Myosin-binding protein C (Mybpc3)-targeted knock-in mice (KI) recapitulate typical aspects of human hypertrophic cardiomyopathy. We evaluated whether these functional alterations can be reproduced in Show more
Myosin-binding protein C (Mybpc3)-targeted knock-in mice (KI) recapitulate typical aspects of human hypertrophic cardiomyopathy. We evaluated whether these functional alterations can be reproduced in engineered heart tissue (EHT) and yield novel mechanistic information on the function of cMyBP-C. EHTs were generated from cardiac cells of neonatal KI, heterozygous (HET) or wild-type controls (WT) and developed without apparent morphological differences. KI had 70% and HET 20% lower total cMyBP-C levels than WT, accompanied by elevated fetal gene expression. Under standard culture conditions and spontaneous beating, KI EHTs showed more frequent burst beating than WT and occasional tetanic contractions (14/96). Under electrical stimulation (6Hz, 37°C) KI EHTs exhibited shorter contraction and relaxation times and a twofold higher sensitivity to external [Ca(2+)]. Accordingly, the sensitivity to verapamil was 4-fold lower and the response to isoprenaline or the Ca(2+) sensitizer EMD 57033 2- to 4-fold smaller. The loss of EMD effect was verified in 6-week-old KI mice in vivo. HET EHTs were apparently normal under basal conditions, but showed similarly altered contractile responses to [Ca(2+)], verapamil, isoprenaline and EMD. In contrast, drug-induced changes in intracellular Ca(2+) transients (Fura-2) were essentially normal. In conclusion, the present findings in auxotonically contracting EHTs support the idea that cMyBP-C's normal role is to suppress force generation at low intracellular Ca(2+) and stabilize the power-stroke step of the cross bridge cycle. Pharmacological testing in EHT unmasked a disease phenotype in HET. The altered drug response may be clinically relevant. Show less
The insertion of double bonds into specific positions of fatty acids is achieved by the action of distinct desaturase enzymes. Here we report the cloning and characterization of three members of the f Show more
The insertion of double bonds into specific positions of fatty acids is achieved by the action of distinct desaturase enzymes. Here we report the cloning and characterization of three members of the fatty acid desaturase (FADS) gene family in humans. Initially identified as cDNA fragments by direct cDNA selection within a defined 1.4-Mb region in 11q12-q13.1, full-length fatty acid desaturase-1 (FADS1) and fatty acid desaturase-2 (FADS2) transcripts were obtained by EST sequence assembly. A third member, fatty acid desaturase-3 (FADS3), was identified in silico revealing 62 and 70% nucleotide sequence identity with FADS1 and FADS2, respectively. The three genes are clustered within 92 kb of genomic DNA located 2 kb telomeric to FEN1 and 50 kb centromeric to VMD2 and are likely to have arisen evolutionarily from gene duplication as they share a remarkably similar exon/intron organization. Protein database searches identified FADS1, FADS2, and FADS3 as fusion products composed of an N-terminal cytochrome b5-like domain and a C-terminal multiple membrane-spanning desaturase portion. Show less