Reduced inhibitor 1 and 2 activity is associated with increased protein phosphatase type 1 activity in left ventricular myocardium of one-kidney, one-clip hypertensive rats

Abstract

In failing hearts, although protein phosphatase type 1 (PP1) activity has increased, information about the regulation and status of PP1 inhibitor-1 (INH-1) and inhibitor-2 (INH-2) is limited. In this study, we examined activity and protein expression of PP1, INH-1 and INH-2 and phosphorylation of sarcoplasmic reticulum (SR) phospholamban (PLB), a substrate of PP1 and modulator of SR Ca2+-ATPase activity, in failing and non-failing hearts. These studies were performed in LV myocardium of seven rats with chronic renal hypertension produced by Goldblatt's one-kidney, one-clip procedure and seven age-matched sham-operated normal controls (CTR). Eight weeks after surgery, LV ejection fraction, LV hypertrophy, and pulmonary congestion were determined in all rats. PP1 activity (nmol 32P/min/mg non-collagen protein) was assessed in LV homogenates using 32 P-labeled phosphorylase a as substrate. INH-1 and INH-2 activity was determined in the immunoprecipitate of LV homogenates and expressed as percentage inhibitory activity. Using a specific antibody, LV tissue levels of PP1C and calsequestrin (CSQ), a SR calcium binding protein, which is not altered in failing hearts, were also determined. Further, total and phosphorylated PLB, INH-1 and INH-2 protein levels were determined in the LV homogenate and phosphoprotein-enriched fraction, respectively. The band density of each protein was quantified in densitometric units and normalized to CSQ. Results: Rats with chronic r enal hypertension exhibited significantly reduced LV ejection fraction and increased LV hypertrophy and pulmonary congestion, characteristics of chronic heart failure (CHF). We found that compared to CTR, (1) both INH-1 (10.2 ± 2 versus 57.5 ± 1; p <0.05) and INH-2 activity (3.8 ± 0.4 versus 36.2 ± 4; p <0.05) were reduced, (2) total and phosphorylated PLB amount reduced, (3) protein level of phosphorylated INH-1 was reduced (2.32 ± 0.1 versus 0.73 ± 0.04; p <0.05) whereas that of phosphorylated INH-2 increased (3.05 ± 0.3 versus 1.42 ± 0.1; p <0.05), and (4) PP1 activity was increased approximately 2.6-fold in rats with CHF (1.59 ± 0.05 versus 0.61 ± 0.01; p <0.05) while protein level of the catalytic subunit of PP1 (PP1C) increased 3.85-fold (0.77 ± 0.05 versus 0.20 ± 0.02; p <0.05). These results suggest that reduced inhibitory INH-1 and INH-2 activity, increased PP1C protein level, and reduced PLB phosphorylation are associated with increased PP1 activity in failing hearts. © Springer Science + Business Media, Inc. 2005.