Browsing School, Graduate by Subject "Hypertrophy"
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Calcium(2+)-signaling in cardiac muscle: From development to heart failureSome changes observed in heart failure include altered ultrastructure, defects in excitation contraction coupling (ECC) and serious changes in beta-adrenergic signaling. I have examined these topics with an emphasis on cellular Ca 2+ signaling. Using immunohistochemical techniques combined with fluorescence confocal laser scanning microscopy (FCLSM) I have examined the localization and distribution of structural components of ECC in isolated ventricular myocytes from neonatal rats, and rat models of hypertrophy (Dahl SS/jr) and heart failure (SHHF). Using whole cell patch clamp techniques to record membrane voltage or currents, and simultaneously measuring intracellular [Ca2+] i using fluorescent Ca2+ sensitive indicators and FCLSM I have examined cardiac function in MLP-/- and transgenic V12HRas murine models of heart failure. I also examined the effectiveness of therapeutic strategies at preventing ECC defects in these models, Even at the early stages of development examined, primitive striations of a number of ECC protein structures were observed. Striations of sarcolemmal membrane (T-tubules) appear to form from the outside inwards, whereas proteins localized more intracellularly are first seen more internally. Structural changes observed in hypertrophic and failing myocytes include altered distribution of the Na 2+/Ca2+ exchanger. Increased Na2+/Ca 2+ exchanger density at the external surface is seen in these myocytes. In Ras myocytes unchanged Ca2+ currents (ICa(L)), decreased [Ca2+]i transients, defective ECC and action potential (AP) alterations were seen. Crossing Ras and PLBKO mice prevented AP alterations. In MLP-/- unchanged ICa(L ) and Ca2+ spark characteristics but decreased [Ca 2+]i transients, contractile responses and defective ECC were seen. Cellular defects were prevented in MLP-/- mice expressing a cardiac-targeted transgene blocking the function of beta-adrenergic receptor kinase-1 (betaARKct). These data suggest both defective SR function and down-regulation/de-sensitization of beta-adrenergic receptors play a pivotal role pathogenesis of heart failure. As such, PLBKO and betaARKct may prove effective therapies for preventing and possibly rescuing the cellular defects seen in heart failure. The mechanism(s) responsible for targeting ECC proteins to specific intracellular localizations remains to be discovered. Further studies on these and other methods for preventing and reversing Ca 2+ signaling defects seen in heart failure may provide valuable therapeutic tools for human heart failure treatment.
Transcriptional regulation of cardiac development and hypertrophyTAZ (transcriptional coactivator with PDZ-binding motif) is composed of a 14-3-3 binding motif, a WW domain which binds Pro-Pro-X-Tyr motifs, a coiled-coil region within a regulatory domain, multiple phosphorylation sites, and PDZ binding motif at the C-term. The mechanism of regulation of TAZ is not fully understood but involves changes in its expression level, phosphorylation states, and subcellular localization. TAZ has been shown to interact with TEF-1 as well as other transcription factors including Runx2, PPARγ, and TBX5. TAZ and the related protein YAP65 are strong transcriptional coactivators. YAP/TAZ interact with target proteins in most, but not all cases, through conserved WW domains which bind (A/L/P)PXY motifs in transcription factors. TBX20, a cardiac t-box family member related to TBX5, also contains a PPXY motif similarly to TBX5. This motif potentially can interact with the WW domain in TAZ proteins. Tbx20 is involved in cardiac development and physically interacts with several cardiac specific transcription factors including but not limited to GATA4 and Nkx2. Mutations in this gene has also been identified in individuals diagnosed with Congenital Heart Defects. There are two forms of TBX20, a long(A) and short(B) isoform in which the long form contains the PPXY motif and the short form does not. I have shown, however, that TAZ interacts with both TBX20long and TBX20short, and this interaction is functionally significant. TAZ has also been shown to interact with the TEF-1 protein. The TEF-1 family of proteins has four members including TEF-1( nominal NTEF-1), RTEF-1(Related to TEF-1), DTEF-1(Divergent TEF-1) and ETF-1(Embryonic TEF-1). The TEF-1 family has significant sequence similarity and all bind to (muscle-CAT) MCAT elements (related to CATTCC(A/T)) in different promoters. I have demonstrated, through the use of Immunocytochemistry, that TEF-1 is critical for the increase in cell size during cardiac hypertrophy as well as increase in sarcomeric organization and increase in ANF expression under normal and hypertrophic conditions in neonatal cardiac muscle cells.