Suppression of Cardiac Troponin T Induces Reduction of Contractility and Structural Disorganization in Chicken Cardiomyocytes

Abstract

ABSTRACT. We herein examine the effect of cardiac troponin T (CTnT) suppression in cultured chicken cardiomyocytes derived from embryonic cardiac ventricular muscle. TnT is an important protein participating in regulation of striated muscle contraction, but it is not clear whether TnT contributes to the formation of sarcomere structure in myofibrils. Double-stranded RNA homologous to the nucleotide sequence of CTnT (CTnTsiRNA) was introduced into cultured muscle cells two days after plating. Transfection efficiency was above 80%. Immunoblot analyses suggested that the expression of CTnT progressively falls for the three consecutive days after transfection, but partly reappears on the fourth day. Maximum suppression occurs three days after transfection, with almost invisible CTnT protein on immunoblots in all the examined conditions: 0.5-2 nmol CTnT-siRNA towards 1-3×10 6 cells. The suppression was specific to CTnT, and the other myofibrillar proteins such as myosin, connectin/titin, tropomyosin, alpha-actinin, and troponin I were all present in transfected cells. The following functional and morphological changes were detected in CTnT-suppressed cells. The population of beating cells decreased significantly after transfection, when compared to control cells. A part of CTnTsuppressed cells showed two non-overlapping types of morphological changes: 1) myofibrils presenting unusually long Z-Z intervals; 2) myofibrils with irregular small striations in cells not connected at their adhesion interfaces of a jagged-appearance. Thus, our results reveal that CTnT is important for stable beating in cultured ventricular muscle cells, and also to some extent, for maintaining myofibrillar structure and cell-to-cell adhesion.

Cardiomyopathy Mutation Alters End-to-End Junction of Tropomyosin and Reduces Calcium Sensitivity

“…Previous reports show that suppression of cTnT causes a reduction in contractility in vivo (48). Furthermore, DCMcausing TnT1 mutations, located close to the Tpm overlap in cardiac thin filaments, also result in a hyposensitization to calcium due to altered TnT-Tpm binding affinity (48).…”

Melatonin treatment induces interplay of apoptosis, autophagy, and senescence in human colorectal cancer cells

“…The number of beating cells was assessed using an inverted phase‐contrast microscope (200 × magnification). The proportion of beating embryoid bodies (%) was calculated as a percentage of the total number of cells (nonbeating + beating) .…”

Morphological Modifications in Myofibrils by Suppressing Tropomyosin 4α in Chicken Cardiac Myocytes

“…Culture of embryonic chicken ventricular muscle cells and siRNA transfection were carried out as described previously (Toyota et al, 2008). Ventricular muscle cells were prepared from 7 or 8-day-old chicken embryos according to DeHaan (1970).…”

“…Protein bands were visualized by reaction with hydrogen peroxide in the presence of 3,3'-diaminobenzidine tetrahydrochloride (Dojindo, Kumamoto, Japan). Cardiac actomyosin was extracted from homogenates of adult ventricular muscle in KCl (final concentration: 0.6 M) as described previously (Toyota et al, 1998(Toyota et al, , 2008.…”

“…The antibodies were purchased or obtained as gift, as indicated below: a) Chicken cardiac muscle TPM was prepared from adult ventricles according to Bailey (1948), and anti-CTPM antibody was prepared according to Toyota and Shimada (1981 (Shimizu and Shimada, 1985;Toyota et al, 2008). f) AntiCTnT was the same antibody used previously (Toyota and Shimada, 1981).…”