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Tropomyosin (TM) is involved in [Ca.sup.2+]-mediated muscle contraction and relaxation in the heart. Striated muscle [alpha]-TM is the major isoform expressed in the heart. The expression of striated muscle [beta]-TM in the murine myocardium results in a decreased rate of relaxation and increased myofilament [Ca.sup.2+] sensitivity. Replacing the carboxyl terminus (amino acids 258-284) of [alpha]-TM with [beta]-TM (a troponin T-binding region) results in decreased rates of contraction and relaxation in the heart and decreased myofilament [Ca.sup.2+] sensitivity. We hypothesized that the putative internal troponin T-binding domain (amino acids 175-190) of [beta]-TM may be responsible for the increased myofilament [Ca.sup.2+] sensitivity observed when the entire [beta]-TM is expressed in the heart. To test this hypothesis, we generated transgenic mice that expressed chimeric TM containing [beta]-TM amino acids 175-190 in the backbone of [alpha]-TM (amino acids 1-174 and 191-284). These mice expressed 16-57% chimeric TM and did not develop cardiac hypertrophy or any other morphological changes. Physiological analysis showed that these hearts exhibited decreased rates of contraction and relaxation and a positive response to isoproterenol. Skinned fiber bundle analyses showed a significant increase in myofilament [Ca.sup.2+] sensitivity. Biophysical experiments demonstrated that the exchanged amino acids did not influence the flexibility of the TM. This is the first study to demonstrate that a specific domain within TM can increase the [Ca.sup.2+] sensitivity of the thin filament and affect sarcomeric performance. Furthermore, these results enhance the understanding of why TM mutations associated with familial hypertrophic cardiomyopathy demonstrate increased myofilament sensitivity to [Ca.sup.2+]. calcium sensitivity; contractile function; genetically altered mice

A key challenge in Parkinson's disease research is to understand mechanisms underlying selective degeneration of dopaminergic neurons mediated by genetic factors such as alpha-synuclein (alpha-Syn). The present study examined whether dopamine (DA)-dependent oxidative stress underlies alpha-Syn-mediated neurodegeneration using Drosophila primary neuronal cultures. Green fluorescent protein (GFP) was used to identify live dopaminergic neurons in primary cultures prepared on a marked photoetched coverslip, which allowed us to repeatedly access preidentified dopaminergic neurons at different time points in a non-invasive manner. This live tracking of GFP-marked dopaminergic neurons revealed age-dependent neurodegeneration mediated by a mutant human alpha-Syn (A30P). Degeneration was rescued when alpha-Syn neuronal cultures were incubated with 1 mm glutathione from Day 3 after culturing. Furthermore, depletion of cytoplasmic DA by 100 microm alpha-methyl-p-tyrosine completely rescued the early stage of alpha-Syn-mediated dopaminergic cell loss, demonstrating that DA plays a major role in oxidative stress-dependent neurodegeneration mediated by alpha-Syn. In contrast, overexpression of a Drosophila tyrosine hydroxylase gene (dTH1) alone caused DA neurodegeneration by enhanced DA synthesis in the cytoplasm. Age-dependent dopaminergic cell loss was comparable in alpha-Syn vs dTH1-overexpressed neuronal cultures, indicating that increased DA levels in the cytoplasm is a critical change downstream of mutant alpha-Syn function. Finally, overexpression of a Drosophila vesicular monoamine transporter rescued alpha-Syn-mediated neurodegeneration through enhanced sequestration of cytoplasmic DA into synaptic vesicles, further indicating that a main cause of selective neurodegeneration is alpha-Syn-induced disruption of DA homeostasis. All of these results demonstrate that elevated cytoplasmic DA is a main factor underlying the early stage of alpha-Syn-mediated neurodegeneration.