A novel variant of cardiac myosin-binding protein-C that is unable to assemble into sarcomeres is expressed in the aged mouse atrium

Cardiac myosin-binding protein-C (MyBP-C), also known as C-protein, is one of the major myosin-binding proteins localizing at A-bands. MyBP-C has three isoforms encoded by three distinct genes: fast-skeletal, slow-skeletal, and cardiac type. Herein, we are reporting a novel alternative spliced form of cardiac MyBP-C, MyBP-C(+), which includes an extra 30 nucleotides, encoding 10 amino acids in the carboxyl-terminal connectin/titin binding region. This alternative spliced form of MyBP-C(+) has a markedly decreased binding affinity to myosin filaments and connectin/titin in vitro and does not localize to A-bands in cardiac myocytes. When MyBP-C(+) was expressed in chicken cardiac myocytes, sarcomere structure was markedly disorganized, suggesting it has possible dominant negative effects on sarcomere organization. Expression of MyBP-C(+) is hardly detected in ventricles through cardiac development, but its expression gradually increases in atria and becomes the dominant form after 6 mo of age. The present study demonstrates an age-induced new isoform of cardiac MyBP-C harboring possible dominant negative effects on sarcomere assembly.     

Fast skeletal muscle isoforms exhibit the highest incorporation level into myofibrils and stress fibers among members of myosin alkali light chain isoform family

Isoproteins of myosin alkali light chain (LC) were co-expressed in cultured chicken cardiomyocytes and fibroblasts and their incorporation levels into myofibrils and stress fibers were compared among members of the LC isoform family. In order to distinguish each isoform from the other, cDNAs of LC isoforms were tagged with different epitopes. Expressed LCs were detected with antibodies to the tags and their distribution was analyzed by confocal microscopy. In cardiomyocytes, the incorporation level of LC into myofibrils was shown to increase in the order from nonmuscle isoform (LC3nm), to slow skeletal muscle isoform (LC1sa), to slow skeletal/ventricular muscle isoform (LC1sb), and to fast skeletal muscle isoforms (LC1f and LC3f). Thus, the hierarchal order of the LC affinity for the cardiac myosin heavy chain (MHC) is identical to that obtained in the rat (Komiyama et al., 1996. J. Cell Sci., 109: 2089-2099), suggesting that this order may be common for taxonomic animal classes. In fibroblasts, the affinity of LC for the nonmuscle MHC in stress fibers was found to increase in the order from LC3nm, to LC1sb, to LC1sa, and to LC1f and LC3f. This order for the nonmuscle MHC is partly different from that for the cardiac MHC. This indicates that the order of the affinity of LC isoproteins for MHC varies depending on the MHC isoform. Further, for both the cardiac and nonmuscle MHCs, the fast skeletal muscle LCs exhibited the highest affinity. This suggests that the fast skeletal muscle LCs may be evolved isoforms possessing the ability to associate tightly with a variety of MHC isoforms.     

Analysis of myosin heavy chain functionality in the heart

Comparison of mammalian cardiac alpha- and beta-myosin heavy chain isoforms reveals 93% identity. To date, genetic methodologies have effected only minor switches in the mammalian cardiac myosin isoforms. Using cardiac-specific transgenesis, we have now obtained major myosin isoform shifts and/or replacements. Clusters of non-identical amino acids are found in functionally important regions, i.e. the surface loops 1 and 2, suggesting that these structures may regulate isoform-specific characteristics. Loop 1 alters filament sliding velocity, whereas Loop 2 modulates actin-activated ATPase rate in Dictyostelium myosin, but this remains untested in mammalian cardiac myosins. Alpha --> beta isoform switches were engineered into mouse hearts via transgenesis. To assess the structural basis of isoform diversity, chimeric myosins in which the sequences of either Loop 1+Loop 2 or Loop 2 of alpha-myosin were exchanged for those of beta-myosin were expressed in vivo. 2-fold differences in filament sliding velocity and ATPase activity were found between the two isoforms. Filament sliding velocity of the Loop 1+Loop 2 chimera and the ATPase activities of both loop chimeras were not significantly different compared with alpha-myosin. In mouse cardiac isoforms, myosin functionality does not depend on Loop 1 or Loop 2 sequences and must lie partially in other non-homologous residues.     

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl,Ambystoma mexicanum

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and -actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, localizes only in skeletal muscle of the axolotl. This slow axolotl isoform (Ax_slow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C₃₁₅ antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Ax_card2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.This work was supported by NIH grants HL-32184 and HL-37702 and a grant-in-aid from the American Heart Association to L.F.L.     

Evidence for inserted sequences in the head region of nonmuscle myosin specific to the nervous system. Cloning of the cDNA encoding the myosin heavy chain-B isoform of vertebrate nonmuscle myosin.

The complete amino acid sequence of a vertebrate nonmuscle myosin heavy chain-B isoform (MHC-B, 1976 amino acids, 229 kDa) has been deduced by using cDNA clones from chicken brain libraries. The chicken nonmuscle MHC-B shows overall similarity in primary structure to other MHCs in the areas contributing to the ATP-binding site and actin-binding site. Similar to other nonsarcomeric MHC IIs, there is a short uncoiled tail sequence at the carboxyl terminus of the molecule. It is in the uncoiled tail sequence that the greatest number of differences in amino acids sequence between MHC-A and B were found, which allowed generation of isoform-specific antibodies. These antibodies were used to determine the relative content of MHC-A and MHC-B in various tissues. During the cloning of the cDNA encoding chicken brain MHC-B, we found a 63-nucleotide insertion encoding 21 amino acids located in the head region of the MHC near to the actin-binding site and a 30 nucleotide insertion encoding 10 amino acids near to the ATP-binding site. Analysis using S-1 nuclease showed that both inserts are expressed in a tissue-dependent manner; mRNA containing the inserts is present in tissues of the nervous system, but is absent from other non-muscle cells, which contain the noninserted isoform of MHC-B. Similar inserts were found in corresponding positions in human cerebellar mRNA. Antibodies raised against a peptide synthesized based on the 21 amino acid insert found in chickens recognize a MHC isoform in the same tissues that are enriched for the mRNA. These insertions appear to be a mechanism for generating additional MHC-B isoforms specific to the nervous system.     

Troponin I chimera analysis of the cardiac myofilament tension response to protein kinase A

Viral-mediated gene transfer of troponin I(TnI) isoforms and chimeras into adult rat cardiac myocytes was used toinvestigate the role TnI domains play in the myofilament tensionresponse to protein kinase A (PKA). In myocytes expressing endogenouscardiac TnI (cTnI), PKA phosphorylated TnI and myosin-binding protein Cand decreased the Ca²⁺ sensitivity of myofilament tension.In marked contrast, PKA did not influence Ca²⁺-activatedtension in myocytes expressing the slow skeletal isoform of TnI or achimera (N-slow/card-C TnI), which lack the unique phosphorylatableamino terminal extension found in cTnI. PKA-mediated phosphorylation ofa second TnI chimera, N-card/slow-C TnI, which has the amino terminalregion of cTnI, caused a decrease in the Ca²⁺ sensitivityof tension comparable in magnitude to control myocytes. Based on theseresults, we propose the amino terminal region shared by cTnI andN-card/slow-C TnI plays a central role in determining the magnitude ofthe PKA-mediated shift in myofilament Ca²⁺ sensitivity,independent of the isoform-specific functional domains previouslydefined within the carboxyl terminal backbone of TnI. Interestingly,exposure of permeabilized myocytes to acidic pH after PKA-mediatedphosphorylation of cTnI resulted in an additive decrease in myofilamentCa²⁺ sensitivity. The isoform-specific, pH-sensitive regionwithin TnI lies in the carboxyl terminus of TnI, and the additiveresponse provides further evidence for the presence of a separatedomain that directly transduces the PKA phosphorylation signal.

     

Molecular genetic characterization of a developmentally regulated human perinatal myosin heavy chain

We have isolated a human cDNA which corresponds to a developmentally regulated sarcomeric myosin heavy chain. RNA hybridization and DNA sequence analysis indicate that this cDNA, called SMHCP, encodes a perinatal myosin heavy chain isoform. The nucleotide and deduced amino acid sequences of the 3.4-kb cDNA insert show strong homology with other sarcomeric myosin heavy chains. The strongest homology is to a previously described 970-bp cDNA encoding a rat perinatal isoform (Periasamy, M., D. F. Wieczorek, and B. Nadal-Ginard. 1984. J. Biol. Chem. 259:13573-13578). The homology between the analogous human and rat perinatal myosin heavy chain cDNAs is maintained through the highly isoform-specific final 20 carboxyl-terminal amino acids, as well as the 3' untranslated region. Ribonuclease protection studies show that the mRNA encoding this isoform is expressed at high levels in 21-wk fetal skeletal tissue and not in fetal cardiac muscle. In contrast to the rat perinatal isoform, which was not found to be expressed in adult hind-leg tissue, the gene encoding SMHCP continues to be expressed in adult human skeletal tissue, but at lower levels relative to fetal skeletal tissue.     

Effects of electrically induced contractile activity on cultured embryonic chick breast muscle cells

Development of chicken breast muscle is characterized by the sequential appearance of six electrophoretically distinct myosin heavy chain (HC) isoforms. Cultured secondary myotubes, derived from 12-day embryonic chick breast muscle, mainly express the early embryonic HC isoform HCemb/e, normally present in 8-day embryonic breast muscle, and the two fast light chain isoforms LC1f and LC2f. Direct low-frequency (2.5 Hz) stimulation of these myotubes via platinum electrodes leads to a shift in myosin HC expression with increases in the late embryonic HC isoform HCemb/l amounting to 35% of total HC in 19-day-stimulated cultures. Measurements of 35S-methionine incorporation and immunohistochemical analyses demonstrate increases in LC3f. This increase is also seen at the mRNA level. These results indicate that induced contractile activity promotes myotube maturation in vitro. The observation that chronic stimulation enhances the expression of the slow isoform LC2s at the RNA, as well as the protein level, suggests an additional effect consisting of a fast-to-slow change in phenotype expression. In view of the fact that muscle maturation and phenotype expression is under neural control during development in vivo, our results on directly stimulated, aneural myotubes indicate that neurally transmitted contractile activity may be an important factor in modulating phenotype expression of secondary myotubes.     

Intracompartmental sorting of essential myosin light chains: molecular dissection and in vivo monitoring by epitope tagging.

The isoprotein-specific intracompartmental sorting of the three essential myosin light chains (LCs), the skeletal muscle LC-1f and LC-3f and the nonmuscle LC-3nm, was investigated. Epitope tagging was used to monitor the intracellular localization to different cytoskeletal structures of the exogenously introduced constructs in adult rat cardiomyocytes (ARCs), which exhibit both stress fibers and regenerating myofibrils. LC-1f and LC-3f bind almost exclusively to the sarcomeric myosin heavy chain (MHC) with high affinity, while the LC-3nm interacts with stress fibers and sarcomeres equally well. Sorting appears to be directed by a hierarchical order of different affinities. Domain mapping by deletion and by construction of a LC-1f/3nm chimera suggests that the LCs are composed of three functionally distinct domains: a basal MHC binding site in the C-terminus; the central part, modulating the preferential interaction with MHC isoforms; and the isoprotein-specific N-terminus of the essential LC, which is probably not involved in the sorting process.     

Blockage of AMV reverse transcriptase by antisense oligodeoxynucleotides.

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca²⁺-calmodulin and binding to actin, actin-tropomyosin, Ca²⁺-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca²⁺-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.     

Amino acid sequence of the 20-kDa regulatory light chain of porcine aorta media smooth muscle myosin.

The amino acid sequence of the 20-kDa regulatory light chain (LC20) of myosin from porcine aorta media smooth muscle was determined. The LC20 consisted of 171 amino acid residues and its N-terminal Ser residue was blocked by an acetyl group. The amino acid sequence was identical with that of chicken gizzard myosin LC20 except that the 60th residue, Met in chicken gizzard LC20, was substituted for Leu in porcine aorta LC20.     

Fast myosin light chain expression in chicken muscles studied by in situ hybridization.

We have studied the fiber type-specific expression of the fast myosin light chain isoforms LC 1f, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and two-dimensional gel electrophoresis. Type II (fast) fibers contain all three fast myosin light chain mRNAs; Types I and III (slow) fibers lack them. The myosin light chain patterns of two-dimensional gels from microdissected single fibers match their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC 1f or LC 2f. Moreover, the ratio between LC 3f mRNA and protein was different in samples from muscles, indicating that LC 3f is regulated somewhat differently than LC 1f and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein mRNAs, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber's cross-sections, and also in the few rare fibers which showed hybridization signals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.     

The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca²⁺-calmodulin and binding to actin, actin-tropomyosin, Ca²⁺-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca²⁺-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.     

An N-terminal sequence targets and tethers Na+ pump alpha2 subunits to specialized plasma membrane microdomains

Sodium pumps (alphabeta dimers) with the alpha1 isoform of the catalytic (alpha) subunit are expressed in all cells. Additionally, most cells express Na+ pumps with a second alpha isoform. For example, astrocytes and arterial myocytes also express Na+ pumps with the alpha2 isoform. The alpha2 pumps localize to plasma membrane (PM) microdomains overlying "junctional" sarco-/endoplasmic reticulum (S/ER), but the alpha1 pumps are more uniformly distributed. To study alpha2 targeting, we expressed alpha1/alpha2 and alpha2/alpha1 chimeras and 1-90 and 1-120 amino acid N-terminal peptides in primary cultured mouse astrocytes. Immunocytochemistry revealed that alpha2/alpha1 (but not alpha1/alpha2) chimeras markedly reduced native alpha2 (i.e. were "dominant negatives"). N-terminal (1-120 and 1-90 amino acids) alpha2 (and alpha3), but not alpha1 peptides also targeted to the PM-S/ER junctions and were dominant negative for native alpha2 in astrocytes and arterial myocytes. Thus alpha2 and alpha3 have the same targeting sequence. Ca2+ (fura-2) signals in astrocytes expressing the 1-90 alpha2 peptide were comparable to signals in cells from alpha2 null mutants (i.e. functionally dominant negative): 1 microM ATP-evoked Ca2+ transients were augmented, and 100 nM ouabain-induced amplification was abolished. Amino acid substitutions in the 1-120 alpha1 and alpha2 constructs, and in full-length alpha1, revealed that Leu-27 and Ala-35 are essential for targeting/tethering the constructs to PM-S/ER junctions.     

Analysis of the chicken fast myosin heavy chain family. Localization of isoform-specific antibody epitopes and regions of divergence.

cDNAs encoding the rod region of four different fast myosin heavy chains (MYCHs) in the chicken were identified, using anti-MYCH monoclonal antibodies, in two expression libraries prepared from 19-day embryonic and adult chicken muscle. These clones were used to determine the amino acid sequences that encompass the epitopes of five anti-MYHC monoclonal antibodies. Additionally, the amino acid sequences were compared to each other and to a full length embryonic MYHC. Although there is extensive homology in the chicken fast myosin rods, sequences within the hinge, within the central portion of the light meromyosin fragment, and at the carboxy terminus exhibit the largest number of amino acid substitutions. We propose that divergence within these subdomains may contribute to isoform-specific properties associated with skeletal myosin rods.     

Isoforms of the small non-catalytic subunit of smooth muscle myosin light chain phosphatase.

Chicken gizzard smooth muscle myosin light chain phosphatase is composed of a approximately 37 kDa catalytic subunit, a approximately 110 kDa myosin binding or targeting subunit and a approximately 20 kDa subunit (MPs) whose function is as yet undefined. It was reported previously that a cloned chicken gizzard MPs cDNA encodes a protein of 186 amino acids (aa) [Y.H. Chen, M.X. Chen, D.R. Alessi, D.G. Gampbell, C. Shanahan, P. Cohen, P.T.W. Cohen, FEBS Lett. 356 (1994) 51-55]. More recently, we obtained by PCR amplification another MPs cDNA that encodes a protein of only 161 aa [Y. Zhang, K. Mabuchi, T. Tao, Biochim. Biophys. Acta 1343 (1997) 51-58]. In this work we obtained cDNAs corresponding to both sequences using a different set of PCR primers, indicating that the two sequences correspond to isoforms that most likely arose from alternative splicing of the same gene. Using two polyclonal antibodies, one raised against the recombinant 161 aa isoform of chicken gizzard MPs and the other against a C-terminal polypeptide that is present only in the 186 aa isoform, we found that while the 161 aa isoform is the predominant one in chicken gizzard, in chicken aorta it is the 186 aa one; in chicken stomach both isoforms are present, and in mammalian tissues such as ferret and rat only the 186 aa isoform is detected. Furthermore, we purified the MPs associated with the chicken gizzard myosin light chain phosphatase holoenzyme and determined its molecular weight, amino acid composition and six residues of its C-terminal sequence. The results from these analyses showed conclusively that the predominant isoform in chicken gizzard is the 161 aa one.