The lung function score and its components as predictors of overall survival and chronic graft-vs-host disease after allogeneic stem cell transplantation.

AIM: To retrospectively assess if the modified lung function score (LFS) and/or its components, forced expiratory volume within the first second (FEV1) and diffusion capacity for carbon monoxide corrected for hemoglobin level (cDLCO), predict overall survival (OS) and chronic graft-vs-host-disease (cGvHD). METHODS: We evaluated 241 patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) at the University of Regensburg Transplant Center between June 1998 and July 2005 in relation to their LFS, FEV1 and cDLCO, before and after HSCT. RESULTS: Decreased OS after allo-HSCT was related to decreased pre-transplantation values of FEV1<60% (P=0.040), cDLCO<50% of predicted value (P=0.025), and LFS≥III (P=0.037). It was also related to decreased FEV1 at 3 and 12 months after HSCT (P<0.001 and P=0.001, respectively) and increased LFS at 3 and 12 months after HSCT (P=.028 and P=0.002, respectively), but not to changes of cDLCO. A higher incidence of cGvHD was related to decreased FEV1 at 6, 12, and 18 months (P=0.069, P=0.054, and P=0.009, respectively) and increased LFS at 12 months (P=0.002), but not to changes in cDLCO. CONCLUSIONS: OS was related to both LFS and FEV1, but cGvHD had a stronger relation to FEV1 than to cDLCO or LFS. FEV1 alone offered more information on the outcome after allo-HSCT than LFS or cDLCO, suggesting limited value of LFS for the patients' assessment after allo-HSCT.

Role of calcineurin and protein phosphatase-2A in the regulation of phosphatase inhibitor-1 in cardiac myocytes.

Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), the predominating Ser/Thr phosphatase in the heart. Non-phosphorylated I-1 is inactive, whereas I-1 phosphorylated by protein kinase A (PKA) at Thr35 is a potent PP1 inhibitor. The phosphatases that dephosphorylate I-1Thr35 and thus deactivate I-1 in the heart are not established. Here we overexpressed I-1 in neonatal rat cardiac myocytes with recombinant adenovirus and determined phosphorylation of I-1, and one of the major target proteins of PKA/PP1 in the heart, phospholamban (PLB), by Western blot with phospho-specific antibodies. Incubation with the calcineurin inhibitor cyclosporine A or okadaic acid, used at a concentration preferentially inhibiting phosphatase 2A (PP2A), increased significantly I-1Thr35 (approximately 2- to 6-fold) and PLB Ser16 phosphorylation (approximately 2-fold). The results indicate that calcineurin and PP2A act to maintain a low basal level of phosphorylated (active) I-1 in living cardiac myocytes. Calcineurin may constitute a cross-talk between calcium- and cAMP-dependent pathways.

Decreased protein and phosphorylation level of the protein phosphatase inhibitor-1 in failing human hearts.

OBJECTIVE: The protein phosphatase inhibitor-1 (I-1) is a highly specific and potent inhibitor of type 1 phosphatases (PP1) that is active only in its protein kinase A (PKA)-phosphorylated form. I-1 ablation decreases, I-1 overexpression sensitizes beta-adrenergic signaling in the heart. It is controversial whether I-1 expression is altered in human heart failure (HF), likely because its detection in heart is difficult due to its low abundance. METHODS AND RESULTS: I-1 was >500-fold enriched from left ventricular myocardium (LVM) from patients with terminal HF (n=16) and non-failing controls (NF, n=5) and quantified with an affinity-purified I-1 and a I-1 phosphospecific antiserum. In non-failing I-1 protein levels amounted to 126 fmol/mg protein. In failing hearts, I-1 protein levels were reduced by 58% and I-1 phosphorylation by 77% (P<0.001 vs. NF). I-1 phosphorylation correlated well with serine-16 phosphorylation of phospholamban (PLB) in the same hearts (P<0.001). In contrast, PLB, troponin I (TnI) and PP1 protein and TnI phosphorylation levels did not differ between HF and NF. CONCLUSIONS: The results suggest that the reduction in I-1 protein and phosphorylation in failing human hearts leads to increased phosphatase activity which in turn may result in reduced phosphorylation of cardiac proteins such as PLB.

Evidence for protein phosphatase inhibitor-1 playing an amplifier role in beta-adrenergic signaling in cardiac myocytes.

The protein phosphatase inhibitor-1 (PPI-1) inhibits phosphatase type-1 (PP1) only when phosphorylated by protein kinase A and could play a pivotal role in the phosphorylation/dephosphorylation balance. Rat cardiac PPI-1 was cloned by reverse transcriptase-polymerase chain reaction, expressed in Eschericia coli, evaluated in phosphatase assays, and used to generate an antiserum. An adenovirus was constructed encoding PPI-1 and green fluorescent protein (GFP) under separate cytomegalovirus promotors (AdPPI-1/GFP). A GFP-only virus (AdGFP) served as control. Engineered heart tissue (EHT) from neonatal rat cardiomyocytes and adult rat cardiac myocytes (ARCMs) were used as model systems. PPI-1 expression was determined in human ventricular samples by Northern blots. Compared with AdGFP, AdPPI-1/GFP-infected neonatal rat cardiomyocytes displayed a 73% reduction in PP1 activity. EHTs infected with AdPPI-1/GFP exhibited a fivefold increase in isoprenaline sensitivity. AdPPI-1/GFP-infected ARCMs displayed enhanced cell shortening as well as enhanced phospholamban phosphorylation when stimulated with 1 nM isoprenaline. PPI-1 mRNA levels were reduced by 57+/-12% in failing hearts with dilated and ischemic cardiomyopathy (n=8 each) compared with nonfailing hearts (n=8). In summary, increased PPI-1 expression enhances myocyte sensitivity to isoprenaline, indicating that PPI-1 acts as an amplifier in beta-adrenergic signaling. Decreased PPI-1 in failing human hearts could participate in desensitization of the cAMP pathway.