Derek Milner | University of Illinois (original) (raw)
Papers by Derek Milner
Cell and tissue research, Jun 1, 2018
Advances in stem cell biology and materials science have provided a basis for developing tissue e... more Advances in stem cell biology and materials science have provided a basis for developing tissue engineering methods to repair muscle injury. Among stem cell populations with potential to aid muscle repair, adipose-derived mesenchymal stem cells (ASC) hold great promise. To evaluate the possibility of using porcine ASC for muscle regeneration studies, we co-cultured porcine ASC with murine CC myoblasts. These experiments demonstrated that porcine ASC display significant myogenic potential. Co-culture of ASC expressing green fluorescent protein (GFP) with CC cells resulted in GFP myotube formation, indicating fusion of ASC with myoblasts to form myotubes. The presence of porcine lamin A/C positive nuclei in myotubes and RTqPCR analysis of porcine myogenin and desmin expression confirmed that myotube nuclei derived from ASC contribute to muscle gene expression. Co-culturing GFPASC with porcine satellite cells demonstrated enhanced myogenic capability of ASC, as the percentage of labele...
Journal of Molecular and Cellular Cardiology, 2008
We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen spec... more We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen species (ROS) as a probe to detect myofibrillar protein (MP) oxidation. In particular we found that (i) oxidation of the cysteinyl residue of Tm caused the formation of high molecular weight complexes (HMC) due to disulfide cross-bridges in microembolized pigs hearts and (ii) this modification was related to contractile dysfunction (Eur. Heart. J. 27, 875-881, 2006). The present study investigated the occurrence and the extent of MP oxidation in human end stage (NYHA class IV) heart failure. To this aim 50 left ventricular (LV) biopsies from HF patients undergoing heart transplantation (HF group) were compared with LV biopsies from non-failing donor hearts (n = 24, control group) in a blinded manner. Tissue samples were analyzed by means of SDS/PAGE under non-reducing conditions. The amount of HMC in Tm was quantified by densitometry and was found to be significantly greater in the HF group (0.055 ± 0.014 vs 0.010 ± 0.001 in the control group, p b 0.05). Tm appeared especially susceptible to oxidation, since actin and desmin did not undergo HMC formation, although this type of modification can be induced in these proteins in vitro. Besides changes due to ROS, we tested whether MP are also targets of reactive nitrogen species. By using the biotin-switch technique we found that S-nitrosylation affects several proteins among which we could identify Tm. The extent of S-nitrosylation was higher in HF samples.
American Journal of Pathology, 2007
Transgenic expression of the ␣71 integrin in the dystrophic mdx/utr ؊/؊ mouse decreases developm... more Transgenic expression of the ␣71 integrin in the dystrophic mdx/utr ؊/؊ mouse decreases development of muscular dystrophy and enhances longevity. To explore the possibility that elevating ␣71 integrin expression could also ameliorate different forms of muscular dystrophy , we used transgenic technology to enhance integrin expression in mice lacking ␦-sarcoglycan (␦ sgc) , a mouse model for human limb girdle muscular dystrophy type 2F. Unlike ␣7 transgenic mdx/utr ؊/؊ mice , enhanced ␣71 integrin expression in the ␦ sgc-null mouse did not alleviate muscular dystrophy in these animals. Expression of the transgene in the ␦ sgc-null did not alleviate dystrophic histopathology , nor did it decrease cardiomyopathy or restore exercise tolerance. One hallmark of integrin-mediated alleviation of muscular dystrophy in the mdx/utr ؊/؊ background is the restoration of myotendinous junction integrity. As assessed by atomic force microscopy , myotendinous junctions from normal and ␦ sgc-null mice were indistinguishable , thus suggesting the important influence of myotendinous junction integrity on the severity of muscular dystrophy and providing a possible explanation for the inability of enhanced integrin expression to alleviate dystrophy in the ␦ sgc-null mouse. These results suggest that distinct mechanisms underlie the development of the diseases that arise from deficiencies in dystrophin and sarcoglycan.
The Journal of Cell Biology, 1994
The muscle-specific intermediate filament
The Journal of Cell Biology, 2000
Ultrastructural studies have previously suggested potential association of intermediate filaments... more Ultrastructural studies have previously suggested potential association of intermediate filaments (IFs) with mitochondria. Thus, we have investigated mitochondrial distribution and function in muscle lacking the IF protein desmin. Immunostaining of skeletal muscle tissue sections, as well as histochemical staining for the mitochondrial marker enzymes cytochrome C oxidase and succinate dehydrogenase, demonstrate abnormal accumulation of subsarcolemmal clumps of mitochondria in predominantly slow twitch skeletal muscle of desmin-null mice. Ultrastructural observation of desmin-null cardiac muscle demonstrates in addition to clumping, extensive mitochondrial proliferation in a significant fraction of the myocytes, particularly after work overload. These alterations are frequently associated with swelling and degeneration of the mitochondrial matrix. Mitochondrial abnormalities can be detected very early, before other structural defects become obvious. To investigate related changes in mitochondrial function, we have analyzed ADP-stimulated respiration of isolated muscle mitochondria, and ADPstimulated mitochondrial respiration in situ using sapo-nin skinned muscle fibers. The in vitro maximal rates of respiration in isolated cardiac mitochondria from desmin-null and wild-type mice were similar. However, mitochondrial respiration in situ is significantly altered in desmin-null muscle. Both the maximal rate of ADPstimulated oxygen consumption and the dissociation constant ( K m ) for ADP are significantly reduced in desmin-null cardiac and soleus muscle compared with controls. Respiratory parameters for desmin-null fast twitch gastrocnemius muscle were unaffected. Additionally, respiratory measurements in the presence of creatine indicate that coupling of creatine kinase and the adenine translocator is lost in desmin-null soleus muscle. This coupling is unaffected in cardiac muscle from desmin-null animals. All of these studies indicate that desmin IFs play a significant role in mitochondrial positioning and respiratory function in cardiac and skeletal muscle.
The Journal of Cell Biology, 1996
Desmin, the muscle specific intermediate filament (IF) protein encoded by a single gene, is expre... more Desmin, the muscle specific intermediate filament (IF) protein encoded by a single gene, is expressed in all muscle tissues. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. To investigate the function of desmin in all three muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, desmin null mice are viable and fertile. However, these mice demonstrated a multisystern disorder involving cardiac, skeletal, and smooth muscle. Histological and electron microscopic analysis in both heart and skeletal muscle tissues revealed se-1. Abbreviations used in this paper: ES, embryonic stem; IF, intermediate filament; pc, post coitum.
Molecular Biology of the Cell, 2002
The sarcolemma of fast-twitch muscle is organized into "costameres", structures that are oriented... more The sarcolemma of fast-twitch muscle is organized into "costameres", structures that are oriented transversely, over the Z and M lines of nearby myofibrils, and longitudinally, to form a rectilinear lattice. Here we examine the role of desmin, the major intermediate filament protein of muscle in organizing costameres. In control mouse muscle, desmin is enriched at the sarcolemmal domains that lie over nearby Z lines and that also contain -spectrin. In tibialis anterior muscle from mice lacking desmin due to homologous recombination, most costameres are lost. In myofibers from desmin -/-quadriceps, by contrast, many costameric structures are stable. Alternatively, Z line domains may be lost while domains oriented longitudinally or lying over M lines are retained. Experiments with pan-specific antibodies to intermediate filament proteins and to cytokeratins suggest that control and desmin -/-muscles express similar levels of cytokeratins. Cytokeratins concentrate at the sarcolemma at all three domains of costameres when the latter are retained in desmin -/-muscle and redistribute with -spectrin at the sarcolemma when costameres are lost. Our results suggest that desmin associates with and selectively stabilizes the Z line domains of costameres, but that cytokeratins associate with all three domains of costameres, even in the absence of desmin.
Medicine & Science in Sports & Exercise, 1999
Medicine & Science in Sports & Exercise, 1999
Journal of Molecular and Cellular Cardiology, 2008
We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen spec... more We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen species (ROS) as a probe to detect myofibrillar protein (MP) oxidation. In particular we found that (i) oxidation of the cysteinyl residue of Tm caused the formation of high molecular weight complexes (HMC) due to disulfide cross-bridges in microembolized pigs hearts and (ii) this modification was related to contractile dysfunction (Eur. Heart. J. 27, 875-881, 2006). The present study investigated the occurrence and the extent of MP oxidation in human end stage (NYHA class IV) heart failure. To this aim 50 left ventricular (LV) biopsies from HF patients undergoing heart transplantation (HF group) were compared with LV biopsies from non-failing donor hearts (n = 24, control group) in a blinded manner. Tissue samples were analyzed by means of SDS/PAGE under non-reducing conditions. The amount of HMC in Tm was quantified by densitometry and was found to be significantly greater in the HF group (0.055 ± 0.014 vs 0.010 ± 0.001 in the control group, p b 0.05). Tm appeared especially susceptible to oxidation, since actin and desmin did not undergo HMC formation, although this type of modification can be induced in these proteins in vitro. Besides changes due to ROS, we tested whether MP are also targets of reactive nitrogen species. By using the biotin-switch technique we found that S-nitrosylation affects several proteins among which we could identify Tm. The extent of S-nitrosylation was higher in HF samples.
Journal of Molecular and Cellular Cardiology, 2008
We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen spec... more We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen species (ROS) as a probe to detect myofibrillar protein (MP) oxidation. In particular we found that (i) oxidation of the cysteinyl residue of Tm caused the formation of high molecular weight complexes (HMC) due to disulfide cross-bridges in microembolized pigs hearts and (ii) this modification was related to contractile dysfunction (Eur. Heart. J. 27, 875-881, 2006). The present study investigated the occurrence and the extent of MP oxidation in human end stage (NYHA class IV) heart failure. To this aim 50 left ventricular (LV) biopsies from HF patients undergoing heart transplantation (HF group) were compared with LV biopsies from non-failing donor hearts (n = 24, control group) in a blinded manner. Tissue samples were analyzed by means of SDS/PAGE under non-reducing conditions. The amount of HMC in Tm was quantified by densitometry and was found to be significantly greater in the HF group (0.055 ± 0.014 vs 0.010 ± 0.001 in the control group, p b 0.05). Tm appeared especially susceptible to oxidation, since actin and desmin did not undergo HMC formation, although this type of modification can be induced in these proteins in vitro. Besides changes due to ROS, we tested whether MP are also targets of reactive nitrogen species. By using the biotin-switch technique we found that S-nitrosylation affects several proteins among which we could identify Tm. The extent of S-nitrosylation was higher in HF samples.
Journal of Molecular and Cellular Cardiology, 2008
Journal of Molecular and Cellular Cardiology, 1999
Developmental Biology, 1995
cardiac, skeletal, and smooth muscle myogenesis of mouse embryos was developed and used to invest... more cardiac, skeletal, and smooth muscle myogenesis of mouse embryos was developed and used to investigate the effects of the disruption of the desmin gene on muscle cell differentiation. Wild-type, heterozygous, and homozygous cell lines with the mutated desmin allele were evaluated. Skeletal myogenesis was totally inhibited in desmin null mutant EBs, as manifested by the absence of myotube formation, contractility, and myoD, myogenin, myf5, and myosin heavy chain expression. Smooth muscle formation was also completely blocked in the absence of desmin. On the other hand, there were no obvious effects on cardiomyocyte differentiation in these desmin null mutant EBs. However, reduced desmin expression in EBs heterozygous for the desmin mutation leads to partial inhibition of cardiac muscle formation. These data suggest that in contrast to early cardiocyte differentiation, desmin is indispensable for skeletal and smooth muscle formation. ᭧
Cell Structure and Function, 1997
Desmin, the muscle-specific member of the intermediate filament (IF) family, is one of the earlie... more Desmin, the muscle-specific member of the intermediate filament (IF) family, is one of the earliest known myogenic markers in both skeletal muscle and heart. Its expression precedes that of all known muscle proteins including the members of the MyoD family of myogenic helix-loop-helix (mHLH) regulators with the exception of myf5. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. In vitro studies using both antisense RNA and homologous recombination techniques in embryonic stem (ES) cells demonstrated that desmin plays a crucial role during myogenesis, as inhibition of desmin expression blocked myoblast fusion and myotube formation. Both in C2C12 cells and differentiating embryoid bodies, the absence of desmin interferes with the normal myogenic program, as manifested by the inhibition of the mHLH transcription regulators. To investigate the function of desmin in all muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, a considerable number of these mice are viable and fertile, potentially due to compensation by vimentin, nestin or synemin. However, desmin null mice demonstrate a multisystem disorder involving cardiac, skeletal and smooth muscle, beginning early in their postnatal life. Histological and electron microscopic analysis in both heart and skeletal muscle tissues reveals severe disruption of muscle architecture and degeneration. Structural abnormalities include loss of lateral alignment of myofibrils, perturbation of myofibril anchorage to the sarcolemma, abnormal mitochondrial number and organization, and loss of nuclear shape and positioning. Loose cell adhesion and increased intercellular space are prominent defects. The consequences of these abnormalities are most severe in the heart, which exhibits progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. There is a direct correlation between severity of damage and muscle usage, possibly due to increased susceptibility to normal mechanical damage and/or to repair deficiency in the absence of desmin. In conclusion, the studies so far have demonstrated that though desmin is absolutely necessary for muscle differentiation in vitro, muscle development can take place in vivo in the absence of this intermediate filament protein. However, desmin seems to play an essential role in the maintenance of myofibril, myofiber and whole muscle tissue structural and functional integrity.
The American Journal of Pathology, 2005
We previously reported that enhanced expression of the ␣71 integrin ameliorates the development ... more We previously reported that enhanced expression of the ␣71 integrin ameliorates the development of muscular dystrophy and extends longevity in ␣7BX2mdx/utr ؊/؊ transgenic mice (Burkin DJ , Wallace GQ, Nicol KJ , Kaufman DJ , Kaufman SJ: Enhanced expression of the ␣71 integrin reduces muscular dystrophy and restores viability in dystrophic mice. J Cell Biol 2001 , 152:1207-1218). We now report on the mechanism by which these mice were rescued by the integrin. As a result of increased integrin in ␣7BX2-mdx/ utr ؊/؊ mice the structural integrity of the myotendinous and neuromuscular junctions are maintained. A twofold increase in satellite cells in ␣7BX2-mdx/utr ؊/؊ skeletal muscle was detected by immunofluorescence using the satellite cell marker c-met. These cells enhanced the regenerative capacity of muscle in the transgenic animals as determined by fusion of BrdUrd-labeled cells into muscle fibers. Increased integrin also leads to hypertrophy. Finally, transgenic expression of ␣7BX2 integrin chain in skeletal muscle secondarily reduces the development of cardiomyopathy , the ultimate cause of death in these animals. We believe this multiplicity of responses to increased ␣71 integrin collectively inhibits the development of muscle disease and increases longevity in these mice.
The American Journal of Pathology, 2007
Transgenic expression of the ␣71 integrin in the dystrophic mdx/utr ؊/؊ mouse decreases developm... more Transgenic expression of the ␣71 integrin in the dystrophic mdx/utr ؊/؊ mouse decreases development of muscular dystrophy and enhances longevity. To explore the possibility that elevating ␣71 integrin expression could also ameliorate different forms of muscular dystrophy , we used transgenic technology to enhance integrin expression in mice lacking ␦-sarcoglycan (␦ sgc) , a mouse model for human limb girdle muscular dystrophy type 2F. Unlike ␣7 transgenic mdx/utr ؊/؊ mice , enhanced ␣71 integrin expression in the ␦ sgc-null mouse did not alleviate muscular dystrophy in these animals. Expression of the transgene in the ␦ sgc-null did not alleviate dystrophic histopathology , nor did it decrease cardiomyopathy or restore exercise tolerance. One hallmark of integrin-mediated alleviation of muscular dystrophy in the mdx/utr ؊/؊ background is the restoration of myotendinous junction integrity. As assessed by atomic force microscopy , myotendinous junctions from normal and ␦ sgc-null mice were indistinguishable , thus suggesting the important influence of myotendinous junction integrity on the severity of muscular dystrophy and providing a possible explanation for the inability of enhanced integrin expression to alleviate dystrophy in the ␦ sgc-null mouse. These results suggest that distinct mechanisms underlie the development of the diseases that arise from deficiencies in dystrophin and sarcoglycan.
The Journal of cell biology, Jan 2, 2008
We explored the involvement of the muscle-specific intermediate filament protein desmin in the mo... more We explored the involvement of the muscle-specific intermediate filament protein desmin in the model of tumor necrosis factor alpha (TNF-alpha)-induced cardiomyopathy. We demonstrate that in mice overexpressing TNF-alpha in the heart (alpha-myosin heavy chain promoter-driven secretable TNF-alpha [MHCsTNF]), desmin is modified, loses its intercalated disk (ID) localization, and forms aggregates that colocalize with heat shock protein 25 and ubiquitin. Additionally, other ID proteins such as desmoplakin and beta-catenin show similar localization changes in a desmin-dependent fashion. To address underlying mechanisms, we examined whether desmin is a substrate for caspase-6 in vivo as well as the implications of desmin cleavage in MHCsTNF mice. We generated transgenic mice with cardiac-restricted expression of a desmin mutant (D263E) and proved that it is resistant to caspase cleavage in the MHCsTNF myocardium. The aggregates are diminished in these mice, and D263E desmin, desmoplakin, ...
Reproduction Fertility and Development
Regenerative medicine has long sought to develop therapies for articular cartilage repair and for... more Regenerative medicine has long sought to develop therapies for articular cartilage repair and for enhancing endochondral ossification to address complications of long bone healing. The objective of this study was to determine the chondrogenic potential of porcine primary cell cultures for possible utility in orthopedic tissue engineering applications. Adipose-derived mesenchymal stem cell (ASC), chondrocyte (positive control), periosteal cell, and fibroblast (negative control) primary cell cultures from 8- to 12-month-old Yorkshire pigs were plated at 5000 to 10000cellscm(-2) in 75-cm(2) cell culture flasks using high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with NaHCO3, 10% fetal bovine serum (FBS), and antimicrobials (penicillin-streptomycin, gentamicin sulfate, and amphotericin B), then incubated at 37°C, 5% CO2, and 18% O2. Cells were trypsinized at ~80% confluency and transferred into 15-mL conical tubes at 500000 cellstube(-1). Suspensions were wa...
Cell and tissue research, Jun 1, 2018
Advances in stem cell biology and materials science have provided a basis for developing tissue e... more Advances in stem cell biology and materials science have provided a basis for developing tissue engineering methods to repair muscle injury. Among stem cell populations with potential to aid muscle repair, adipose-derived mesenchymal stem cells (ASC) hold great promise. To evaluate the possibility of using porcine ASC for muscle regeneration studies, we co-cultured porcine ASC with murine CC myoblasts. These experiments demonstrated that porcine ASC display significant myogenic potential. Co-culture of ASC expressing green fluorescent protein (GFP) with CC cells resulted in GFP myotube formation, indicating fusion of ASC with myoblasts to form myotubes. The presence of porcine lamin A/C positive nuclei in myotubes and RTqPCR analysis of porcine myogenin and desmin expression confirmed that myotube nuclei derived from ASC contribute to muscle gene expression. Co-culturing GFPASC with porcine satellite cells demonstrated enhanced myogenic capability of ASC, as the percentage of labele...
Journal of Molecular and Cellular Cardiology, 2008
We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen spec... more We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen species (ROS) as a probe to detect myofibrillar protein (MP) oxidation. In particular we found that (i) oxidation of the cysteinyl residue of Tm caused the formation of high molecular weight complexes (HMC) due to disulfide cross-bridges in microembolized pigs hearts and (ii) this modification was related to contractile dysfunction (Eur. Heart. J. 27, 875-881, 2006). The present study investigated the occurrence and the extent of MP oxidation in human end stage (NYHA class IV) heart failure. To this aim 50 left ventricular (LV) biopsies from HF patients undergoing heart transplantation (HF group) were compared with LV biopsies from non-failing donor hearts (n = 24, control group) in a blinded manner. Tissue samples were analyzed by means of SDS/PAGE under non-reducing conditions. The amount of HMC in Tm was quantified by densitometry and was found to be significantly greater in the HF group (0.055 ± 0.014 vs 0.010 ± 0.001 in the control group, p b 0.05). Tm appeared especially susceptible to oxidation, since actin and desmin did not undergo HMC formation, although this type of modification can be induced in these proteins in vitro. Besides changes due to ROS, we tested whether MP are also targets of reactive nitrogen species. By using the biotin-switch technique we found that S-nitrosylation affects several proteins among which we could identify Tm. The extent of S-nitrosylation was higher in HF samples.
American Journal of Pathology, 2007
Transgenic expression of the ␣71 integrin in the dystrophic mdx/utr ؊/؊ mouse decreases developm... more Transgenic expression of the ␣71 integrin in the dystrophic mdx/utr ؊/؊ mouse decreases development of muscular dystrophy and enhances longevity. To explore the possibility that elevating ␣71 integrin expression could also ameliorate different forms of muscular dystrophy , we used transgenic technology to enhance integrin expression in mice lacking ␦-sarcoglycan (␦ sgc) , a mouse model for human limb girdle muscular dystrophy type 2F. Unlike ␣7 transgenic mdx/utr ؊/؊ mice , enhanced ␣71 integrin expression in the ␦ sgc-null mouse did not alleviate muscular dystrophy in these animals. Expression of the transgene in the ␦ sgc-null did not alleviate dystrophic histopathology , nor did it decrease cardiomyopathy or restore exercise tolerance. One hallmark of integrin-mediated alleviation of muscular dystrophy in the mdx/utr ؊/؊ background is the restoration of myotendinous junction integrity. As assessed by atomic force microscopy , myotendinous junctions from normal and ␦ sgc-null mice were indistinguishable , thus suggesting the important influence of myotendinous junction integrity on the severity of muscular dystrophy and providing a possible explanation for the inability of enhanced integrin expression to alleviate dystrophy in the ␦ sgc-null mouse. These results suggest that distinct mechanisms underlie the development of the diseases that arise from deficiencies in dystrophin and sarcoglycan.
The Journal of Cell Biology, 1994
The muscle-specific intermediate filament
The Journal of Cell Biology, 2000
Ultrastructural studies have previously suggested potential association of intermediate filaments... more Ultrastructural studies have previously suggested potential association of intermediate filaments (IFs) with mitochondria. Thus, we have investigated mitochondrial distribution and function in muscle lacking the IF protein desmin. Immunostaining of skeletal muscle tissue sections, as well as histochemical staining for the mitochondrial marker enzymes cytochrome C oxidase and succinate dehydrogenase, demonstrate abnormal accumulation of subsarcolemmal clumps of mitochondria in predominantly slow twitch skeletal muscle of desmin-null mice. Ultrastructural observation of desmin-null cardiac muscle demonstrates in addition to clumping, extensive mitochondrial proliferation in a significant fraction of the myocytes, particularly after work overload. These alterations are frequently associated with swelling and degeneration of the mitochondrial matrix. Mitochondrial abnormalities can be detected very early, before other structural defects become obvious. To investigate related changes in mitochondrial function, we have analyzed ADP-stimulated respiration of isolated muscle mitochondria, and ADPstimulated mitochondrial respiration in situ using sapo-nin skinned muscle fibers. The in vitro maximal rates of respiration in isolated cardiac mitochondria from desmin-null and wild-type mice were similar. However, mitochondrial respiration in situ is significantly altered in desmin-null muscle. Both the maximal rate of ADPstimulated oxygen consumption and the dissociation constant ( K m ) for ADP are significantly reduced in desmin-null cardiac and soleus muscle compared with controls. Respiratory parameters for desmin-null fast twitch gastrocnemius muscle were unaffected. Additionally, respiratory measurements in the presence of creatine indicate that coupling of creatine kinase and the adenine translocator is lost in desmin-null soleus muscle. This coupling is unaffected in cardiac muscle from desmin-null animals. All of these studies indicate that desmin IFs play a significant role in mitochondrial positioning and respiratory function in cardiac and skeletal muscle.
The Journal of Cell Biology, 1996
Desmin, the muscle specific intermediate filament (IF) protein encoded by a single gene, is expre... more Desmin, the muscle specific intermediate filament (IF) protein encoded by a single gene, is expressed in all muscle tissues. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. To investigate the function of desmin in all three muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, desmin null mice are viable and fertile. However, these mice demonstrated a multisystern disorder involving cardiac, skeletal, and smooth muscle. Histological and electron microscopic analysis in both heart and skeletal muscle tissues revealed se-1. Abbreviations used in this paper: ES, embryonic stem; IF, intermediate filament; pc, post coitum.
Molecular Biology of the Cell, 2002
The sarcolemma of fast-twitch muscle is organized into "costameres", structures that are oriented... more The sarcolemma of fast-twitch muscle is organized into "costameres", structures that are oriented transversely, over the Z and M lines of nearby myofibrils, and longitudinally, to form a rectilinear lattice. Here we examine the role of desmin, the major intermediate filament protein of muscle in organizing costameres. In control mouse muscle, desmin is enriched at the sarcolemmal domains that lie over nearby Z lines and that also contain -spectrin. In tibialis anterior muscle from mice lacking desmin due to homologous recombination, most costameres are lost. In myofibers from desmin -/-quadriceps, by contrast, many costameric structures are stable. Alternatively, Z line domains may be lost while domains oriented longitudinally or lying over M lines are retained. Experiments with pan-specific antibodies to intermediate filament proteins and to cytokeratins suggest that control and desmin -/-muscles express similar levels of cytokeratins. Cytokeratins concentrate at the sarcolemma at all three domains of costameres when the latter are retained in desmin -/-muscle and redistribute with -spectrin at the sarcolemma when costameres are lost. Our results suggest that desmin associates with and selectively stabilizes the Z line domains of costameres, but that cytokeratins associate with all three domains of costameres, even in the absence of desmin.
Medicine & Science in Sports & Exercise, 1999
Medicine & Science in Sports & Exercise, 1999
Journal of Molecular and Cellular Cardiology, 2008
We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen spec... more We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen species (ROS) as a probe to detect myofibrillar protein (MP) oxidation. In particular we found that (i) oxidation of the cysteinyl residue of Tm caused the formation of high molecular weight complexes (HMC) due to disulfide cross-bridges in microembolized pigs hearts and (ii) this modification was related to contractile dysfunction (Eur. Heart. J. 27, 875-881, 2006). The present study investigated the occurrence and the extent of MP oxidation in human end stage (NYHA class IV) heart failure. To this aim 50 left ventricular (LV) biopsies from HF patients undergoing heart transplantation (HF group) were compared with LV biopsies from non-failing donor hearts (n = 24, control group) in a blinded manner. Tissue samples were analyzed by means of SDS/PAGE under non-reducing conditions. The amount of HMC in Tm was quantified by densitometry and was found to be significantly greater in the HF group (0.055 ± 0.014 vs 0.010 ± 0.001 in the control group, p b 0.05). Tm appeared especially susceptible to oxidation, since actin and desmin did not undergo HMC formation, although this type of modification can be induced in these proteins in vitro. Besides changes due to ROS, we tested whether MP are also targets of reactive nitrogen species. By using the biotin-switch technique we found that S-nitrosylation affects several proteins among which we could identify Tm. The extent of S-nitrosylation was higher in HF samples.
Journal of Molecular and Cellular Cardiology, 2008
We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen spec... more We have previously characterized the oxidation of Tropomyosin (Tm) caused by reactive oxygen species (ROS) as a probe to detect myofibrillar protein (MP) oxidation. In particular we found that (i) oxidation of the cysteinyl residue of Tm caused the formation of high molecular weight complexes (HMC) due to disulfide cross-bridges in microembolized pigs hearts and (ii) this modification was related to contractile dysfunction (Eur. Heart. J. 27, 875-881, 2006). The present study investigated the occurrence and the extent of MP oxidation in human end stage (NYHA class IV) heart failure. To this aim 50 left ventricular (LV) biopsies from HF patients undergoing heart transplantation (HF group) were compared with LV biopsies from non-failing donor hearts (n = 24, control group) in a blinded manner. Tissue samples were analyzed by means of SDS/PAGE under non-reducing conditions. The amount of HMC in Tm was quantified by densitometry and was found to be significantly greater in the HF group (0.055 ± 0.014 vs 0.010 ± 0.001 in the control group, p b 0.05). Tm appeared especially susceptible to oxidation, since actin and desmin did not undergo HMC formation, although this type of modification can be induced in these proteins in vitro. Besides changes due to ROS, we tested whether MP are also targets of reactive nitrogen species. By using the biotin-switch technique we found that S-nitrosylation affects several proteins among which we could identify Tm. The extent of S-nitrosylation was higher in HF samples.
Journal of Molecular and Cellular Cardiology, 2008
Journal of Molecular and Cellular Cardiology, 1999
Developmental Biology, 1995
cardiac, skeletal, and smooth muscle myogenesis of mouse embryos was developed and used to invest... more cardiac, skeletal, and smooth muscle myogenesis of mouse embryos was developed and used to investigate the effects of the disruption of the desmin gene on muscle cell differentiation. Wild-type, heterozygous, and homozygous cell lines with the mutated desmin allele were evaluated. Skeletal myogenesis was totally inhibited in desmin null mutant EBs, as manifested by the absence of myotube formation, contractility, and myoD, myogenin, myf5, and myosin heavy chain expression. Smooth muscle formation was also completely blocked in the absence of desmin. On the other hand, there were no obvious effects on cardiomyocyte differentiation in these desmin null mutant EBs. However, reduced desmin expression in EBs heterozygous for the desmin mutation leads to partial inhibition of cardiac muscle formation. These data suggest that in contrast to early cardiocyte differentiation, desmin is indispensable for skeletal and smooth muscle formation. ᭧
Cell Structure and Function, 1997
Desmin, the muscle-specific member of the intermediate filament (IF) family, is one of the earlie... more Desmin, the muscle-specific member of the intermediate filament (IF) family, is one of the earliest known myogenic markers in both skeletal muscle and heart. Its expression precedes that of all known muscle proteins including the members of the MyoD family of myogenic helix-loop-helix (mHLH) regulators with the exception of myf5. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. In vitro studies using both antisense RNA and homologous recombination techniques in embryonic stem (ES) cells demonstrated that desmin plays a crucial role during myogenesis, as inhibition of desmin expression blocked myoblast fusion and myotube formation. Both in C2C12 cells and differentiating embryoid bodies, the absence of desmin interferes with the normal myogenic program, as manifested by the inhibition of the mHLH transcription regulators. To investigate the function of desmin in all muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, a considerable number of these mice are viable and fertile, potentially due to compensation by vimentin, nestin or synemin. However, desmin null mice demonstrate a multisystem disorder involving cardiac, skeletal and smooth muscle, beginning early in their postnatal life. Histological and electron microscopic analysis in both heart and skeletal muscle tissues reveals severe disruption of muscle architecture and degeneration. Structural abnormalities include loss of lateral alignment of myofibrils, perturbation of myofibril anchorage to the sarcolemma, abnormal mitochondrial number and organization, and loss of nuclear shape and positioning. Loose cell adhesion and increased intercellular space are prominent defects. The consequences of these abnormalities are most severe in the heart, which exhibits progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. There is a direct correlation between severity of damage and muscle usage, possibly due to increased susceptibility to normal mechanical damage and/or to repair deficiency in the absence of desmin. In conclusion, the studies so far have demonstrated that though desmin is absolutely necessary for muscle differentiation in vitro, muscle development can take place in vivo in the absence of this intermediate filament protein. However, desmin seems to play an essential role in the maintenance of myofibril, myofiber and whole muscle tissue structural and functional integrity.
The American Journal of Pathology, 2005
We previously reported that enhanced expression of the ␣71 integrin ameliorates the development ... more We previously reported that enhanced expression of the ␣71 integrin ameliorates the development of muscular dystrophy and extends longevity in ␣7BX2mdx/utr ؊/؊ transgenic mice (Burkin DJ , Wallace GQ, Nicol KJ , Kaufman DJ , Kaufman SJ: Enhanced expression of the ␣71 integrin reduces muscular dystrophy and restores viability in dystrophic mice. J Cell Biol 2001 , 152:1207-1218). We now report on the mechanism by which these mice were rescued by the integrin. As a result of increased integrin in ␣7BX2-mdx/ utr ؊/؊ mice the structural integrity of the myotendinous and neuromuscular junctions are maintained. A twofold increase in satellite cells in ␣7BX2-mdx/utr ؊/؊ skeletal muscle was detected by immunofluorescence using the satellite cell marker c-met. These cells enhanced the regenerative capacity of muscle in the transgenic animals as determined by fusion of BrdUrd-labeled cells into muscle fibers. Increased integrin also leads to hypertrophy. Finally, transgenic expression of ␣7BX2 integrin chain in skeletal muscle secondarily reduces the development of cardiomyopathy , the ultimate cause of death in these animals. We believe this multiplicity of responses to increased ␣71 integrin collectively inhibits the development of muscle disease and increases longevity in these mice.
The American Journal of Pathology, 2007
Transgenic expression of the ␣71 integrin in the dystrophic mdx/utr ؊/؊ mouse decreases developm... more Transgenic expression of the ␣71 integrin in the dystrophic mdx/utr ؊/؊ mouse decreases development of muscular dystrophy and enhances longevity. To explore the possibility that elevating ␣71 integrin expression could also ameliorate different forms of muscular dystrophy , we used transgenic technology to enhance integrin expression in mice lacking ␦-sarcoglycan (␦ sgc) , a mouse model for human limb girdle muscular dystrophy type 2F. Unlike ␣7 transgenic mdx/utr ؊/؊ mice , enhanced ␣71 integrin expression in the ␦ sgc-null mouse did not alleviate muscular dystrophy in these animals. Expression of the transgene in the ␦ sgc-null did not alleviate dystrophic histopathology , nor did it decrease cardiomyopathy or restore exercise tolerance. One hallmark of integrin-mediated alleviation of muscular dystrophy in the mdx/utr ؊/؊ background is the restoration of myotendinous junction integrity. As assessed by atomic force microscopy , myotendinous junctions from normal and ␦ sgc-null mice were indistinguishable , thus suggesting the important influence of myotendinous junction integrity on the severity of muscular dystrophy and providing a possible explanation for the inability of enhanced integrin expression to alleviate dystrophy in the ␦ sgc-null mouse. These results suggest that distinct mechanisms underlie the development of the diseases that arise from deficiencies in dystrophin and sarcoglycan.
The Journal of cell biology, Jan 2, 2008
We explored the involvement of the muscle-specific intermediate filament protein desmin in the mo... more We explored the involvement of the muscle-specific intermediate filament protein desmin in the model of tumor necrosis factor alpha (TNF-alpha)-induced cardiomyopathy. We demonstrate that in mice overexpressing TNF-alpha in the heart (alpha-myosin heavy chain promoter-driven secretable TNF-alpha [MHCsTNF]), desmin is modified, loses its intercalated disk (ID) localization, and forms aggregates that colocalize with heat shock protein 25 and ubiquitin. Additionally, other ID proteins such as desmoplakin and beta-catenin show similar localization changes in a desmin-dependent fashion. To address underlying mechanisms, we examined whether desmin is a substrate for caspase-6 in vivo as well as the implications of desmin cleavage in MHCsTNF mice. We generated transgenic mice with cardiac-restricted expression of a desmin mutant (D263E) and proved that it is resistant to caspase cleavage in the MHCsTNF myocardium. The aggregates are diminished in these mice, and D263E desmin, desmoplakin, ...
Reproduction Fertility and Development
Regenerative medicine has long sought to develop therapies for articular cartilage repair and for... more Regenerative medicine has long sought to develop therapies for articular cartilage repair and for enhancing endochondral ossification to address complications of long bone healing. The objective of this study was to determine the chondrogenic potential of porcine primary cell cultures for possible utility in orthopedic tissue engineering applications. Adipose-derived mesenchymal stem cell (ASC), chondrocyte (positive control), periosteal cell, and fibroblast (negative control) primary cell cultures from 8- to 12-month-old Yorkshire pigs were plated at 5000 to 10000cellscm(-2) in 75-cm(2) cell culture flasks using high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with NaHCO3, 10% fetal bovine serum (FBS), and antimicrobials (penicillin-streptomycin, gentamicin sulfate, and amphotericin B), then incubated at 37°C, 5% CO2, and 18% O2. Cells were trypsinized at ~80% confluency and transferred into 15-mL conical tubes at 500000 cellstube(-1). Suspensions were wa...