Fig 2: 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).

Fig 3: Possible mechanism of hyperglycemia in change of cardiac troponin I expression in type-1 diabetic rats. Hyperglycemia-induced ROS activates the MEK/ERK pathway to increase GATA-4 phosphorylation and nuclear translocation. Also, MEK/ERK activation causes GSK3ß phosphorylation, lowering the export of GATA-4 and leading to GATA-4 preservation in the nucleus, which finally leads to an increase in the expression level of cTnI.

Fig 4: 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 ?-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 ?-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.

Fig 5: Determination of the cTnI site targeted by PKD. A, Phos-tag gels of mouse ?-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 ?-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.