Mutual rescues between two dominant negative mutations in cardiac troponin I and cardiac troponin T

Abstract

Troponin T (TnT) and troponin I (TnI) are two evolutionarily and functionally linked subunits of the troponin complex that regulates striated muscle contraction. We previously reported a single amino acid substitution in the highly conserved TnT-binding helix of cardiac TnI (cTnI) in wild turkey hearts in concurrence with an abnormally spliced myopathic cardiac TnT (cTnT) (Biesiadecki, B. J., Schneider, K. L., Yu, Z. B., Chong, S. M., and Jin, J. P. (2004) J. Biol. Chem. 279, 13825-13832). To investigate the functional effect of this cTnI mutation and its potential value in compensating for the cTnT abnormality, we developed transgenic mice expressing the mutant cTnI (K118C) in the heart with or without the deletion of the endogenous cTnI gene to mimic the homozygote and heterozygote of wild turkeys. Double and triple transgenic mice were created by crossing the cTnI-K118C lines with transgenic mice overexpressing the myopathic cTnT (exon 7 deletion). Functional studies of ex vivo working hearts found that cTnI-K118C alone had a dominantly negative effect on diastolic function and blunted the inotropic responses of cardiac muscle to beta-adrenergic stimuli without abolishing the protein kinase A-dependent phosphorylation of cTnI. When co-expressed with the cTnT mutation, cTnI-K118C corrected the significant depression of systolic function caused by cTnT exon 7 deletion, and the co-existence of exon 7-deleted cTnT minimized the diastolic abnormality of cTnI-K118C. Characterization of this naturally selected pair of mutually rescuing mutations demonstrated that TnI-TnT interaction is a critical link in the Ca(2+) signaling and beta-adrenergic regulation in cardiac muscle, suggesting a potential target for the treatment of troponin cardiomyopathies and heart failure.

FIGURE 1.

Transgenic mouse models expressing exon 7-deleted cTnT and/or K118C-cTnI in the heart. A, locations of exon 7-encoded segment in cTnT and the K118C mutation in cTnI are illustrated in this model structure of troponin complex. The high resolution portion was adapted from the crystal structure of partial human cardiac troponin complex (7). The two tropomyosin (Tm)-binding sites in TnT (27) are outlined. B, CT3 and TnI-1 Western blots showed the expression of exon 7-deleted (ΔE7) cTnT and/or cTnI-K118C in the transgenic (TG) mouse hearts in the presence (upper panel) or absence (lower panel) of endogenous wild type cTnI. C, Western blots using anti-cTnT mAb CT3 and anti-TnI mAb TnI-1 demonstrated the postnatally up-regulated expression of exon 7-deleted cTnT and/or cTnI-K118C in the transgenic mouse cardiac muscle along with wild type (WT) controls that showed the developmental switching of cTnT and TnI isoforms. D, densitometry analysis showed that the total levels of cTnT and cTnI were not different in the transgenic mouse hearts as compared with that in wild type controls. Error bars indicate S.D.

FIGURE 2.

Unchanged morphology and myosin isoform expression in 3-month-old transgenic mouse hearts expressing exon 7-deleted cTnT and/or cTnI-K118C. A, low and high magnification images of hematoxylin and eosin-stained cross-sections of wild type and the single, double, and triple transgenic mouse hearts. B, heart weight:body weight ratio, the cross area of left and right ventricular chambers, and the thickness of left ventricular free wall did not show statistical differences. Error bars indicate S.D. C, glycerol-SDS-PAGE showed only α-MHC in wild type, exon 7-deleted (ΔE7) cTnT, and cTnI-K118C single and double transgenic (TG) mouse hearts. A failing ventricular muscle sample from G_sα knock-out mouse heart (15) was used as control for the adaptive expression of β-MHC.

FIGURE 3.

Function of the transgenic mouse hearts overexpressing exon 7-deleted cTnT. Contractile and relaxation velocities, LVP development, and stroke volume were measured in ex vivo working heart preparations under different afterloads with preload fixed at 10 mm Hg (A) or under different preloads with afterload fixed at 55 mm Hg (B). The results demonstrated that all of the parameters indicated significantly decreased function of the exon 7-deleted (ΔE7) cTnT transgenic mouse hearts as compared with wild type controls. *, p < 0.05 compared with wild type using two-tailed Student's t test. Error bars indicate S.D.

FIGURE 4.

Function of the transgenic mouse hearts expressing cTnI-K118C. Contractile and relaxation velocities, LVP development, and stroke volume were measured in ex vivo working heart preparations under different afterloads with preload fixed at 10 mm Hg (A) or under different preloads with afterload fixed at 55 mm Hg (B). The results demonstrated that cTnI-K118C decreased cardiac function, specifically the relaxation velocity and LVP development, in the transgenic mouse hearts as compared with wild type controls. The 100% cTnI-K118C hearts also exhibited decreased contractile velocity when preload was increased. *, p < 0.05 compared with wild type using two-tailed Student's t test. Error bars indicate S.D.

FIGURE 5.

Mutually rescued cardiac function when exon 7-deleted cTnT and cTnI-K118C were co-expressed in transgenic mouse hearts. Contractile and relaxation velocities, LVP development, and stroke volume were measured in ex vivo working heart preparations under different afterloads with preload fixed at 10 mm Hg (A) or under different preloads with afterload fixed at 55 mm Hg (B). The exon 7-deleted (ΔE7) cTnT/cTnI-K118C double (∼50% cTnI-K118C) and triple (100% cTnI-K118C) transgenic mouse hearts exhibited apparently normal function similar to that of the wild type controls (dashed curves). *, p < 0.05 compared with wild type using two-tailed Student's t test. Error bars indicate S.D.

FIGURE 6.

Responses of cardiac function to β-adrenergic stimulation. A, effects of 10 nm isoproterenol on contractile and relaxation velocities, LVP development, stroke volume, and stroke work were examined in ex vivo working hearts at 10 mm Hg preload and 55 mm Hg afterload. In the histograms, the open boxes indicate the extent of functional change upon isoproterenol treatment. The results showed that isoproterenol induced profound increases in cardiac function from the decreased base line in exon 7 (E7)-deleted cTnT hearts to reach the wild type level. In contrast, cTnI-K118C hearts had blunted responses to isoproterenol treatment as compared with the wild type controls. The double (∼50% cTnI-K118C) and triple (100% cTnI-K118C) transgenic mouse hearts co-expressing exon 7-deleted cTnT and cTnI-K118C reached wild type level of function in response to 10 nm isoproterenol treatment. Error bars indicate S.D. B, representative P-V loops outline the β-adrenergic responses of the wild type and single, double, and triple transgenic mouse hearts. LVEDV, left ventricular end diastolic volume. #, p < 0.05 comparing the base-line cardiac function with the wild type control; *, p < 0.05 after 10 nm isoproterenol treatment, using two-tailed Student's t test.

FIGURE 7.

PKA-dependent phosphorylation of cTnI was preserved in the transgenic mouse hearts. A, Pro-Q Diamond phosphoprotein staining of SDS gels was used to measure phosphorylated cTnI in mouse cardiac muscle in vivo and after 10 nm isoproterenol treatment of ex vivo working hearts. The total levels of cTnI and cTnT were measured by Western blots using mAbs TnI-1 and CT3. B, densitometry quantification of the Pro-Q-stained gels and Western blots for the relative levels of the phosphorylated versus total proteins showed that the cTnI-K118C mutation did not reduce the level of cTnI phosphorylation in vivo or upon β-adrenergic treatment ex vivo as compared with the wild type (WT) controls. The presence of exon 7-deleted (ΔE7) cTnT alone or in combination with cTnI-K118C did not affect the level of cTnI phosphorylation. Error bars indicate S.D.