1. Cardiac Myofilaments Slow cardiac myosin isoforms Cardiac troponin
Titin

2. Anisotropic Isotropic to polarized light ~ 2.0 μm
The Sarcomere
Anisotropic Isotropic to polarized light ~ 2.0 μm

3. Hexagonal Arrangement of Myofilaments in Cross-Section

4. The Sarcomere

5. Myofilaments Thick myosin filaments (180 myosin molecules polymerized)
Thin actin filaments
They interdigitate to form the SARCOMERE

6. Actin F-actin molecule formed from G-actin
Twisted with a period of 70 nm - double helix
Two strands of tropomyosin
Globular troponin molecules (with a strong affinity for Ca2+) attached to tropomyosin strands, every 7 G-actin monomers

7. Myosin Subdomains: Myosin Heavy Chain Myosin Light Chain
S1 Head
S2 Tail
Myosin Light Chain
Myosin leads protrude in pairs at 180º
There are about 50 pairs of cross-bridges at each end of the thick filament
The filament makes a complete twist 310º every 3 pairs
14.3 nm internal between pairs

8. Myosin Light Chains
Description: Part of the myosin structure, atoms in the heavy chain are colored red on the left-hand side, and atoms in the light chains are colored orange and yellow. (image PDB)
Author: David S. Goodsell of The Scripps Research Institute
(Palermo et al. 1995, Sweeney et al. 1993)

9. Myosin binding to actin (from Rayment et al.,1993)

10. Simplified Crossbridge Cycle
Pi should be released during C

11. Cyclical actin and myosin interactions converts energy in ATP to force/motion
From SDSU: 11

12. Cyclical actin and myosin interactions converts energy in ATP to force/motion
From the website of the Michael Geeves' Laboratory:

13. Cyclical actin and myosin interactions converts energy in ATP to force/motion
Color Key for Figure
Actin monomers, Orange
Myosin head, Green
Light chains, Yellow
Myosin S2 & thick filament, Black
ATP/ADP, Blue/Pink
Pi, Pink
Contributed by Stefan Weiss & Mike Geeves
ATP binding to either a resting length myosin head (c) or to a head bearing a load (b) results a change in conformation in the myosin head, causing a rapid, almost irreversible dissociation of the myosin head from actin (d).
Following detachment from actin, the ATP is hydrolysed to ADP and Pi, both of which remain very tightly bound to the myosin head (e).
The hydrolysis is relatively rapid (taking about about 10 msec) and reversible (Keq = 10). The small value of the Keq indicates that the free energy of ATP hydrolysis is not released but remains within the structure of the M.ADP.Pi complex.
Hydrolysis is accompanied by a major conformational change which represents the reversal or a repriming of the power stroke.
ADP and Pi will remain bound to the myosin head until the myosin binds to an actin site. The affinity of M.ADP.Pi for actin is significantly higher than that of M.ATP.
If an actin site is within reach of the myosin head, it will bind rapidly and reversibly to the actin site.
Once bound Pi is released and XB undergoes powerstroke
From Cambridge: 13

14. Primary Determinants of Force Production in Cardiac Muscle
Intracellular calcium concentration
Sarcomere length (SL)
Rate of change in SL (velocity)
[Ca2+](t)
Cardiac Muscle
Force(t)
SL(t)
dSL/dt

15. Ca2+ Force is highly sensitive to [Ca2+] in cardiac muscle
The steep relation between steady-state force and [Ca2+] is known as ‘cooperative’
Cooperative activation of force by [Ca2+] is critical to cardiac function
Dobesh et al. 2001
Bers, 2001

16. Cooperative Activation of Myofilaments by Ca2+
Thought to be mediated by overlap of adjacent tropomyosin molecules
Inhibits force at diastolic [Ca2+]
Enhances force during twitch and greatly impacts twitch dynamics

17. Cooperative Activation of Myofilaments by Ca2+
Thought to be mediated by overlap of adjacent tropomyosin molecules
Inhibits force at diastolic [Ca2+]
Enhances force during late twitch and greatly impacts twitch dynamics

18. Isometric Tension in Skeletal Muscle: Sliding Filament Theory
(a) Tension-length curves for frog sartorius muscle at 0ºC
(b) Developed tension versus length for a single fiber of frog semitendinosus muscle

19. Evidence for sliding filaments and cycling force generators
The smallest contractile unit is the sarcomere
The sarcomere is composed of actin & myosin
Filaments slide but do not change length
Force proportional to degree of overlap
The maximum sarcomere length for contractile force development is ~3.6 µm - above this length no active force is generated
Maximum unloaded velocity is independent of SL
When the myosin head binds actin the interaction with actin can promote a major change in conformation (the power stroke) which is accompanied by the dissociation of Pi. Crystal structures suggest that the power stroke consists of a reorientation of part of the myosin head distal to the actin-binding site and includes the 'converter' region and the light chain-binding domain (LCBD). This results in the displacement of the tip of the LCBD by up to 10 nm. The structural changes in the actin-myosin interface that produce the power stroke remain undefined.
If the filaments carry an external load then the power stroke results in the distortion of an elastic element (b). The location and nature of the elastic element is unknown but may represent a distortion in the myosin head or of the LCBD. For simplicity, the elastic element is drawn here as part of the connection between the myosin head and the thick filament. While the myosin head carries a load and is elastically distorted, the dissociation of Pi is a reversible event and Pi can rebind to reverse the power stroke (and also go back through intermediates e & d).
If the external load is small, then the power stroke results in the relative sliding of the actin and myosin filaments by a distance of up to 10 nm. Following the sliding, ADP is released very quickly (within 2 msec) to be replaced within a msec by ATP, and the myosin head dissociates once more to complete the cycle. The final element of the mechanochemical coupling is believed to be a mechanism to limit the rate of release of ADP until the sliding motion is complete. Thus ADP release from b is much slower than from c. The mechanism of the strain-limited ADP release is currently under investigation, but appears to be a key event which differs between myosins designed for efficient fast shortening vs efficient load bearing. The structural changes observed on binding ADP to smooth actomyosin and BBMI might reflect this strain-sensing mechanism. 19

20. Muscle testing Tissue preparation right ventricular muscle
isolated rat trabecula
4000x200x90 mm
Force transducers
piezoresistive 1 V/mN
capacitive 0.1 V/mN
Displacement
variable mutual inductance transducer
Sarcomere length, L
1 mW He-Ne laser (l = 632 nm)
L = kl/d
Diffraction spacing, d
linear photodiode array
lateral effect photodetector

21. Cardiac Muscle Testing
Cardiac muscle is much more difficult to test than skeletal muscle:
tissue structure is complex and 3-D
long uniform preparations with tendons attached are not available
the best preparations are isolated papillary muscles (which hold atrioventricular valves closed during systole) and isolated trabeculae, which are more uniform but very small
cardiac muscle branching scatters light making laser diffraction more difficult
intact cardiac muscle can not be tetanized so it must be tested dynamically or artificially tetanized

22. Isometric testing
At constant muscle length, muscle preparation shortens in the middle at the expense of lengthening at the damaged ends

23. Isometric Testing Sarcomere length, mm Sarcomere isometric 2.1 2.0
Muscle isometric
1.9
Tension, mN
2.0
1.0
time, msec
100
200
300
400
500
600
700

24. Isometric Length-Tension Curve
Peak developed isometric twitch tension (total-passive)
High calcium
Low calcium
sarcomere isometric
muscle isometric
Passive

25. Length-Dependent Activation
Isometric peak twitch tension in cardiac muscle continues to rise at sarcomere lengths >2 mm due to sarcomere-length dependent increase in myofilament calcium sensitivity

26. Sarcomere Length
Sarcomere length determines myofilament overlap, which is proportional to force.
SL also changes sensitivity of myofilaments to Ca2+
Increasing SL
Dobesh et al. 2001

27. Force-velocity relations27

28. Factors affecting the measurement of Vo
A) time of release. Vo (open circles) is independent of time when twitch force (solid line) is >70% of F0 .
B) sarcomere length on Fo. Active and passive force development as function of sarcomere length
C) sarcomere length on Vo. Sarcomere length does not affect Vo above slack length (~1.90 µm).
D) speed of release. Increasing the rate of muscle release does not affect sarcomere shortening velocity over a wide range. This demonstrates that the actual maximal shortening velocity of the sarcomeres is rate limiting, and thus allows for the direct measurement of Vo. 28

29. Isotonic Testing
Isovelocity release experiment conducting during a twitch
Cardiac muscle force-velocity relation corrected for viscous forces of passive cardiac muscle which reduce shortening velocity

30. Small Length Step Response
Tetanized single frog muscle fiber at 0ºC during a 1% shortening step lasting 1 ms

31. Instantaneous and Plateau Tension
Solid lines: sarcomere length = 2.2 mm (near maximal myofilament overlap). Broken lines: sarcomere length = 3.1 mm (39% myofilament overlap). Thus instantaneous tension T1 reflects crossbridge stiffness and number of attached crossbridges which varies with myofilament overlap.