In this paper, we describe the effects of voluntary cage wheel exercise on mouse cardiac and skeletal muscle. Inbred male C57/Bl6 mice (age 6-8 wk; n = 12) [corrected] ran an average of 4.3 h/24 h, for an average distance of 6.8 km/24 h, and at an average speed of 26.4 m/min. A significant increase in the ratio of heart mass to body mass (mg/g) was evident after 2 wk of voluntary exercise, and cardiac atrial natriuretic factor and brain natriuretic peptide mRNA levels were significantly increased in the ventricles after 4 wk of voluntary exercise. A significant increase in the percentage of fibers expressing myosin heavy chain (MHC) IIa was observed in both the gastrocnemius and the tibialis anterior (TA) by 2 wk, and a significant decrease in the percentage of fibers expressing IIb MHC was evident in both muscles after 4 wk of voluntary exercise. The TA muscle showed a greater increase in the percentage of IIa MHC-expressing fibers than did the gastrocnemius muscle (40 and 20%, respectively, compared with 10% for nonexercised). Finally, the number of oxidative fibers as revealed by NADH-tetrazolium reductase histochemical staining was increased in the TA but not the gastrocnemius after 4 wk of voluntary exercise. All results are relative to age-matched mice housed without access to running wheels. Together these data demonstrate that voluntary exercise in mice results in cardiac and skeletal muscle adaptations consistent with endurance exercise.

The ancient MYH7b gene, expressed in striated muscle and brain, encodes a sarcomeric myosin and the intronic microRNA miR-499. We find that skipping of an exon introduces a premature termination codon in the transcript that downregulates MYH7b protein production without affecting microRNA expression. Among other genes, endogenous miR-499 targets the 3 untranslated region of the transcription factor Sox6, which in turn acts as a repressor of MYH7b transcriptional activity. Thus, concerted transcription and alternative splicing uncouple the level of expression of MYH7b and miR-499 when their coexpression is not required.Skeletal and cardiac muscle exhibit a broad range of contractile characteristics and adaptive responses that are controlled, at least in part, by sarcomeric myosins, a multigene family of actin-based motors that convert chemical energy released from ATP hydrolysis into mechanical force. In addition to the eight well-characterized myosin heavy chain (MYH) isoforms expressed in heart and skeletal muscle, three more ancient myosin genes, MYH7b (MYH14), MYH15, and MYH16, have been identified (13). In humans, the MYH16 gene is a pseudogene by virtue of a frameshift mutation and consequent premature termination codon (PTC). Remarkably, the gene is not mutated in other nonhuman primates and appears to regulate the fiber size of the masticatory muscles (35). Phylogenetic analysis suggests that MYH7b is most closely related to ␣-and ␤-cardiac myosins, with 69% amino acid identity. Moreover, these three related myosin genes encode microRNAs (miRNAs) in one of their introns (MYH-␣, miR-208a; MYH-␤, miR-208b; and MYH7b, miR-499) (40, 41). In particular, miR-208a has been shown to be a key mediator of the stress response and a regulator of hypertrophy and electrical conduction in the mouse heart (6, 41). Since MYH7b mRNA levels appear much lower than those of other muscle myosins, the role of MYH7b protein in vertebrate muscle remains unclear.Here, we show that MYH7b expression is controlled transcriptionally by different stimuli, as well as posttranscriptionally, through a unique alternative splicing event. Moreover, we show that miR-499 acts in a regulatory feedback circuit to control MYH7b activity. Our data reveal the complexity of MYH7b gene regulation and unveil a novel mechanism that uncouples host gene-microRNA coexpression. MATERIALS AND METHODSAnimal use and care. C57/BL6 mice used in the experiments were allowed access to standard soy-based rodent chow and water ad libitum. Unless otherwise noted, C57/BL6 male mice 12 weeks of age were used in the experiments. For altered thyroid hormone conditions, mice were fed an iodine-deficient chow supplemented with 0.15% propylthiouracil ([PTU] Harlan Teklad diet composition TD.95125) ad libitum for 30 days. Mice treated with PTU and triiodothyronine (T 3 ) were injected intraperitoneally with 0.2 g/g T 3 on days 29 and 30 of PTU diet ingestion. Four mice from each thyroid condition were sacrificed at 12 weeks of age, and tissues were rapidly excised and froze...

Previous studies of the Sleeping Beauty (SB) transposon system, as an insertional mutagen in the germline of mice, have used reverse genetic approaches. These studies have led to its proposed use for regional saturation mutagenesis by taking a forward-genetic approach. Thus, we used the SB system to mutate a region of mouse Chromosome 11 in a forward-genetic screen for recessive lethal and viable phenotypes. This work represents the first reported use of an insertional mutagen in a phenotype-driven approach. The phenotype-driven approach was successful in both recovering visible and behavioral mutants, including dominant limb and recessive behavioral phenotypes, and allowing for the rapid identification of candidate gene disruptions. In addition, a high frequency of recessive lethal mutations arose as a result of genomic rearrangements near the site of transposition, resulting from transposon mobilization. The results suggest that the SB system could be used in a forward-genetic approach to recover interesting phenotypes, but that local chromosomal rearrangements should be anticipated in conjunction with single-copy, local transposon insertions in chromosomes. Additionally, these mice may serve as a model for chromosome rearrangements caused by transposable elements during the evolution of vertebrate genomes.

. Skeletal muscle adaptations in response to voluntary wheel running in myosin heavy chain null mice. J Appl Physiol 92: 313-322, 2002; 10.1152/ japplphysiol.00832.2001.-To examine the effects of gene inactivation on the plasticity of skeletal muscle, mice null for a specific myosin heavy chain (MHC) isoform were subjected to a voluntary wheel-running paradigm. Despite reduced running performance compared with nontransgenic C57BL/6 mice (NTG), both MHC IIb and MHC IId/x null animals exhibited increased muscle fiber size and muscle oxidative capacity with wheel running. In the MHC IIb null animals, there was no significant change in the percentage of muscle fibers expressing a particular MHC isoform with voluntary wheel running at any time point. In MHC IId/x null mice, wheel running produced a significant increase in the percentage of fibers expressing MHC IIa and MHC I and a significant decrease in the percentage of fibers expressing MHC IIb. Muscle pathology was not affected by wheel running for either MHC null strain. In summary, despite their phenotypes, MHC null mice do engage in voluntary wheel running. Although this wheel-running activity is lessened compared with NTG, there is evidence of distinct patterns of muscle adaptation in both null strains. myosin heavy chain; endurance exercise; muscle plasticity SKELETAL MUSCLE SHOWS a remarkable adaptive ability, which is exemplified by alterations in the expression of a wide range of muscle-specific genes. Paradigms such as endurance exercise increase muscle activity and result in changes within the muscle that allow the elevated metabolic demands to be met more effectively. Specifically, there are shifts in myosin heavy chain (MHC) isoform expression that result in a decrease in the percentage of muscle fibers expressing the faster MHC IIb isoform and an increase in the percentage of muscle fibers expressing the slower MHC IIa isoform (3,6,7,14,19,27,32,33,40,44). These changes in MHC isoform expression can also be accompanied by increased levels of key oxidative enzymes (3,6,8,14,24,25,37) and skeletal muscle fiber hypertrophy (3,14,27,44).Although the plasticity of skeletal muscle in response to endurance exercise is well documented for a number of animal models using both treadmill-based and voluntary wheel running-based exercise programs, relatively few studies have combined endurance exercise and a transgenic rodent model

The sarcomeric myosin gene, Myh7b, encodes an intronic microRNA, miR-499, which regulates cardiac and skeletal muscle biology, yet little is known about its transcriptional regulation. To identify the transcription factors involved in regulating Myh7b/miR-499 gene expression, we have mapped the transcriptional start sites and identified an upstream 6.2 kb region of the mouse Myh7b gene whose activity mimics the expression pattern of the endogenous Myh7b gene both in vitro and in vivo. Through promoter deletion analysis, we have mapped a distal E-box element and a proximal Ikaros site that are essential for Myh7b promoter activity in muscle cells. We show that the myogenic regulatory factors, MyoD, Myf5 and Myogenin, bind to the E-box, while a lymphoid transcription factor, Ikaros 4 (Eos), binds to the Ikaros motif. Further, we show that through physical interaction, MyoD and Eos form an active transcriptional complex on the chromatin to regulate the expression of the endogenous Myh7b/miR-499 gene in muscle cells. We also provide the first evidence that Eos can regulate expression of additional myosin genes (Myosin 1 and β-Myosin) via the miR-499/Sox6 pathway. Therefore, our results indicate a novel role for Eos in the regulation of the myofiber gene program.