Monophosphorylation of cardiac troponin-I at Ser-23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis

Abstract

The acceleration of myocardial relaxation produced by β-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca²⁺ sensitivity. Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca²⁺ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca²⁺ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated (using sequential incubations in λ-phosphatase, PKD, and PKA, respectively). Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca²⁺ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca²⁺ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics (k_tr). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca²⁺ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca²⁺ sensitivity.

Keywords: Ca2+ sensitivity; calpain; cardiac muscle; cardiomyocyte; myofibril; phosphorylation; protein kinase D (PKD); sarcomere; troponin.

Figure 1.

PKD monophosphorylates cTnI in the sarcomere. A, mouse cardiac myofibrils were incubated in PKD for different times, separated in SDS-PAGE with Phos-tag reagent, and blotted with a specific antibody against cTnI. Top panel, representative Western blot. Bottom panel, relative abundance of the three bands of cTnI in each lane. Symbols show the data from individual experiments; vertical bars show the mean ± S.D. (n = 3 experiments; *, p < 0.05 versus nontreated). IB, immunoblot. B, as A, except isolated myofibrils were incubated with λ-PP before incubation in PKD (n = 4; *, p < 0.05 versus nontreated). Note that incubation with PKD produces monophosphorylation of cTnI almost exclusively. C, top-down high-resolution MS analysis of cTnI from mouse myofibrils. Top panel, representative total ion chromatogram showing separation and elution of cTnI. Bottom panel, high-resolution mass spectra of cTnI from λ-PP–treated myofibrils before and after 1-h incubation of the myofibrils with PKD. Horizontal arrows indicate the position of unphosphorylated (cTnI), mono-phosphorylated (_pcTnI), and bisphosphorylated cTnI (_ppcTnI). Insets, isotopically resolved molecular ions with the calculated most abundant molecular weight (Calc'd) based on the amino acid sequence and experimental most abundant molecular weight (Expt'l). Circles represent the theoretical isotopic abundance distribution of the isotopomer peaks corresponding to the assigned mass.

Figure 2.

Determination of the cTnI site targeted by PKD. A, Phos-tag gels of mouse λ-PP–treated myofibrils after incubation in PKD alone (1 h) or PKD (1 h) followed by PKA (1 h). Western blots of Phos-tag gels used specific antibodies against total TnI or pSer-23/24 TnI. IB, immunoblot. B, comparison of results using myofibrils from WT mice or cTnI-Ala2 transgenic mice. Other conditions were as for A. C, Western blots using human λ-PP–treated cardiac myofibrils incubated with PKD alone or PKD followed by PKA and then probed for TnI or pSer-24 cTnI. Other conditions were as for A. All gels are representative of at least three experiments.

Figure 3.

Comparison of the effects of PKD and PKA on the force–Ca²⁺ relationship of mouse skinned trabeculae. A, forces were measured in λ-PP–treated skinned muscles before (Pre-kinase) and after incubations in PKD (PKD) and then PKA (PKD+PKA). Forces expressed relative to that at 30 μmol/liter Ca²⁺. Inset, mean pCa₅₀ (−log[Ca²⁺]) values. Symbols show the data from individual experiments; vertical bars show the mean ± S.D. B, WT time-matched controls (no kinase) corresponding to each incubation in A. C, experiments as for A but using skinned muscles from cTnI-Ala2 mice. D, changes in Ca²⁺ sensitivity (ΔpCa₅₀) produced by PKD with or without PKA compared with WT time-matched controls. Data labeled PKD show the difference in pCa₅₀ from the corresponding prekinase pCa₅₀. Data labeled +PKA show the difference in pCa₅₀ from the corresponding PKD pCa₅₀. WT(t-m) are the time-matched controls (as in B). E, the same data as in D but after subtraction of the mean ΔpCa₅₀ of the corresponding time-matched controls to illustrate the time-independent effects of the kinases. For all panels, n = 10 for the pre-kinase and PKD incubation groups in WT muscles, and n = 5 for all other groups. ‡, p < 0.0001; *, p < 0.05; ns, nonsignificant (all paired t tests, n = 5 or n = 10).

Figure 4.

Phosphorylation of sarcomeric cTnI by either PKD or PKA reduces its proteolysis by calpain. A, λ-PP–treated isolated myofibrils were incubated with PKD alone and then treated with calpain-1 at the activities shown (as units per 100 μl). Shown is representative Western blotting (WB) using an antibody against full-length TnI and loading control (Coomassie stain). B, as A, except the myofibrils were incubated in PKD and then PKA before incubation in calpain. C, summary of calpain-dependent cTnI degradation after no kinase addition (n = 7), PKD alone (n = 4), or PKD+PKA (n = 3). Symbols show the data from individual experiments; vertical bars show the mean ± S.D. D--F, as A–C but using an antibody that recognizes a sequence in the C terminus of TnI. Symbols show the data from individual experiments; vertical bars show the mean ± S.D. (n = 6 for no kinase, n = 4 for PKD alone, n = 3 for PKD+PKA). *, p < 0.05 versus no calpain; #, p < 0.05 PKD or PKD+PKA versus no kinase addition.

Figure 5.

PKD can monophosphorylate cTnI in rat adult cardiomyocytes. Cardiomyocytes infected with adenovirus carrying PKDWT/EGFP (+) or control EGFP (−) were stimulated with ET-1 (100 nmol/liter for 5, 10, or 20 min), PDBu (200 nmol/liter, 20 min), or isoproterenol (Iso, 10 nmol/liter, 10 min). A, representative immunoblots (IB) of p744/748 PKD and total PKD (top panel; the vertical line shows where an empty lane has been removed) and relative levels of p744/748 PKD normalized to total PKD (bottom panel). B, representative Phos-tag gels of total TnI and phosphoserine 23/24 cTnI (top panel) and proportion of cTnI unphosphorylated, monophosphorylated, and bisphosphorylated cTnI after the various treatments. Symbols show the data from individual experiments; vertical bars show the mean ± S.D. (n = 3–5).