Cardiac development and disease affect cardiac contractility and troponin T (TnT) isoform expression. This study of the molecular basis and functional effects of TnT expression is motivated by results that suggest TnT isoforms and contractility are related. Molecular, morphological, biochemical, and physiological differences will be used to test these hypotheses: 1) Cardiac TnT isoforms modulate the sensitivity of myofilaments to calcium; 2) the heterogeneity in cardiac TnT isoforms modulate the sensitivity of myofilaments to calcium; 2) the heterogeneity in cardiac TnT isoforms has a molecular basis, involving alternative splicing of the primary transcript of a cardiac TnT gene; 3) the sarcomere length dependence of cardiac myofilament sensitivity to calcium is acquired with maturation and is related to TnT isoform expression.
Aim 1. The force-pCa relation of fetal and postnatal rabbit myocardium will be measured at different sarcomere lengths. The Hill coefficient and pCa for half-maximal activation (pCa50) will be related to the TnT isoforms in the myofilaments, quantitated on Western blots, to sarcomere length, and to the results of Aims 2 & 4.
Aim 2. cDNAs of rabbit cardiac TnT isoforms will be isolated and cloned. The primary structure will be deduced from cDNA sequences.
Aim 3. TnT cDNAs will be co-transfected with a neomycin resistance gene into C2C12 mouse skeletal muscle cell myoblasts. Myofilament organization and incorporation of cardiac TnT will be examined with fluorescent and electron immunocytochemistry in differentiating myotubes.
Aim 4. The force-pCa relation of control myotubes and those transfected with expression vector alone or with cardiac TnT cDNAs will be measured. The Hill coefficient and pCa50 will be used as in Aim 1. The results of Aims 1-4 will be used to propose structure-function relationships.
Aim 5. TnT cDNA will be mutated to alter regions of potential functional importance and used as in Aims 3 & 4.
Aim 6. Human cardiac TnT cDNAs from normal and failing human hearts will be isolated and cloned. They will be compared to rabbit cardiac TnT cDNA and used, as in Aims 3 & 4, to test the functional significance of preferential isoform expression in normal and failing hearts. These studies will establish the molecular basis of cardiac TnT isoform expression. These results are important in the understanding of the regulation of cardiac contraction by thin filament protein interaction.
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