Constitutively active phosphatase inhibitor-1 improves cardiac contractility in young mice but is deleterious after catecholaminergic stress and with aging
Katrin Wittköpper, Larissa Fabritz, Stefan Neef, Katharina R. Ort, Clemens Grefe, Bernhard Unsöld, Paulus Kirchhof, Lars S. Maier, Gerd Hasenfuss, Dobromir Dobrev, Thomas Eschenhagen, Ali El-Armouche
Katrin Wittköpper, Larissa Fabritz, Stefan Neef, Katharina R. Ort, Clemens Grefe, Bernhard Unsöld, Paulus Kirchhof, Lars S. Maier, Gerd Hasenfuss, Dobromir Dobrev, Thomas Eschenhagen, Ali El-Armouche
View: Text | PDF
Research Article Cardiology

Constitutively active phosphatase inhibitor-1 improves cardiac contractility in young mice but is deleterious after catecholaminergic stress and with aging

Abstract

Phosphatase inhibitor-1 (I-1) is a distal amplifier element of β-adrenergic signaling that functions by preventing dephosphorylation of downstream targets. I-1 is downregulated in human failing hearts, while overexpression of a constitutively active mutant form (I-1c) reverses contractile dysfunction in mouse failing hearts, suggesting that I-1c may be a candidate for gene therapy. We generated mice with conditional cardiomyocyte-restricted expression of I-1c (referred to herein as dTGI-1c mice) on an I-1–deficient background. Young adult dTGI-1c mice exhibited enhanced cardiac contractility but exaggerated contractile dysfunction and ventricular dilation upon catecholamine infusion. Telemetric ECG recordings revealed typical catecholamine-induced ventricular tachycardia and sudden death. Doxycycline feeding switched off expression of cardiomyocyte-restricted I-1c and reversed all abnormalities. Hearts from dTGI-1c mice showed hyperphosphorylation of phospholamban and the ryanodine receptor, and this was associated with an increased number of catecholamine-induced Ca2+ sparks in isolated myocytes. Aged dTGI-1c mice spontaneously developed a cardiomyopathic phenotype. These data were confirmed in a second independent transgenic mouse line, expressing a full-length I-1 mutant that could not be phosphorylated and thereby inactivated by PKC-α (I-1S67A). In conclusion, conditional expression of I-1c or I-1S67A enhanced steady-state phosphorylation of 2 key Ca2+-regulating sarcoplasmic reticulum enzymes. This was associated with increased contractile function in young animals but also with arrhythmias and cardiomyopathy after adrenergic stress and with aging. These data should be considered in the development of novel therapies for heart failure.

Authors

Katrin Wittköpper, Larissa Fabritz, Stefan Neef, Katharina R. Ort, Clemens Grefe, Bernhard Unsöld, Paulus Kirchhof, Lars S. Maier, Gerd Hasenfuss, Dobromir Dobrev, Thomas Eschenhagen, Ali El-Armouche

×

Figure 7

Characterization of I-1S67A–expressing mice.

Options: View larger image (or click on image) Download as PowerPoint
Characterization of I-1S67A–expressing mice. (A) Doxycycline-depende...
(A) Doxycycline-dependent I-1S67A expression and a lack of leakiness in single transgenic I-1S67A responders (I-1S67A). I-1S67A mRNA amount in dTGS67A ON mice is 8-fold higher compared with Ppp1r1a mRNA from WT mice (n = 5 each). *P < 0.05 versus WT. (B) I-1S67A enhances cardiac contractility in vivo (n = 6 for each group). *P < 0.05 versus tTA. (C) Phosphorylation state of PLB (PLBSer16) and RyR2 (RyRSer2815) in tTA and dTGS67A ON hearts (n ≥ 6 for each group). Samples were run on the same gel. *P < 0.05 versus tTA. (D) Number of tTA and dTGS67A ON mice that developed VTs (black bar) after arrhythmia provocation in freely moving mice. Doxycycline application completely repressed the development of VTs (dTGS67A OFF). *P < 0.05 by χ2 test. (E) Representative longitudinal line-scan images of tTA and dTGS67A ON mice and Ca2+ spark frequency, respectively (10 nM isoprenaline). The numbers in the columns represent the number of myocytes and hearts from each group. *P < 0.05 versus tTA. (F) Isoprenaline-infusion accelerated the decrease in contractility in dTGS67A ON versus tTA mice after 4 and 14 days. Exaggerated decline of contractility in dTGS67A ON mice could be stopped by doxycycline feeding from day 4 to 14 (red line). The dashed line represents the I-1c double-transgenic mice kept on water at any time. *P < 0.05 tTA versus dTGS67A ON on day 4; #P < 0.05 dTGS67A ON versus dTGS67A 10d OFF and tTA. (G) Aging I-1S67A double-transgenic mice show an exaggerated decrease in FAS and increase in LVEDD (n ≥ 5 each) compared with tTA mice at the age of 15 months. *P < 0.05 versus tTA. Representative M-mode views from tTA and dTGS67A ON mice. Experiments were done in parallel to the I-1c double-transgenic mice (C–E).