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Growth and muscle defects in mice lacking adult myosin heavy chain genes.

Acakpo-Satchivi LJ, Edelmann W, Sartorius C, Lu BD, Wahr PA, Watkins SC, Metzger JM, Leinwand L, Kucherlapati R - J. Cell Biol. (1997)

Bottom Line: The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical.Both strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product.Most striking is that while both strains exhibit physiological defects in isolated muscles, the defects are distinct.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Albert Einstein College of Medicine, New York 10461, USA.

ABSTRACT
The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical. We describe mice carrying mutations in each of two predominant adult fast MyHC genes, IIb and IId/x. Both strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product. For example, despite the fact that MyHC-IIb accounts for >70% of the myosin in skeletal muscle and shows the broadest distribution of expression, the phenotypes of IIb mutants are generally milder than in the MyHC-IId/x strain. In addition, in a muscle which expresses both IIb and IId/x MyHC in wild-type mice, the histological defects are completely different for expression of the two genes. Most striking is that while both strains exhibit physiological defects in isolated muscles, the defects are distinct. Muscle from IIb mice has significantly reduced ability to generate force while IId mouse muscle generates normal amounts of force, but has altered kinetic properties. Many of the phenotypes demonstrated by these mice are typical in human muscle disease and should provide insight into their etiology.

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MyHC-IIb protein  expression. Myofibrils were  prepared from tibialis anterior muscles of MyHC-IIb+/+,  MyHC-IIb+/−, and MyHC-IIb−/− mice. (A) Myofibrils  were separated by SDS-PAGE and stained with Coomassie blue. Lane 1, MW;  lane 2, IIb+/+; lane 3, IIb+/−;  lane 4, IIb−/−. (B) Myofibrils  were blotted onto nitrocellulose and probed with an antibody against MyHC-IIb  (BFF3). (C) High resolution  gel electrophoresis of MyHC  from tongue from +/+ and  −/− IId mice.
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Figure 3: MyHC-IIb protein expression. Myofibrils were prepared from tibialis anterior muscles of MyHC-IIb+/+, MyHC-IIb+/−, and MyHC-IIb−/− mice. (A) Myofibrils were separated by SDS-PAGE and stained with Coomassie blue. Lane 1, MW; lane 2, IIb+/+; lane 3, IIb+/−; lane 4, IIb−/−. (B) Myofibrils were blotted onto nitrocellulose and probed with an antibody against MyHC-IIb (BFF3). (C) High resolution gel electrophoresis of MyHC from tongue from +/+ and −/− IId mice.

Mentions: We used two different methods to determine the effect of the genetic modification on MyHC-IIb and IId/x gene expression. RNase protection was chosen for the analysis of MyHC mRNA expression levels. RNA samples from five different mouse muscle tissues (heart, diaphragm, whole lower leg, masseter, and tongue) were studied using this method. Representative results are shown in Fig. 2 of RNase protection analysis of mRNA from masseter and lower-leg tissue using a probe specific to the 3′-UTRs of the two MyHC genes. These results show that while +/+ and +/− animals express the MyHC-IIb and IId/x genes, MyHC-IIb and IId/x−/− animals had little or no detectable MyHC-IIb or IId/x mRNA expression, respectively. This was true for all five muscle sources tested (data not shown). Analysis of the same samples with a β-actin probe revealed approximately equal expression levels of these mRNAs in samples from all three groups of animals (Fig. 2). To confirm that the homologous recombination event resulted in a allele for protein expression, myofibrils were purified from tibialis anterior, which in +/+ mice expresses high levels of MyHC-IId/x and IIb. To confirm that the IIb mice are for IIb protein, immunoblot analysis was carried out with a mAb specific for MyHC-IIb. Fig. 3 A shows myofibrils from the tibialis anterior muscles of IIb mice separated by SDS-PAGE. The same amounts of protein as in Fig. 3 A (lanes 2–4) were blotted onto nitrocellulose and probed with an antibody specific to MyHC-IIb (Fig. 3 B). Whereas IIb+/+ and IIb+/− mice contain IIb protein, there is no detectable MyHC-IIb protein in −/− mice. An additional point shown by Fig. 3 A is that the normal actin/ myosin ratio appears normal. Because there is no antibody specific for MyHC-IId/x, high resolution protein gel expression was carried out on myosin from tongue, which in +/+ mice, contains MyHC-IIB (faster migrating species) and MyHC-II/x (slower migrating species). As shown in Fig. 3 C, there is no detectable MyHC-IId/x in −/− mice.


Growth and muscle defects in mice lacking adult myosin heavy chain genes.

Acakpo-Satchivi LJ, Edelmann W, Sartorius C, Lu BD, Wahr PA, Watkins SC, Metzger JM, Leinwand L, Kucherlapati R - J. Cell Biol. (1997)

MyHC-IIb protein  expression. Myofibrils were  prepared from tibialis anterior muscles of MyHC-IIb+/+,  MyHC-IIb+/−, and MyHC-IIb−/− mice. (A) Myofibrils  were separated by SDS-PAGE and stained with Coomassie blue. Lane 1, MW;  lane 2, IIb+/+; lane 3, IIb+/−;  lane 4, IIb−/−. (B) Myofibrils  were blotted onto nitrocellulose and probed with an antibody against MyHC-IIb  (BFF3). (C) High resolution  gel electrophoresis of MyHC  from tongue from +/+ and  −/− IId mice.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140209&req=5

Figure 3: MyHC-IIb protein expression. Myofibrils were prepared from tibialis anterior muscles of MyHC-IIb+/+, MyHC-IIb+/−, and MyHC-IIb−/− mice. (A) Myofibrils were separated by SDS-PAGE and stained with Coomassie blue. Lane 1, MW; lane 2, IIb+/+; lane 3, IIb+/−; lane 4, IIb−/−. (B) Myofibrils were blotted onto nitrocellulose and probed with an antibody against MyHC-IIb (BFF3). (C) High resolution gel electrophoresis of MyHC from tongue from +/+ and −/− IId mice.
Mentions: We used two different methods to determine the effect of the genetic modification on MyHC-IIb and IId/x gene expression. RNase protection was chosen for the analysis of MyHC mRNA expression levels. RNA samples from five different mouse muscle tissues (heart, diaphragm, whole lower leg, masseter, and tongue) were studied using this method. Representative results are shown in Fig. 2 of RNase protection analysis of mRNA from masseter and lower-leg tissue using a probe specific to the 3′-UTRs of the two MyHC genes. These results show that while +/+ and +/− animals express the MyHC-IIb and IId/x genes, MyHC-IIb and IId/x−/− animals had little or no detectable MyHC-IIb or IId/x mRNA expression, respectively. This was true for all five muscle sources tested (data not shown). Analysis of the same samples with a β-actin probe revealed approximately equal expression levels of these mRNAs in samples from all three groups of animals (Fig. 2). To confirm that the homologous recombination event resulted in a allele for protein expression, myofibrils were purified from tibialis anterior, which in +/+ mice expresses high levels of MyHC-IId/x and IIb. To confirm that the IIb mice are for IIb protein, immunoblot analysis was carried out with a mAb specific for MyHC-IIb. Fig. 3 A shows myofibrils from the tibialis anterior muscles of IIb mice separated by SDS-PAGE. The same amounts of protein as in Fig. 3 A (lanes 2–4) were blotted onto nitrocellulose and probed with an antibody specific to MyHC-IIb (Fig. 3 B). Whereas IIb+/+ and IIb+/− mice contain IIb protein, there is no detectable MyHC-IIb protein in −/− mice. An additional point shown by Fig. 3 A is that the normal actin/ myosin ratio appears normal. Because there is no antibody specific for MyHC-IId/x, high resolution protein gel expression was carried out on myosin from tongue, which in +/+ mice, contains MyHC-IIB (faster migrating species) and MyHC-II/x (slower migrating species). As shown in Fig. 3 C, there is no detectable MyHC-IId/x in −/− mice.

Bottom Line: The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical.Both strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product.Most striking is that while both strains exhibit physiological defects in isolated muscles, the defects are distinct.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Albert Einstein College of Medicine, New York 10461, USA.

ABSTRACT
The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical. We describe mice carrying mutations in each of two predominant adult fast MyHC genes, IIb and IId/x. Both strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product. For example, despite the fact that MyHC-IIb accounts for >70% of the myosin in skeletal muscle and shows the broadest distribution of expression, the phenotypes of IIb mutants are generally milder than in the MyHC-IId/x strain. In addition, in a muscle which expresses both IIb and IId/x MyHC in wild-type mice, the histological defects are completely different for expression of the two genes. Most striking is that while both strains exhibit physiological defects in isolated muscles, the defects are distinct. Muscle from IIb mice has significantly reduced ability to generate force while IId mouse muscle generates normal amounts of force, but has altered kinetic properties. Many of the phenotypes demonstrated by these mice are typical in human muscle disease and should provide insight into their etiology.

Show MeSH
Related in: MedlinePlus