Cardiac Transgenic and gene transfer strategies converge to support an important role for troponin I in regulating relaxation in cardiac myocytes

Elucidating the relative roles of cardiac troponin I ( cTnI) and phospholamban ( PLN) in beta- adrenergic - mediated hastening of cardiac relaxation has been challenging and controversial. To test the hypothesis that beta- adrenergic phosphorylation of cTnI has a prominent role in accelerating cardiac myocyte relaxation performance we used transgenic ( Tg) mice bearing near complete replacement of native cTnI with a beta- adrenergic phospho- mimetic of cTnI whereby tandem serine codons 23/ 24 were converted to aspartic acids ( cTnI S23/ 24D). Adult cardiac myocytes were isolated and contractility determined at physiological temperature under unloaded and loaded conditions using micro- carbon fibers. At baseline, cTnI S23/ 24D myocytes had significantly faster relaxation times relative to controls, and isoproterenol stimulation ( Iso) had only a small effect to further speed relaxation in cTnI S23/ 24D myocytes ( delta Iso: 7.2 ms) relative to the maximum Iso effect ( 31.2 ms) in control. The Ca2+ transient decay rate was similarly accelerated by Iso in Tg and nontransgenic ( Ntg) myocytes. Gene transfer of cTnI S23/ 24D to myocytes in primary culture showed comparable findings. Gene transfer of cTnI with both serines 23/ 24 converted to alanines ( cTnI S23/ 24A), or gene transfer of slow skeletal TnI, both of which lack PKA phosphorylation sites, significantly blunted Iso-mediated enhanced relaxation compared with controls. Gene transfer of wild- type cTnI had no effect on relaxation. These findings support a key role of cTnI in myocyte relaxation and highlight a direct contribution of the myofilaments in modulating the dynamics of myocardial performance.