The role of PKC phosphorylation in modulating cardiac contractility
There are significant differences between a healthy heart and one that is in end stage failure. There is, however, little known of the molecular mechanisms that cause a sound muscular pump to become weak and inefficient. One of the characteristics of a failing heart is a chronic increase in the expression of the alpha- and beta- isoforms of protein kinase C (PKC). This altered cell signaling pathway is thought to be at least partially responsible for the enhanced lusitrophy, and compensatory hypertrophy that are hallmarks of the maladaptive changes that lead to heart failure. Two targets for phosphorylation by PKC in the cardiac myocyte are the thin filament proteins, troponin I (TnI) and troponin T (TnT). These, along with troponin C (TnC), comprise the troponin complex (Tn). TnI and TnT are involved in transmitting the Ca2+ activation signal from TnC to the rest of the contractile element. This signal transmission is thought to be modulated by PKC phosphorylation and results in a decrease in Ca2+ sensitivity and MgATPase activity. A chronic expression of PKC has been suggested to impair the ability of the myocardium to modulate Ca2+ activation. There are two goals of this research project. The first is to characterize how the phosphorylation of TnI and TnT by PKC alters the Ca2+ activation of isolated Tn, and then determine how this translates into changes in MgATPase activity and the kinetics of Ca2+ activated force generation. The second goal is to determine the sensitivity of the contractile element to increasing levels of Tn phosphorylation by PKC. These experiments are utilizing steady-state Ca2+ binding measurements and stopped flow kinetic analysis to measure the Ca2+ affinity of Tn in solution and the rate of Ca2+ disassociation (koff), respectively. In addition, we are characterizing the influence of PKC phosphorylation on the kinetics of Ca2+ activation using skinned cardiac trabeculae.

Functional characterization of trout cardiac troponin I and cardiac troponin T:
The Ca2+ sensitivity of force generation by trout cardiac tissue is ~10 fold that of mammalian cardiac tissue. It is thought that this high Ca2+ sensitivity is responsible for enabling cardiac function in trout at their comparatively low physiological temperature. We have previously demonstrated that the Ca2+ affinity of trout cardiac troponin C (ScTnC) is 2.3 fold that of human cTnC. cTnC is the Ca2+ activated trigger that initiates myocyte contraction. We also identified the residues responsible for the high Ca2+ affinity of ScTnC. By replacing native cTnC in rabbit cardiac myocytes with a McTnC containing these identified residues we were able to incease the Ca2+ sensitivity of force generation by 2 fold. This clearly demonstrates that ScTnC is partly responsible for the comparatively high Ca2+ sensitivity of trout cardiac myocytes. As the Ca2+ sensitivity of trout cardiac tissue is 10 fold that of mammalian cardiac tissue it is thought that it is functional differences of trout cardiac troponin I (cTnI) and trout cardiac troponin T (cTnT) that is responsible for the remaining difference in Ca2+ sensitivity. To test this hypothesis Kelly Kirkpatrick is currently cloning the genes for these proteins. Once cloned and sequenced these genes will be expressed using E. coli and purified using column chromatography. These proteins will then be complexed with ScTnC. The function of this troponin complex (ScTn) will then be characterized using Ca2+ binding studies and stopped flow kinetic analysis. ScTn will then be exchanged into mammalian cardiac tissue and its influence on the Ca2+ activation of force generation characterized.

Influence of cold acclimation on contractile protein expression in the trout heart:
We are interested in the abilities of the vertebrate heart to respond to a stressor and we are using the trout heart as a model for these studies. Trout remain active at low temperatures and cold acclimation causes cardiac hypertrophy in these fish. This hypertrophy is due to an increase in cell size, not cell number. It is thought the hypertrophy is in response to an increase in blood viscosity that requires the heart to work harder. We are interested in how the structure and function of trout cardiac myocytes change during this compensatory response. To characterize how cold acclimation influences the proteins expressed within the heart, Jordan Klaiman is using 2D DIGE to analyze the protein composition of cardiac myofilaments from fish that have been acclimated to 4 C, 12 C and 17 C. Through this work we will be able to identify any changes in protein isoform expression as well as changes in the phosphorylation state of individual proteins. Through integrating these results with measurements of actin myosin ATPase activity we will be able to correlate changes in cross-bridge cycling rates with changes in the proteome of the myofilaments. Through this work we will identify specific mechanisms utilized to alter the physiological capabilities of the heart.

Influence of hypoxia on aerobic metabolism and cardiac development in trout embryos:
Hypoxia represents a stress routinely experienced by developing trout embryos in their natural environment. We are interested in how hypoxia exposure impacts aerobic metabolism and cardiac development/function in embryos. This project is being done in collaboration with Dr. Pat Wright in Integrative Biology. Silvana Miller has recently completed a study where she characterized changes in metabolic rate and oxygen levels within the developing embryo during chronic and acute hypoxia. She has demonstrated that the oxygen consumption of chronically exposed embryos is less than that of acutely exposed embryos in the latter stages of development. This difference in oxygen consumption is due to the smaller size of the chronically exposed embryos as a result of an impaired growth rate. We are also characterizing the influence of chronic hypoxia on heart rate and cardiac gene expression. Heart rate is monitored through a microscope and gene expression is monitored using Q-PCR. The specific genes we are monitoring are used as markers for cardiac growth and metabolism. Through this study we are working to characterize the adaptive ability of the vertebrate heart during early development.