P. Bryant Chase, Ph.D. - US grants

The purpose of the Programming and Instrumentation Core will be to develop and maintain equipment and software for detectors, signal processing, data acquisition, experimental control, data analysis, and data backup and archiving. This is a necessary unit since the successful completion of all projects depends on sophisticated instrumentation, computer-based data acquisition and control, and streamlined analysis of data.

Vertebrate cardiac and striated muscle contraction has been hypothesized to be regulated by altering either the number of myosin-binding sites on actin or the kinetics of the force generating reaction. In addition, this regulation has also been hypothesized to be modulated not just by the primary effector, Ca2+ binding to troponin C (TnC), but also in turn by crossbridge binding to the thin filament. Our recent experimental observations have led to a new and simpler model to explain the Ca2+ regulation of actomyosin binding and kinetics (outlined in the Introduction): that the molecular dynamics of thin filament regulatory units directly influence the apparent kinetics of actomyosin function at submaximal activation. In Project II, we will use permeabilized preparations to examine the molecular regulatory mechanism of dynamic processes during muscle contraction as indicated by three mechanical kinetic parameters: the rate of isometric tension redevelopment (k/TR); unloaded shortening velocity (V/US); and the myosin 'power stroke,' assessed by phase 2 of tension transients in response to small amplitude length steps. Three chemomechanical parameters have been chosen to test the regulatory mechanism in addition to steady state isometric force to examine on multiple facets of the crossbridge cycle and because each of these parameters exhibits an apparent variation with thin filament activation level, insofar as they have been examined. The first specific hypothesis to be tested is that k/TR at submaximal Ca2+-activation reflects dynamic properties of individual regulatory units. Directly related to this hypothesis, as supported by our Preliminary data, is that the type of TnC in a regulatory unit is a major determinant of the dynamics of that unit during submaximal activations. The second hypothesis is that the kinetics of the myosin unitary 'power stroke,' as reflected by millisecond time scale tension transients following a step change in length, are regulated by Ca2+. At the very least, these experiments will allow us to distinguish between hypotheses that phase 2 kinetics are modulated by either (a) a transient population of an initial attached, low force producing actomyosin intermediate, vs. (b) cooperative interactions between crossbridges such that the back rate constant of the force-producing transition decreases as the fraction of attached crossbridges increases. The third hypothesis is that unloaded shortening velocity is primarily regulated by different mechanisms in cardiac vs. skeletal muscle. These experiments using skinned fiber preparations will be complemented with measurements of filament sliding velocity in Project IV using in vitro motility assays on purified proteins. In total, we will be testing the validity and universality of a new hypothesis--that the dynamic properties of individual regulatory units play a major and heretofore unrecognized role in determining the macroscopic kinetics of muscle contraction.

DESCRIPTION (the applicant's description verbatim): Mutations in cardiac thin filament protein troponin I (cTnI) have been identified as causal in some forms of familial hypertrophic cardiomyopathy (FHC). The overall goal of this proposal is to characterize functional consequences of these six disease-related mutations in the C-terminus of cTnI. Specific predictions about their effects on steady-state force-pCa relationship derive from the localization of mutations in binding of TnI's C-terminus to: (i) the N-terminus of cTnC (R145G and R145Q mutations in the inhibitory peptide and R162W); or (ii) actin-Tm (R145G and R145Q mutations in the inhibitory peptide which is part of actin-Tm binding site I and K183delta which is part of actin-Tm binding site II). In addition, C-terminal truncation studies of TnI predict that R162W, K183delta, G203S and K206Q could all compromise the ability of cTn to relax the myocardium during diastole. Recombinant cTnI constructs will be altered with mutations found in FHC. Mutant and wild type (WT) constructs will be incorporated into permeabilized muscle preparations-for measurements of sarcomere mechanics-and into regulated actin filaments-for measurements on single actin filaments using in vitro motility assays. Mutant proteins will be tested, first by complete substitution of mutant for WT, secondly in varying proportions of WT and mutant as occurs in the diseased myocardium, and thirdly with additional mutations that mimic phosphorylation of Ser22 & Ser23 or Thr143 (introduction of acidic residues). For each mutation, we will determine the effects on Ca2+ sensitivity of steady-state isometric force, filament sliding, and the rate of tension redevelopment (kTR; a parameter that is important for evaluating cardiac function). The results will aid understanding normal Ca2+ regulation of the heart, pathological mechanism(s) of hypertrophy, and the assays will he useful for identifying treatments.

[unreadable] DESCRIPTION (provided by applicant): This is a new R21 application in response to PAR-03-045 "Nanoscience and Nanotechnology in Biology and Medicine." The major goal is to develop a new format of micro- to nano-scale assays for biomolecular interactions such as DNA-DNA, protein-protein, and protein-DNA; we focus exclusively on sequence-specific DNA hybridization for this R21 project. These assays will utilize high-sensitivity Hall crosses, a magnetic sensing technology that has very recently reached the point where we can pursue its use in biomedical applications. An interdisciplinary research team will accomplish this goal through high-risk/high-reward research on three specific aims. (1) Demonstrate selective DNA hybridization by micro-Hall magnetometry with commercially available magnetic beads; feasibility will be established through sequence-specific binding of multiple magnetic particles, each via multiple DNA-DNA linkages. (2) Develop and test nano-scale format Hall magnetometers; sensitivity and signal-to-noise of the assay will be improved upon by parallel development of magnetic nano-particles and further miniaturization of the Hall magnetometer. Nano-particle composition and size will be varied to optimize output of the Hall device. (3) Determine experimentally the limits of bio-molecular detection by Hall magnetometry; detection of sequence-specific DNA hybridization will be tested using the nano-scale Hall crosses and magnetic particles from Aim 2. Nano-particles with optimal composition and size for magnetometry will be tested first for compatibility with bio-molecule functionalization (DNA attachment) and sequence-specific hybridization. Nano-particles that yield optimal combinations of signal-to-noise for Hall magnetometry and sequence-specific DNA hybridization will be tested further in dilute solutions of analyte to determine the limit of detection. Nano-scale magneto-sensing is anticipated for detection of a small number of analyte molecules-one or two molecules in the limit as are needed for single cell analyses. Further, magneto-sensing assays could be used to determine activity of some enzymes such as nucleases and proteases, possibly at the single molecule level. The format should also be amenable to determining the melting temperature for double stranded DNA, again perhaps at the level of a single molecule. Magneto-sensing is also compatible with magneto-control systems, opening up the possibility of controlling (via an externally applied electromagnetic field) the location of bio-molecules attached to beads. [unreadable] [unreadable] [unreadable]