MECHANISM OF CARDIAC MUSCLE REGULATION BY TROPONIN

  • Potter, James D., (PI)
  • Szczesna-Cordary, Danuta (PI)

Project: Research project

Description

The major questions being addressed deal with determining the state of the
single Ca2+ regulatory site on cardiac TnC in skinned muscle fibers and how
it is modulated by various physiological and pathological states of the
heart. This project will focus on the static and dynamic calcium binding
properties of troponin in skinned fibers using techniques recently
developed in this laboratory. Much is know about the role of Ca2+ binding
to troponin in the regulation of cardiac muscle contraction, based
primarily upon solution studies of the various proteins involved an these
results have been extrapolated to intact muscle. However, recent evidence
suggests that the Ca2+ binding properties of the regulatory sites of
troponin are altered when troponin is incorporated into thin filaments and,
of equal importance, other studies suggest that myosin crossbridge
interaction with the actin filament may also affect Ca2+ binding. In
addition, on recent preliminary report suggests a role for cardiac TnC in
the length dependent autoregulation of the hart. With our new techniques,
it is possible to study these question directly. Cardiac TnC (CTnC) can be
selectively extracted from skinned fibers and replaced with exogenous
fluorescently labeled CTnC. The Ca2+ dependence of the fluorescence change
of the incorporated CTnC can be correlated directly with Ca2+ or Sr2+
binding to the single Ca2+-specific regulatory site on CTnC. Through the
use of another new technique we can directly measure the Sr2+ affinity of
this site using Fura-2 in skinned fibers and thus validate unequivocally
the CTnC fluorescence results. Using these techniques we can then follow
Ca2+binding, force development and ATPase activity (fluorescence linked
enzyme assay) using microspectrofluorometry. This approach has already
made it possible to learn much about the regulation of skeletal muscle
contraction. In this application we will address questions which are
unique to cardiac muscle where the Ca2+ binding properties of CTnC are
thought to be modulated by various alterations in the heart (e.g., beta-
adrenergic stimulation, ischemia, etc.). Specifically, we will study the
effect of troponin I phosphorylation, myosin crossbridge state (e.g.,
rigor, ADP, Pi, ADP-Pi), sarcomere length and pH on the Ca2+ affinity of
the single regulatory site in cardiac muscle. We will also study the
mechanism of the unique Sr2+ sensitivity of cardiac muscle. Using the new
technique of photolysis of the caged compounds Ca2+ (Nitr- 5 and DM-
nitrophen) and Ca2+ -chelator, we will be able to study the various kinetic
steps involved in the regulation of force development and relaxation. For
example, the rate of Ca2+ dissociation from CTnC and the rate of force
development and relaxation can be measured simultaneously upon liberation
of caged Ca2+ -chelator and yield important new information about the
relationship of bound Ca2+ to relaxation and its modulation by
phosphorylation, etc. All of the proposed experiments, which were not
previously possible, should lead to a clearer view of the molecular events
involved in cardiac muscle regulation and their time course.
StatusFinished
Effective start/end date4/1/892/28/13

Funding

  • National Institutes of Health: $382,500.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $203,557.00
  • National Institutes of Health: $184,845.00
  • National Institutes of Health: $242,753.00
  • National Institutes of Health: $304,833.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $197,784.00
  • National Institutes of Health: $367,090.00
  • National Institutes of Health: $376,670.00
  • National Institutes of Health: $366,334.00
  • National Institutes of Health: $298,025.00
  • National Institutes of Health
  • National Institutes of Health: $269,797.00
  • National Institutes of Health: $382,500.00
  • National Institutes of Health: $373,996.00
  • National Institutes of Health: $311,843.00
  • National Institutes of Health: $375,991.00

Fingerprint

Troponin
Myocardium
Familial Hypertrophic Cardiomyopathy
Muscle Contraction
Troponin T
Mutation
Myosins
Fluorescence
Restrictive Cardiomyopathy
Chelating Agents
Muscles
Dilated Cardiomyopathy
Adenosine Triphosphatases
Protein Isoforms
Sarcomeres
Troponin I
Fura-2
Photolysis
Actin Cytoskeleton
Phosphorylation

ASJC

  • Medicine(all)