SUMMARYThe trout heart is 10-fold more sensitive to Ca 2+ than the mammalian heart. This difference is due, in part, to cardiac troponin C (cTnC) from trout having a greater Ca 2+ affinity than human cTnC. To determine what other proteins are involved, we cloned cardiac troponin I (cTnI) from the trout heart and determined how it alters the Ca 2+ affinity of a cTn complex containing all mammalian components (mammalian cTn). Ca 2+ activation of the complex was characterized using a human cTnC mutant that contains anilinonapthalenesulfote iodoacetamide attached to Cys53. When the cTn complex containing labeled human cTnC was titrated with Ca 2+, its fluorescence changed, reaching an asymptote upon saturation. Our results reveal that trout cTnI lacks the N-terminal extension found in cTnI from all other vertebrate groups. This protein domain contains two targets (Ser23 and Ser24) for protein kinase A (PKA) and protein kinase C. When these are phosphorylated, the rate of cardiomyocyte relaxation increases. When rat cTnI in the mammalian cTn complex was replaced with trout cTnI, the Ca 2+ affinity was increased ~1.8-fold. This suggests that trout cTnI contributes to the high Ca 2+ sensitivity of the trout heart. Treatment of the two cTn complexes with PKA decreased the Ca 2+ affinity of both complexes. However, the change for the complex containing rat cTnI was 2.2-fold that of the complex containing trout cTnI. This suggests that the phosphorylation of trout cTnI does not play as significant a role in regulating cTn function in trout.

Skeletal tropomyosin, (Tm), is an a-helical coiled-coil which binds to actin, and with troponin, regulates muscle contraction. We previously demonstrated that a conserved Asp 137 in the hydrophobic interface produces a dynamic region in the middle of Tm, and that this region is involved in the myosin dependent activation of the thin filament at high Ca 2þ , (Sumida et. al. JBC 283 2008). The current work characterizes a long-range interaction between positions 137 and 190. The thermodynamic properties of wild type (WT) Tm and two single mutants, C190A and D137L, are compared with those of the double mutant, D137L/ C190A, using differential scanning calorimetry, (DSC), and circular dichroism, (CD). CD measurements show that Ala 190 increases the fraction of helix unfolding in the 40 o C pre-transition, before the main transition. DSC measurements support this finding, indicating a large enthalpic pre-transition, (DH¼150kcal/mol), for the C190A mutant relative to D137L, D137L/ C190A, or WT, (average DH¼20kcal/mol). Additionally, Ala 190 increases the DCp, (heat capacity), of Tm ~5 fold, reflecting an increase in solvent exposure of hydrophobic residues in the pre-transition during unfolding. Since the D137L/C190A and D137L mutants do not exhibit the large enthalpic pre-transition observed for C190A, the Leu mutation at 137 must stabilize the alanine effect observed for C190A mutation 77A ˚away. This demonstrates how a locally dynamic region near 137 is able to produce global effects along the thin filament, and in this manner provide the proper regulation of the myosin dependent activation of the thin filament. This observation may also contribute to our understanding about the manner in which single point mutations significantly affect function in cardio-myopathies such as FHC and DCM.

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