Cardiac Hypertrophy and Impaired Relaxation in Transgenic Mice Overexpressing Triadin 1

Abstract

Triadin 1 is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum (SR), which forms a quaternary complex with the ryanodine receptor (Ca 2؉ release channel), junctin, and calsequestrin. To better understand the role of triadin 1 in excitation-contraction coupling in the heart, we generated transgenic mice with targeted overexpression of triadin 1 to mouse atrium and ventricle, employing the ␣-myosin heavy chain promoter to drive protein expression. The protein was overexpressed 5-fold in mouse ventricles, and overexpression was accompanied by cardiac hypertrophy. The levels of two other junctional SR proteins, the ryanodine receptor and junctin, were reduced by 55% and 73%, respectively, in association with triadin 1 overexpression, whereas the levels of calsequestrin, the Ca 2؉ -binding protein of junctional SR, and of phospholamban and SERCA2a, Ca 2؉ -handling proteins of the free SR, were unchanged. Cardiac myocytes from triadin 1-overexpressing mice exhibited depressed contractility; Ca 2؉ transients decayed at a slower rate, and cell shortening and relengthening were diminished. The extent of depression of cell shortening of triadin 1-overexpressing cardiomyocytes was rate-dependent, being more depressed under low stimulation frequencies (0.5 Hz), but reaching comparable levels at higher frequencies of stimulation (5 Hz). Spontaneously beating, isolated work-performing heart preparations overexpressing triadin 1 also relaxed at a slower rate than control hearts, and failed to adapt to increased afterload appropriately. The fast time inactivation constant, 1 , of the L-type Ca 2؉ channel was prolonged in transgenic cardiomyocytes. Our results provide evidence for the coordinated regulation of junctional SR protein expression in heart independent of free SR protein expression, and furthermore suggest an important role for triadin 1 in regulating the contractile properties of the heart during excitation-contraction coupling.

A beneficial role of cardiac P2X4 receptors in heart failure: rescue of the calsequestrin overexpression model of cardiomyopathy

“…Hearts from 3-mo-old CSQ, P2X 4R, CSQ/P2X4R, and nontransgenic (NTG) mice were isolated, blotted dry, weighed, and homogenized in ice-cold buffer containing 0.25 M sucrose and 10 mM MOPS, pH 7.2 (16 ml/g wt), using a tissue homogenizer (PowerGen Model 125, Fisher Scientific; Pittsburgh, PA). After solublization in sample buffer, SDS-PAGE and immunoblotting were conducted as recently described (13). Twenty-five micrograms of homogenate protein were electrophoresed per gel lane using 8% polyacrylamide and transferred to nitrocellulose membranes.…”

“…Twenty-five micrograms of homogenate protein were electrophoresed per gel lane using 8% polyacrylamide and transferred to nitrocellulose membranes. For detection of sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA2a), CSQ, and phospholamban (PLB), monoclonal antibody 2A7-A1, affinity-purified rabbit polyclonal antiserum, and monoclonal antibody 2D12 were used, respectively (13). Antibodies bound to proteins were detected with 125 I-labeled protein A and quantified using a PhosphorImager (GS-250 Molecular Imager, Bio-Rad; Hercules, CA) (13).…”

Suppression of Early and Late Afterdepolarizations by Heterozygous Knockout of the Na + /Ca 2+ Exchanger in a Murine Model

“…Single ventricular cardiomyocytes were isolated as reported previously 15 with modifications as reported in the Data Supplement.…”

Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A

“…Ventricular myocytes were isolated by collagenase digestion using an established protocol (20). After digestion, the ventricles were cut into several pieces and subjected to gentle agitation through a nylon mesh to separate the myocytes.…”