Myofilament incorporation and contractile function after gene transfer of cardiac troponin I Ser43/45Ala

Abstract

Phosphorylation of cardiac troponin I serines 43/45 (cTnISer43/45) by protein kinase C (PKC) is associated with cardiac dysfunction and yet there is disagreement about the role this cluster plays in modulating contractile performance. The present study evaluates the impact of phospho-null Ala substitutions at Ser43/45 (cTnISer43/45Ala) on contractile performance in intact myocytes. Viral-based gene transfer of cardiac troponin I (cTnI) or cTnISer43/45Ala resulted in time-dependent increases in expression, with 70-80% of endogenous cTnI replaced within 4 days. Western analysis of intact and permeabilized myocytes along with immunohistochemistry showed each exogenous cTnI was incorporated into the sarcomere of myocytes. In contractile function studies, there were no differences in shortening and re-lengthening for cTnI and cTnISer43/45Ala-expressing myocytes 2 days after gene transfer. However, more extensive replacement with cTnISer43/45Ala after 4 days diminished peak shortening amplitude and accelerated re-lengthening measured as the time to 50% re-lengthening (TTR50%). A decrease in myofilament Ca2+ sensitivity of tension also was observed in permeabilized myocytes expressing cTnISer43/45Ala and is consistent with accelerated re-lengthening observed in intact myocytes under basal conditions. Phosphorylation of cTnI Ser23/24 and the Ca2+ transient were not changed in these myocytes. These results demonstrate extensive sarcomere expression of cTnISer43/45Ala directly modulates myofilament function under basal conditions. In further work, the accelerated re-lengthening observed in control or cTnI-expressing myocytes treated with the PKC agonist, endothelin-1 (ET, 10nM) was slowed in myocytes expressing cTnISer43/45Ala. This outcome may indicate Ser43/45 is targeted for phosphorylation by ET-activated PKC and/or influences transduction of this agonist-activated response.

Independent modulation of contractile performance by cardiac troponin I Ser43 and Ser45 in the dynamic sarcomere

“…For cTnIS43 and/or cTnIS45, site-directed mutagenesis was used to substitute S with D in rat cTnI [29,34]. The cTnI phosphomimetics generated include cTnIS43D, cTnIS45D, and cTnIS43/45D with and without FLAG [29].…”

“…The cTnI phosphomimetics generated include cTnIS43D, cTnIS45D, and cTnIS43/45D with and without FLAG [29]. The primers used to produce the phosphomimetics were: (substitutions are underlined) 5’-atgccaagaaaaagtctaa gat cgatgcctccagaaaacttcagttgaag-3’ (sense) and 5’-cttcaactgaagttttctggaggc atc gatcctagactttttcttggcat-3’ (anti-sense) for cTnIS43D, and 5’-gatctccgcc ga cagaaagcttcagttgaag-3’ (sense) and 5’-cttcaactgaa gc tttct gtcg gcggagatc-3’ (anti-sense) for cTnIS45D.…”

“…Primers for cTnIS43/45D were 5’-gccaagaaaaagtctaagatc gatg cc ga cagaaaacttcag-3’ (sense) and 5’-ctgaagttttct gtcgg c atcg atcttagactttttcttggc-3’ (antisense). Recombinant adenoviruses were prepared using the pDC315 shuttle vector and pBHGLoxΔE1,3Cre (Microbix Biosystems Inc, Mississauga,ON) in HEK293 cells [29]. …”

“…The isolation of Ca 2+ -tolerant myocytes from adult Sprague Dawley rats was performed as previously described in detail [29]. Briefly, myocytes were plated on laminin coated coverslips in DMEM containing 5% fetal bovine serum (FBS) and antibiotics (P/S; penicillin 50 U/ml; streptomycin 50μg/ml) for 2 hrs followed by incubation with a multiplicity of infection (MOI) of 50-500 recombinant adenoviral constructs (e.g.…”

“…Cardiac TnI with phosphomimetic S43/45 substitutions also reduces maximum sliding speed during in vitro motility assays [24], and maximum unloaded shortening velocity in permeabilized myocytes [26]. Moreover, non-phosphorylatable cTnIS43/45A attenuates the PKC activation response [16,27-29]. Based on this in vitro work, reduced pressure development and accelerated relaxation rate could be expected in animal models expressing phosphomimetic cTnIS43/45.…”