Protein kinase C and A sites on troponin I regulate myofilament Ca2+ sensitivity and ATPase activity in the mouse myocardium

Abstract

Cardiac troponin I (cTnI) is a phosphoprotein subunit of the troponin-tropomyosin complex that is thought to inhibit cardiac muscle contraction during diastole. To investigate the contributions of cTnI phosphorylation to cardiac regulation, transgenic mice were created with the phosphorylation sites of cTnI mutated to alanine. Activation of protein kinase C (PKC) by perfusion of hearts with phorbol-12-myristate-13-acetate (PMA) or endothelin-1 (ET-1) inhibited the maximum ATPase rate by up to 25 % and increased the Ca2+ sensitivity of ATPase activity and of isometric tension by up to 0.15 pCa units. PKC activation no longer altered cTnI phosphorylation, depressed ATPase rates or enhanced myofilament Ca2+ sensitivity in transgenic mice expressing cTnI that could not be phosphorylated on serines43/45 and threonine144 (PKC sites). Modest changes in myosin regulatory light chain phosphorylation occurred in all mouse lines, but increases in myofilament Ca2+ sensitivity required the presence of phosphorylatable cTnI. For comparison, the beta-adrenergic agonist isoproterenol caused a 38 % increase in maximum ATPase rate and a 0.12 pCa unit decrease in myofilament Ca2+ sensitivity. These beta-adrenergic effects were absent in transgenic mice expressing cTnI that could not be phosphorylated on serines23/24 (protein kinase A, PKA, sites). Overall, the results indicate that PKC and PKA exert opposing effects on actomyosin function by phosphorylating cTnI on distinct sites. A primary role of PKC phosphorylation of cTnI may be to reduce the requirements of the contractile apparatus for both Ca2+ and ATP, thereby promoting efficient ATP utilisation during contraction.

Figure 1. Mouse genotyping

A, cardiac TnI constructs and the PCR primers (horizontal arrows and trivial names UTF, KK1, KK2, NP, PGK and 1IR) used to detect them. Genotyping required four separate PCR reactions on DNA isolated from mouse tails. Reaction 1 (Rxn 1) generated a 390 base pair (bp) fragment for the cTnI-knockout allele (cTnI-KO). Reaction 2 (Rxn 2) generated a 630 bp fragment for the wild-type cTnI gene (WT-cTnI). The Ala₂ and Ala₅ transgenes containing the α-myosin heavy chain (MHC) promoter were both detected by reaction 3 (Rxn 3; 304 bp fragment) and reaction 4 (Rxn 4; 1041 bp fragment). B, a typical agarose gel showing PCR fragments for the four reactions performed on three different mouse lines. Mouse genotypes (indicated above each set of four lanes) were determined by the pattern of PCR fragments. Ala₅^nb/Ala₂^nb mice were positive for the transgene and the cTnI-knockout allele but negative for wild-type cTnI. MW = molecular weight.

Figure 6. Effects of MLCK treatment on tension and myofilament protein phosphorylation

The data points represent the mean ±s.e.m. from six separate experiments, with solid lines (in A and B) illustrating non-linear least-squares fits to the Hill equation. A, pCa-tension curves in wild-type myocytes before and after MLCK (0.5 units ml⁻¹, 10 min, room temperature). The pCa₅₀ values were 5.56 ± 0.03 (control) and 5.67 ± 0.04 (MLCK). Maximum active tension and resting tension in mN mm⁻² were 16.2 ± 2 and 1.1 ± 0.2, respectively (control), and 14.7 ± 2 and 1.5 ± 0.2, respectively (MLCK). B, pCa-tension curves in cTnI-Ala₅^nb before and after the same MLCK treatment. The pCa₅₀ values were 5.24 ± 0.03 (control) and 5.2 ± 0.07 (MLCK). Maximum active tension and resting tension in mN mm⁻² were 21.3 ± 3 and 1.0 ± 0.2, respectively (control), and 20.3 ± 3 and 1.4 ± 0.3, respectively (MLCK). C, SDS-PAGE analysis of skinned myocytes treated with MLCK. Lane 1: wild-type, no MLCK; lane 2: wild-type, with MLCK; lane 3: cTnI-Ala₅^nb, no MLCK; lane 4: cTnI-Ala₅^nb, with MLCK. The right panel summarises data from four gels obtained by excising LC₂, counting and normalising to microgrammes of protein in the LC₂ band.

Figure 5. Agonist effects on ³²P-phosphate incorporation into myofilament proteins of intact myocytes

A, autoradiograms of ventricular myocytes from wild-type, cTnI-Ala₂^nb and cTnI-Ala₅^nb mice treated with standard doses of the indicated agonists. B, summary of data from a minimum of four autoradiograms. The left panel summarises the effects of 10 nm Iso on cTnI phosphorylation. The other three panels summarise the effects of 10 nm ET-1 on cTnI, LC₂ and cardiac troponin T (TnT). The effects of PMA were similar to those of ET-1. *P < 0.05 compared to untreated controls.

Figure 2. Characterisation of TnI expression

A, Western blot of myofibrils from wild-type (WT), cTnI-Ala₂ transgenic founder, and cTnI-Ala₂^nb mice at 1 month of age. Myofibrils from 15-day-old cTnI-KO mice (lane 2) and purified bovine cTnI (std cTnI, lane 6) are included as controls. ssTnI is slow skeletal TnI. B, autoradiogram of PKA-phosphorylated myofilament proteins. Incorporation of ³²P in the absence (left three lanes, labelled −) and presence (right three lanes, labelled +) of a PKA catalytic subunit. ³²P-incorporation in cTnI of Ala₂ was 14 ± 5 % that of wild-type and in Ala₂^nb was undetectable. Tissue samples were the same as in A.

Figure 4. Agonist effects on the pCa versus isometric tension relationship

A, wild-type, B, cTnI-Ala₂^nb and C, cTnI-Ala₅^nb. Data points represent the mean from six separate experiments in which active tension was determined by subtracting the resting tension at pCa 9 from the measured tension at each pCa value. Active tension at each pCa value was then normalised to maximum tension at pCa 4.5 for that experiment. Solid lines represent non-linear least-squares fits to the Hill equation. pCa₅₀ values are given in Table 2, and maximum active tensions and resting tension are given in Table 1.

Figure 3. Agonist effects on pCa versus MgATPase activity

A, wild-type, B, cTnI-Ala₂^nb and C, cTnI-Ala₅^nb. Data points represent the mean of six separate experiments in which the ATPase activity at each pCa value was performed in triplicate. The basal ATPase activity measured at pCa 9 was subtracted and the activity at each pCa value was normalised to the maximum at pCa 4 for that experiment. Typical basal and maximum ATPase activities are illustrated and summarised in the on-line data supplement. Solid lines represent non-linear least-squares fits to the Hill equation. pCa₅₀ values are given in Table 2.