BackgroundDiet-induced obesity (DIO) is a significant health concern which has been linked to structural and functional changes in the gut microbiota. Exercise (Ex) is effective in preventing obesity, but whether Ex alters the gut microbiota during development with high fat (HF) feeding is unknown.ObjectiveDetermine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO.MethodsMale C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation.ResultsHF diet resulted in significantly greater body weight and adiposity as well as decreased glucose tolerance that were prevented by voluntary Ex (p<0.05). Visualization of Unifrac distance data with principal coordinates analysis indicated clustering by both diet and Ex at week 12. Sequencing demonstrated Ex-induced changes in the percentage of major bacterial phyla at 12 weeks. A correlation between total Ex distance and the ΔCt Bacteroidetes: ΔCt Firmicutes ratio from qPCR demonstrated a significant inverse correlation (r2 = 0.35, p = 0.043).ConclusionEx induces a unique shift in the gut microbiota that is different from dietary effects. Microbiota changes may play a role in Ex prevention of HF-DIO.

To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant alpha-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the alpha-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous alpha-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain alpha-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC alpha-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

Abstract-Tropomyosin (TM) is an integral component of the thin filament in muscle fibers and is involved in regulating actin-myosin interactions. TM is encoded by a family of four alternatively spliced genes that display highly conserved nucleotide and amino acid sequences. To assess the functional and developmental significance of ␣-TM, the murine ␣-TM gene was disrupted by homologous recombination. Homozygous ␣-TM null mice are embryonic lethal, dying between 8 and 11.5 days post coitum. Mice that are heterozygous for ␣-TM are viable and reproduce normally. Heterozygous knockout mouse hearts show a 50% reduction in cardiac muscle ␣-TM mRNA, with no compensatory increase in transcript levels by striated muscle ␤-TM or TM-30 isoforms. Surprisingly, this reduction in ␣-TM mRNA levels in heterozygous mice is not reflected at the protein level, where normal amounts of striated muscle ␣-TM protein are produced and integrated in the myofibril. Quantification of ␣-TM mRNA bound in polysomal fractions reveals that both wild-type and heterozygous knockout animals have similar levels. These data suggest that a change in steady-state level of ␣-TM mRNA does not affect the relative amount of mRNA translated and amount of protein synthesized. Physiological analyses of myocardial and myofilament function show no differences between heterozygous ␣-TM mice and control mice. The present study suggests that translational regulation plays a major role in the control of TM expression. (Circ Res. 1998;82:116-123.)Key Words: tropomyosin Ⅲ knockout mouse Ⅲ translational regulation T ropomyosin, an essential thin filament protein, binds to actin and the troponin complex to regulate the Ca 2ϩ -sensitive interaction of actin and myosin. TM assembles into an ␣-helical coiled-coil dimer, with each molecule interacting with six or seven actin monomers. The TMs bind to themselves in a head-to-tail manner and wrap around the actin molecule to stabilize thin filament assembly. Although the exact role of TM is still not completely understood, the TM-troponin complex inhibits the actin-myosin interaction in the resting state; with an increase of Ca 2ϩ in the myofilament space and binding of Ca 2ϩ to troponin, this inhibition is released and leads to muscle contraction.TM is encoded by a small multigene family consisting of the ␣, ␤, TM-30, and TM-4 genes. These genes, and associated proteins, exhibit a very high degree of conservation across species ranging from Drosophila to humans. For example, there is 86% amino acid conservation between the striated muscle ␣-and ␤-TM isoforms.1 Previous work conducted by our laboratory 2,3 and others 4,5 has demonstrated that TM isoforms are generated through alternative exon splicing and are regulated in a developmental and tissue-specific manner. The functional significance of the alternatively spliced TM domains and the physiological role of the TM isoforms produced by the various multigene family members remain to be determined.During murine cardiac development, ␣-and ␤-TMs are expressed at different le...

We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.