Inhibitor-2 prevents protein phosphatase 1-induced cardiac hypertrophy and mortality

Abstract

Cardiac-specific overexpression of the catalytic subunit of protein phosphatase type 1 (PP1) in mice results in hypertrophy, depressed contractility, propensity to heart failure, and premature death. To further address the role of PP1 in heart function, PP1 mice were crossed with mice that overexpress a functional COOH-terminally truncated form of PP1 inhibitor-2 (I-2(140)). Protein phosphatase activity was increased in PP1 mice but was normalized in double transgenic (DT) mice. The maximal rates of contraction (+dP/dt) and of relaxation (-dP/dt) were reduced in catheterized PP1 mice but normalized in DT mice. Similar contractile abnormalities were observed in isolated, perfused work-performing hearts and in whole animals by means of echocardiography. The increased absolute and relative heart weights observed in PP1 mice were normalized in DT mice. Histological analyses indicated that PP1 mice had significant cardiac fibrosis, which was absent in DT mice. Furthermore, PP1 mice exhibited an age-dependent increase in mortality, which was abrogated in DT mice. These results indicate that I-2 overexpression prevents the detrimental effects of PP1 overexpression in the heart and further underscore the fundamental role of PP1 in cardiac function. Therefore, PP1 inhibitors such as I-2 could offer new therapeutic options to ameliorate the deleterious effects of heart failure.

Fig. 1.

Expression of protein phosphatase (PP)1 and inhibitor-2 (I-2) in wild-type (WT) and transgenic mice. WT, COOH-terminally truncated I-2-overexpressing (I-2¹⁴⁰), PP1, and double transgenic (DT) heart extract samples were immunoblotted with I-2 (A)- and PP1 catalytic subunit (PP1c; B)-specific antibodies as described in materials and methods. For Western blots of I-2, 13 μg of protein was loaded for samples from WT and PP1c hearts, whereas only 2.6 μg of protein was loaded for I-2¹⁴⁰ and DT hearts. Therefore, the endogenous I-2 [mouse (m)I-2] cannot be detected in the lanes where less protein was loaded. For Western blotting of PP1 13 μg of protein was loaded for all samples. Fold overexpression is calculated with respect to the level in the WT samples and taking into account the protein loaded, and is given at bottom. Molecular mass markers are indicated on left. Tg, transgenic.

Fig. 2.

Total phosphatase activity. Phosphatase (PP) activity was determined in homogenates prepared from WT, PP1, I-2¹⁴⁰, and DT mouse hearts (30 wk of age) with ³²P-labeled phosphorylase a as substrate. Values are means ± SE. The elevated PP activity in PP1 mice is normalized in DT mice. *P < 0.05 vs. WT.

Fig. 3.

Immunohistochemical analysis of I-2. Longitudinal sections from paraffin-embedded hearts of WT, I-2¹⁴⁰, PP1, and DT mice were immunoreacted with mouse monoclonal antibody raised against human I-2 as described in materials and methods. Note that the monoclonal antibody used here does not cross-react with the endogenous mouse I-2. A representative set of sections is shown. Magnification, ×100. Note detection of I-2 in cardiomyocytes of I-2¹⁴⁰ and DT mice.

Fig. 4.

Histochemical detection of fibrosis. Longitudinal sections from paraffin-embedded hearts of WT, I-2¹⁴⁰, PP1, and DT mice at 30 wk of age were stained with Sirius red. Representative sections are shown. Note fibrosis in PP1 and great attenuation of fibrosis in DT.

Fig. 5.

Representative 2-dimensional parasternal axis view echocardiographic images from littermates aged 24 wk with left ventricular (LV) diameters indicated in diastole and systole (top) and values from motion mode (M-mode) measurements (bottom). Values are means ± SE for n mice. LVEDd and LVEDs, LV end-diastolic and end-systolic diameters; IVSd, diastolic thickness of the septum; LVPWd, diastolic posterior wall thickness of LV; LV mass, calculated LV mass {[(IVSd + LVEDd + LVPWd)³ − LVEDd] × 1.055}; BW, body weight; FS, % of fractional shortening calculated as (LVEDd − LVESd)/LVEDd × 100; EF, ejection fraction (Teichholz); Vcf, velocity of circumferential fiber shortening {FS/LV ejection time [circumference (circ)/s]}; bpm, beats/min. *P < 0.05 vs. WT.

Fig. 6.

Survival curves of WT, PP1, I-2¹⁴⁰, and DT mice. Note the enhanced mortality in PP1 mice, which is abrogated in DT mice, indicating protection against death by the overexpressed I-2¹⁴⁰. n = 35–68 mice.