Structural basis of the super-relaxed state in human cardiac muscle - Myosin filaments in muscle exhibit an energy-saving “super-relaxed” (SRX) state that is thought to be
fundamental to the energetics and regulation of contraction. In cardiac muscle (including human), the SRX state
contributes to energy economy by sequestering a proportion of myosin heads away from actin, to be released
as needed when cardiac activity increases. Pathologic alterations to the SRX state are thought to underlie many
inherited cardiomyopathies, and therapeutic drugs appear to work by reversing these changes. Despite its
ubiquity and importance, the structural basis of the SRX has not been defined, leaving a crucial gap in our
understanding of cardiac contraction and the mechanism of disease and its treatment. A widely held view is that
SRX is structurally related to another ubiquitous feature of muscle myosin: the “interacting-heads motif” (IHM),
in which myosin’s two heads (blocked and free) interact with each other and with the proximal myosin tail (S2),
inhibiting their activity and conserving ATP. However, recent studies suggest that SRX may be a property of the
myosin heads themselves, and not require head interactions. In this grant we will use single particle electron
microscopy (EM), cryo-EM, and other biophysical techniques to define the structural basis of the SRX state and
the impact of key hypertrophic (HCM) and dilated (DCM) cardiomyopathy-inducing mutations and therapeutic
drugs on the structure of the IHM.
Aim 1 will define the basis of SRX in the isolated cardiac myosin head (S1) and heavy meromyosin
(HMM) by assessing if: (A) the SRX results directly from a specific conformation of the myosin heads, and (B)
the IHM correlates with the SRX state. Myosin constructs comprising single heads (S1) or two heads with 15
heptads of tail (15-hep), enough to form the IHM, will be expressed and characterized by our collaborator, Dr.
Christopher Yengo. Controls will have both heads but only 2 heptads of tail (2-hep), which cannot form a full
IHM. Negative staining EM and class averaging will reveal S1 and IHM conformations, and cryo-EM will show
for the first time the near-atomic resolution structure revealing the interactions within the IHM that underlie cardiac
relaxation. Aim 2 will define the structural basis of the SRX in native thick filaments by determining the near-
atomic cryo-EM structure of filaments isolated from cardiac muscle. Aim 3 will reveal the structural impact of key
HCM- and DCM-inducing mutations and therapeutic drugs on myosin head and IHM structure, using cryo-EM
and single particle imaging.
The SRX is now widely recognized as a fundamental state of normal relaxed muscle, but its structural
basis is not understood. Our high-resolution structural studies will reveal the near-atomic structures of cardiac
S1 and the IHM and their relationship to the SRX in both molecules and filaments. This will provide new insights
into the fundamental mechanism of relaxation (diastole) in cardiac muscle, and an improved understanding of
the structural basis of cardiomyopathies and their treatment.