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1.  Breaking Down Protein Degradation Mechanisms In Cardiac Muscle: Therapeutic Outlooks For Disease 
Trends in molecular medicine  2013;19(4):239-249.
Regulated protein degradation through the ubiquitin-proteasome and lysosomal-autophagy systems is critical for homeostatic protein-turnover in cardiac muscle, and for proper cardiac function. The discovery of muscle-specific components in these systems has illuminated how aberrations in their levels are pivotal to the development of cardiac stress and disease. New evidence suggests that equal importance in disease development should be given to ubiquitously expressed degradation components. These are compartmentalized within cardiac muscles and, when mislocalized, can be critical in the development of specific cardiac diseases. Here, we discuss how alterations in the compartmentalization of degradation components affect disease states, the tools available to investigate these mechanisms, as well as recent discoveries that highlight the therapeutic value of targeting these pathways in disease.
PMCID: PMC3622835  PMID: 23453282
cardiac muscle; ubiquitin-proteasome; lysosomal-autophagy; protein turnover; cardiac disease
2.  Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity 
The Journal of Cell Biology  2013;200(4):523-536.
Obscurin contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity in skeletal muscle fibers.
Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and β-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of β-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity.
PMCID: PMC3575540  PMID: 23420875
3.  Thymosin Beta 4 is Dispensable for Murine Cardiac Development and Function 
Circulation research  2011;110(3):456-464.
Thymosin beta 4 (Tβ4) is a 43 amino acid factor encoded by an X-linked gene. Recent studies have suggested that Tβ4 is a key factor in cardiac development, growth, disease, epicardial integrity and blood vessel formation. Cardiac specific shRNA knockdown of tβ4 has been reported to result in embryonic lethality at E14.5-16.5, with severe cardiac and angiogenic defects. However, this shRNA tβ4-knockdown model did not completely abrogate Tβ4 expression. To completely ablate Tβ4 and to rule out the possibility of off-target effects associated with shRNA gene silencing, further studies of global or cardiac specific knockouts are critical.
Here, we examined the role of Tβ4 in developing and adult heart via global and cardiac specific tβ4-knockout mouse models.
Methods and Results
Global tβ4-knockout mice were born at Mendelian ratios and exhibited normal heart and blood vessel formation. Furthermore, in adult global tβ4-knockout mice, cardiac function, capillary density, expression of key cardiac fetal and angiogenic genes, epicardial marker expression, and extracellular matrix deposition were indistinguishable from that of controls. Tissue specific tβ4-deficient mice, generated by crossing tβ4-floxed mice to Nkx2.5-Cre and αMHC-Cre, were also found to have no phenotype.
Therefore, we conclude that Tβ4 is dispensable for embryonic viability, heart development, coronary vessel development and adult myocardial function.
PMCID: PMC3739283  PMID: 22158707
Cardiac Development; Cardiac Function; Epicardium; Thymosin Beta 4
4.  Obscurin and KCTD6 regulate cullin-dependent small ankyrin-1 (sAnk1.5) protein turnover 
Molecular Biology of the Cell  2012;23(13):2490-2504.
Small ankyrin-1 isoform 5 (sAnk1.5) turnover is regulated by posttranslational modification (ubiquitylation, neddylation, and acetylation), the presence of obscurin, and KCTD6 (a novel tissue-specific interaction partner). KCTD6 links sAnk1.5 to cullin-3. The absence of obscurin results in translocation of sAnk1.5/KCTD6 to the Z-disk and loss of sAnk1.5 on the protein level.
Protein turnover through cullin-3 is tightly regulated by posttranslational modifications, the COP9 signalosome, and BTB/POZ-domain proteins that link cullin-3 to specific substrates for ubiquitylation. In this paper, we report how potassium channel tetramerization domain containing 6 (KCTD6) represents a novel substrate adaptor for cullin-3, effectively regulating protein levels of the muscle small ankyrin-1 isoform 5 (sAnk1.5).
Binding of sAnk1.5 to KCTD6, and its subsequent turnover is regulated through posttranslational modification by nedd8, ubiquitin, and acetylation of C-terminal lysine residues. The presence of the sAnk1.5 binding partner obscurin, and mutation of lysine residues increased sAnk1.5 protein levels, as did knockdown of KCTD6 in cardiomyocytes. Obscurin knockout muscle displayed reduced sAnk1.5 levels and mislocalization of the sAnk1.5/KCTD6 complex. Scaffolding functions of obscurin may therefore prevent activation of the cullin-mediated protein degradation machinery and ubiquitylation of sAnk1.5 through sequestration of sAnk1.5/KCTD6 at the sarcomeric M-band, away from the Z-disk–associated cullin-3. The interaction of KCTD6 with ankyrin-1 may have implications beyond muscle for hereditary spherocytosis, as KCTD6 is also present in erythrocytes, and erythrocyte ankyrin isoforms contain its mapped minimal binding site.
PMCID: PMC3386213  PMID: 22573887
6.  Formin follows function: a muscle-specific isoform of FHOD3 is regulated by CK2 phosphorylation and promotes myofibril maintenance 
The Journal of Cell Biology  2010;191(6):1159-1172.
Phosphorylation of the muscle-specific formin splice variant FHOD3 by CK2 regulates its stability, myofibril targeting, and myofibril integrity.
Members of the formin family are important for actin filament nucleation and elongation. We have identified a novel striated muscle–specific splice variant of the formin FHOD3 that introduces a casein kinase 2 (CK2) phosphorylation site. The specific targeting of muscle FHOD3 to the myofibrils in cardiomyocytes is abolished in phosphomutants or by the inhibition of CK2. Phosphorylation of muscle FHOD3 also prevents its interaction with p62/sequestosome 1 and its recruitment to autophagosomes. Furthermore, we show that muscle FHOD3 efficiently promotes the polymerization of actin filaments in cardiomyocytes and that the down-regulation of its expression severely affects myofibril integrity. In murine and human cardiomyopathy, we observe reduced FHOD3 expression with a concomitant isoform switch and change of subcellular targeting. Collectively, our data suggest that a muscle-specific isoform of FHOD3 is required for the maintenance of the contractile structures in heart muscle and that its function is regulated by posttranslational modification.
PMCID: PMC3002041  PMID: 21149568
7.  Nesprin 1 is critical for nuclear positioning and anchorage 
Human Molecular Genetics  2009;19(2):329-341.
Nesprin 1 is an outer nuclear membrane protein that is thought to link the nucleus to the actin cytoskeleton. Recent data suggest that mutations in Nesprin 1 may also be involved in the pathogenesis of Emery-Dreifuss muscular dystrophy. To investigate the function of Nesprin 1 in vivo, we generated a mouse model in which all isoforms of Nesprin 1 containing the C-terminal spectrin-repeat region with or without KASH domain were ablated. Nesprin 1 knockout mice are marked by decreased survival rates, growth retardation and increased variability in body weight. Additionally, nuclear positioning and anchorage are dysfunctional in skeletal muscle from knockout mice. Physiological testing demonstrated no significant reduction in stress production in Nesprin 1-deficient skeletal muscle in either neonatal or adult mice, but a significantly lower exercise capacity in knockout mice. Nuclear deformation testing revealed ineffective strain transmission to nuclei in muscle fibers lacking Nesprin 1. Overall, our data show that Nesprin 1 is essential for normal positioning and anchorage of nuclei in skeletal muscle.
PMCID: PMC2796894  PMID: 19864491
8.  Simple and High Yielding Method for Preparing Tissue Specific Extracellular Matrix Coatings for Cell Culture 
PLoS ONE  2010;5(9):e13039.
The native extracellular matrix (ECM) consists of a highly complex, tissue-specific network of proteins and polysaccharides, which help regulate many cellular functions. Despite the complex nature of the ECM, in vitro cell-based studies traditionally assess cell behavior on single ECM component substrates, which do not adequately mimic the in vivo extracellular milieu.
Methodology/Principal Findings
We present a simple approach for developing naturally derived ECM coatings for cell culture that provide important tissue-specific cues unlike traditional cell culture coatings, thereby enabling the maturation of committed C2C12 skeletal myoblast progenitors and human embryonic stem cells differentiated into cardiomyocytes. Here we show that natural muscle-specific coatings can (i) be derived from decellularized, solubilized adult porcine muscle, (ii) contain a complex mixture of ECM components including polysaccharides, (iii) adsorb onto tissue culture plastic and (iv) promote cell maturation of committed muscle progenitor and stem cells.
This versatile method can create tissue-specific ECM coatings, which offer a promising platform for cell culture to more closely mimic the mature in vivo ECM microenvironment.
PMCID: PMC2946408  PMID: 20885963
9.  An FHL1-containing complex within the cardiomyocyte sarcomere mediates hypertrophic biomechanical stress responses in mice 
The Journal of Clinical Investigation  2008;118(12):3870-3880.
The response of cardiomyocytes to biomechanical stress can determine the pathophysiology of hypertrophic cardiac disease, and targeting the pathways regulating these responses is a therapeutic goal. However, little is known about how biomechanical stress is sensed by the cardiomyocyte sarcomere to transduce intracellular hypertrophic signals or how the dysfunction of these pathways may lead to disease. Here, we found that four-and-a-half LIM domains 1 (FHL1) is part of a complex within the cardiomyocyte sarcomere that senses the biomechanical stress–induced responses important for cardiac hypertrophy. Mice lacking Fhl1 displayed a blunted hypertrophic response and a beneficial functional response to pressure overload induced by transverse aortic constriction. A link to the Gαq (Gq) signaling pathway was also observed, as Fhl1 deficiency prevented the cardiomyopathy observed in Gq transgenic mice. Mechanistic studies demonstrated that FHL1 plays an important role in the mechanism of pathological hypertrophy by sensing biomechanical stress responses via the N2B stretch sensor domain of titin and initiating changes in the titin- and MAPK-mediated responses important for sarcomere extensibility and intracellular signaling. These studies shed light on the physiological regulation of the sarcomere in response to hypertrophic stress.
PMCID: PMC2575833  PMID: 19033658
10.  “Z”eroing in on the Role of Cypher in Striated Muscle Function, Signaling, and Human Disease 
Trends in cardiovascular medicine  2007;17(8):258-262.
The striated muscle Z-line, a multiprotein complex at the boundary between sarcomeres, plays an integral role in maintaining striated muscle structure and function. Multiple Z-line-associated proteins have been identified and shown to play an increasingly important role in the pathogenesis of human muscle disease. Cypher/ZASP, a PDZ-LIM protein in the Z-line, binds to α-actinin (via its PDZ domain) and has been suggested to function as a linker-strut to maintain cytoskeletal structural integrity during contraction. Cypher may also participate in signaling pathways by binding to protein kinase C (PKC) via its LIM domains. Analysis of Cypher deficient mice has revealed that Cypher plays an integral role in Z-line maintenance/integrity of striated muscles and the pathogenesis of congenital myopathies, including cardiomyopathy. These studies have led to the subsequent discovery of Cypher mutations in human patients with dilated cardiomyopathy, hypertrophic cardiomyopathy as well as skeletal muscle myopathies, which have been recently termed Zaspopathies. The recent discovery of various alternatively spliced isoforms of Cypher with potentially distinct structural and signaling roles brings a different level of complexity to the mechanisms underlying Cypher-based human myopathies. This review will focus on recent developments on the role of Cypher and its isoforms in striated muscle structure, signaling, and disease to provide insights into the mechanisms involved in the pathogenesis of Z-line-associated human myopathies.
PMCID: PMC2134983  PMID: 18021935

Results 1-10 (10)