Analysis of Myosin Heavy Chain Functionality in the Heart
2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês
10.1074/jbc.m210804200
ISSN1083-351X
AutoresMaike Krenz, Atsushi Sanbe, Florence Bouyer-Dalloz, James Gulick, Raisa Klevitsky, Timothy E. Hewett, Hanna Osińska, John N. Lorenz, Christine Brosseau, Antonio Federico, Norman R. Alpert, David M. Warshaw, M. Benjamin Perryman, Steve M. Helmke, Jeffrey Robbins,
Tópico(s)Cardiovascular Effects of Exercise
ResumoComparison of mammalian cardiac α- and औ-myosin heavy chain isoforms reveals 937 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. α → औ 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 α-myosin were exchanged for those of औ-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 α-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. Comparison of mammalian cardiac α- and औ-myosin heavy chain isoforms reveals 937 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. α → औ 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 α-myosin were exchanged for those of औ-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 α-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. myosin heavy chain human growth hormone Loop 1 Loop 2 non-transgenic muscle lengths per second transgenic propylthiouracil untranslated region Myosin, the molecular motor of the heart, generates force and motion by coupling its ATPase activity to its cyclic interaction with actin. Myosin is a hexameric protein and is composed of two heavy chains (MHC)1 and two essential and two regulatory myosin light chains. Structurally, MHC is composed of a number of discrete domains: a helical rod necessary for thick filament formation, and a globular head that contains the actin-binding site, catalytic, and motor domains (1Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Science. 1993; 261: 58-65Crossref PubMed Scopus (1451) Google Scholar). In the mammalian heart, two functionally distinct MHC isoforms, termed V1 and V3, are present. V1 is a homodimer of two α-MHC molecules, whereas V3 is a औऔ-homodimer. Expression of V1 and V3 is controlled both developmentally and hormonally. In the mouse, औ-MHC expression in the ventricles predominates prenatally. However, via thyroid hormone regulation, औ-MHC expression is silenced at birth, and α-MHC is transcribed (2Morkin E. Circulation. 1993; 87: 1451-1460Crossref PubMed Scopus (177) Google Scholar). The functional differences between V1 and V3 myosin in terms of shortening velocity, force generation, and ATPase activity are profound. For example, rabbit V1 myosin has a 2–3-fold faster actin filament sliding velocity than V3, but generates only half the average isometric force (3Harris D.E. Work S.S. Wright R.K. Alpert N.R. Warshaw D.M. J. Muscle Res. Cell Motil. 1994; 15: 11-19Crossref PubMed Scopus (152) Google Scholar, 4Palmiter K.A. Tyska M.J. Dupuis D.E. Alpert N.R. Warshaw D.M. J. Physiol. 1999; 519: 669-678Crossref PubMed Scopus (100) Google Scholar). Likewise, both the Ca2+-stimulated and actin-activated ATPase activities of rabbit V1 myosin are ∼2–3 times greater than for V3 myosin (3Harris D.E. Work S.S. Wright R.K. Alpert N.R. Warshaw D.M. J. Muscle Res. Cell Motil. 1994; 15: 11-19Crossref PubMed Scopus (152) Google Scholar, 5Litten 3rd, R.Z. Martin B.J. Low R.B. Alpert N.R. Circ. Res. 1982; 50: 856-864Crossref PubMed Scopus (131) Google Scholar). Similar differences in actin velocity and myofibrillar ATPase activity have been observed between mouse V1 and V3 myosin, but there is no difference in their average force generation (6Alpert N.R. Brosseau C. Federico A. Krenz M. Robbins J. Warshaw D.M. Am. J. Physiol. Heart Circ. Physiol. 2002; 283: H1446-H1454Crossref PubMed Scopus (88) Google Scholar). Although the proteins are functionally distinct, the primary amino acid sequences of mouse α- and औ-MHC are 937 identical. Thus cardiac isoform diversity must lie in the non-identical residues (127 of 1938 amino acids in mice). The differences in the enzyme kinetics and mechanics of the myosin interactions that are observed between the two cardiac isoforms are believed to reside in two, hypervariable "loops," so called because their structures cannot be defined via x-ray crystallography due to their relative disorder. Loop 1 (L1), which is located between residues 213 and 223, is at the mouth of the nucleotide pocket while Loop 2 (L2), at positions 624–646, cradles the long cleft running from the actin binding site to the nucleotide binding pocket (1Rayment I. Holden H.M. Whittaker M. Yohn C.B. Lorenz M. Holmes K.C. Milligan R.A. Science. 1993; 261: 58-65Crossref PubMed Scopus (1451) Google Scholar, 7Murphy C.T. Spudich J.A. J. Muscle Res. Cell Motil. 2000; 21: 139-151Crossref PubMed Scopus (39) Google Scholar). Comparison of MHC sequences within the human sarcomeric MHC family shows that these domains of sequence variability are conserved (8Weiss A. Schiaffino S. Leinwand L.A. J. Mol. Biol. 1999; 290: 61-75Crossref PubMed Scopus (183) Google Scholar). Spudich and co-workers (9Spudich J.A. Nature. 1994; 372: 515-518Crossref PubMed Scopus (423) Google Scholar, 10Murphy C.T. Spudich J.A. Biochemistry. 1998; 37: 6738-6744Crossref PubMed Scopus (83) Google Scholar, 11Uyeda T.Q. Ruppel K.M. Spudich J.A. Nature. 1994; 368: 567-569Crossref PubMed Scopus (189) Google Scholar) have proposed that Loop 1 modulates velocity through ADP release, whereas Loop 2 helps regulate the actin-activated ATPase rate. Data obtained from studies with chimeric Dictyosteliumand smooth muscle myosins corroborated the model. For example, chimeras were constructed in which 9 amino acids in the L2 region ofDictyostelium myosin II were substituted with the corresponding residues from other myosins such as rabbit skeletal muscle myosin, chicken smooth muscle myosin, or rat cardiac myosin (11Uyeda T.Q. Ruppel K.M. Spudich J.A. Nature. 1994; 368: 567-569Crossref PubMed Scopus (189) Google Scholar). The actin-activated ATPase activities of the chimeras correlated well with the activities of the myosin from which the Loop 2 sequence was derived. Thus, myosin's ATPase activity could be specifically modulated depending on the sequence of Loop 2. However, a number of studies indicate that the loops may not influence myosin kinetics and mechanics as proposed and thus have varying roles depending on the structure of the myosin backbone. Rat and pig औ-MHC, which have identical Loop 1 sequences apart from a single conservative substitution, have 3–4-fold differences in ATPase activity and ADP dissociation (12Pereira J.S. Pavlov D. Nili M. Greaser M. Homsher E. Moss R.L. J. Biol. Chem. 2001; 276: 4409-4415Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Sweeney et al. (13Sweeney H.L. Rosenfeld S.S. Brown F. Faust L. Smith J. Xing J. Stein L.A. Sellers J.R. J. Biol. Chem. 1998; 273: 6262-6270Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) showed that the properties of Loop 1 chimeras with a smooth muscle backbone are a function of loop size/flexibility rather than related to the properties of the myosin from which Loop 1 was derived. Furthermore, chimeric myosins that consisted of a Dictyostelium MHC backbone with carp loop sequences did not exhibit changes in sliding velocity if Loop 1 was substituted, although Loop 2 substitution did lead to the expected modulation of actin-activated ATPase activity (14Hirayama Y. Sutoh K. Watabe S. Biochem. Biophys. Res. Commun. 2000; 269: 237-241Crossref PubMed Scopus (19) Google Scholar). Taken together, these studies indicate that the role of the surface loops for MHC functionality depends on the interplay of the surface loops with other regions important for myosin mechanics and kinetics. In contrast to the abundance of detailed studies on in vitrofunction of various MHC isoforms, our current knowledge of how differences found on the single molecule level are reflected inin vivo cardiac function is limited. Cardiac isoform shifts can be achieved by endocrine intervention but hypothyroidism not only results in a nearly complete V1 → V3 shift in rodent hearts, it also induces a number of structural changes including mitochondrial swelling, as well as rupture and loss of continuity of the myofilaments (15Lopes A.C. Furlanetto R. Sasso W.S. Didio L.J. J. Submicrosc. Cytol. Pathol. 1993; 25: 263-266PubMed Google Scholar). Thus one cannot dissect MHC isoform shift induced functional changes from contractile impairment due to structural damage. Transgenesis avoids these issues. To date, only a single transgenic study has dealt directly with cardiac myosin isoform substitution, showing that contractile function of TG mouse hearts with low-level expression of Myc-tagged rat औ-MHC was reduced by 157 (16Tardiff J.C. Hewett T.E. Factor S.M. Vikstrom K.L. Robbins J. Leinwand L.A. Am. J. Physiol. Heart Circ. Physiol. 2000; 278: H412-H419Crossref PubMed Google Scholar). This disproportionate impairment of contractile function might be due to the presence of a heterologous species (rat) cDNA being placed into the mouse context, resulting in a dominant negative effect of the TG protein. The present study is the first to investigate directly the functional significance of cardiac isoform diversity by using TG mice in which ventricular V1 is largely replaced by V3. This approach has the advantage of effecting isoform replacement in the heart without the pleiotropic stimuli that are normally used to induce MHC isoform transitions, such as pressure-overload induced hypertrophy or changes in hormonal status (17Chien K.R. Knowlton K.U. Zhu H. Chien S. FASEB J. 1991; 5: 3037-3046Crossref PubMed Scopus (696) Google Scholar, 18Imamura S. Matsuoka R. Hiratsuka E. Kimura M. Nakanishi T. Nishikawa T. Furutani Y. Takao A. Am. J. Physiol. 1991; 260: H73-H79PubMed Google Scholar). A significant V1→ V3 shift resulted in the expected changes in the heart at the single motor and biochemical levels as well as in fiber mechanics and kinetics. However, in contrast to a hypothyroidism-effected replacement, cellular structure appeared normal and whole organ function was preserved with relatively minor effects on systolic and diastolic hemodynamics in the intact animal. Furthermore, we generated transgenic (TG) mice in which we substituted the sequences of Loop 1 and/or Loop 2 of mouse α-MHC with the respective sequences of औ-MHC and assessed the mechanical and enzymatic characteristics of the chimeric MHCs. These experiments were designed to test whether a sequence substitution in the Loop 1 and/or Loop 2 region is sufficient to confer औ-like activity to the α-MHC molecule. Complete replacement of the endogenous α-MHC protein with the chimeric myosin resulted in surprisingly minor differences in enzyme kinetics indicating that, for these cardiac isoforms, other variable regions or residues must play a predominant role in determining overall ATPase activity and velocity of shortening. TG mice expressing full-length mouse औ-MHC were generated. औ-MHC cDNA was produced using a combination of a cDNA containing the 3′ one-third of the RNA (19Sanchez A. Jones W.K. Gulick J. Doetschman T. Robbins J. J. Biol. Chem. 1991; 266: 22419-22426Abstract Full Text PDF PubMed Google Scholar), and RT-PCR with औ-MHC-specific primers (GenBankTMaccession number AY056464). The product contained the entire औ-MHC cDNA and was linked to the mouse α-MHC promoter (Fig. 1A). Multiple TG founders (FVB/N) were generated. Line 102 with the highest degree of MHC replacement (∼407) was bred to homozygosity resulting in 737 replacement of α- with औ-MHC (Fig.1D). Neither the heterozygous or homozygous TG animals showed an overt phenotype, and all animals had a normal life span. To shift isoform expression in non-transgenic (NTG) hearts from α- to औ-MHC, adult mice received an iodine-deficient diet containing 0.157 propylthiouracil (PTU) for 8 weeks. Chimeric myosins in which either only the sequence of L2 or of both Loop 1 and Loop 2 (L1+L2) of α-MHC was exchanged for the respective sequence of औ-MHC were subsequently constructed. A diagram of the TG constructs for the chimeric MHCs is shown in Fig. 5A. Constructs for L2, and L1+L2 chimeric MHC were made using standard PCR methodology. Inner sets of oligonucleotides were designed so that the overlap encoded the amino acids that form Loop 1 or Loop 2 of the औ-isoform (Fig. 5B). Fragments generated by PCR amplification were cloned back into full-length α-MHC and placed into the mouse α-MHC promoter cassette (20Subramaniam A. Gulick J. Neumann J. Knotts S. Robbins J. J. Biol. Chem. 1993; 268: 4331-4336Abstract Full Text PDF PubMed Google Scholar). Finally, the DNA was excised free of plasmid sequence and used to generate multiple TG founders (FVB/N). Hearts were fixed in 47 paraformaldehyde. 3–4-ॖm cryostat sections were probed with a custom-made anti-Loop 2 (औ-L2) polyclonal antibody (Genemed Synthesis Inc., San Francisco, CA) followed by incubation with Alexa 488-conjugated secondary antibody and co-labeled with phalloidin-Alexa 594 (Molecular Probes, Eugene, OR). Specimens were examined using confocal microscopy. MHC was purified from individual mouse hearts (21Tyska M.J. Hayes E. Giewat M. Seidman C.E. Seidman J.G. Warshaw D.M. Circ. Res. 2000; 86: 737-744Crossref PubMed Scopus (181) Google Scholar). F-actin was prepared from acetone powder of chicken pectoralis muscle according to a protocol modified from Pardee and Spudich (22Pardee J.D. Spudich J.A. Methods Cell Biol. 1982; 24: 271-289Crossref PubMed Scopus (340) Google Scholar). Actin-activated ATPase activity was measured at actin concentrations ranging from 10 to 80 ॖm (21Tyska M.J. Hayes E. Giewat M. Seidman C.E. Seidman J.G. Warshaw D.M. Circ. Res. 2000; 86: 737-744Crossref PubMed Scopus (181) Google Scholar). Ca2+-stimulated Mg2+-ATPase activity of myofibrillar preparations from ventricular tissue (23McAuliffe J.J. Gao L.Z. Solaro R.J. Circ. Res. 1990; 66: 1204-1216Crossref PubMed Scopus (91) Google Scholar) was determined as previously described (24Powers F.M. Solaro R.J. Am. J. Physiol. 1995; 268: C1348-C1353Crossref PubMed Google Scholar). Transcript analysis was performed with RNA blots with transcript-specific probes as described previously (19Sanchez A. Jones W.K. Gulick J. Doetschman T. Robbins J. J. Biol. Chem. 1991; 266: 22419-22426Abstract Full Text PDF PubMed Google Scholar). The two cardiac myosins were separated on denaturing gels in the presence of glycerol, essentially as described by Reiser and co-workers (25Blough E.R. Rennie E.R. Zhang F. Reiser P.J. Anal. Biochem. 1996; 233: 31-35Crossref PubMed Scopus (108) Google Scholar, 26Reiser P.J. Kline W.O. Am. J. Physiol. 1998; 274: H1048-H1053PubMed Google Scholar). Fiber isolation and their analyses have been described in detail (27Sanbe A. Gulick J. Fewell J. Robbins J. J. Biol. Chem. 2001; 276: 32682-32686Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The in vitro motility assays (28Nguyen T.T. Hayes E. Mulieri L.A. Leavitt B.J. ter Keurs H.E. Alpert N.R. Warshaw D.M. Circ. Res. 1996; 79: 222-226Crossref PubMed Scopus (41) Google Scholar) and determination of cardiac hemodynamics in the isolated working heart model and the closed chest intact mouse model were carried out as described (29Lorenz J.N. Robbins J. Am. J. Physiol. 1997; 272: H1137-H1146PubMed Google Scholar, 30Gulick J. Hewett T.E. Klevitsky R. Buck S.H. Moss R.L. Robbins J. Circ. Res. 1997; 80: 655-664Crossref PubMed Scopus (80) Google Scholar). All data are expressed as mean ± S.E. Comparisons between NTG and TG littermates were evaluated using Student's t test, and a p value of 12-fold overexpression) can be lethal, presumably because of the relatively insoluble nature of the intact protein. TG mice were analyzed for ventricular MHC protein content at 10–12 weeks. The α- and औ-MHC proteins can be separated on 57 glycerol gels (Fig.1D). Hypothyroidism resulting from PTU treatment resulted in nearly complete replacement (907) of α- with औ-MHC. Our previous contractile protein-based TG studies showed that the cardiomyocyte rigidly controls the overall stoichiometry of the contractile protein pool, such that TG overexpression at the mRNA level does not lead to increases of overall protein content (30Gulick J. Hewett T.E. Klevitsky R. Buck S.H. Moss R.L. Robbins J. Circ. Res. 1997; 80: 655-664Crossref PubMed Scopus (80) Google Scholar, 32James J. Osinska H. Hewett T.E. Kimball T. Klevitsky R. Witt S. Hall D.G. Gulick J. Robbins J. Transgenic Res. 1999; 8: 9-22Crossref PubMed Scopus (30) Google Scholar, 33Palermo J. Gulick J. Colbert M. Fewell J. Robbins J. Circ. Res. 1996; 78: 504-509Crossref PubMed Scopus (117) Google Scholar). That is, there is no "overexpression." The steady state levels of endogenous protein are down-regulated and replaced proportionally by the TG protein. Therefore, we could achieve partial or even complete replacement of the endogenous MHC with TG proteins. When the highest expressing line (Line 102) was bred to homozygosity, 737 of the total MHC was औ-MHC (Fig.1D). We reasoned that if a phenotype were likely to be present, it would be most easily detected in line 102, and this line became the focus of our analyses. Immunohistochemical staining using a V3-specific antibody derived from the hypervariable Loop 2 region of औ-MHC (Fig.5B) showed only traces of this isoform in NTG ventricles (Fig. 2A). Confocal analysis confirmed that the PTU-treated animals showed significant accumulation of औ-MHC (Fig. 2B), but the characteristic striated pattern was somewhat blunted, consistent with the major effects that hypothyroidism has on cardiomyocyte morphology. In contrast with the PTU-treated mice, striated morphology was well conserved in the औ-MHC TG cardiomyocytes, with the pattern of staining confirming the correct incorporation of TG protein into the sarcomere (Fig. 2C). Cardiac histology was examined using young adult animals (8–12 weeks) (Fig. 2, D and E) and aged animals (1 and 2 years). 2J. Robbins and M. Krenz, unpublished observations. No significant differences in the gross morphology of either heterozygous or homozygous TG hearts were observed, and no differences in heart rate or chamber weight could be detected (TableI). Quantitation of the molecular markers of hypertrophy, which we have found to be a very sensitive marker of any response at the cellular level, was carried out at the transcript level as described previously (34Jones W.K. Grupp I.L. Doetschman T. Grupp G. Osinska H. Hewett T.E. Boivin G. Gulick J. Ng W.A. Robbins J. J. Clin. Invest. 1996; 98: 1906-1917Crossref PubMed Scopus (165) Google Scholar), and no differences could be detected.2 Similarly, we could not detect any obvious differences in the cardiomyocytes from homozygous TG mouse hearts using either light or electron microscopy (Fig. 2,D–G). No early deaths or overt ill health was noted in any of the TG animals during the first year and a half of life as compared with the NTG experimental cohorts. We conclude that the α → औ transition is benign in terms of the animals overall cardiac morphology of the animals and that no early mortality or morbidity presents under normal animal husbandry conditions.Table IHeart:body and chamber:body weightsNTGTG (heterozygous)TG (homozygous)Body weight (g)25.2 ± 0.623.7 ± 0.823.8 ± 0.6Left ventricle (mg)77.6 ± 2.870.8 ± 5.071.3 ± 2.4Right ventricle (mg)22.0 ± 0.821.2 ± 1.423.4 ± 0.9Atria (mg)8.7 ± 0.610.0 ± 0.210.5 ± 0.5Heart weight (mg)108.0 ± 2.3102.0 ± 4.5105.2 ± 2.6LV/BW ratio3.1 ± 0.13.0 ± 0.13.0 ± 0.1RV/BW ratio0.9 ± 0.10.9 ± 0.11.0 ± 0.1Atria/BW ratio0.11 ± 0.020.14 ± 0.010.15 ± 0.01(LV + RV)/BW ratio4.0 ± 0.13.9 ± 0.14.0 ± 0.1HW/BW ratio4.3 ± 0.14.3 ± 0.14.4 ± 0.2Results were obtained from line 102 TG mice and age-matched NTG controls at 2 months of age, n = 3 in all groups. Only males were used. The values represent the mean ± S.E. for each group. No statistically significant differences were observed. BW, body weight; LV, left ventricle; RV, right ventricle; HW, heart weight. Open table in a new tab Results were obtained from line 102 TG mice and age-matched NTG controls at 2 months of age, n = 3 in all groups. Only males were used. The values represent the mean ± S.E. for each group. No statistically significant differences were observed. BW, body weight; LV, left ventricle; RV, right ventricle; HW, heart weight. In light of the unremarkable phenotype at the whole animal level, we wished to confirm that changes in isoform content had affected the mechanical and kinetic properties of the skinned myofibers. Ventricular papillary muscles were isolated from line 102, PTU-treated and NTG mice. Line 102 heterozygotes have ∼407 औ-MHC while the homozygotes show ∼737 replacement. The skinned fiber is a complex system in which the contractile machinery operates against the internal cytoskeletal structures in both the cardiomyocytes and connective tissue. Therefore,Vmax in a fiber is never truly unloaded, as is assumed to be the case in the in vitro actin motility assay (see below). We first wished to compare the effects of ∼407 replacementversus the fibers derived from PTU-treated animals, in which ∼957 of the cardiac myosin consisted of औ-MHC. Fibers were isolated from 9-week-old animals in order to minimize the effects of any secondary pathology that might develop later in life, and the unloaded shortening and maximum shortening velocities, as well as the relative power that the fibers developed, were measured (Fig.3). As expected on the basis of the degree of α-MHC replaced by औ-MHC, the values derived from line 102 heterozygotes were intermediate between the NTG (1007 α-MHC) and PTU (907 औ-MHC) data. Significant, graded decreases in the unloaded shortening velocity were noted (Fig. 3A) from NTG (3.80 ± 0.14 m. l./s, n = 7) to line 102 (2.72 ± 0.26 m. l./s, n = 4) to the PTU-derived fibers (1.51 ± 0.24 m. l./s, n = 3). The same gradual decreases were also observed in the force-velocity data used to derive the maximum shortening velocities (Fig. 3B). The power-force relationships, and maximum power produced followed the same trend (Fig. 3C), and the data show that the shift in MHC isoform content leads directly to changes in cross-bridge cycling rates. We confirmed both the trend and stability of these changes by developing a cohort of heterozygotes and homozygotes over the course of a year and subsequently carrying out fiber measurements comparing these two populations to NTG fibers (Fig. 4). Similar graded decreases in the unloaded shortening velocity (Fig.4A), maximum shortening velocity (Fig. 4B), and maximum power produced (Fig. 4C) were noted when the NTG, heterozygotes, and homozygotes were compared. No changes in the calcium-force relationship could be observed in any of the TG fibers.2 The above data clearly showed that the cardiomyocyte is tolerant of significant MHC isoform shifts that are transgenically imposed. We next explored the structural basis of the different cardiac myosins' unique functionalities by replacing the endogenous MHC with α/औ chimeras, the working hypothesis being that the functional differences between the isoforms presumably are caused by the different loop sequences. The structure of Loop 1 is thought to modulate the rate of Mg2+-ATP binding and Mg2+-ADP release while the structure of Loop 2 affects the rate of myosin attachment to actin (9Spudich J.A. Nature. 1994; 372: 515-518Crossref PubMed Scopus (423) Google Scholar, 10Murphy C.T. Spudich J.A. Biochemistry. 1998; 37: 6738-6744Crossref PubMed Scopus (83) Google Scholar, 11Uyeda T.Q. Ruppel K.M. Spudich J.A. Nature. 1994; 368: 567-569Crossref PubMed Scopus (189) Google Scholar). Two constructs, in which the sequences of either Loop 1 and Loop 2 (L1+L2) or only Loop 2 (L2) of mouse α-MHC were substituted by the corresponding औ-MHC sequences were made and used to generate TG mice (Fig.5A). In order to detect the TG protein, an antibody to the औ-MHC Loop 2 sequence was generated (Fig.5B). Quantification of protein replacement in hearts from L1+L2- and L2-TG mice by Western blotting (Fig. 5C) showed nearly complete replacement (L1+L2, 1007; L2, 847). This was confirmed by mass spectroscopy, in which the tryptic peptide of the endogenous protein (LMATLFSTYASADTGDSGK, mono-isotopic mass 1935.90) was replaced by the respective fragment containing औ-MHC-sequence (LLSNLFANYAGADAPADK, mono-isotopic mass 1850.93). To determine the effects on motor function, in vitro actin motility assays were performed using MHC that had been isolated from heterozygous line 102 औ-TG mice (407 replacement), from high-replacement (1007) L1+L2 TG hearts, and from NTG as well as from PTU-treated hearts (Fig. 6). V1 → V3 replacement had a clear effect on molecular motor velocity, with the isoform switches in the PTU and औ-MHC TG preparations significantly decreasing the sliding velocity of myosin. For the 407 replacement of V1 with V3 in line 102 heterozygotes, the observed values were intermediate between the NTG and PTU-derived samples, as expected considering the results of our previous studies in which we compared filament sliding velocities of mouse V1/V3mixtures in varying proportions, and observed a linear relationship between relative isoform content and filament sliding velocit
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