Myocardial proteins/contraction cycle

Question 1

Ultrastructure of cardiac muscle

Answer 1

• Myo¢ contain numerous myofibrils
• Myofibrils = longitudinally repeating sarcomeres

Question 2

Define sarcomere

Answer 2

from Z line to Z line, 1.6-2.2um
o Thin filament: Z line to center
 Form I band
o Thick filament: center (M line) => Z line
 Form H band
o Overlap of filaments = darker zone = A band

Question 3

Structure of thick filaments

Answer 3

MYOSIN
* Tail: confer stability
o Heavy chains form the A helical tail
o Light chains: link tail => heads
 4 lights chains/ bilobed head
 2 types: MLC-1 and MLC-2
* 2 heads: 2 distinct sites to interact w actin and hydrolyze ATP
o S1 subfragment: head
 ATP binding => conformational change
o S2 subfragment: neck connecting the head to filament

• Myosin molecules form myosin filaments: 300-400 myosin heads

Question 4

Myosine isoenzymes

Answer 4

2 isoforms of heavy chains = Aand B
o V1: 2 A chains = high ATPase activity => fast muscle contraction
o V2: AB heterodimer
o V3: 2 B chains = low ATPase activity => slow muscle contraction
 Primarily in normal adult myocardium

Question 5

Regulation of myosin ATP ase

Answer 5

 Short term: incr [Ca2+] => incr myosin ATPase activity => V1
 Long term: gene changes with CHf, hypertrophy -> V3

Question 6

Role of myosin binding protein C

Answer 6

o Prevent interaction of actin and myosin decr [Ca2+]
o Stabilize S2 subfragment

Question 7

Thin filaments structure

Answer 7

• 2 strains of fibrous actin => double A helix filament
  o G actin monomere (multiple globular units) that assemble into filament (F-actin) at physio [ATP/Mg]
  o Interact w globular head of myosin => form cross bridges

Question 8

What are the regulatory proteins around contractile proteins

Answer 8

Ca2+ dependent regulation
o Troponin: globular prot
 TnC: binds Ca2+ => conformational changes in tropomyosin
 TnI: inhibit Mg stimulated actomyosin ATPase
* incr[Ca2+] => firmly bind to TnC (and not to actin) to allow contraction
 TnT: attach troponin to tropomyosin
* incr Ca2+ => activate actomyosin ATPase
o Tropomyosin: double strand A helical chain
 Lie in 2 major groove along actin filament in diastole

Question 9

Titin role

Answer 9

• Link the myosin molecule to the Z line and stabilize filaments
• Provides elasticity: molecular spring modulating systole and diastole
  o Expand and contract as sarcomere stretch
  o incr systolic contraction in response to stretch and deformation
• Role in transducing stretch signal into growth
  o Interact w 2 prot of Z disc

Question 10

Steps of excitation-contraction coupling

Answer 10

Resting state: tropomyosin block myosin binding sites on actin
a) Ca2+ bind to TnC => displace TnI => conformational change of tropomyosin
b) Exposition of actin sites => myosin heads binding sites
a. decr [ATP] => high affinity (severe hypoxia, ischemia, death)
b. Need ATP + Ca2+
c) ATP cleaved by myosin ATPase => ADP + Pi => bind myosin + actin => cross bridge
a. Formation of strong binding state
b. E derived from rx used to generate movement (ratchet like)
c. Rigor state
d) Release ADP + Pi => bind new ATP => myosin conformation change => dissociate actin/myosin
a. Conformation change: strong binding state to weak binding state until cycle starts again

Question 11

Sarcomere shortening depend on

Answer 11

Cycle repeated to have significant sarcomere shortening
* If incr [Ca2+] => tropomyosin block active sites of actin
* Contraction force and rate of shortening => related to # cross bridges + [Ca2+]
* Cycling rate is dependent on the rate of attachment (F) and detachment (G) of the cross bridges

Question 12

Control of contractile cycle by Ca2+

Answer 12

incr force development
* incr activation of cross bridges by Ca2+
o Binding Ca2+ to TnC
 Molecular signal in actin filament => incr rate of cross bridges
 TnC bind TnI => relieve inhibition
 incr [Ca2+] => incr Ca2+ binding to TnC =>incr # cross bridges
o B adrenergic stimulation: cAMP => PKA
 incr lusitropy (see below): phospholamban Pi + decr [Ca2+) will incr TnC dissociation
* Pi or dPi of contractile proteins (see below)
* Starling mechanism

Question 13

Intracell Ca2+ normal

Answer 13

• Intra¢ [Ca2+] > extra¢ [Ca2+] => large chemical gradient for Ca2+ entry
  o Ca2+ transient: cyclical variation in cytosolic [Ca2+]
   incr by B adrenergic stimulation

Question 14

Sarcolemmal channels important for excitation contraction coupling

Answer 14

L type channels Ca2+ channels
Na+/Ca2+ exchanger
Na+/K+ ATPase
Ca2+ pump

Question 15

Structure of L type Ca2+ channels

Answer 15

• Structure: 5 subunits
  o A1 => active unit, pore to enter Ca2+
  o 4 repeated units containing 5 hydrophobic helices + 1 charged hydrophilic helix

Question 16

Role of L type ca2+ channel

Answer 16

• Will sense change in voltage during depolarization in phase 2 of AP
• Opening => small amount of Ca2+ enters => stimulate release of larger amounts

Question 17

Structure of SR and important channels

Answer 17

• Membrane limited structure intracell surrounding myofibrils

SERCA Ca2+ ATP ase pump
Ca2+ release channels RYR

Question 18

Functions of SR

Answer 18

o Actively pump Ca2+ in its lumen during diastole
o Store Ca2+ in diastole
o Release Ca2+ in systole
 incr with incr [Ca2+] triggering or incr amount of Ca2+ in SR
* incr # of channels or incr amount of Ca2+ released
 Controlled by local [Ca2+]

Question 19

Role of SERCA channels

Answer 19

o SERCA-2a is the isoform present in cardiac myo¢
 Bind Ca2+ and ATP
o Activated by incr intra[Ca2+] => actively pump Ca2+ into SR lumen in diastole
 Remove 75% of Ca2+
 1ATP => moves 2 Ca2+ into SR

Question 20

Regulation of SERCA channels

Answer 20

o Inhibited by phospholamban (monomer)  Pi relieve inhibition
 B adrenergic stimulation: cAMP => PKA
 incr [Ca2+] promote Pi at 2nd site => incr rate of relaxation and Ca2+ stores

Question 21

Structure Ca2+ release channels Ryanodine R

Answer 21

4 ryanodine R protein to form 1 RyR

Question 22

Control of RyR

Answer 22

 B adrenergic stimulation => PKA Pi
* inrc rate of incr/decr of intra¢ [Ca2+]
* Pi RyR => incr rate of Ca2+ release
* Pi phospholamban => incr SR Ca2+
 A adrenergic stimulation => IP3
* incr Ca2+ release via IP3 R on RyR
 Caffeine: incr probability of opening of RyR => can empty SR
o Closure: 3 possibilities
 Continuing incr in local [Ca2+] at supraoptimal levels
 Local decr or SR Ca2+
 Stochastic attrition: spontaneous decay of active clusters due to random channel closure => all L type + release channels close at same time => decr [Ca2+]
o Ca2+ sparks: small amount of Ca2+ locally/spontaneously released w/o L type opening
 Not enough to induce depol

Question 23

Role of RyR

Answer 23

Stimulated by Ca2+ entering ¢ by L type channels
 Conduit for rapid Ca2+ release from SR in systole

Question 24

Ca2+ regulatory proteins/factors

Answer 24

• Calmodulin (CaM): regulatory protein
  o Activated w Ca2+ binding
• Calcineurin: CaM activated growth factor => sustained incr [Ca2+] => promote hypertrophy
• Calsequestrin: prot binding Ca2+ w/i SR (strong – charge)
  o Allow rapid Ca2+ mvt
  o Calreticulin is another storage protein similar in structure/fct
  o Low affinity, high capacity
• Phospholamban: on SR membrane => inhibit Ca2+ pump in dePi state

Question 25

Structure of transverse tubular system (T system)

Answer 25

* Continuous w ¢ membrane, in close association w SR * T tubule + junctional SR (terminal section) => form triads o Foot proteins = 7-29 Ca2+ release channels close to 1 Ca2+ L type channels  Arrival of Ca2+ ions from L type channels => conformational change in foot => open release channels

Question 26

Na+/Ca2+ exchanger role

Answer 26

3Na+ in for 1Ca2+ out o Function: efflux of Ca2+ in diastole (25% of Ca2+ released by SR)  Activated by ↑ Ca2+ and ATP  Inactivated by Na+ o Can move Ca2+ both direction depending on [Na+] o Transient Ca2+ influx can occur at depol state after Na+ channel opening => Na+ accumulate close to channels => transient activation of exchanger in reverse direction => incr Ca2+ => incr contraction

Question 27

Na+/K+ ATPase role

Answer 27

maintain low [Na+] to allow Ca2+ efflux o Activated by incr [Ca2+], ATP o Inactivated by incr [Na+]

Question 28

Ca2+ pump role

Answer 28

Ca2+ efflux w ATP o Respond to incr [Ca2+] in diastole to maintain Ca2+ homeostasis

Question 29

What determines contraction force

Answer 29

of active cross bridges => determine contraction force * Normal heart: Ca2+ released for 10-25% myofilament activation

Question 30

Cellular regulation of myocardial force generation

Answer 30

a) Amount of Ca2+ entering through L type channels b) Amount of Ca2+ stored in SR c) Myofilaments proteins => phosphorylation d) Starling’s law e) ATP availability: deficiency impairs performance

Question 31

How amount of Ca2+ going into L type channels influence force of contraction

Answer 31

* incr activity with: o B adrenergic stimulation: cAMP prot kinase phosphorylation  incr release by RyR o Calmomodulin mediated prot kinase => activate cGMP  Respond to transient incr in [Ca2+] from hormones, NT, depol * incr activity with acidosis => slow channel

Question 32

how amount of Ca2+ stored into SR influence force of contraction

Answer 32

* Autoregulation: o incr SR [Ca2+] => incr RyR release => decr Ca2+ entry and incr efflux o decr SR [Ca2+] => incr rapid refilling => SERCA activation * B adrenergic stimulation => G prot coupled => Gi or Gs o Gs: activate AC => cAMP stimulat cAMP dependent prot kin => phospholamban phosphorylation => incr Ca2+ pump activity

Question 33

How myofilament protein phosphorylation influence force of contraction

Answer 33

* TnI Pi: by cAMP dependent prot kin o decr affinity of TnC for Ca2+ => incr relaxation by incr rate of Ca2+ dissociation * Myosin binding prot C Pi * Myosin light chains Pi: calmomodulin kin, myosin light chain kin, PCK o incr responsiveness to Ca2+ o incr force generation at low Ca2+ * TnT isoforms will change o Myofibrillar ATPase activity o Sensitivity of myofilaments to Ca2+ o Sarcomere length dependance of myofilament sensitivity to Ca2+

Question 34

How starling laws influence force of contraction

Answer 34

incr contraction force with incr sarcomere length * incr SV w incr end diastolic volume/pressure * incr affinity of TnC for Ca2+ as sarcomere length incr => incr cross bridge #R

Question 35

Role of mitochondria in intracell ca2+ control

Answer 35

*Mitochondria play no role in beat to beat control of intra Ca2+ * ↑ [Ca2+] w/ increase in intracell cytosolic Ca2+ → may stimulate citric acid and produce more ATP * Buffer by storing excess Ca2+

Question 36

Effect of CHF on pumps

Answer 36

* decr activity of SERCA + incr activity of Na+/Ca2+ exchanger => decr Ca2+ SR stores => decr contractility * HyperPi of RyR => incr Ca2+ leaks * decr # or mRNAs for RyR, SERCA and phospholamban

Question 37

Effect of arrhythmias on intra cell Ca2+

Answer 37

Ca2+ overload * 2nd to myocardial ischemia, reperfusion, incr catecholamine stimulation * incr O2 demand by mitochondria o Attempt to buffer incr intra¢ [Ca2+] => mitochondrial Ca2+ overload => ATP waste => incr O2 needs * Stimulation of phospholipase enzymes => incr membrane breakdown * Can cause excess sustained contraction => contracture * Excess Ca2+ cycling in/out of SR => substrate for arrhythmias