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MOLECULAR BIOPHYSICS OF FORCE GENERATION: Structure and Dynamics II lectures by Joe Mure.a, [email protected] Learning Goals: Describe a general structure based model for muscle contracGon. Explore current topics in the structural biophysics of muscle contracGon. Explore current models of muscle diseases. Lecture Outline 1.  Structure and dynamics of myosin
2.  Structure and dynamics of actin, the thin filament, and actomyosin
3.  Current Problems
4.  Health and disease
Searching for drugs to directly modulate contractility.
Omecamtiv mecarbil: a small molecule in clinical trials for treating systolic
heart failure.
Reading. Malik 2011. h.p://www.sciencedirect.com/science/arCcle/pii/S0022282811001854 How to treat heart failure?
Hypertrophic Dilated Approximation: Ftot = funi * X * N
Ftot = Total Muscle force
is the product of…
funi = force generated by one myosin
single molecule weak-to-strong actin binding free energy change
X = Duty cycle
controlled by biochemical kinetics
N = Total number of active myosin
controlled by Ca and by filament overlap, phosphorylation etc, allostery.
MichaelJay.allaboutrowing.blogspot.com
The myosin duty cycle
tc = 1/Vmax
Lets discover some drugs! Steady-state ATPase cycling
+ ATP +Ca +
ATP in
ADP out
Phosphate out
Fady I. Malik , Bradley P. Morgan
Journal of Molecular and Cellular Cardiology, Volume 51, Issue 4, 2011, 454 - 461
Thin-filament activated ATPase screen
Fady I. Malik , Bradley P. Morgan
Journal of Molecular and Cellular Cardiology, Volume 51, Issue 4, 2011, 454 - 461
Lead compound optimization
Fady I. Malik , Bradley P. Morgan
Journal of Molecular and Cellular Cardiology, Volume 51, Issue 4, 2011, 454 - 461
OM activates cardiac myosin to activate contractility
Without changing Calcium handling.
Fady I. Malik , Bradley P. Morgan
Journal of Molecular and Cellular Cardiology, Volume 51, Issue 4, 2011, 454 - 461
OM activates cardiac myosin to activate contractility
Without changing Calcium homeostasis.
Fady I. Malik , Bradley P. Morgan
Journal of Molecular and Cellular Cardiology, Volume 51, Issue 4, 2011, 454 - 461
Except, OM does not activate Cardiac Myosin Actin ATPase…
ATPase ATP/s/HMM
Activity (s-1)
4
HMM
DMSO
OM
3
Model: Hyperbolic Equation: Activity = Vmax[Actin]/(KM + [Actin]) Red. χ2 0.4636 1.54519 0.6164 Adj. R2 0.99768 0.98709 0.99897 Value Std Error Vmax 6.9 0.3 KM 61.5 3.9 Vmax 7.2 1.2 KM 65.5 13.4 Vmax 3.5 0.1 KM 35.1 2.2 2
HMM DMSO 1
OM 0
0
10
20
30
[Actin] (uM)
40
50
Actin-activated NADH-coupled ATPase assay. John Rohde, Joe Muretta DDTlab
100 nM βCM (bovine ventricular) HMM.
10 mM Tris pH 7.5, 3 mM MgCl2, 6 mM KCl, 1.2 mM ATP +/- 1% DMSO +/- 4 uM OM
An independent study.
OM inhibits ATPase cylcing.
OM slows ATP stimulated AM dissociation and ATP binding.
-OM faster
+OM slower
+OM slower
OM accelerates phosphate release.
+OM faster
-OM slower
+OM larger Pi burst
-OM smaller Pi burst
OM slows myosin motility.
tc = 1/Vmax
Displacement* (Duty
Velocity = Cycle * 1/ATPase)-1
Summary: Effects of OM on myosin kinetics.
Effects of OM on myosin kinetics.
Effects of OM on myosin kinetics.
So what’s going on?
Control
+OM
So what’s going on?
+ ATP +Ca +
ATP in
ADP out
Phosphate out
Fady I. Malik , Bradley P. Morgan
Journal of Molecular and Cellular Cardiology, Volume 51, Issue 4, 2011, 454 - 461
What kind of questions will I ask on the exam?
1 A. Draw a cartoon of myosin. Note the predominant structural elements described in this
class.
1 B. Draw the actomyosin ATPase cycle. Include the predominant biochemical species. Not
the predominant structural state of the predominant structural elements described in this
class (Lecture 1).
What kind of questions will I ask on the exam?
2. A mutation in the myosin Relay-loop causes extreme cardiomyopathy in the heart.
Propose three Aims (aka experiments) to test the mechanism of action of this mutation.
Propose a novel treatment strategy for patients with this disease.
What kind of questions will I ask on the exam?
3. Propose a molecular mechanism for the Fenn effect in muscle. Propose 3 Aims to test this
mechanism. Special credit will be given to answers which seek to disprove your hypothesis
rather than prove you hypothesis.
Suggested Term Paper Topics
Identify an unanswered question in muscle structural biophysics. Review the current state of
the scientific literature.
Review the current understanding of how myosin RLC phosphorylation regulates muscle
contraction in heart, skeletal, and smooth muscle. Propose a structural mechanism and 3
experiments to test this mechanism.
Review the lit. on myosin super-relaxed state. What is it? And describe its implications in
health and disease.
Describe in molecular details, the structural changes in contractile proteins in each state of a
left ventricular cardiac pressure volume loop. How are these events altered in hypertrophic
cardiomyopathy. How are they altered in end-stage heart failure?
Review the literature on hypertrophic and dilated cardiomyopathies. What are the current
debates in the field? Describe 3 treatment strategies under clinical development. How are
these treatment strategies related to topics discussed in this class?