Cell transplantation has been shown to improve cardiac function following myocardial infarction (MI). The mechanisms behind improved function are not well-understood, but are correlated with changes in myofilament protein phosphorylation, which likely affect Ca2+- and length-dependent (preload) changes in force. This project was designed to test the hypotheses that 1) responsiveness to ventricular filling (Frank-Starling relationship) will decrease following MI, and 2) neonatal rat cardiomyocyte (NRC) transplantation after infarction will ameliorate this loss of function. One week after induction of MI, fluorescently-labeled NRCs were injected into the area of infarction of some of the animals. Sham-operated rats served as control. After 4-6 weeks, cardiac performance for all groups was preliminarily assessed using echocardiography. Then, whole working heart preparations were used measure cardiac power output, testing the heart’s ability to follow the Frank-Starling relationship. NRC-injected hearts exhibited higher average power and response to ventricular filling than sham (media-only) injected hearts, indicating that NRC-engraftment significantly ameliorates MI-induced decreases in preload responsiveness. Force and Ca2+ sensitivity (pCa50) of demembranated myocardial strips from these hearts were then assessed. NRC-injected hearts strips had a sarcomere length associated change in pCa50 that was similar to uninfarcted hearts (0.07 ± .02 vs. 0.10 ± .03, respectively), which was significantly improved as compared to strips from untreated, infarcted animals (0.0 ± .03). These results indicate that loss of preload dependence following MI is, at least in part, due to altered myofilament function and this is reversed following NRC engraftment. Current work involves investigating myofilament phosphorylation profiles to assess the mechanism of myocardial function changes. Preliminary results show decreased phosphorylation levels of cardiac troponin T (cTnT) in NRC-injected hearts compared to sham-operated and infarcted hearts. These changes in phosphorylation may be associated with the changes in force production following cell therapy.

Muscle contraction begins when Ca2+ binds to sTnC, which removes the inhibition of myosin binding to actin, allowing force development or shortening. Our laboratory has engineered sTnC variants with differential Ca2+ binding affinities to investigate how it affects healthy and diseased muscle. Chief among these variants are I60Q and M80Q, which have lower and higher Ca2+ binding affinities than the native protein, respectively. Expression of these variants in diseased or damaged muscle as a therapeutic treatment will be accomplished by viral transduction with cDNA. This method does not achieve 100% replacement of the native protein. Thus, my project is to determine the minimum variant level that needs to be replaced to have a signficant effect on contraction to help design treatment studies. Replacing native sTnC with I60Q or M80Q is also expected to gradually produce reduced or greater contraction when exposed to higher [Ca2+] prior to sTnC extraction and following reconstitution with variant:WT mixtures to determine force outputs. pCa50 is compared between pre-extraction, and reconstitution. Variants used in this study also contained polyhistidine tags, allowing us to quantify the amount of reconstitued protein incorporated into the fiber. These data show us the the effect of a particular reconstitution ratio and the amount of protein necessary to produce this effect. My preliminary data suggest that there is a progressive effect of increasing I60Q in muscle fibers to reduce the calcium sensitivity of force, thus reducing contractile strength. This variant may therefore be effective in reducing contraction in muscular dystrophy, thus maintaining muscle integrity and function.

Monocrops, genetically engineered organisms, pesticides, dumping, hunger; these ominous words haunt the conscience of the modern food critic and have become increasingly widespread with a renewed focus on local organic farming. In contrast to the menacing tones of conventional agriculture, the alternative agriculture movement offers its own lexicon of inviting adverbs: sustainable, local, environmental, organic, healthy, etc. However, the average age of the American farmer is 57. If family farms and the alternative agriculture movement are to continue, they need to attract young people to take on the physically demanding work of farming. Young, socially conscious, and driven apprentices offer some of the best possibility of continuing and growing these types of farms. How apprentices learn to farm directly informs their knowledge of agriculture and their desire to continue participating as farmers or food activists. My research, focused in the Puget Sound Region, asks whether or not apprenticeships provide participants with the skills and encouragement to continue their involvement in the movement. Over the last five months, I conducted ethnographic research in farms, conferences, and workshops. Additionally, I conducted eleven interviews with young farmers transitioning away from apprenticeships and four interviews with farmers who host apprentices. Each hour-long interview was either conducted in person or via phone. In some cases, I followed up with interviewees via email to clarify certain points. Participants were selected based on their involvement as apprentices on Western Washington farms smaller than 50 acres. To analyze data, I transcribed the interviews and coded participants’ motivations and experiences. The results have revealed how apprenticeships entail a complex political commitment that exists at the intersection of race, class, and gender. My presentation will explore the political nature of apprenticeships to help think about how they represent a new way to understand agricultural labor relations.