The Significance of Parvalbumin Isoform Switching in Determining Muscle Performance

Recently it was found carp skeletal muscle has mRNA coding for at least eight isoforms of the intracellular Ca2+ binding protein parvalbumin (PARV) and these isoforms change with the temperature at which the fish is maintained (Gracey et al 2004). This was a very significant discovery in relation to understanding how muscle properties are programmed at the molecular level. Muscle contraction is initiated by calcium (Ca2+) release into the intracellular fluid from an extensive sac-like organelle: the sarcoplasmic reticulum (SR). As the Ca2+ concentration rises, crossbridges form between the contractile proteins and force is generated. Relaxation occurs when this Ca2+ is pumped back into the SR or is bound by Ca2+ intracellular proteins. The most important of these intracellular Ca2+ binding proteins is PARV. The amount of PARV in muscle correlates closely with its maximum contraction speed: toadfish swimbladder muscle, one of the fastest contracting muscles, has the highest concentration of PARV. Indeed, PARV is so good at binding Ca2+ that it can produce muscle relaxation in the absence of all other Ca2+ sequestration mechanisms. It has also been demonstrated that transferring PARV gene into heart muscle that is unable to relax can help restore function. This illustrates the clinical importance of understanding the role of PARV and how it might be used to help repair the function in failing muscles. While it is well known that the amount of PARV in muscle is important, the significance of the different PARV isoforms has largely been overlooked, perhaps because the technology to identify and quantify groups of highly similar proteins is only now readily accessible. We believe that changing the mixture of PARV isoforms by varying their expression is an important adaptive process, yielding rapid changes in muscle mechanical properties in response to different functional demands. The huge body of gene sequence data is only useful if we understand the function of those genes but discovery of gene function is often confounded by the poor correlation between transcriptome (the mRNA transcribed from the DNA of a cell) and proteome (the proteins present in a cell under a particular set of conditions). Studies that close the gap between genome are sorely needed. In this study we will use transcriptome data, novel proteomic technologies, cellular and whole muscle physiology to determine the function of this small, but critically important, group of gene products (PARV isoforms) that are pivotal in determining the contractile properties of muscle. We will measure the mechanical properties, the time course of the rise and fall of intracellular Ca2+ (a 'Ca2+ transient') and PARV isoform distribution of muscle acclimated to 10 Celsius and 30 Celcius, corresponding to two endpoint temperatures in the Gracey et al (2004) study. In addition, we will feed fish on a diet incorporating a heavy stable isotope of valine and acclimate them to the same temperatures. By periodically sampling the group of fish we can measure the amount of stable isotope incorporated in a PARV isoform (or any muscle protein for that matter) and determine its rate of synthesis and degradation, therefore, its turnover throughout acclimation. This first part of our study provides a comprehensive picture how PARV isoforms change during muscle remodelling and the mechanism by which that change is achieved. However, observing the shift in PARV isoform distribution only provides a priori evidence of its functional role. To prove that changing the amount of a given PARV isoform illicits a particular change in muscle properties we need to directly manipulate its expression. We will do this by transfecting muscle with cDNA coding for a PARV isoform, quantifying the resulting isoform expression and measuring the change in muscle properties and Ca2+ transient. Adjacent untransfected muscle and other untransfected fish provide internal and external controls.

The myoplasmic concentration of intracellular Ca2+ binding protein parvalbumin (PARV) is pivotal in determining the shape and duration of the muscle Ca2+ transient, and correlates with the maximum speed at which a muscle can contract, e.g. toadfish swimbladder muscle, one of the fastest contracting muscles also has the highest measured concentration of PARV. Further, PARV gene transfer into failing heart muscle, where it is not usually expressed, can restore myocardial relaxation. This illustrates the clinical importance of understanding the role of PARV and a possible therapeutic use. Carp skeletal muscle possesses mRNA for at least eight PARV isoforms and their expression changed with acclimation temperature (Gracey et al. 2004). Temperature changes induce major adaptive changes in fish muscle performance. While it is known the amount of PARV is important in setting muscle properties, the significance of the different PARV isoforms has remained largely unexplored. We hypothesise that PARV isoform switching is a key mechanism in the adaptive change of muscle performance. We will determine the mechanical properties and the Ca2+ kinetics of muscle at 10Deg.C and 30Deg.C and correlate this to absolute amount of each PARV isoform in the same muscle. We will measure changes in mechanical properties due to thermal induced remodelling using both traditional physiological techniques and the 'work-loop' technique, which models muscle function in vivo. Absolute quantification of each PARV isoform will be performed using a novel 'QCAT' approach. This strategy involves producing a synthetic protein comprised of concatenated tryptic peptides (Q-peptides) each a signature peptide for an individual PARV isoform. The QCAT protein is expressed in E. coli and can be readily produced in either an unlabelled or labelled form by growth of the bacteria in defined medium containing the chosen label. Through this process a range of representative proteolytic peptides of multiple proteins can be simultaneously generated for use in quantitative mass spectrometric analyses. Whilst a comparison of the absolute quantities of the PARV isoforms in the different temperature induced phenotypes allows us to correlate protein expression with muscle function, it fails to address the dynamics of the proteome and provides little insight into the mechanism of change between these biological states. We need to explain the changes in protein expression in terms of changes in protein synthesis or degradation. We will use a stable isotope labelling approach and mass isotopomer distribution analysis to determine the rate of turnover of individual PARV isoforms in carp skeletal muscle. A stable isotope labelled amino acid (2[H]8-lysine) will be administered to the fish via the diet and its incorporation into the individual muscle proteins will be monitored by MALDI-TOF-MS or LC-MS analysis of complex peptide mixtures. The outcome will provide an integrative view of from gene transcription, through PARV expression and turnover to muscle performance. This will enable us to more fully characterise/investigate the functional roles of PARV and examine how changes in change PARV isoform expression and turnover affects the function of the whole muscle Establishing the relationship between the amount of each PARV isoform and muscle performance would provide persuasive evidence for the role of PARV isoform switching in determining muscle performance. However, to prove that changing the amount of a given PARV isoform produces a given change in muscle properties we need to directly manipulate its expression. We will do this by transfecting muscle by direct injection of cDNA coding for each PARV isoform, quantifying the resulting isoform expressed and determining the change in muscle properties and Ca2+ transient, thereby directly linking changes at the level of the transcriptome, through protein expression to changes in muscle function.