Divergent off-target effects of RSK N-terminal and C-terminal kinase inhibitors in cardiac myocytes.

P90 ribosomal S6 kinases (RSK) are ubiquitously expressed and regulate responses to neurohumoral stimulation. To study the role of RSK signalling on cardiac myocyte function and protein phosphorylation, pharmacological RSK inhibitors were tested. Here, the ATP competitive N-terminal kinase domain-targeting compounds D1870 and SL0101 and the allosteric C-terminal kinase domain-targeting FMK were evaluated regarding their ability to modulate cardiac myocyte protein phosphorylation. Exposure to D1870 and SL0101 significantly enhanced phospholamban (PLN) Ser16 and cardiac troponin I (cTnI) Ser22/23 phosphorylation in response to D1870 and SL0101 upon exposure to phenylephrine (PE) that activates RSK. In contrast, FMK pretreatment significantly reduced phosphorylation of both proteins in response to PE. D1870-mediated enhancement of PLN Ser16 phosphorylation was also observed after exposure to isoprenaline or noradrenaline (NA) stimuli that do not activate RSK. Inhibition of ß-adrenoceptors by atenolol or cAMP-dependent protein kinase (PKA) by H89 prevented the D1870-mediated increase in PLN phosphorylation, suggesting that PKA is the kinase responsible for the observed phosphorylation. Assessment of changes in cAMP formation by FRET measurements revealed increased cAMP formation in vicinity to PLN after exposure to D1870 and SL0101. D1870 inhibited phosphodiesterase activity similarly as established PDE inhibitors rolipram or 3-isobutyl-1-methylxanthine. Assessment of catecholamine-mediated force development in rat ventricular muscle strips revealed significantly reduced EC50 for NA after D1870 pretreatment (DMSO/NA: 2.33 µmol/L vs. D1870/NA: 1.30 µmol/L). The data reveal enhanced cardiac protein phosphorylation by D1870 and SL0101 that was not detectable in response to FMK. This disparate effect might be attributed to off-target inhibition of PDEs with impact on muscle function as demonstrated for D1870.

I-1-deficiency negatively impacts survival in a cardiomyopathy mouse model.

AIMS: Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis. Current treatment is based on beta-adrenoceptor (AR) and calcium channel blockers. Since mice deficient of protein phosphatase-1 inhibitor-1 (I-1), an amplifier in beta-AR signalling, were protected from pathological adrenergic stimulation in vivo, we hypothesized that I-1 ablation could result in an improved outcome in a HCM mouse model. METHODS AND RESULTS: We crossed mice deficient of I-1 with homozygous myosin-binding protein C knock-out (Mybpc3 KO) mice exhibiting cardiac dilatation and reduced survival. Unexpectedly, survival time was shorter in double I-1/Mybpc3 KO than in single Mybpc3 KO mice. Longitudinal echocardiographic assessment revealed lower fractional area change, and higher diastolic left ventricular inner dimensions and end-diastolic volumes in Mybpc3 KO than in WT mice. In comparison to Mybpc3 KO, double I-1/Mybpc3 KO presented higher left ventricular end-diastolic volumes, inner dimensions and ventricular surface areas with increasing differences over time. Phosphorylation levels of PKA-downstream targets and mRNA levels of hypertrophic markers did not differ between I-1/Mybpc3 KO and single Mybpc3 KO mice, except a trend towards higher beta-myosin heavy chain levels in double I-1/Mybpc3 KO. CONCLUSION: The data indicate that interference with beta-AR signalling has no long-term benefit in this severe MYBPC3-related cardiomyopathy mouse model.

Development of phosphatase inhibitor-1 peptides acting as indirect activators of phosphatase 1.

Phosphatase inhibitor-1 (I-1) inhibits the catalytic subunit of protein phosphatase type 1 (PP1c) in its protein kinase A (PKA)-phosphorylated form (I-1(P)). It thereby amplifies PKA signaling, which, in the heart, mediates both beneficial (acute) and adverse (chronic) effects of catecholamines. Genetic deletion of I-1 was associated with protection against catecholamine toxicity, making the PP1c-I-1(P) complex a potential therapeutic target for chronic heart disease. Here, we sought to define targetable interaction sites of I-1 and PP1c, concentrating on the N-terminal domain of I-1 which includes the PP1c binding motif ((9)KIQF(12)) as well as a poly-Arg stretch. Substitution of (9)KIQ(11) residues for analogous amino acids, (9)RLN(11), resulted in doubling of the IC50 values, deletion of (9)KIQF(12) prevented I-1 PKA-dependent phosphorylation and thus activation. Mutation of the Arg residues preceding the PKA phosphorylation site (Thr35) to Ala (R/A(30-33)) abolished I-1 phosphorylation and its binding to and inhibition of PP1c. A series of synthetic peptides (4-11 residues) indicated that the KIQF motif as well as the surrounding anchoring residues was essential for interfering with the inhibitory effect of I-1(P) on PP1c, whereas the four Arg residues were not. Unexpectedly, the most effective nonapeptide (SPRKIQFTV) also antagonized the inhibitory effect of the non-conditional PP1 inhibitor-2 with similar affinity. Incubation of neonatal rat cardiac myocytes with a poly-Arg-modified SPRKIQFTV (10 µM) reduced catecholamine-induced phosphorylation of phospholamban, a well-known PKA downstream target sensitive to PP1c. Our data reiterate the importance of the KIQF motif and provide a tool for antagonizing I-1 inhibitory effects on PP1c, i.e., activating PP1 in vivo.

Guanabenz interferes with ER stress and exerts protective effects in cardiac myocytes.

Endoplasmic reticulum (ER) stress has been implicated in a variety of cardiovascular diseases. During ER stress, disruption of the complex of protein phosphatase 1 regulatory subunit 15A and catalytic subunit of protein phosphatase 1 by the small molecule guanabenz (antihypertensive, α2-adrenoceptor agonist) and subsequent inhibition of stress-induced dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in prolonged eIF2α phosphorylation, inhibition of protein synthesis and protection from ER stress. In this study we assessed whether guanabenz protects against ER stress in cardiac myocytes and affects the function of 3 dimensional engineered heart tissue (EHT). We utilized neonatal rat cardiac myocytes for the assessment of cell viability and activation of ER stress-signalling pathways and EHT for functional analysis. (i) Tunicamycin induced ER stress as measured by increased mRNA and protein levels of glucose-regulated protein 78 kDa, P-eIF2α, activating transcription factor 4, C/EBP homologous protein, and cell death. (ii) Guanabenz had no measurable effect alone, but antagonized the effects of tunicamycin on ER stress markers. (iii) Tunicamycin and other known inducers of ER stress (hydrogen peroxide, doxorubicin, thapsigargin) induced cardiac myocyte death, and this was antagonized by guanabenz in a concentration- and time-dependent manner. (iv) ER stressors also induced acute or delayed contractile dysfunction in spontaneously beating EHTs and this was, with the notable exception of relaxation deficits under thapsigargin, not significantly affected by guanabenz. The data confirm that guanabenz interferes with ER stress-signalling and has protective effects on cell survival. Data show for the first time that this concept extends to cardiac myocytes. The modest protection in EHTs points to more complex mechanisms of force regulation in intact functional heart muscle.

Development of a colorimetric and a fluorescence phosphatase-inhibitor assay suitable for drug discovery approaches.

Protein phosphatases (PP) are interesting drug targets. However, their ubiquitous presence and involvement in different, partially opposing signal pathways suggest that specificity may be achieved rather by targeting their interaction with subunits determining substrate specificity than the enzyme itself. An interesting subunit is phosphatase inhibitor-1 (I-1), which, in its protein kinase A-phosphorylated form (I-1(P)), inhibits the catalytic subunit of type 1 phosphatase (PP1c). In the current study, we established a colorimetric and a fluorescence-based assay system for the identification of compounds interfering with the inhibitory effect of I-1(P) on PP1c. The fluorescence assay exhibited 500-fold higher sensitivity toward PP1c. A nine-residue peptide containing the PP1c-binding motif (RVxF) of I-1 stimulated PP1c activity in the presence of I-1(P) (EC50 27 µM and 2.3 µM in the colorimetric and fluorescence assay, respectively). This suggests that the peptide interfered with the inhibitory effect of I-1(P) on PP1c and represents a proof-of-principle. The calculated Z' factor for PP1c (0.84) and the PP1c-I-1(P) complex (0.73) confirmed the suitability of the fluorescence assay for high-throughput screenings (HTS). By testing several thousand small molecules, we suggest the advantages of kinetic measurements over single-point measurements using the fluorescence-based assay in an HTS format.