Abstract

Impairments in skeletal muscle are a major contributor to the work intolerance and morbidity of patients with congestive heart failure (CHF). The fast-twitch skeletal muscle sarcoplasmic reticulum calcium ATPase (SERCA1) is a calcium transport pump whose expression is markedly decreased in CHF. The goal of this research is to elucidate the molecular mechanism and functional significance of decreased SERCA1 expression in skeletal muscle of rats with CHF.
Aim 1 is to determine the contribution of decreased SERCA 1 transcription rate to decreases in SERCA 1 mRNA expression in skeletal muscle from rats with CHF. Transcription rate will be measured using a new, highly sensitive approach in which the SERCA precursor mRNA/mature mRNA ratio will be compared in tibialis anterior and diaphragm muscle from control and CHF rats using reverse transcriptase-polymerase chain reaction.
Aim 2 is to study the mechanism by which SERCA 1 gene transcription is regulated by CHF. The cis-elements involved in SERCA 1 downregulation will be identified by deletion analysis. This will involve injecting the tibialis anterior muscle with plasmid DNA reporter constructs of the 5' flanking region of the SERCA 1 gene. Gel shift assay will detect and characterize the DNA binding protein(s) involved in transcriptional regulation and one-hybrid assays will identify novel DNA binding protein(s). If decreases in SERCA 1 mRNA expression are not predominately mediated by decreased transcription rate, then other pretranslational sites of control such as SERCA mRNA stability will be studied using in vitro assays of mRNA degradation and RNA-protein interactions.
Aim 3 is to determine the functional significance of decreased SERCA expression in CHF. SERCA 1 expression will be """"""""knocked down"""""""" in control muscles and overexpressed in CHF muscles by in vivo injection of appropriate plasmid DNA. SERCA1 protein expression will be quantified and localized by immunochemical assays and then related to parameters that reflect SERCA 1 function such as SERCA activity, SR calcium uptake rate and muscle relaxation time. Basic knowledge of the molecular regulation of SERCA gene expression in CHF can be applied towards more effective therapeutics in addition to furthering our understanding of the influence of SERCA 1 gene expression on skeletal muscle function.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR041705-08
Application #
6124138
Study Section
Respiratory and Applied Physiology Study Section (RAP)
Program Officer
Lymn, Richard W
Project Start
1992-06-25
Project End
2001-05-31
Budget Start
1999-12-01
Budget End
2001-05-31
Support Year
8
Fiscal Year
2000
Total Cost
$208,276
Indirect Cost

  Institution

Name
Boston University
Department
Other Health Professions
Type
Schools of Allied Health Profes
DUNS #
042250712
City
Boston
State
MA
Country
United States
Zip Code
02215

  Publications

Wu, Chia-Ling; Cornwell, Evangeline W; Jackman, Robert W et al. (2014) NF-?B but not FoxO sites in the MuRF1 promoter are required for transcriptional activation in disuse muscle atrophy. Am J Physiol Cell Physiol 306:C762-7
Jackman, Robert W; Cornwell, Evangeline W; Wu, Chia-Ling et al. (2013) Nuclear factor-?B signalling and transcriptional regulation in skeletal muscle atrophy. Exp Physiol 98:19-24
Wu, Chia-Ling; Kandarian, Susan C (2012) Protein overexpression in skeletal muscle using plasmid-based gene transfer to elucidate mechanisms controlling fiber size. Methods Mol Biol 798:231-43
Jackman, Robert W; Wu, Chia-Ling; Kandarian, Susan C (2012) The ChIP-seq-defined networks of Bcl-3 gene binding support its required role in skeletal muscle atrophy. PLoS One 7:e51478
Yamaki, Takuo; Wu, Chia-Ling; Gustin, Michael et al. (2012) Rel A/p65 is required for cytokine-induced myotube atrophy. Am J Physiol Cell Physiol 303:C135-42
Wu, Chia-Ling; Kandarian, Susan C; Jackman, Robert W (2011) Identification of genes that elicit disuse muscle atrophy via the transcription factors p50 and Bcl-3. PLoS One 6:e16171
Reed, S A; Senf, S M; Cornwell, E W et al. (2011) Inhibition of IkappaB kinase alpha (IKK?) or IKKbeta (IKK?) plus forkhead box O (Foxo) abolishes skeletal muscle atrophy. Biochem Biophys Res Commun 405:491-6
Jackman, Robert W; Rhoads, Mary G; Cornwell, Evangeline et al. (2009) Microtubule-mediated NF-kappaB activation in the TNF-alpha signaling pathway. Exp Cell Res 315:3242-9
Van Gammeren, Darin; Damrauer, Jeffrey S; Jackman, Robert W et al. (2009) The IkappaB kinases IKKalpha and IKKbeta are necessary and sufficient for skeletal muscle atrophy. FASEB J 23:362-70
Koncarevic, Alan; Jackman, Robert W; Kandarian, Susan C (2007) The ubiquitin-protein ligase Nedd4 targets Notch1 in skeletal muscle and distinguishes the subset of atrophies caused by reduced muscle tension. FASEB J 21:427-37

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