Diversity of the troponin C genes during chordate evolution

To elucidate the diversity of troponin C (TnC) during chordate evolution, we determined the organization of TnCs from the amphioxus, the lamprey, and the frog. Like the ascidian, the amphioxus possesses a single gene of TnC, and the fundamental gene structure is identical with the ascidian TnC. However, because alternative splicing does not occur in amphioxus, the potential for generation of TnC isoforms through this event arises only in the ascidian lineage. From the frog Xenopus laevis, two distinct cDNAs encoding fTnC isoforms and a single s/cTnC cDNA were determined. The duplication of the fTnC gene may be a character of only Xenopus or closely related species. The lamprey possesses two cDNAs each encoding fTnC and s/cTnC. The lamprey is the earliest diverged species among vertebrates, and thus it is supposed that the presence of both fTnC and s/cTnC is universal among vertebrate species, and that the gene duplication might have occurred at a vertebrate ancestor after the protochordate/vertebrate divergence. The position of the 4th intron is 3.24/0 in protochordate TnC genes, but at 3. 11/2 in vertebrate fTnCs and s/cTnCs. It is suggested that the 4th intron sliding might have occurred prior to the gene duplication.     

Troponin C modulates the activation of thin filaments by rigor cross-bridges

Extraction of troponin C (TnC) from skinned muscle fibers reduces maximum Ca2+ and rigor cross-bridge (RXB)-activated tensions and reduces cooperativity between neighboring regulatory units (one troponin-tropomyosin complex and the seven associated actins) of thin filaments. This suggests that TnC has a determining role in RXB, as well as in Ca(2+)-dependent activation processes. To investigate this possibility further, we replaced fast TnC (fTnC) of rabbit psoas fibers with either CaM[3,4TnC] or cardiac TnC (cTnC) and compared the effects of these substitutions on Ca2+ and RXB activation of tension. CaM[3,4TnC] substitution has the same effect on Ca(2+)- and RXB-activated tensions; they are reduced 50%, and cooperativity between regulatory units is reduced 40%. cTnC substitution also reduces the maximum Ca(2+)-activated tension and cooperativity. But with RXB activation the effects on tension and cooperativity are opposite; cTnC substitution potentiates tension but reduces cooperativity. We considered whether tension potentiation could be explained by increased activation by cycling cross-bridges (CXBs), but the concerted transition formalism predicts fibers will fail to relax in high substrate and high pCa when CXBs are activator ligands. It predicts resting tension, which is not observed in either control or cTnC-substituted fibers. Rather, it appears that cTnC facilitates RXB activation of fast fibers more effectively than fTnC. The order of RXB-activated tension facilitation is cTnC > fTnC > CaM[3,4TnC] > empty TnC-binding sites. Comparison of the structures of fTnC, CaM[3,4TnC], and cTnC indicates that the critical region for this property lies in the central helix or N-terminal domain, including EF hand motifs 1 and 2.     

Permeability and stability in buffer and in human serum of fluorinated phospholipid-based liposomes

Calsequestrin is the major Ca(2+)-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its alpha-helical content and the intrinsic fluorescence upon binding of Ca2+. Calsequestrin binds Ca2+ with high-capacity and with moderate affinity and it functions as a Ca2+ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca2+ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsequestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.     

Dynamics and thermodynamics of the regulatory domain of human cardiac troponin C in the apo- and calcium-saturated states

The contraction of cardiac and skeletal muscles is triggered by the binding of Ca2+ to their respective troponin C (TnC) proteins. Recent structural data of both cardiac and skeletal TnC in both the apo and Ca2+ states have revealed that the response to Ca2+ is fundamentally different for these two proteins. For skeletal TnC, binding of two Ca2+ to sites 1 and 2 leads to large changes in the structure, resulting in the exposure of a hydrophobic surface. For cardiac TnC, Ca2+ binds site 2 only, as site 1 is inactive, and the structures show that the Ca2+-induced changes are much smaller and do not result in the exposure of a large hydrophobic surface. To understand the differences between regulation of skeletal and cardiac muscle, we have investigated the effect of Ca2+ binding on the dynamics and thermodynamics of the regulatory N-domain of cardiac TnC (cNTnC) using backbone 15N nuclear magnetic resonance relaxation measurements for comparison to the skeletal system. Analysis of the relaxation data allows for the estimation of the contribution of changes in picosecond to nanosecond time scale motions to the conformational entropy of the Ca2+-binding sites on a per residue basis, which can be related to the structural features of the sites. The results indicate that binding of Ca2+ to the functional site in cNTnC makes the site more rigid with respect to high-frequency motions; this corresponds to a decrease in the conformational entropy (TdeltaS) of the site by 2.2 kcal mol(-1). Although site 1 is defunct, binding to site 2 also decreases the conformational entropy in the nonfunctional site by 0.5 kcal mol(-1). The results indicate that the Ca2+-binding sites in the regulatory domain are structurally and energetically coupled despite the inability of site 1 to bind Ca2+. Comparison between the cardiac and skeletal isoforms in the apo state shows that there is a decrease in conformational entropy of 0.9 kcal mol(-1) for site 1 of cNTnC and little difference for site 2.     

Heart-specific targeting of beta-galactosidase by the ventricle-specific cardiac myosin light chain 2 promoter using adenovirus vectors

Adenoviruses are attractive vectors for gene transfer into cardiac muscle. However, their promiscuous tissue tropism, which leads to an ectopic expression of the transgene, is a considerable limitation. To restrict expression to cardiomyocytes, we have constructed two recombinant adenoviruses (Ad-MLC2-250betagal and Ad-MLC2-2100betagal) containing the beta-galactosidase reporter gene under the control of the 250- or 2100-bp rat ventricle-specific cardiac myosin light chain-2v promoter (MLC-2v). Our in vitro and in vivo data have evidenced that the 2100-bp promoter allows stronger beta-galactosidase activity than the 250-bp promoter and that the deleted promoter allows a weak beta-galactosidase expression in skeletal muscle-derived cells in vitro. In contrast to the in vitro results, the highly deleted MLC-2v promoter of 250 pb conserved its heart specificity in in ovo and in vivo when introduced into the adenovirus genome, indicating that the specificity of this promoter is neither altered by the inverted terminal repeat nor by the enhancer of the Ela promoter, both of which located in the 5' flanking region of the promoter. Systemic injections of both recombinant adenoviruses into chicken embryos showed beta-galactosidase expression mainly in the right ventricle of the heart. We have confirmed the cardiac specificity of both promoters in mammalian species after injection of both recombinant adenoviruses into the heart of adult rats in vivo. The comparison of both promoters in vitro and in vivo has shown that the 250-bp MLC-2v promoter is 80% less active than the 2100-bp MLC-2v promoter and has enabled us to conclude that the MLC-2v promoter of 2100 bp is the most appropriate for efficient expression of a reporter gene or a therapeutic cardiac gene (e.g., SERCA2a or minidystrophin gene).     

Localization of the gene for rapidly progressive autosomal dominant parkinsonism and dementia with pallido-ponto-nigral degeneration to chromosome 17q21

The distribution of calcyclin in some chicken tissues was studied by Western blotting using polyclonal antibodies raised against calcyclin purified from chicken gizzard. The protein was found in gizzard muscle and in a lesser amount in skeletal and cardiac muscle. No immunological reaction was observed in chicken liver. Immunohistochemical studies of chicken gizzard tissue revealed the presence of calcyclin only in muscle fibers. Ca(2+)-dependent interaction of chicken gizzard calcyclin with potential protein targets was also examined. By gel overlay method it was found that calcyclin bound to three proteins with molecular masses of approximately 35 kDa, 25 kDa and 15 kDa present in the cytosolic fraction derived from chicken gizzard muscle. The chicken gizzard calcyclin was also shown to interact with lysozyme.     

Molecular cardiomyoplasty: potential cardiac gene therapy for chronic heart failure

In this study, we evaluated the feasibility of converting cardiac fibroblasts into skeletal muscle cells by forced expression of the MyoD gene, one of the basic helix-loop-helix myogenic factors. Primary cardiac fibroblasts, isolated from newborn rats, were infected with retrovirus-carrying sense or antisense MyoD gene. Ten days after infection, expression of MyoD protein was demonstrated in 95% of cells infected with sense MyoD virus by intense nuclear immunostaining with a MyoD polyclonal antibody. In contrast, none of the cells infected with antisense MyoD virus showed staining. On withdrawal of serum, 95% of MyoD positive cells became elongated and, in the presence of appropriate cell density, fused to form multinucleated myotubes, morphologically similar to striated muscle cell. Expression of downstream myogenic differentiation markers, myosin heavy chain and myocyte-specific enhancer factor 2, in 95% of these myotubes were detected by intense cytoplasmic and nuclear immunostaining, respectively, with specific antibodies. In contrast, no detectable staining was noted in MyoD negative cells. Spontaneous contractile movements were noted in a few clusters of myotubes. In summary, cardiac fibroblasts were able to be converted into bonafide potentially functional skeletal myocytes as shown by definitive morphologic and biochemical changes. Further studies with in vivo models are needed to explore this unique molecular strategy to treat patients with chronic heart failure.     

Identification and expression of two novel CLIP-170/Restin isoforms expressed predominantly in muscle

CLIP-170 and Restin, microtubule-binding proteins originally cloned from human cells, are identical except for a stretch of 35 amino acids present in Restin, but missing from CLIP-170. Here we present the discovery of two novel isoforms of the CLIP-170/Restin gene in both chickens and humans. One of the new isoforms, named CLIP-170(11), contains an 11 amino acid insert instead of the 35 amino acid insert found in Restin. Eight of these 11 amino acids, including a helix-breaking proline residue, are perfectly conserved between chickens and humans. The second new isoform, named CLIP-170(11+35), contains both the 11 and 35 amino acid inserts in tandem. PCR analysis of chicken genomic DNA revealed that all four isoforms result from differential splicing of two exons in a region of the CLIP-170 gene that contains approximately 8.6 kb of intervening sequence. We found that the CLIP-170(11) and CLIP-170(11+35) are expressed preferentially in muscle tissues. Chicken and human skeletal muscle express predominantly CLIP-170(11) and to a lesser extent CLIP-170 and CLIP-170(11+35). Adult chicken cardiac and smooth muscles also express CLIP-170(11) and CLIP-170(11+35), but CLIP-170 is the predominant isoform in these muscles as it is in all other tissues except brain. The ratios of CLIP-170 isoform expression found in embryonic and adult chicken cardiac muscles reveal that isoform expression is regulated differentially in different developmental stages as well as in different tissues.     

Sex difference in distribution and iron responsiveness of the two ferritins of rat cardiac and skeletal muscle

The presence of two electrophoretically and structurally distinguishable forms of ferritin ("fast" and "slow") in cardiac and skeletal muscle (diaphragm) of the rat was confirmed. Although the total amount of cardiac ferritin showed no difference in concentration in male and female rats, the distribution between the fast and slow species was markedly different in the two sexes, the fast form predominating in the cardiac muscle and diaphragm of the female. In agreement with this, the rates of synthesis and of degradation of the fast species were greater in the female, while the opposite obtained for the male. Iron administration stimulated synthesis of each ferritin species in the cardiac muscle and diaphragm of both sexes. Induction of cardiac connective tissue hypertrophy with isoproterenol inverted the ratio of slow to fast ferritin in female rats, while iron administration along with isoproterenol restored this to normal. It is concluded that the metabolism of ferritin in cardiac and skeletal muscle is sensitive both to sexual status and to iron administration.