Zhao Bo Li

The mTOR/p70S6K Signal Transduction Pathway Plays a Role in Cardiac Hypertrophy and Influences Expression of Myosin Heavy Chain Genes in vivo

AbstractObjective: Rapamycin (R) inhibits p70 S6 kinase (p70S6K) activity and hypertrophy of cultured neonatal rat cardiac myocytes. The purpose of the present study was to determine whether rapamycin inhibits left ventricular (L V) hypertrophy in intact rats and whether it alters cardiac gene expression.nMethods: 300 g rats were subjected to aortic constriction (AC) or sham-operation (SH) and studied 2 and 3 days after surgery. Beginning 1 day prior to surgery, rats were injected with rapamycin (1.5 mg/kg, i.p.) or carboxymethylcellulose vehicle (V), yielding 4 groups (SH-V, SH-R, AC-V, AC-R). Total RNA was extracted for determination of mRNA levels by Northern blotting.nResults: L V dry weight/body weight ratios were 0.43 ± 0.04 (mean ± SE) for SH-V, 0.46 ± 0.02 for SH-R, 0.56 ± 0.02 for AC-V, and 0.53 ± 0.03 for AC-R. R inhibited cardiac hypertrophy induced by pressure overload (ANOVA; p < 0.05). Rapamycin had no effect on the expression of atrial natriuretic factor mRNA, but increased the levels of β-myosin heavy chain mRNA 6-fold in hearts of SH-R and AC-R compared to SH-V. Rapamycin also increased the expression of α-myosin heavy chain mRNA in SH-R by 3-fold compared with SH-V, but had no effect on the AC-R group.nConclusion: The data suggest that an intact mTOR signaling pathway is required for rapid hypertrophic growth of the heart in vivo. Moreover, the data suggest a novel link between the mTOR/p70S6K signal transduction pathway and pretranslational control of myosin gene expression in the heart.

Myosin Heavy Chain Composition in Normal and Atrophic Equine Laryngeal Muscle

The myosin heavy chain (MHC) composition of a given muscle determines the contractile properties and, therefore, the fiber type distribution of the muscle. MHC isoform expression in the laryngeal muscle is modulated by neural input and function, and it represents the cellular level changes that occur with denervation and reinnervation of skeletal muscle. The objective of this study was to evaluate the pattern of MHC isoform expression in laryngeal muscle harvested from normal cadavers and cadavers with naturally occurring left laryngeal hemiplegia secondary to recurrent laryngeal neuropathy. Left and right thyroarytenoideus (TA) and cricoarytenoideus dorsalis (CAD) were obtained from 7 horses affected with left-sided intrinsic laryngeal muscle atrophy and from 2 normal horses. Frozen sections were evaluated histologically for degree of atrophy and fiber type composition. MHC isoform expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of muscle protein. Histologic atrophy was seen in all atrophic muscles and some right-sided muscles of 3 affected horses, as well as the left TA of 1 normal horse. Fiber type grouping or loss of type I muscle fibers was observed in the left-sided laryngeal muscles in all but 1 affected horse, as well as in the right muscles of 2 affected horses, and the left TA of 1 normal horse. SDS-PAGE showed 2 bands corresponding to the type I and type IIB myosin isoforms in the CAD and TA of the 2 normal horses. Affected horses demonstrated a trend toward increased expression of the type IIB isoform and decreased expression of the type I isoform in atrophic muscles. This study confirmed the presence of histologic abnormalities in grossly normal equine laryngeal muscle, and it demonstrated an increased expression of type IIB MHC with a concurrent decreased expression of type I MHC in affected muscles. Evaluation of muscle fiber changes at the cellular level under denervated and reinnervated conditions may aid in assessing future strategies for reinnervation or regeneration of atrophic laryngeal muscle.

High efficiency gene delivery into laryngeal muscle with bidirectional electroporation

Objective The impact of polarity change on the efficiency of in vivo electroporative (EP) gene transfection was assessed in rat laryngeal muscle. Study Design and Setting High (HV) and low field voltage (LV) were combined with polarity changes to determine transfection in 5 different conditions: 1) without EP (EP[-]), 2) HV+LV (HL), 3) HV+LV followed by HV+LV with no change in polarity (HLHL unidirectional), 4) HV+LV followed by HV+LV with opposite polarity (HLHL bidirectional), 5) HV+LV followed by LV with opposite polarity (HLL bidirectional). Results HLL bidirectional sequence showed the best result with less interindividual variability and extended expression period. With the exception of repeated high voltage sequences, EP parameters were not likely to induce cell injury or inflammation. Conclusion HLL bidirectional electroporative gene delivery produces high transfection rates with limited tissue trauma. Significance Bidirectional EP provides a safe and highly efficient method for therapeutic gene delivery into skeletal muscle.

Laryngeal muscle surface receptors identified using random phage library

Objectives: The ultimate goal of this study is to improve the efficiency of gene transfer in mammalian muscle by developing targeted adenoviral vectors. Altering the tropism of viral vectors to recognize tissue specific antigens is one method to achieve this goal. This approach requires identification of cell‐surface receptors and the insertion of target peptide sequences into the adenoviral fiber protein. In this study, phage biopanning was performed on cultured rat skeletal and laryngeal muscle to identify cell‐surface receptors.