Length dependent modulation of cardiac force

Question 1

Define cardiac output

Answer 1

Cardiac Output = Heart Rate x Stroke Volume

Question 2

How does the length dependence alter cardiac output?
What law determines change in force due to stretch?
Why?
Is it mono or bi-phasic?

Answer 2

Stretch (filling) affects the force (contractility) and the rate (electrical activity)

Frank-Starling Law of the Heart

• Increase in end-diastolic volume of the ventricle leads to an increase in stroke volume
• Could be due to increase in venous return (exercise) or aortic resistance (hypertension)
• More the heart fills during diastole, the greater the force of contraction during systole
• Allows heart to respond to demands of circulation

Response to stretch is biphasic:
-	Rapid response
o	Due to increased Ca sensitivity
-	Slow response
o	Due to increased [Ca2+]i

Question 3

What evidence is there for the biphasic nature of the response?

Answer 3

Alvarez et al., 1999
Rat trabeculae isolated from the right ventricle then mounted and stretched to maximal twitch force.
Myocardial stretch produces an increase in force that occurs in two phases:
1. rapidly occurring is generally attributed to an increase in myofilament calcium sensitivity
2. gradually developing to an increase in [Ca2+]i.

Question 4

What is the rapid response due to?

What accounts for 20% of the rapid response?

Answer 4

Relationship between force and sarcomere length is too steep and too variable to be explained by myofilament overlap solely, only contributes to 20% of the rapid response.
Using Bers, 2001 diagram

Question 5

Describe the theories of increased Ca sensitivity

Answer 5

1) Increased Muscle Length Facilitates Cross-bridge Formation
2) Increased muscle length alters binding of Ca to troponin/tropomyosin on thin filament

Question 6

Describe the mechanism of cross bridge formation

Which parts show co-operativity?

Answer 6

Ca binds to TnC – changes configuration as TnI moves from TnT towards TnC and tropomyosin moves into the actin groove to allow myosin to bind to myosin binding site on actin and cross bridge formation and force development occurs

Myofilament activation is highly co-operative so:

1) Formation of one strongly bound cross bridge promotes the binding of other cross bridges
2) The binding of calcium promotes more binding of calcium to Troponin C

Question 7

Give the two theories for this process

Answer 7

Lattice Spacing and Titin

Question 8

What is the lattice spacing theory of cross bridge formation?

Answer 8

Stretch reduces distance between thin and thick filaments

Closer the myosin head is to the actin filament and more likely a CB will form at a given (Ca2+)

Question 9

What evidence supports the theory of lattice spacing? 1

Answer 9

McDonald & Moss, 1995
Skinned cardiac myocytes from rat enzymatically attached to a force transducer and piezoelectric translator.
Tested at short sarcomere length (SL), at the same short SL in the presence of 2.5% dextran (mimics decrease in lattice spacing associated with long SL), and at long SL.
Decrease in pCa is an increase in concentration. The pCa50 increased when the length of the sarcomere increased, and osmotic compression (Dextran) also increased this value.
Supports the idea that the length dependence of Ca2+ sensitivity of tension in cardiac muscle arises from the changes in inter-filament lattice spacing that accompany changes in SL.
LIMITATION
Change in myocyte width after osmotic compression versus myocyte lengthening is different. Due to shape of the myocyte was altered differently by mechanical stretch compared with osmotic compression with dextran.

Question 10

What further studies contradicted lattice spacing theory? 2

Answer 10

Konhilas et al., 2002

Directly measured lattice spacing by x-ray diffraction in skinned rat cardiac trabeculae bathed in dextran solutions.
Increasing SL significantly increased Ca2+ sensitivity using EC50 but similar reduction in myofilament lattice spacing by 1% dextran did not alter Ca2+ sensitivity.

Effect of different amounts of osmotic compression on muscle width is not equivalent to the effect on lattice spacing which could explain the discrepancy between results as McDonald et al. (1995).

Question 11

Describe the mechanism of titin in cross bridge formation?

Answer 11

Titin theory of Cross-Bridge formation
Titin – largest protein in body and run from Z line to the M band and has been shown to alter the relationship between length and myofilament sensitivity. May be due to lattice spacing changes or increasing the disorder of myosin heads.

Question 12

What evidence is there for titin playing a role in cross bridge formation? 1

Answer 12

Fukuda et al., 2001
Rat skinned right ventricular trabeculae, Ca activated tension at increased sarcomere lengths decreased after treatment of the preparation with trypsin which degraded titin without affecting other structural proteins.
Suggests the length dependent activation is modulated by titin as well as lattice spacing.
Cazorla et al., 1999 however found that titin was invovled in passive tension only which disagrees however could be due to the lesser amounts of collagen in ventricular cells than trabecular preparations.

Question 13

Describe the mechanism of increased stretch resulting in altered binding of Ca to troponin - second theory

Answer 13

Inhibition of strong cross-bridge attachment with blebbistatin had no effect on the length-dependent modulation of Ca2+ sensitivity suggesting it is something upstream affecting Ca sensitivity

Question 14

What evidence is there for alterations upstream affecting Ca sensitivity? 1

Answer 14

Farman et al., 2010
Replaced WT cTnC with a mutant cTnC (DM-TnC) that is incapable of binding Ca2+ in skinned rat cardiac trabeculae.
Mutant TnC decreases Ca sensitivity, but effects are less pronounced at long length
At short SL, Ca2+ binding to TnC works co-operatively to activate neighbouring TnC units.
At long SL, Ca-bound TnC activates more distant TnC units
Stretching the SL shifts the requirement for Ca binding from every 1-2 Tn complexes to every 3-4 Tn complexes – more binding sites are activated by the same level of Ca2+
However, further studies will be needed to assess the molecular basis for the enhanced propagation of Ca2+ with increased SL.

Question 15

Describe the slow response to stretch

Answer 15

2. Slow Response to Stretch
   Refer to previous figure, by Cingolani which demonstrates the slow response and it is due to an increase in the Ca2+ transient

Question 16

Increase in transient has been shown to be due to what?
Two papers for evidence of this process
1 - describe process after

Answer 16

SAC and NHE channels
Calaghan & White, 2004
Intact ventricular myocytes were stretched using carbon fibres.
Inhibition of NHE with HOE 642 and the SAC blocker streptomycin significantly reduced the magnitude of the slow force response and there was a decrease in [Ca2+]i transient amplitude.
Slow response relies on activation of two pathways, NHE and SACs.
Activation of NaH exchanger means more Na in cells, leads to reverse mode NCX which brings more Ca into the cell. Which can be seen in the Figure with the pathway of how it works….

Caldiz et al., 2007
Used ROS scavengers to reduce the activation of the NHE in isolated cat papillary muscles. This led to a decrease in intracellular sodium and ultimately less Ca transient, so the force difference was decreased.

Question 17

What further evidence supports that TRPC6 channel is involved in response?

Answer 17

Dyachenko et al., 2009
Murine ventricular myocytes were attached to a glass coverslip and sheared
Voltage clamp showed stretch induced inward currents that increased with the extent of stretch and attenuated with TRPC6 block.
However, it is difficult to extrapolate whether and to what extent shear stress compares with mechanical stimuli encountered by the myocyte in vivo.

Question 18

What evidence for role of SR calcium release have in slow response?
Why can this not be the only way?

Answer 18

Vila-Petroff et al., 2001
Found that stretching of single rat cardiac myocytes produces NO most probably through S-nitrosylation of the RyR channels and leads to an increase in calcium release by increasing the open probability.
Abolished by inhibiting NOS.
Increased RyR open probability cannot cause a maintained response due to autoregulation which means there must be another mechanism for slow release to be maintained.

Question 19

Draw both the mechanism for the fast and slow responses and also the mechanism for NHE activation

Answer 19

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