Theme 1: Signalling in the Cardiac Myocyte Flashcards

Question

What are ratiometric and non-ratiometric calcium dyes?

Answer 1

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).

Answer 2

Ratiometric

Answer 3

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.

Answer 4

Non-radiometric dyes have better temporal resolution, which is important in fast events, such as cardiac myocyte contraction.

Answer 5

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.

Answer 6

(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

Answer 7

* 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

Answer 8

* 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.

Answer 9

(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.

Answer 10

* 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.

Answer 11

(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

Answer 12

1. Ca²⁺ flux through L-type calcium channels is about 0.3pA, while it is around 0.4 - 1.0pA through ryanodine receptors 2. Each L-type calcium channel leads to the opening of multiple ryanodine recptors 3. Ryanodine receptors open for longer than L-type calcium channels

Answer 13

Calcium spark

Answer 14

They can be both: * They can be evoked by cardiac mycoyte depolarisation * They can also occur spontaneously when ryanodine receptors open spotaneously

Answer 15

* 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

Answer 16

The summation of multiple calcium sparks

Answer 17

* 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

Answer 18

* 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

Answer 19

Yes, although this is controversial. The evidence for it is quite strong.

Answer 20

(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

Answer 21

* 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.

Answer 22

All 3 isoforms.

Answer 23

(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

Answer 24

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

Answer 25

* 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.

Answer 26

(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

Answer 27

(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

Answer 28

* Primary pacemaker * Subsidiary pacemaker

Answer 29

Caveolae, which are smallninvaginations of the plasma membrane.

Answer 30

* HCN4 (hyperpolarisation activated cyclic nucleotide gated) -\> Responsible for the funny current upon hyperpolarisation (sodium current) * Beta-2 receptor * NCX * Ca_v1.2 (L-type calcium channel) * Ca_v3 (T-type calcium channel) The sarcoplasmic reticulum is closely apposed to this, such that most of the RyR are found near the sarcolemma.

Answer 31

* 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

Answer 32

* The membrane is gradually depolarised by 3 main inwards currents (phase 4): 1. Sodium currents are partly background currents and ‘funny’ currents (I_f) through non-selective channels that open upon hyperpolarisation, allowing sodium and potassium to flow. 2. 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. 3. 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.

Answer 33

(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.

Answer 34

* 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.

Answer 35

The upstroke is determined by calcium, rather than sodium, which means that it is intrinsically inter-connected with calcium release from the SR.

Answer 36

(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

Answer 37

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

Answer 38

(Ju, 2011): * IP₃R2 are found at the sarcolemma and also slightly along the tubules

Answer 39

(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

Answer 40

* 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

Answer 41

(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

Answer 42

* 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.

Answer 43

* 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)

Answer 44

* Dihydropyridines (nifedipine, nimodipine) * Phenylalkylamines (verapamil) * Benzothiazapines (diltiazem)

Answer 45

* 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

Answer 46

* 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

Answer 47

* Calmodulin * Calmodulin kinase II * cAMP / Protein kinase (related to beta agonists)

Answer 48

* 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

Answer 49

* It is the largest ion channel in the body -\> This allows there to be lots of modulatory sites * It is a homotetramer

Answer 50

No, they can be in vesicles that are then inserted into the membrane.

Answer 51

* 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

Answer 52

On cytosolic side: * Ca²⁺/calmodulin-dependent protein kinase (CMKII) * Calmodulin * Phosphodiesterase 4D * PKA * FKBP12/12.6 * PP1 * PP2A On SR lumen side: * Calsequestrin

Answer 53

CaM regulates RyR2 and many other proteins in the heart that are involved in calcium signalling (including LTCC and other kinases).

Answer 54

It has 4 EF hands that are important for binding.

Answer 55

They result in cardiac arrhythmias.

Answer 56

Ca²⁺/calmodulin-dependent protein kinase (It can also be referred to as CaM kinase)

Answer 57

* 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

Answer 58

* 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

Answer 59

* Noradrenaline and adrenaline bind to β₁ adrenoceptors, which are G_s-coupled GPCRs that stimulate adenylate cyclase and increase intracellular cAMP, and therefore protein kinase A. * cAMP stimulates the **funny current** (I_f) -\> 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

Answer 60

* 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

Answer 61

* 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

Answer 62

* 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

Answer 63

* 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.

Answer 64

(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

Answer 65

* 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.

Answer 66

* 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

Answer 67

* 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

Answer 68

* SERCA pump * NCX * Membrane ATPase * Mitochondrial ATPase

Answer 69

(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

Answer 70

* The SERCA pump moves 2 Ca²⁺ per ATP * The NCX moves 1 Ca²⁺ per ATP Thus, the SERCA pump is more efficient.

Answer 71

(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

Answer 72

The larger the amplitude, the stronger the contraction (Bers, 2002).

Answer 73

Troponin C

Answer 74

They stretch along the mytocyte, so they can take up calcium from multiple T-tubules.

Answer 75

(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.

Answer 76

(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

Answer 77

* 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)

Answer 78

* 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.

Answer 79

Note how the mitochondria act as a buffer when the calcium reaches a certain level.

Answer 80

(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

Answer 81

(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

Answer 82

* 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)

Answer 83

* 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

Answer 84

* Heart failure with reduced ejection fraction (systolic heart failure) * Heart failure with preserved ejection fraction (diastolic heart failure)

Answer 85

* 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.

Answer 86

* 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

Answer 87

* 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 IP₃signalling driven by ET-1 overall leads to hypertrophy and heart failure.

Answer 88

(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

Answer 89

(Gaskell, 1880): * Showed that an ex vivo heart contracts less when it is exposed to acid (Swietach, 2009): * Studied an isolated guinea pig myocyte * Found that the percentage shortening upon stimulation was less with increased acidity

Answer 90

Essentially all of them.

Answer 91

(Saegusa, 2011): * Extracellular acidosis blocks calcium currents, shortening the action potential * Intracellular acidosis promotes calcium currents, lengthening the action potential

Answer 92

(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

Answer 93

(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

Answer 94

It is produced continuously in cardiac cells in large amounts (several mmol/min/L of cell). Most other small ions are just shifted between compartments.

Answer 95

It is transported to across the membrane, then diffuses to blood vessels, where perfusion happens.

Answer 96

(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

Answer 97

* CO₂ can diffuse freely across the cell membrane * Lactate and H⁺ are transported together by the H⁺-monocarboxylate transporter (MCT1)

Answer 98

(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.

Answer 99

* BCECF * cSNARF Both of these are ratiometric dyes.

Answer 100

* 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.

Answer 101

It depends on these three questions: 1. Is the signal evoked in a physiological context? 2. Does it produce a robust response? 3. Is the signal tightly regulated?

Answer 102

(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.)

Answer 103

(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

Answer 104

(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

Answer 105

* 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.

Answer 106

* NHE -\> Sodium/H⁺exchanger * NBC -\> Sodium/bicarbonate co-transporter

Answer 107

* CHE -\> Chloride/OH^- exchanger * CBE -\> Chloride/bicarbonate exchanger

Answer 108

NHE1 is found in almost all of the cells in your body.

Answer 109

* 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.

Answer 110

(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

Answer 111

These essentially change the set point of the pH.

Answer 112

At the intercalated discs.

Answer 113

* NBCn1: Na⁺-HCO₃^- (non-electrogenic) * NBCe1: Na⁺-2HCO₃^- (electrogenic)

Answer 114

* DIDS * S0859

Answer 115

* 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.

Answer 116

* Since it is electrogenic, it means that pH and membrane potential are related. * This means that changes in pH can alter action potentials and thus cardiac contraction. * For example, when NBCe1 is activated, it leads to shorter action potentials.

Answer 117

Acid loaders utilise the chloride gradient, while acid extruders utilise the sodium gradient.

Answer 118

* 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.

Answer 119

No, because a large amount of the H⁺ is buffered within the cell and this buffered H⁺ must also be removed.

Answer 120

* 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

Answer 121

* Buffers are a passive storage system for H⁺ ions * There is no active transport and so they are only a temporary solution for pH disturbances * You can this of them like bins in your room. You can clean up your room by putting the rubbish in the bin but ultimately the only way to get rid of the rubbish is to take the rubbish out of your room.

Answer 122

* 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.

Answer 123

For every 400,000 H⁺ions, only 1 is free.

Answer 124

* H⁺ ions cannot diffuse freely * H⁺ ions diffuse as fast as the buffers that carry them * To allow adequate diffusive coupling, some buffers must be small molecules

Answer 125

* 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

Answer 126

* 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

Answer 127

* 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

Answer 128

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).

Answer 129

* 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.

Answer 130

* 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)

Answer 131

(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

Answer 132

* Intracellular pH exerts multiple effects on Ca²⁺ signalling, including: * H⁺/Ca²⁺ competition for common buffers (influences diastolic Ca²⁺) * H⁺ modulation of Ca²⁺ currents (e.g. effect on LTCC) * Coupling involving Na⁺ as an intermediate (e.g. NHE-NCX coupling) * Dysregulated pH can, directly or via Ca²⁺ signals, affect inotropic state, trigger arrhythmias and activate pro-hypertrophic signalling.

Answer 133

In most cases, it is the alpha subunit, but with G_i G-proteins, the beta-gamma subunit also activates the K⁺ channels.

Answer 134

* 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

Answer 135

Note: EPAC is exchange protein directly activated by cAMP. It is involved in cytoskeletal remodelling.

Answer 136

* β1 adrenergic receptor -\> Involved in modulating contractility via G_s * β2 adrenergic receptor -\> Involved in regulation of cardiac function [ADD DETAIL] via G_s and G_i * α₁ adrenergic receptor -\> Involved in adverse remodelling and arrythmias via G_q * Angiotensin II type 1 receptor -\> Involved in adverse remodelling and arrythmias via G_q * Other GPCRs -\> Coupled to G_s, but not involved in modulating contractility or function

Answer 137

ACE inhibitors have been used with high efficacy to treat heart failure.

Answer 138

* 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.

Answer 139

* 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 G_i subunits * This counteracts the activity of the G_s subunits

Answer 140

No, they are detrimental in the long term. This is likely to be because of the pro-apoptotic effects of chronic beta stimulation.

Answer 141

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

Answer 142

* Effectiveness is limited in some patients (e.g. in heart failure with preserved ejection fraction, where the problem is not heart contraction, but heart filling) * Side effects are significant

Answer 143

* 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.

Answer 144

GRK phosphorylates GPCRs that are chronically activated, so that β-arrestin can bind: * This leads to the desensitisation of the receptor * β-arrestin can also activate different signalling pathways, such as the MAP kinase pathway

Answer 145

* When a GPCR is bound to by a ligand, it can lead to activation of either the normal G_s, G_i, G_q, 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.

Answer 146

* 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)

Answer 147

* 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

Answer 148

(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.

Answer 149

* 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)

Answer 150

* cAMP * IP₃ (to a lesser extent)

Answer 151

* 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

Answer 152

It activates PKA, which can influence: * Learning and memory * Cell growth * Cell differentiation * Secretion * Migration * Metabolism * Gene transcription

Answer 153

(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.

Answer 154

Signal specificity is achieved by spatial control of cAMP signalling: * Receptors are confined to specific parts of the membrane * They can be in caveolae or on the rest of the membrane * PKA is anchored close to its targets * AKAPs tether the PKAs * cAMP is confined to specific compartments within the cell * Phosphodiesterases prevent diffusion of the cAMP away from the original location This means that when an agonist binds to a receptor, cAMP production is confined to a certain part of a cell and this only activates PKA in proximity. This PKA can then only activate the targets that it is anchored close to.

Answer 155

* Different G_s-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.

Answer 156

(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

Answer 157

* 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

Answer 158

(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.

Answer 159

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

Answer 160

(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

Answer 161

* 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

Answer 162

* 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.

Answer 163

(Beca, 2013): * Phosphodiesterase 3A coimmunoprecipitates with SERCA and various other proteins * This shows that AKAPs tether various proteins, creating functional signalosomes

Answer 164

(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.

Answer 165

* 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)

Answer 166

(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 β₂receptor localisation on the membrane. * Also found that a β₂stimulus 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. β₂signalling is usually protective in the heart so the disruption could potentially lead to uncoupling of the protective function.

Answer 167

(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

Answer 168

Fatty acids

Answer 169

* Energy generation * Generation of ROS

Answer 170

* They are generated as a byproduct of metabolism, mainly in the mitochondria. * Complexes I and III of the ETC are the main sites of ROS generation. * 0.2-2% of the electrons in the ETC leak out and interact with oxygen to produce superoxide or hydrogen peroxide

Answer 171

* In excess, they are harmful and lead to the damage of lipids, protein and DNA * However, cardiac myocytes require a certain level of ROS for proper function. ROS modify proteins, enabling them to carry out important physiological functions.

Answer 172

ROS regulate the activity of the proteins shown in the diagram.

Answer 173

(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

Answer 174

* In the kidneys * By renal peritubular fibroblasts

Answer 175

(Paliege, 2010): * Co-localisation = Observation of the spatial overlap between two different fluorescent labels, each having a separate emission wavelength, to see if the different "targets" are located in the same area of the cell or very near to one another. * EPO co-localises with HIF-2α

Answer 176

All cell types

Answer 177

* HIF-1α -\> All tissues * HIF-2α -\> Kidney, Liver, Heart, Lung, Carotid body, Intestine * HIF-3α -\> Adipose tissue, Cerebral Cortex, Hippocampus, Lung

Answer 178

Note: The alpha molecules need to form heterodimers with the beta molecules.

Answer 179

* 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

Answer 180

* Oxygen * 2OG * Iron * Ascorbate

Answer 181

* Prolyl Hydroxylase Domain (PHD) -\> Hydroxylates the ODDD domain, targeting HIF for degradation by the VHL E3 ligase pathway. * Factor Inhibiting HIF (FIH) -\> Hydroxylates asparaginyl residues on the TAD domain. This leads to inhibition of HIF transactivation by p300, which is necessary for HIF to promote transcription.

Answer 182

The HIF complex binds consensus sequences known as Hypoxia Response Elements (HREs) distributed throughout the genome.

Answer 183

HREs may be found in promoter regions, intronic regions and even distant regions outside the target gene.

Answer 184

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

Answer 185

* 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

Answer 186

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

Answer 187

* 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.

Answer 188

The shift from oxidative phosphorylation to glycolysis in cardiac myocytes during ischaemia.

Answer 189

* In the short term it is beneficial because it enables continued production of ATP in the absence of oxygen. * In the long term it is detrimental because: 1. Energetic insufficiency -\> Glycolysis is less efficient than oxidative phosphorylation 2. Ionic imbalance -\> Glycolysis produced lactate, which raises intracellular pH. This excess H⁺ drives the activity of the NHE, which in turn drives the activity of the NCX, leading to increased calcium influx. * Thus, glycolytic shift in the long term can lead to contractile dysfunction of the heart.

Answer 190

* 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

Answer 191

* 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

Answer 192

* 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

Answer 193

* 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

Answer 194

* Calcium is a charge carrier (which makes sense because excitation involves currents, so it makes sense for calcium to be charged) * Calcium is a small ion with a large charge, so it has high polarising power * The cell has a way of keeping free intracellular calcium very low -\> This means that a calcium signal is very clear and there is little background interference

Answer 195

* ET coupling is the way in which excitation of a cardiac myocyte can lead to transcription changes * ET coupling is hard to study because: * Transcription is a delayed process, so there is a lot of error and it is difficult to keep cells alive (as opposed to EC coupling, for example) * It is difficult to measure transcription. It is also hard to know what is being transcribed.

Answer 196

* No * This conclusion is model-dependent since the model is an assumption for the heart failure to occur this way

Answer 197

* Use antibodies. Repeat with an IP₃R knockout to show that the antibodies are binding specifically to the IP₃Rs. * Use RNA sequencing. * Introduce IP₃ into the cell to see if there is a response.

Answer 198

Calcium ions are very strongly buffered, so they diffuse very slowly across the cell. Their buffering ratio is about 100:1.

Answer 199

Abstract: Previous work showed that calmodulin (CaM) andCa2+-CaM–dependent protein kinase II(CaMKII) are somehow involved in cardiac hypertrophic signaling,thatinositol 1,4,5-trisphosphate receptors (InsP3Rs)in ventricular myocytes are mainly in the nuclear envelope, where they associate with CaMKII, and that class II histone deacetylases (e.g., HDAC5) suppress hypertrophic gene transcription. Furthermore, HDAC phosphorylation in response to neurohumoral stimuli that induce hypertrophy, such as endothelin-1 (ET-1), activates HDAC nuclear export, thereby regulating cardiac myocyte transcription. Here we demonstrate a detailed mechanistic convergence of these 3 issues in adult ventricular myocytes. We show that ET-1, which activates plasmalemmal G protein–coupled receptors and InsP3 production, elicits local nuclear envelope Ca2+ release via InsP3R. This local Ca2+ release activates nuclear CaMKII, which triggers HDAC5 phosphorylation and nuclear export (derepressing transcription). Remarkably, this Ca2+-dependent pathway cannot be activated by the global Ca2+ transients that cause contraction at each heartbeat. This novel local Ca2+ signaling in excitation-transcription coupling is analogous to but separate (and insulated) from that involved in excitation-contraction coupling. Thus, myocytes can distinguish simultaneous local and global Ca2+ signals involved in contractile activation from those targeting gene expression. Summary: Showed that ET-1 activation of GPCR leads to IP₃ production and thus IP₃R activation. This leads to calcium release from the nuclear envelope. The calcium activates CaMKII, which in turn leads to the phosphorylation of HDAC5 and its export from the nucleus. This reduces transcription. Thus, this is an example of excitation-transcription coupling, where excitation of the cell leads to changes in transcription. Interestingly, this effect cannot be elicited by the calcium transients that trigger contraction, showing that myocytes can distinguish simultaneous local and global Ca2+ signals involved in contractile activation from those targeting gene expression.

Answer 200

* The cardiac myocytes were infected with an exogenous HDAC5 that was labelled with GFP * Drawbacks: * The GFP could influence the function of the HDAC5 * There is overexpression of the HDAC because the endogenous HDAC is also being expressed -\> This could alter function * Alternatively, you could just use a wild type cell and use antibodies to label the HDAC. However, this experiment is run over a period of time, whereas antibodies just give a 'snapshot' of the cell because they are terminal.

Answer 201

* HDAC translocation was triggered by administering an agonist (endothelin-1) * The issue with this is that it was administered at 100nm, which is not necessarily the physiological concentration. * Ideally, you would plot a dose-response curve on which you can mark the typical physiological concentration of ET-1

Answer 202

* 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

Answer 203

It is possible that the HDAC5-GFP is being degraded in the nucleus, changing the fluorescence ratio.

Answer 204

* 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

Answer 205

* 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.

Answer 206

* 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.