Removal of the N-terminal Extension of Cardiac Troponin I as a Functional Compensation for Impaired Myocardial β-Adrenergic Signaling

Han-Zhong Feng,Min Chen,Lee S Weinstein,Jian-Ping Jin

doi:10.1074/jbc.m803302200

Abstract

Although beta-adrenergic stimuli are essential for myocardial contractility, beta-blockers have a proven beneficial effect on the treatment of heart failure, but the mechanism is not fully understood. The stimulatory G protein alpha-subunit (Gsalpha) couples the beta-adrenoreceptor to adenylyl cyclase and the intracellular cAMP response. In a mouse model of conditional Gsalpha deficiency in the cardiac muscle (Gsalpha-DF), we demonstrated heart failure phenotypes accompanied by increases in the level of a truncated cardiac troponin I (cTnI-ND) from restricted removal of the cTnI-specific N-terminal extension. To investigate the functional significance of the increase of cTnI-ND in Gsalpha-DF cardiac muscle, we generated double transgenic mice to overexpress cTnI-ND in Gsalpha-DF hearts. The overexpression of cTnI-ND in Gsalpha-DF failing hearts increased relaxation velocity and left ventricular end diastolic volume to produce higher left ventricle maximum pressure and stroke volume. Supporting the hypothesis that up-regulation of cTnI-ND is a compensatory rather than a destructive myocardial response to impaired beta-adrenergic signaling, the aberrant expression of beta-myosin heavy chain in adult Gsalpha-DF but not control mouse hearts was reversed by cTnI overexpression. These data indicate that the up-regulation of cTnI-ND may partially compensate for the cardiac inefficiency in impaired beta-adrenergic signaling.

Highlights

The ␤-adrenoreceptor (␤-AR)3 signaling pathway plays an important role in the regulation of heart function [1,2,3]
In a mouse model of conditional Gs␣ deficiency in the cardiac muscle (Gs␣-DF), we demonstrated heart failure phenotypes accompanied by increases in the level of a truncated cardiac troponin I from restricted removal of the cTnI-specific N-terminal extension
These results show that cTnI-ND overexpression partially corrected the heart failure in Gs␣-DF mice and support the hypothesis that the increase in cTnI-ND expression in Gs␣-DF hearts is a compensatory adaptation against the heart failure phenotype originating from impaired ␤-adrenergic signaling

Summary

EXPERIMENTAL PROCEDURES

Production of Gs␣-DF in Mouse Cardiac Muscle—Mice with floxed Gs␣ exon 1 allele (E1fl) [26] were bred with the muscle creatine kinase (MCK)-cre mice (Taconic, Hudson, NY) to induce striated muscle-specific disruption of the Gs␣ gene (MCK-cre,E1fl/fl; MGsKO). SDS-PAGE and Immunoblot Analysis—Gs␣ protein expression in the cardiac muscle was measured on total tissue protein extracts by SDS-PAGE and Western immunoblotting using a Gs␣-specific antibody [27]. Myosin heavy chain isoforms were resolved using 8% polyacrylamide gel with a 50:1 ratio of acrylamide/bisacrylamide containing 30% glycerol prepared in 200 mM Tris-HCl, 100 mM glycine (pH 8.8), and 0.4% SDS. The high concentration of D-glucose [32] and sodium pyruvate effectively prevents contractile cycling (cyclic fluctuations) [33] caused by insufficient metabolic substrates for ex vivo working mouse heart [34], allowing the hearts to be functionally stable in working mode for over 2 h. Stroke volume (␮l/mg heart tissue) was calculated from the sum of aortic flow and coronary effluent, normalized to heart rate

Cardiac Function at Various

Isolated working heart function at increased afterload

Findings

DISCUSSION