myocardium, but are prominent in conducting and pacemaking cells.
What is unusual about the SAN action potential?
The upstroke is determined by calcium, rather than sodium, which means that it is intrinsically inter-connected with calcium release from the SR.
Describe an experiment that shows the importance of calcium in the SAN action potential.
(Maltsev, 2017):
- Used a fluorescent calcium dye and patch clamping to track intracellular calcium concentration and Em in SAN cells
- This showed that calcium concentration and Em are linked, although depolarisation precedes increases in calcium
- Thus, calcium fluxes are an important contributor to the SAN action potential
Does the SAN require RyR? What is the evidence for this?
Yes, RyR are still involved in spontaneous calcium transients.
(Kapoor, 2015):
- Fluorescent calcium dye use shows that there are whole cell calcium transients (seen as green lines)
- Between these, there are small burst of calcium activity due to RyR, which can summate to produce the larger calcium transients
- The importance of RyR in producing these calcium transients is demonstrated by administration of ryanodine, which blocks this effect
- Caffeine (ryanodine recptor agonist) administration leads to mass calcium release
What IP3R isoform is found in the SAN?
IP3R2
Describe the distribution of IP3R2 in SAN cells. Give some experimental evidence.
(Ju, 2011):
- IP3R2 are found at the sarcolemma and also slightly along the tubules
What are the effects of IP3 in SAN cells? Give some experimental evidence.
(Ju, 2011):
- Used IP3-BM to introduce IP3 into SAN cells
- The IP3 caused an increase in calcium spark frequency and an increase in the action potential frequency and amplitude
Draw a diagram of the currents contributing to the SAN action potential, as well as the phases of the calcium clock.
What factors influence the speed of the calcium clock in the SAN?
- Factors that influence local calcium release between each cell-wide calcium transient can lead to faster depolarisation such that the threshold potential is reached more quickly.
- For example:
- More leaky RyR
- IP3
- NCX overactivity
- Beta receptors can also increase the amplitude of the calcium transient
Give a summary of calcium signalling in the heart.
How are LTCC on cardiac myocytes affected by calcium? Give some experimental evidence.
(Josephson, 2009):
- Patch clamped ventricular myocytes in 2mM calcium and 2mM barium at -50mV
- Then depolarised to -10mV
- With the barium, the ventricular myocytes are maintained in the open state for longer and there is a much larger current compared to the calcium
- This is because there is calcium-dependent inactivation of the LTCC
Describe the structure of LTCCs.
- Made up of 5 different subunits: α1, α2, δ, β and γ subunits (the gamma is only found in skeletal and cardiac muscle).
- The α1 subunit is the pore-forming unit where there is also a drug-binding domain for modulatory drugs.
Describe the pore-forming α1 subunit of the LTCC.
- Made up of 4 domains, each containing 6 transmembrane segments.
- S1-S4 form the voltage-sensing module.
- S5-S6 form the pore.
(Note: The diagram only shows two of the 4 domains)
Name some types of LTCC blockers.
- Dihydropyridines (nifedipine, nimodipine)
- Phenylalkylamines (verapamil)
- Benzothiazapines (diltiazem)
How does nimodipine (dihydropyridine) block calcium channels? Give some experimental evidence.
- Nimodipine binds between outer domains, displacing a lipid molecule
- This triggers allosteric changes at the selectivity filter
- (Tang, 2016):
- Used patch-clamping to study LTCCs and applied depolarising pulses to study the effects of nimodipine on calcium currents
- The higher the concentration of nimodipine, the more the current falls with each pulse
- Since the current falls with each pulse and then plateaus, this is a state-dependent block
- When the LTCC is mutated with the I199S substitution, it reduces the efficacy of nimodipine and the current does not fall as quickly with each pulse
How does verapamil (phenylalkylamine) block calcium channels? Give some experimental evidence.
- Verapamil binds in the pore region of the LTCC
- It enters the pore from the cytosolic side
- (Tang, 2016):
- Used patch-clamping to study LTCCs and applied depolarising pulses to study the effects of verapamil on calcium currents
- The current falls with each pulse when verapamil is applied
- Since the current falls with each pulse and then plateaus, this is a state-dependent block
- When the LTCC is mutated with the T206S substitution, it reduces the efficacy of verapamil and the current does not fall as quickly with each pulse
What are some factors that can modulate LTCCs on the cytosolic side?
- Calmodulin
- Calmodulin kinase II
- cAMP / Protein kinase (related to beta agonists)
Which ryanodine receptor isotype is expressed in cardiac muscle?
RyR2
Describe the permeability properties of the cardiac calcium-release channels (RyR and IP3R).
- They are ideally selective for cations
- But they are relatively non-selective between monovalent and divalent cations
- For example, they have a very high conductance for both calcium and potassium
Describe the structure of the RyR2 in the heart.
- It is the largest ion channel in the body -> This allows there to be lots of modulatory sites
- It is a homotetramer
Are RyR2 always in the membrane?
No, they can be in vesicles that are then inserted into the membrane.
How can you study the biophysical properties of the RyR2?
- The RyR2 in a membrane are clamped between two chambers
- The ion properties of these chambers can be controlled
- The currents through the RyRs can be measured
What factors modulate the opening of RyR2?
On cytosolic side:
- Ca²⁺/calmodulin-dependent protein kinase (CMKII)
- Calmodulin
- Phosphodiesterase 4D
- PKA
- FKBP12/12.6
- PP1
- PP2A
On SR lumen side:
- Calsequestrin
What is the role of calmodulin?
CaM regulates RyR2 and many other proteins in the heart that are involved in calcium signalling (including LTCC and other kinases).
Describe the structure of calmodulin.
It has 4 EF hands that are important for binding.
What is the result of calmodulin mutations?
They result in cardiac arrhythmias.
What is CAMK short for?
Ca²⁺/calmodulin-dependent protein kinase
(It can also be referred to as CaM kinase)
Describe how the activity of CaMKII is controlled.
- Under resting conditions, the catalytic domain is constrained by the regulatory domain
- When intracellular calcium rises and complexes with calmodulin, the Ca2+/CaM binds to the regulatory domain, which activates it
- When there is sustained calcium or increased oxidation, the CaMKII can become a Ca2+/CaM-autonomous active enzyme after autophosphorylation (at Thr287) or oxidation (at Met281/282) of amino acids in the regulatory domain
What are the actions of CaMKII in the heart?
- Increases the circulation of calcium:
- Increases mean open time of LTCCs
- Increases activity of SERCA pump
- Increases opening of RyRs
- Increases activity of Na+ and K+ channels
- Affects calcium uniporter
- Affects myofilaments
Describe the mechanism by which sympathetic stimulation affects the contraction and relaxation the heart.
- Noradrenaline and adrenaline bind to β1 adrenoceptors, which are Gs-coupled GPCRs that stimulate adenylate cyclase and increase intracellular cAMP, and therefore protein kinase A.
- cAMP stimulates the funny current (If) -> Increases the heart rate (at SAN)
- PKA phosphorylates 3 things:
- L-type calcium channels and RyRs -> Helps to speed up decay of pacemaker potential (at SAN), so heart rate is increased and contraction is strong due to more calcium entry
- Delayed rectifier potassium channels -> Enabling faster repolarisation (so max heart rate is increased)
- Phospholamban -> Stops it inhibiting the Ca2+-ATPase on the sarcoplasmic reticulum, so uptake into the SR is increased (disinhibition) -> Relaxation occurs more quickly and increases amount of calcium stored in SR (for stronger contraction)
- Myofilaments
- The higher heart rate is also enabled by faster firing at the atrioventricular node
What are the effects of FKBP in cardiac myocytes? Give some experimental evidence.
- The effects of FKBP are controversial
- (Guo, 2010):
- Expressed fluorescent FKBP12.6 in cardiac myocytes
- Fluorescence microscopy showed that the FKBP binds at the Z-lines (where the RyRs are)
- Overlayed the calcium sparks in the cardiac myocytes over the FKBP and found that calcium sparks originate from the FKBP binding sites (i.e. the RyR)
- FKBP12.6 but not FKBP12 inhibits the frequency of calcium sparks
What is calsequestrin and what is its role?
- Calsequestrin (CASQ) is a low-affinity, high-capacity calcium-binding protein
- It is found in the SR lumen
- CASQ increases the luminal calcium sensitivity of single RyRs
How can calcium signals in the heart go wrong?
- Calcium sparks (which occur spontaneously between action potentials) can sometimes summate to produce a calcium transient
- This can be the cause of arrythmias
- These are known as either early after depolarisations (EADs) if they are during an action potential, or delayed after depolarisations (DADs) if they are after the action potential
Give a possible mechanism by which after depolarisations and spontaneous calcium waves can occur.
- Delayed after depolarisations (DADs) may be caused by overloading of the sarcoplasmic reticulum.
- This causes calcium sparks, where the calcium is removed by the NCX.
- This has an electrogenic effect that depolarises the cell and thus there is opening of calcium channels.
Give an example of a time when calcium waves may occur abnormally. Give experimental evidence.
(Mattiazzi, 2015):
- Studied intracellular events in a Langendorff-perfused contraction-blocked heart
- Exposed the heart to ischaemia and then reperfusion (as might occur in a myocardial infarction)
- Before ischaemia, calcium sparks and calcium waves are both rare
- During ischaemia, calcium sparks are frequent and calcium waves occur slightly more frequently
- After ischaemia, calcium sparks are rare and calcium waves occur very frequently
- This suggests that ischaemia followed by reperfusion may drive abnormal calcium waves that are the cause of arrhythmias
What mechanism underlies atrial fibrillation? Give experimental evidence.
- Abnormal intracellular calcium control appears to contribute.
- (Hove-Madsen, 2004):
- Studied atrial myocytes from healthy patients and those with atrial fibrillation.
- Patients with atrial fibrillation showed higher frequency of calcium sparks and calcium waves than healthy patients.
- This supports the idea that abnormal intracellular calcium leak can produce after depolarisations and thus dysrhythmias.
- (Voigt, 2012) [SEE DIAGRAM]:
- Studied atrial myocytes from healthy patients and those with atrial fibrillation.
- Fired action potentials in the myocytes for 30 seconds, then wateched for spontaneous activity for the next minute -> measured intracellular calcium and currents produced by the NCX.
- The atrial fibrillation myocytes showed much higher rates and sizes of calcium events (sparks/waves), shorter latency after the action potentials and larger currents produced by the NCX.
How are RyR implicated in disease?
- In disease, RyR channel regulation by binding proteins can become disrupted.
- For example:
- RyR2 phosphorylation state is altered in heart failure
- Binding of CaM and CaMKII are altered in heart failure
- Mutations to CASQ can lead to sudden cardiac death
How can dysregulated RyR in disease be targeted therapeutically?
- Heart failure and CVPT (catecholaminergic polymorphic ventricular tachycardia) are both characterised by leaky RyRs
- In heart failure, beta-blockers and ARBs can stabilise the RyR by inhibiting the hyperphosphorylation of the RyR and subsequent FHBP12.6 dissociation
- In CPVT, JTV519 also stabilises channel gating but this time it works by acting on the channel directly
What are the 4 main ways in which calcium is removed from the cardiac myocyte cytosol after contraction?
- SERCA pump
- NCX
- Membrane ATPase
- Mitochondrial ATPase
What are the different contributions of the various calcium removal methods to removing calcium from cardiac myocytes after contraction? Give experimental evidence.
(Bers, FIND REFERENCE):
- Studied cardiac myocytes when they were relaxing after contraction
- Blocked various proteins and observed how that affected the rate of relaxation
- With a SERCA block, relaxation is 3-fold slower
- With a SERCA and NCX block, relaxation is 20-fold slower than the previous
- With only the plasma ATPase or mitochondrial uptake functioning, relaxation is 2-3 slower than previous
- Thus, SERCA and NCX dominate calcium removal, while the plasma ATPase and mitochondrial uptake are much slower
(Bassani, 1994):
- Studied ventricular myocytes from both rabbits and rats
- Measured the calcium fluxes through each of the 4 removal methods after contraction
- In rabbits, the SERCA pump had 70% of the flux, the NCX had 28% and the plasma ATPase/mitochondria had 2%
- In rats, the SERCA pump had 92% of the flux, the NCX had 7% and the plasma ATPase/mitochondria had 1%
- The rabbit appears to be more similar to the human heart
Is the SERCA pump or NCX more efficient in removing calcium from the cytosol after cardiac myocyte contraction?
- The SERCA pump moves 2 Ca2+ per ATP
- The NCX moves 1 Ca2+ per ATP
Thus, the SERCA pump is more efficient.
How is calcium removal from the cytosol of cardiac myocytes after contraction affected in heart failure? Give experimental evidence.
(Bassani, 1994):
- Compared rabbit ventricular myocytes during health and heart failure
- The calcium fluxes through the NCX were proportionally larger in heart failure
- The NCX is less energetically efficient than the SERCA pump, so the process requires more energy and it is more slow
- Therefore, heart failure is exacerbated because the process of calcium removal requires more energy
How is calcium transient amplitude related to contraction?
The larger the amplitude, the stronger the contraction (Bers, 2002).
How are mitochondria positioned within myocytes?
They stretch along the mytocyte, so they can take up calcium from multiple T-tubules.
Give some experimental evidence for the role of the mitochondria in cardiac myocyte contraction.
(Lu, 2013):
- Used MityCam (a genetic fluorescent calcium sensor) to study the levels of calcium in the mitochondria of cardiac myocytes
- Stimulated the cells at 0.1, 0.2 and then 0.5Hz
- The higher the frequency of stimulation, the smaller and faster the calcium transients in the mitochondria become. Also, the average calcium concentration increases.
- When isoprenaline is added, the calcium transients become larger and the average calcium concentration increases.
- This shows that the calcium concentration of the mitochondria varies in proportion to the degree of stimulation of the cell, so that the mitochondria are stimulated to produce a proportional amount of ATP.
Give some experimental evidence for where calcium enters mitochondria in cardiac myocytes.
(Lu, 2013):
- Studied calcium concentration at two points within the mitochondria -> Near the Z-line and near the M-line
- Upon a twitch, the calcium spike is larger and faster near the Z-line
- This is explained by the fast that the Z-line is where the T-tubules are so that is where more calcium is
How is mitochondrial energy production linked to calcium concentration in the cardiac myocyte?
- As calcium in the cytosol increases, it enters mitochondria through a uniporter
- This activates dehydrogenases in the citric acid cycle that produce NADH
- This NADH is the source of H+ that cross the mitochondrial membrane and are used by ATP synthase to produce ATP
- Thus, ATP production is matched to calcium concentration in the cell (and therefore metabolic demand)
What happens when too much calcium enters the mitochondria in cardiac myocytes? How is this dealt with?
- When too much calcium enters the mitochondria, it can affect the negative membrane potential across the mitochondrial membrane
- This reduces the driving force for H+ passing through ATP synthase, so less ATP is produced.
- If calcium is elevated for too long, permeability transition pores (mPTPs) open, leading to loss of ATP production and cell death.
- To prevent this, the NCX is used to remove the excess calcium, but this is electrogenic
- A sodium-hydrogen exchanger is used to remove the sodium that enters and this maintains the membrane potential
- This means that hydrogen is moved across the membrane, bypassing the ATP synthase. Therefore, the probem is that this makes ATP generation less efficient.
Draw a graph of how cytosolic calcium affects mitochondrial calcium.
Note how the mitochondria act as a buffer when the calcium reaches a certain level.
How do mitochondria in cardiac myocytes prevent widespread damage? Give experimental evidence.
(Glancy, 2017):
- Used UV radiation to cause localised damage to mitochondria in cardaic myocytes
- The cardiac myocytes are linked in networks with intermitochondrial junctions
- These junctions close when damage occurs and if there is sustained damage then physical separation occurs
- This prevents damage spreading to other parts of the cell
Give an example of a condition that may be caused by mitochondrial damage. Give experimental evidence.
(Sabbah, 1992 OR 2017):
- Used electron microscopy to study the electron density of cardiac myocytes (as a measure of the mitochondrial damage)
- Lower ejection fraction was associated with more mitochondrial damage and higher levels of circulating catecholamines
Describe the basic calcium-related mechanisms underlying heart failure and how they affect contraction.
- Leaky RyRs cause calcium sparks that lead to increased cytosolic calcium concentration
- This can lead to some contraction that is not related to action potentials
- It also leads to increased activity of the NCX, which is electrogenic and can lead to the eventual triggering of an action potential (delayed afterdepolarisation-triggered action potential)
- However, the SR is drained from calcium at this point, so the resulting contraction is weak
(SEE GRAPH ON DIAGRAM)
Describe the different cellular proteins involved in heart failure and how they contribute.
- The NCX is overactive, the SR Ca2+-ATPase is underactive and the RyR is leaky:
- This leads to decreased SR calcium load, so that contraction is weaker (systolic heart failure)
- There is also increased cytosolic calcium during diastole, so that there is decreased relaxation (diastolic heart failure)
- The overactive NCX also causes increased sodium entry, so there is greater depolarisation and the cell may not return to the resting potential -> This increases the risk of arrhythmias
- There is decreased activity and expression of potassium channels, so there is more delayed after depolarisations -> This increases the risk of arrythmias
- Beta-agonists are released in heart failure, leading to increased spontaneous calcium release -> This increases the risk of arrythmias
What are some factors that can cause the autonomous activation of CaMKII in heart failure?
- Oxidation (e.g. by ROS)
- Nitrosylation (by nitric oxide)
- Glycation (adding glucose to a residue)
These factors contribute to why the heart does not function as well in heart failure and diabetes.
Describe how CaMKII can affect the cardiac action potential.
- CaMKII increases RyR calcium leak, which activates the NCX -> This can lead to delayed after-depolarisations (DADs)
- CaMKII increases the opening of LTCCs and Na+ channels -> This can lead to prolongation of the action potential and early after-depolarisations (EADs). This leads to long QT.
- CaMKII increases the opening of K+ channels -> This leads to dispersion of repolarisation between the endocardium and epicardium, which predisposes to arrythmias
How does calcium in the nucleus affect heart function?
- Calcium in the nucleus is related to IP3R in the nucleus
- IP3 is released by GPCRs, such as endothelin receptors
- It then binds to IP3R in the nucleus
- IP3R facing inwards into the nucleus:
- IP3 binding to the receptor leads to release of calcium locally, which activates CaM and CaM kinase
- This leads to phosphorylation and export of HDAC (histone deacetylase), which usually represses the transcription of hypertrophic genes
- Hence, the hypertrophic genes are derepressed.
- IP3R facing outwards into the cytosol:
- IP3 binding to the receptor leads to release of calcium, which activates CaM
- CaM dephosphorylates NFAT, which moves to the nucleus. NFAT in the nucleus promotes the transcription of hypertrophic genes. This hypertrophy can contribute to heart failure.
- So IP3 signalling driven by ET-1 overall leads to hypertrophy and heart failure.
Give some experimental evidence for how calcium in the nucleus affects heart function.
(Wu, 2006):
- Expressed HDAC5 tagged with GFP in cardiac myocytes
- Under control conditions, the fluorescence remains in the nucleus
- When exposed to endothelin, the fluorescence spreads out of the nucleus because the HDAC5-GFP is exported from the nucleus
- When an IP3R2 knockout is used, this export does not occur, suggesting that this is the receptor involved in the nucleus
Write the Ka equation for an acid buffering reaction.
Give a summary of the basic concepts in acid-base chemistry.
What effect does acidity have on cardiac calcium currents? Give experimental evidence.
(Saegusa, 2011):
- Extracellular acidosis blocks calcium currents, shortening the action potential
- Intracellular acidosis promotes calcium currents, lengthening the action potential
What effect does acidity have on cardiac contractile apparatus? Give experimental evidence.
(Solaro, 1988):
- Plotted a graph of relative contractile force against calcium concentration at a pH of 6.5 and 7
- At each calcium concentration, there a greater relative contractile force at a pH of 7
- Thus, acidity has a negative effect on cardiac contractile apparatus
- This is because acidity is thought to weaken the interaction between troponin C and troponin I, so that troponin C cannot “pull” troponin I away and allow cross-bridge cycling
Compare and explain the effect that acidity has on cardiac and skeletal muscle. Give experimental evidence.
(Robertson, 2012):
- Compared the amino acid sequence of the cardiac and skeletal troponin I
- Found that cardiac TnI lacks a critical histidine, which strengthens the interaction with TnC at low pH in skeletal muscle
- This histidine is important because it can acquire a positive charge at low pH, so it is more attracted to the TnC
- This means that cardiac contractile apparatus is much more affected by pH than skeletal contractile apparatus
Describe how H+ ions are produced under aerobic and anaerobic conditions.
Give some experimental evidence for the importance of perfusion in removing acid from the heart.
(Garlick, 1979):
- Used phosphorus nuclear magnetic resonance to study the acidity of the heart (since phosphorus emits differently when in the presence of acid)
- When the blood flow was stopped, the pH of the heart dropped
- This shows that perfusion is important in removing acid from the heart and that ischaemia can quickly acidify the heart
Write an equation for the rate of trans-membrane acid venting.
Describe how acid is vented out across the cardiac myocyte membrane.
- CO2 can diffuse freely across the cell membrane
- Lactate and H+ are transported together by the H+-monocarboxylate transporter (MCT1)
Give some experimental evidence for the role of MCT1 in transporting lactate and H+ across the cell membrane.
(Wang, 1994):
- Administered a pH-sensitive fluorescent indicator into cardiac myocytes
- Administered lactate externally
- Observed a rapid fall of intracellular pH, which was largely inhibited by 5 mM alpha-cyano-4-hydroxycinnamate (CHC), a specific inhibitor of the lactate carrier.
Name some advanced pH-sensitive dyeing techniques.
- Nuclear pH / Cytoplasmic pH -> Dyeing just one cellular compartment.
- Permeabilised myocyte -> Where the whole cell is dyed and then the cytoplasmic dye is removed. This leaves mostly just the mitochondria stained.
- Sarcolemmal-targeted pH reporter protein (GFP) -> GFP can be made targeted to a certain part of the cell.
Give some experimental evidence that pH changes occur in the heart physiologically.
(Bountra Kaila, 1998):
- Stimulated a sheep Purkinje fibre at different frequencies
- The pH fell when the frequency of stimulation increased
- This shows that pH “signals” occur physiologically (i.e. not just when acid is applied, etc.)
Draw a diagram showing the way in which acid in the heart leads to feedback.
Give some experimental evidence that intracellular pH is regulated.
(Sun, 1996):
- Acidified the extracellular pH, causing it to drop by 1
- The drop in intracellular pH was smaller than this
- When the extracellular pH is reverted, the intracellular pH also returns to normal
- This suggests that intracellular pH is regulated
- This is done by active transport
Give some experimental evidence for the mechanism by which intracellular pH is regulated.
(Thomas, 1977):
- Injected hydrochloric acid into snail neurons
- Under normal conditions, the pH quickly returned to normal
- In the absence of CO2/HCO3-, the pH returned to normal much more slowly
- This suggested that there were two processes involved in pH control:
- Process A -> This involves active transport of H+ out of the cell
- Process B -> This involves active transport of HCO3- into the cell, so that it reacts with H+ to produce CO2 that diffuses out of the cell
What are the different types of transporter involved in intracellular pH control? When is each active?
- pH can be controlled by either transporting H+ or bases (OH- and HCO3-)
- There are two types of transporters:
- Acid extruders -> These work by either pumping H+ out or OH-/HCO3- into the cell.
- Acid loaders -> These work by either pumping H+ into or OH-/HCO3- out of the cell
- Acid extruders are most active below 7.2 pH, while acid loaders are most active above 7.2 pH.
How can the function of NHE1 be studied?
- When ammonium is administered outside cells, it enters the cell as ammonia. The ammonia inside cells then picks up H+ ions, leading to a rise in pH.
- When the extracellulra ammonium is removed, the opposite happens and the intracellular pH drops.
- The NHE1 then returns the pH to a set point.
- The action of NHE1 can be studied using inhibitors, such as dimethylamiloride, cariporide and zoniporide.
How does NHE1 know how to return intracellular pH to a set point? Give experimental evidence.
(Webb, 2016):
- Studied the C terminus of the NHE1 in different species
- Found that there are 4 highly conserved histidines in close proximity
- Histidines can be protonated/deprotonated so they are good at sensing pH (see previous flashcards) -> This means that these histidines are likely to be responsible for activating the NHE1 when they are protonated in acidic conditions
- This was confirmed by mutation studies
- When the histidine was mutated to the permanently unprotonated alanine, the pH remains low because the NHE1 thinks the cell is alkaline
- When the histidine was mutated to the permanently protonated arginine, the pH remains high because the NHE1 thinks the cell is acidic
Name some positive and negative regulators of NHE1.
These essentially change the set point of the pH.
Where is the NHE1 found in cardiac myocytes?
At the intercalated discs.
How can the function of NBC be studied? Give experimental evidence.
- When ammonium is administered outside cells, it enters the cell as ammonia. The ammonia inside cells then picks up H+ ions, leading to a rise in pH.
- When the extracellulra ammonium is removed, the opposite happens and the intracellular pH drops.
- The NBC then returns the pH to a set point.
- The action of NBC can be studied using inhibitors, such as DIDS and S0859.
(Leem, 1999) did this using amiloride as the inhibitor.
How can the function of CBE (chloride-bicarbonate exchange) be studied? Give experimental evidence.
- An acetate pre-pulse is used to create an alkaline intracellular pH.
- The CBE then returns the pH to a set point.
- The action of NBC can be studied using inhibitors.
A pH of 6 is equivalent to just 1µM (1 x 10-6M). But this graph shows that the NHE works at a rate of around 30mM (3 x 10-2M) at this pH in order to return to the resting pH. Is this not overkill?
No, because a large amount of the H+ is buffered within the cell and this buffered H+ must also be removed.
When an intracellular pH disturbance happens, how can you calculate the corrective H+ flux at any given pH?
- Flux can be written as dH/dt
- dH/dt can be written as (-dpH/dt) x (dH/dpH)
- This is the rate of pH change x the buffering capacity
- This can be calculated at various pH values to plot a graph of the flux against pH
What are the two main types of buffer?
- Extrinsic buffer -> The CO2/HCO3- buffer system. It is the most important buffer system. It is called extrinsic because the CO2/HCO3- can travel in and out of the cell.
- Intrinsic buffer -> Other buffers. They are considered intrinsic because they are confined to the cytoplasm.
What are the main types of mobile buffer in cells?
- CO2/HCO3- (the extrinsic buffer) is a mobile buffer, since the molecules are small
- There is also an intrinsic mobile buffer, which consists of histidyl-dipeptides (dipeptides containing histidine)
- Protein buffers are not mobile
How can we investigate experimentally the rate of H+ diffusion across the cytoplasm? What is the consequence of this rate?
- You can shine UV light on an aldehyde, which causes it to become an acid
- This acid releases a proton that can diffuse across the cell
- It takes around 30 seconds for a proton to diffuse the length of a myocyte, which is around 100 microns
- This is unusual because H+ is very small but is diffuses more slowly than other small ions
- The consequence is that there may be microdomains within the cell with different pH’s
How can H+ travel between cells? Give experimental evidence.
- It can travel through gap junctions attached to a mobile buffer
- (Swietach, 2010):
- Applied UV flashing to neonatal myocytes grown in a stretch of cells
- This UV flashing caused a drop in pH that over time was observed to spread throughout the stretch of myocytes
Describe the effects that intracellular acidosis has on diastolic and systolic calcium levels. Give experimental evidence.
Diastolic - (Swietach, 2013):
- Infused acetate to rapidly acidify rat cardiac myocytes
- This led to a slow increase in intracellular calcium
- This is because the H+ displaces Ca2+ from common buffers, so that there is more free calcium during diastole
Systolic:
- A drop in pH leads to increased activity of the Na+/H+ exchanger (NHE) and NBC
- This leads to an increase in intracellular Na+, which reduces the activity of the NCX
- Thus, intracellular Ca2+ increases and it is stored in the SR
- Since there is an increased SR calcium load, there is more calcium release during systole
- This effect can be inhibited by inhibiting the NHE/NBC
Note that these effects are separate from the effects of acidosis on the LTCCs (see flashcard).
Summarise the effects that acidosis has on the heart’s inotropy.
- Acidosis experimentally leads to decreased inotropy (due to, for example, negative effects on the contractile apparatus)
- However, acidosis can also increase inotropy due to the NBC and NHE transporters (and H+ displacing calcium from buffers), which lead to increased cytosolic calcium over time.
Thus, the NHE and NBC appear to play important roles in sustaining cardiac inotropy by increasing cytosolic calcium.
NHE and NBC activity during intracellular acidosis is usually protective against negative inotropy because it increases cytosolic calcium. When can it become problematic?
- When the NHE/NBC are overloaded, calcium retention in the SR (see flashcard) becomes excessive
- This can lead to spontaneous calcium waves that are not linked to an electrical events
- This must be dealt with by the NCX which is electrogenic and therefore can predispose to DADs (delayed afterdepolarisations)
Give an example of a condition in which the NHE and NBC may be implicated. Give experimental evidence.
(Yamamoto, 2007):
- Tied a knot around the aorta of mice to increase the afterload
- This produced hypertrophy
- Mice with the hypertrophy showed higher levels of NBC and NHE than the normal mice
- This suggests that NHE and NBC are involved in hypertrophy, but it is not clear whether this is the cause or effect of the hypertrophy
Draw how Gs and Gi GPCRs work.
Draw how Gq GPCRs work.
How do GPCRs work in general?
- When the ligand binds, the GPCR undergoes a conformational change
- This leads to the dissociation of the alpha and beta-gamma subunits
- The alpha subunit associates with GTP instead of GDP, allowing it to interact with its target
Draw the downstream pathway of beta-adrenergic receptors.
Note: EPAC is exchange protein directly activated by cAMP. It is involved in cytoskeletal remodelling.
Summarise the main GPCRs in the heart and their functions.
- β1 adrenergic receptor -> Involved in modulating contractility via Gs
- β2 adrenergic receptor -> Involved in regulation of cardiac function [ADD DETAIL] via Gs and Gi
- α1 adrenergic receptor -> Involved in adverse remodelling and arrythmias via Gq
- Angiotensin II type 1 receptor -> Involved in adverse remodelling and arrythmias via Gq
- Other GPCRs -> Coupled to Gs, but not involved in modulating contractility or function
Describe how adrenergic receptors in the heart are involved in heart failure.
- In response to insult, such as myocardial infarction, there is increased adrenergic drive and increased myocyte size
- Chronic stimulation of beta adrenergic receptors leads to increased cardiac workload, myocyte apoptosis and adverse signalling pathways.
- Consequently, there are the markers of heart failure.
How is cAMP-mediated signalling implicated in heart failure? Give experimental evidence.
- During heart failure, there is a marked alteration in cAMP-mediated signalling.
- (Bristow, 1982):
- Examined β-adrenergic receptor density in normal and heart failure patients
- Found a lower density of β-adrenergic receptors in the heart failure patients
- Heart failure is thought to involve a fall in β1 receptor density, so that the β1:β2 ratio decreases from 80:20 to 60:40
- (Ungerer, 1993):
- Found increased levels of GRK (GPCR kinases)
- GRK phosphorylate GPCR and desensitise them
- (Feldmanet, 1988):
- Found upregulation of Gi subunits
- This counteracts the activity of the Gs subunits
What are the two main treatments for heart failure and how do they work?
Beta-blockers:
- Prevents excessive beta adrenoreceptor stimulation (which is pro-apoptotic)
- Reverses beta adrenoreceptor down-regulation
- Improve coronary blood flow and improve left ventircular function
- Others (see diagram)
ACE inhibitors and ARBs:
- Reverse the effects of upregulated angiotensin receptors in heart failure
How does GPCR desensitisation work? Why is this important?
- GRK (GPCR kinases) phosphorylate GPCR
- This marks them out for binding by β-arrestin
- This sterically inhibits G protein activation and also leads to the internalisation and degradation of the GPCR
This is important because chronic activation of beta adrenergic receptors can lead to apoptosis of cardiac myocytes.
Describe the concept of GPCR biased signalling.
- When a GPCR is bound to by a ligand, it can lead to activation of either the normal Gs, Gi, Gq, etc. pathway or the pathway downstream of β-arrestin
- These pathways can be activated to different extents
- This gives rise to several concepts:
- Balanced ligand = A ligand that activates both the G protein and β-arrestin equally
- Biased ligand = A ligand that activates either the G protein or β-arrestin more. Some ligands can activate one pathway and inhibit the other.
- Biased receptor = When the same ligand leads to different pathway activation depending on the receptor it binds to.
Give an example of GPCR biased signalling at the β1 adrenergic receptor.
- Carvedilol is a non-selective beta blocker
- However, it is a biased agonist because it activates β-arresting, leading to downstream pathways (shown in diagram)
Describe the pathway by which certain ligands at β1 adrenergic receptors can have protective effects on cardiac myocyte survival.
- Molecules similar to carvedilol bind to β1 adrenergic receptors
- GRK5 and GRK6 phosphorylate the GPCR
- This leads to β-arrestin binding, which signals via SRC to activate a membrane metalloproteinase (MMP)
- This hydrolyses a membrane epidermal growth factor (EGF)
- The soluble domain of the EGF that is released goes on to transactivate the membrane EGF receptor
- This EGFR signals via the Ras/Raf pathway, which has a cardioprotective effect
- Evidence for this pathway comes from transgenic mice that express β1 receptors lacking the phosphorylation sites (so the GPCR cannot be phosphorylated by GRK), which show increased cardiac myocyte apoptosis
Draw a diagram to show GPCR biased signalling at the angiotensin II receptor.
What is the effect on the cardiovascular system of β-arrestin recruitment at angiotensin II receptors? Give some experimental evidence.
(Violin, 2010):
- Used two β-arrestin biased ligands of the angiotensin II type 1 receptor (TRV120027 and TRV120023)
- These competitively antagonise G protein signalling, but stimulate β-arrestin recruitment
- Set of results 1:
- Graph A shows how β-arrestin (black) and G-protein (red) activation change with angiotensin II concentration
- Graph B shows how valsartan blocks both of these responses by antagonising the receptor
- Graph C shows how TRV120027 and TRV120023 antagonise the G-protein activation but allow β-arrestin recruitment
- Set of results 2:
- Graph A shows that angiotensin II and TRV120027/TRV120023 both lead to phosphorylation of ERK1/2 -> Thus β-arrestin signalling is occuring here (since it activates the MAP kinase pathway)
- Set of results 3:
- Graph A shows that TRV120027 and TRV120023 reduce blood pressure more effectively than losortan (ARB), which shows that they effectively block the G-protein signalling
- Graph B shows that TRV120027 and TRV120023 lead to increases in cardiac contractility relative to baseline and losortan -> This suggests that the β-arrestin is cardioprotective and improves cardiac performance
This is clinically very promising because TRV120027 and TRV120023 have the useful ability to simultaneously promote vasodilation and cardiac contractility.
Describe a mechanism for how GPCR biased signalling might work.
- GPCRs can be thought of as oscillating
- This means that they can take on a variety of different conformations, each with different functional properties
- When each ligand binds, it stabilises the receptor and increases the likelihood of a given conformation (and thus affects whether the G-protein or β-arrestin pathways are activated)
Who came up with the idea of second messengers?
- Earl Sutherland
- He considered the hormone in the blood to be the first messenger and then an intracellular messenger like cAMP to be the second messenger
- The diagram shows the schematic he used in his Nobel prize lecture
Draw a simple model for how cAMP signalling works.
How is cAMP able to activate so many functions?
It activates PKA, which can influence:
- Learning and memory
- Cell growth
- Cell differentiation
- Secretion
- Migration
- Metabolism
- Gene transcription
Give some experimental evidence for the fact that cAMP can have different effects depending on the receptor that was activated to cause the rise in cAMP.
(Hayes, 1979):
- Studied a cardiac myocyte by either exposing it to isoproterenol (a beta agonist) or PGE1
- Compared to control, both agonists lead to significant and similar increases in cAMP and protein kinase activity
- However, only the isoproterenol produced an increase in contractility compared to the control. The PGE1 did not.
- This showed that isoproterenol and PGE1 are able to produce different end effects despite both activating cAMP and PKA.
Describe how Gs GPCR signal specificity is achieved by receptors being confined to specific parts of the cardiac myocyte membrane.
- Different Gs-coupled GPCRs have specific locations on either the cell membrane or on caveolae
- For example, β1 receptors are both in caveolae and the membrane, while β2 receptors are mostly in caveolae. The prostaglandin EP2 receptor is mostly on the membrane.
- This means that the resulting cAMP rise occurs in a specific part of the cell.
- However, the location of receptors is dynamic. For example, β2 receptor activation leads to the β2 moving out of the caveloae.
Give some experimental evidence for the importance of caveloae in GPCR signalling.
(Rybin, 2000):
- Fig 1:
- Divided the cell membrane into several sections
- Used a Western blot to identify the proteins found in each section
- The sections with caveolin-3 (i.e. the caveolae) had high levels of the various beta adrenoceptors, G protein subunits, adenyl cyclase and PKA -> This suggests that the caveolae are important centres for GPCR signalling
- β2 receptors are not found outside of the caveolae -> This shows evidence of membrane compartmentalisation of receptors
- Fig 6:
- Carried out a Western blot of caveolae and non-caveolae membrane before and after application of isoproterenol (beta agonist)
- There was evidence of β2 receptors in the caveolae before the isoproterenol but no after, suggesting that the β2 receptors are dynamic and move out of the caveolae
- Fig 11:
- Applied cyclodextrin to deplete the cholesterol that holds the shape of caveolae (i.e. breaking the caveolae down)
- Found that application of isoproterenol and zinterol produced much greater increases in cAMP with the cyclodextrin than without it
- This suggests that the caveolae somewhat restrict the action of the GPCRs in them
How is spatial confinement of PKA done in cardiac myocytes and what is the significance of this?
- PKA molecules are not free but anchored close to targets by AKAP (A kinase anchoring protein)
- This enables signal specificity
- In other words, although different receptor agonists all lead to increases in cAMP, since this cAMP is spatially confined and so is the PKA, they can have different end effects based on the location of the receptor in the membrane
Give some experimental evidence for the importance of AKAPs.
(Lygren, 2007):
- Used siRNA to knockout AKAP18δ, which is the AKAP that tethers PKA close to phospholamban
- Sarcoplasmic reticulum calcium was depleted using BHQ and then calcium was applied to see how long it takes for the sarcoplasmic reticulum to refill with calcium
- AKAP knockout showed much slower refilling of the SR compared to control, while noradrenaline showed much faster refilling of the SR
- This shows that AKAPs are essential for enabling PKA to activate the nearby target and that a lack of this cannot be compensated by other tethered or free PKA molecules.
How can the distribution of cAMP in the cell be studied?
FRET imaging:
- Two fluorophores are linked by a cAMP-sensitive region
- One fluorophore is cyan (the donor) and the other is yellow (the acceptor)
- When the two fluorophores are in proximity, the donor fluorophore can cause the acceptor fluorophore to be able to emit light also
- However, when the cAMP binds, the two fluorophores move further apart and only the donor emits light
- This means that the ratio of the cyan to yellow emission can be used to quantify the amount of cAMP in that part of the cell
Give some experimental evidence that cAMP is compartmentalised within parts of the cell.
(Zaccolo, 2002):
- Used FRET imaging to observe cAMP signals in cells
- When noradrenaline was administered, it resulted in cAMP spikes within the cell, but these appeared to be compartmentalised within parts of the cell
- When a phosphodiesterase inhibitor was administerd, it resulted in the cAMP spikes no longer being compartmentalised as clearly
- This suggests that phosphodiesterases may be responsible for the compartmentalisation of cAMP in cells
How is compartmentalisation of cAMP within cardiac myocytes enabled?
- Cardiac myocytes express different isoforms of phosphodiesterases (PDE1-9 except PDE6), which have different properties
- These have different amino termini, which contain different targeting domains
- These domains enable each phosphodiesterase to be confined to a particular part of the cell
- These phosphodiesterases prevent diffusion of the cAMP away from the part of the cell in which it is produced
- This is critical in enabling signal specificity of receptors
Aside from tethering PKA, what is another function of AKAPs?
- AKAPs serve as multiscaffolding proteins
- This means that they also tether various other proteins, including the target of the PKA, as well as phosphodiesterases and phosphatases.
- This creates local signalosomes.
- The phosphodiesterases and phosphatases enable negative feedback on the cAMP signalling and therefore enable short-lived localised signalling.
Give some experimental evidence for the existence of local signalosomes on AKAPs.
(Beca, 2013):
- Phosphodiesterase 3A coimmunoprecipitates with SERCA and various other proteins
- This shows that AKAPs tether various proteins, creating functional signalosomes
Are phosphodiesterase inhibitors useful in treating heart failure? Give experimental evidence.
(Anderson, 1991):
- Milirone (PDE inhibitor) improves the short term markers of cardiac performance, including cardiac index and pulmonary wedge capillary pressure
- This is to be expected because heart failure is typically marked by reduced cAMP signalling
(Packer, 1991):
- Studied the survival of severe chronic heart failure patients over 21 months
- Mortality was 53% higher in the milirone (PDE inhibitor) group compared to the placebo
Thus, although PDE inhibitors are effective in the short-term, they are ineffective in the long-term. Paradoxically, beta-antagonists are beneficial in the long-term, even though cAMP signalling is already reduced in heart failure.
Why do PDE inhibitors increase mortality from heart failure in the long term? Give experimental evidence.
- PDE3 inhibitors are not selective and therefore inhibit multiple isoforms of PDE3 (e.g. PDE3A1, PDE3A2, etc.)
- Since the different PDE3 isoforms are compartmentalised within the cell, this means that the increase in cAMP occurs in various parts of the cell and can therefore affect various functions
- Thus, the cAMP also activates ICER (inducible cAMP early repressor), which has been linked to pro-apoptotic signals and early death (Ding, 2005)
Give some experimental evidence for the idea that cAMP compartmentalisation may be disrupted in heart failure.
(Nikolaev, 2010):
- Used scanning ion-conductance microscopy (SCIM) to study the surface of cardiac myocytes and FRET imaging to study intracellular cAMP
- SCIM uses a nanopipette that enables stimulation of either the crest or trough of T-tubules
- In the control:
- A β1 stimulus produced spikes in cAMP when applied at either the crest or the T tubule
- A β2 stimulus produced spikes in cAMP when applied at the T tubule, but not when applied at the crest
- In heart failure:
- A β1 stimulus produced spikes in cAMP when applied at either the crest or the T tubule
- A β2 stimulus produced spikes in cAMP when applied at either the crest or the T tubule
- Thus, heart failure is characterised by disrupted β2 receptor localisation on the membrane.
- Also found that a β2 stimulus produced a more widespread increase in cAMP throughout the heart failure cell than the control.
- This shows that cAMP compartmentalisation is disrupted in heart failure. However, it is not clear whether this is a cause or consequence of heart failure. β2 signalling is usually protective in the heart so the disruption could potentially lead to uncoupling of the protective function.
Are other components of the beta signalling pathway also disrupted in heart failure (apart from the beta receptors)? Give experimental evidence.
(Aye, 2012):
- Generated a lysate from both a normal heart and a failing heart
- Passed the lysate through a solution containing beads bound to cAMP (so that any cAMP-binding proteins and anything bound to that would precipitate out)
- Used mass spectrometry to analyse the components
- Each panel shows a comparison of the amounts of the different components in the control and failing heart
- The results show clearly that there is disruption in the PKA, PDE and AKAP proteins in heart failure
What is the function of oxygen in cardiac myocytes?
- Energy generation
- Generation of ROS
Describe the role of ROS in cardiac myocytes.
ROS regulate the activity of the proteins shown in the diagram.
Give experimental evidence for the existence of a hormone that controls production of red blood cells.
(Carnot, 1906):
- Transfused blood from an anaemic rabbit into a non-anaemic rabbit
- This led to increased RBC production in the non-anaemic rabbit
- Thus, this is evidence that an oxygen-dependent hormone is released into the blood that drives RBC productiion
Describe the structures of the different isoforms of HIF.
Note: The alpha molecules need to form heterodimers with the beta molecules.
Describe the general idea of how HIFs work.
- HIFs are continually produced by the cell
- In the presence of oxygen, HIFs are degraded by PHD (prolyl hydroxylase domain) and FIH (factor inhibiting HIF) enzymes
- PHD hydroxylates the HIF, enabling to to be tagged by VHL for proteasomal degradation
- FIH hydroxylates the HIF, preventing it from activating transcription
- During hypoxia, this degradation/inhibition does not occur, so the HIF can drive transcription
How can Hypoxia Response Elements (HREs) be identified?
By sequence prediction and/or chromatin immunoprecipitation using HIF antibodies:
- The cell is lysed
- The chromatin is fragmented
- Specific antibodies for HIF molecules are used to precipitate out the parts of the chromatin with HIF bound
- The regions where the HIF binds are sequenced to identify HREs
What are the different pathways that HIF can regulate and which ones affect the heart?
How is HIF involved in the heart’s adaptation to hypoxia?
- When the heart is hypoxic (e.g. in ischaemia), there is a shift from oxygen-demanding substrates (e.g. fatty acids) to less oxygen-demanding substrates (e.g. glucose)
- In other words, there is a shift from more oxidative phosphorylation to more glycolysis
- This adaptation is driven by HIF-1α
- HIF-1α drives transcription of genes for: Glucose uptake, Glycolysis and Mitophagy (degradation of mitochondria)
- HIF-1α targets inhibit: TCA cycle, Electron transport chain
How can the rates of glycolysis and oxidative phosphorylation in cardiac myocytes?
Hyperpolarised magnetic resonance spectroscopy (MRS):
- Pyruvate containing C13 is administered into the blood
- This pyruvate can be converted into either lactate (glycolysis) or bicarbonate (oxidative phosphorylation)
- These give off different signals and the ratio of these signals can therefore give an indication of cardiac metabolism
How is HIF-1α implicated in diabetic heart disease and what does this tell us about HIF-1α?
- Cardiac adaptation to hypoxia is impaired in the diabetic heart due to reduced HIF-1α
- In essence, there is the inability to shift towards a more glycolysis-dominated state rather than an oxidative phosphorylation-dominated state -> This shows the importance of HIF-1α in this process
- The reason for reduced HIF-1α is thought to be increased use of fatty acids as metabolic fuel (rather than carbohydrates), which reduces succinate.
- Reduced succinate reduces HIF-1α because succinate is produced by the activity of FIH. Thus, reduced levels of succinate increase the activity of FIH and hence there is less HIF-1α.
How does iron deficiency affect the heart? Give experimental evidence.
- The activity of FIH and PHD is dependent on iron
- Thus, when iron is low, FIH and PHD do not degrade HIF-1α and so there is a glycolytic shift driven by this HIF that can lead to heart failure
- (Lakhal-Littleton, 2016):
- Created genetically engineered mice that have leaky cardiac myocytes (i.e. the mice are iron deficient only in the heart)
- These mice underwent a glycolytic shift that caused fatal heart failure
How does the glycolytic shift resulting from iron deficiency lead to heart failure?
What is ischaemia reperfusion injury and what is the mechanism?
- Ischaemia reperfusion injury occurs after an area of the heart undergoes ischaemia for a prolonged period and is then reperfused
- The reperfusion causes injury by a number of mechanisms:
- Generation of excess ROS
- Opening of MPTP channels in the inner mitochondrial membrane
How is HIF related to ischaemia reperfusion injury?
- When short bursts of ischaemia occur before the main period of ischaemia, this induces HIF, which is protective against ischaemia reperfusion injury
- This ischaemic pre-conditioning is because HIF is thought to inhibit formation of ROS and opening of MPTP channels
How can HIF’s role in ischaemic pre-conditioning be exploited clinically?
- PHD inhibitors could be used to stabilise HIF during normoxia
- This increased HIF could in theory protect against ischaemia reperfusion injury following organ transplant and following post-MI stenting
In Figure 1, what prevents Ca ions released from SR (left, top corner) from entering the nucleus, and vice versa (i.e. cross contamination)?
Calcium ions are very strongly buffered, so they diffuse very slowly across the cell. Their buffering ratio is about 100:1.
In (Bers, 2015), what is the point of diagram 2D?
- The graph shows Rhod-2 fluorescence (which is a calcium dye)
- This dye shows that global calcium within the cell does not increase
- Thus, excitation-transcription coupling is mediated by local calcium signals, not global signals
In (Bers, 2015), table 2C shows that the fluorescence ratio between the nucleus and cytosol changes over time. This suggests that HDAC5 tagged with GFP is being exported out from the nucleus. Are there any alternative explanations?
It is possible that the HDAC5-GFP is being degraded in the nucleus, changing the fluorescence ratio.
In (Bers, 2015), excitation-transcription coupling was studied by stimulating the mouse ventricular myocytes at around 0.5-1 Hz. Comment on this pacing.
- This frequency of stimulation is lower than the typical heart rate in mice
- But it is used because:
- The resolution of the dye is limited and it stops the signals becoming overlapping
- Stimulating single cells at a high rate promotes arrythmias since single cells are already susceptible to this
In (Bers, 2015), what does this diagram show? What is missing?
- This is a Western blot for phosphorylated CaMKII (which indicates autophosphorylation i.e. activation)
- The test is run in presence of ET-1 and adenophostin
- The results show that the amount of phosphorylated CaMKII is increasing over time, indicating the pathway of E-T coupling
- However, the loading control is missing. In this case it would be a Western blot against unphosphorylated CaMKII, since it is the ratio of phosphorylated to unphosphorylated CaMKII that we are interested in.
In (Bers, 2015), graph 6B shows how MEF2 reporter activity changes in the presence of ET-1 (MEF2 is a transcription factor that HDAC5 normally represses). What does the graph show? Why is the experiment done twice?
- MEF2 activity decreases in the presence of ET-1, which suggests that ET-1 elicits E-T coupling by causing HDAC5 to be exported from the nucleus, derepressing MEF2
- The paper relies on viral insertion of a GFP-labelled HDAC into the cell, which leads to overexpression since both the endogenous and viral HDAC are expressed in the cell
- The ET-1 and ET+TG experiments are repeated with and without the overexpression of HDAC5. This allows us to see how much of an effect the overexpression of the HDAC5 is having.
- The large difference between the repeats suggests that the overexpression significantly changes the activity of the HDAC5, which somewhat weakens the rest of the paper. You are essentially not studying the experiment in a wild-type model. However, it is unavoidable and it is not necessarily a problem.
