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Brian R Wamhoff

Publications and source records attributed to Brian R Wamhoff.

14 recordsLinked to original sources
PubMed2006

Exercise training prevents Ca2+ dysregulation in coronary smooth muscle from diabetic dyslipidemic yucatan swine.

Aerobic exercise training is known to have profound cardioprotective effects in disease, yet cellular mechanisms remain largely undefined. We tested the hypothesis that increased sarcoplasmic reticulum Ca(2+) buffering and increased voltage-gated Ca(2+) channel density underlie coronary smooth muscle intracellular Ca(2+) (Ca(2+)(i)) dysregulation in diabetic dyslipidemia and that exercise training would prevent these increases. Yucatan swine were maintained in 1) control, 2) alloxan-induced hyperglycemic, 3) high fat/cholesterol fed, 4) hyperglycemic plus high fat/cholesterol fed (diabetic dyslipidemic), and 5) diabetic dyslipidemic plus exercise-trained (treadmill running) conditions. After 20 wk, the heart was removed and smooth muscle cells isolated from the right coronary artery. We utilized fura-2 imaging of Ca(2+)(i) levels to separate the functional role of the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) from the Na(+)-Ca(2+) exchanger and the plasmalemmal Ca(2+)-ATPase, and whole-cell patch clamp to examine voltage-gated Ca(2+) channel current density (i.e., Ca(2+) influx). Results indicated that diabetic dyslipidemia impaired plasmalemmal Ca(2+) efflux, increased basal Ca(2+)(i) levels, increased SERCA protein and sarcoplasmic reticulum Ca(2+)(i) buffering, and elicited an approximately 50% decrease in voltage-gated Ca(2+) channel current density. Exercise training concurrent with the diabetic dyslipidemic state restored plasmalemmal Ca(2+) efflux, SERCA protein, sarcoplasmic reticulum Ca(2+)(i) buffering, and voltage-gated Ca(2+) channel current density to control levels. Interestingly, basal Ca(2+)(i) levels were significantly lower in the exercise-trained group compared with control. Collectively, these results demonstrate a crucial role for exercise in the prevention of diabetic dyslipidemia-induced Ca(2+)(i) dysregulation.

Animals↗
PubMed2006

Excitation-transcription coupling in arterial smooth muscle.

The primary function of the vascular smooth muscle cell (SMC) is contraction for which SMCs express a selective repertoire of genes (eg, SM alpha-actin, SM myosin heavy chain [SMMHC], myocardin) that ultimately define the SMC from other muscle cell types. Moreover, the SMC exhibits extensive phenotypic diversity and plasticity, which play an important role during normal development, repair of vascular injury, and in vascular disease states. Diverse signals modulate ion channel activity in the sarcolemma of SMCs, resulting in altered intracellular calcium (Ca) signaling, activation of multiple intracellular signaling cascades, and SMC contraction or relaxation, a process known as "excitation-contraction coupling" (EC-coupling). Over the past 5 years, exciting new studies have shown that the same signals that regulate EC-coupling in SMCs are also capable of regulating SMC-selective gene expression programs, a new paradigm coined "excitation-transcription coupling" (ET-coupling). This article reviews recent progress in our understanding of the mechanisms by which ET-coupling selectively coordinates the expression of distinct gene subsets in SMCs by disparate transcription factors, including CREB, NFAT, and myocardin, via selective kinases. For example, L-type voltage-gated Ca2+ channels modulate SMC differentiation marker gene expression, eg, SM alpha-actin and SMMHC, via Rho kinase and myocardin and also regulate c-fos gene expression independently via CaMK. In addition, we discuss the potential role of IK channels and TRPC in ET-coupling as potential mediators of SMC phenotypic modulation, ie, negatively regulate SMC differentiation marker genes, in vascular disease.

Animals↗
PubMed2006

Assessment of contractility of purified smooth muscle cells derived from embryonic stem cells.

The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin-resistance gene under the control of either a smooth muscle alpha-actin (SMalphaAlpha) or smooth muscle-myosin heavy chain (SM-MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and flow cytometry and were designated APSCs (SMalphaAlpha-puromycin-selected cells) or MPSCs (SM-MHC-puromycin-selected cells), respectively. Both SMC lines displayed agonist-induced Ca(2+) transients, expressed functional Ca(2+) channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin-1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC-derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.

Actins↗
PubMed2006

Control of SRF binding to CArG box chromatin regulates smooth muscle gene expression in vivo.

Precise control of SMC transcription plays a major role in vascular development and pathophysiology. Serum response factor (SRF) controls SMC gene transcription via binding to CArG box DNA sequences found within genes that exhibit SMC-restricted expression. However, the mechanisms that regulate SRF association with CArG box DNA within native chromatin of these genes are unknown. Here we report that SMC-restricted binding of SRF to murine SMC gene CArG box chromatin is associated with patterns of posttranslational histone modifications within this chromatin that are specific to the SMC lineage in culture and in vivo, including methylation and acetylation to histone H3 and H4 residues. We found that the promyogenic SRF coactivator myocardin increased SRF association with methylated histones and CArG box chromatin during activation of SMC gene expression. In contrast, the myogenic repressor Kruppel-like factor 4 recruited histone H4 deacetylase activity to SMC genes and blocked SRF association with methylated histones and CArG box chromatin during repression of SMC gene expression. Finally, we observed deacetylation of histone H4 coupled with loss of SRF binding during suppression of SMC differentiation in response to vascular injury. Taken together, these findings provide novel evidence that SMC-selective epigenetic control of SRF binding to chromatin plays a key role in regulation of SMC gene expression in response to pathophysiological stimuli in vivo.

Animals↗
PubMed2005

Mobilization of bone marrow-derived cells enhances the angiogenic response to hypoxia without transdifferentiation into endothelial cells.

Bone marrow-derived cells (BMCs) have been implicated as a modifiers of vascular growth either directly by transdifferentiation into endothelial cells (ECs) or indirectly through growth factor release. To examine these possibilities under physiological conditions, we developed a model of hypoxia-mediated angiogenesis in the mouse spinotrapezius muscle. This allows whole-mount analysis; therefore, the morphology and location of BMCs within the vascular network may be observed along with differentiation markers. We exposed bone marrow transplant chimeric mice to hypoxia and treated a subset with granulocyte macrophage colony-stimulating factor. Exposure to hypoxia caused an 13% increase in capillary density relative to control. Hypoxia did not increase the overall number of muscle-resident BMCs, but did increase the number of rounded BMCs by 25%. There was no discernable BMC contribution to the endothelium, although some BMCs assumed a pericyte morphology around capillaries. Granulocyte macrophage colony-stimulating factor treatment further increased the number of round BMCs within the muscle and caused a 23% increase in angiogenesis. The results of this study suggest a potentially beneficial action of BMCs during hypoxia through paracrine release of growth factors but not transdifferentiation into ECs.

Animals↗
PubMed2005

5' CArG degeneracy in smooth muscle alpha-actin is required for injury-induced gene suppression in vivo.

CC(A/T)6GG-dependent (CArG-dependent) and serum response factor-dependent (SRF-dependent) mechanisms are required for gene expression in smooth muscle cells (SMCs). However, an unusual feature of many SMC-selective promoter CArG elements is that they contain a conserved single G or C substitution in their central A/T-rich region, which reduces binding affinity for ubiquitously expressed SRF. We hypothesized that this CArG degeneracy contributes to cell-specific expression of smooth muscle alpha-actin in vivo, since substitution of c-fos consensus CArGs for the degenerate CArGs resulted in relaxed specificity in cultured cells. Surprisingly, our present results show that these substitutions have no effect on smooth muscle-specific transgene expression during normal development and maturation in transgenic mice. However, these substitutions significantly attenuated injury-induced downregulation of the mutant transgene under conditions where SRF expression was increased but expression of myocardin, a smooth muscle-selective SRF coactivator, was decreased. Finally, chromatin immunoprecipitation analyses, together with cell culture studies, suggested that myocardin selectively enhanced SRF binding to degenerate versus consensus CArG elements. Our results indicate that reductions in myocardin expression and the degeneracy of CArG elements within smooth muscle promoters play a key role in phenotypic switching of smooth muscle cells in vivo, as well as in mediating responses of CArG-dependent smooth muscle genes and growth regulatory genes under conditions in which these 2 classes of genes are differentially expressed.

Actins↗
PubMed2004

Molecular regulation of vascular smooth muscle cell differentiation in development and disease.

The focus of this review is to provide an overview of the current state of knowledge of molecular mechanisms/processes that control differentiation of vascular smooth muscle cells (SMC) during normal development and maturation of the vasculature, as well as how these mechanisms/processes are altered in vascular injury or disease. A major challenge in understanding differentiation of the vascular SMC is that this cell can exhibit a wide range of different phenotypes at different stages of development, and even in adult organisms the cell is not terminally differentiated. Indeed, the SMC is capable of major changes in its phenotype in response to changes in local environmental cues including growth factors/inhibitors, mechanical influences, cell-cell and cell-matrix interactions, and various inflammatory mediators. There has been much progress in recent years to identify mechanisms that control expression of the repertoire of genes that are specific or selective for the vascular SMC and required for its differentiated function. One of the most exciting recent discoveries was the identification of the serum response factor (SRF) coactivator gene myocardin that appears to be required for expression of many SMC differentiation marker genes, and for initial differentiation of SMC during development. However, it is critical to recognize that overall control of SMC differentiation/maturation, and regulation of its responses to changing environmental cues, is extremely complex and involves the cooperative interaction of many factors and signaling pathways that are just beginning to be understood. There is also relatively recent evidence that circulating stem cell populations can give rise to smooth muscle-like cells in association with vascular injury and atherosclerotic lesion development, although the exact role and properties of these cells remain to be clearly elucidated. The goal of this review is to summarize the current state of our knowledge in this area and to attempt to identify some of the key unresolved challenges and questions that require further study.

Aging↗
PubMed2004

Lost in transdifferentiation.

What are the true origins of the smooth muscle cells (SMCs) present in the intimal lesions of transplant arteriosclerosis? A new study in the JCI shows that Sca-1(+) cells purified from the mouse aortic root can migrate through an irradiated vein graft to the neointima of the vessel and transdifferentiate to express the early SMC differentiation marker gene SM22. Do Sca-1(+) cells transdifferentiate into SMC-like cells, or is activation of SMC marker genes a consequence of fusion of these cells with preexisting SMCs, a possibility raised by results of studies of adult stem cells in animal models of liver regeneration ? Or could this be bona fide transdifferentiation that recapitulates the pathologic processes in humans?

Animals↗
PubMed2003

Myocardin is a key regulator of CArG-dependent transcription of multiple smooth muscle marker genes.

The interactions between serum response factor (SRF) and CArG elements are critical for smooth muscle cell (SMC) marker gene transcription. However, the mechanisms whereby SRF, which is expressed ubiquitously, contributes to SMC-specific transcription are unknown. Myocardin was recently cloned as a coactivator of SRF in the heart, but its role in regulating CArG-dependent expression of SMC differentiation marker genes has not been clearly elucidated. In this study, we examined the expression and the function of myocardin in SMCs. In adult mice, myocardin mRNA was expressed in multiple smooth muscle (SM) tissues including the aorta, bladder, stomach, intestine, and colon, as well as the heart. Myocardin was also expressed in cultured rat aortic SMCs and A404 SMC precursor cells. Of particular interest, expression of myocardin was induced during differentiation of A404 cells, although it was not expressed in parental P19 cells from which A404 cells were derived. Cotransfection studies in SMCs revealed that myocardin induced the activity of multiple SMC marker gene promoters including SM alpha-actin, SM-myosin heavy chain, and SM22alpha by 9- to 60-fold in a CArG-dependent manner, whereas myocardin short interfering RNA markedly decreased activity of these promoters. Moreover, adenovirus-mediated overexpression of a dominant-negative form of myocardin significantly suppressed expression of endogenous SMC marker genes, whereas adenovirus-mediated overexpression of wild-type myocardin increased expression. Taken together, results provide compelling evidence that myocardin plays a key role as a transcriptional coactivator of SMC marker genes through CArG-dependent mechanisms.

3T3 Cells↗
PubMed2003

Porcine model of diabetic dyslipidemia: insulin and feed algorithms for mimicking diabetes mellitus in humans.

A weakness of many animal models of diabetes mellitus is the failure to use insulin therapy, which typically results in severe body wasting. Data collected from such studies must be interpreted cautiously to separate the effects of hyperglycemia from those of starvation. We provide several algorithms that were used by us in two long-term (20-week) experiments in which hyperglycemia (300 to 400 mg/dl), dyslipidemia (cholesterol [280 to 405 mg/dl] and triglycerides [55 to 106 mg/dl] concentrations), and positive energy balance were maintained in swine. Yucatan miniature swine groups included control, alloxan-induced diabetes mellitus, diabetes mellitus plus diet-induced dyslipidemia, and exercise-trained diabetic dyslipidemic pigs. The algorithms were developed for the porcine model because of several similarities to humans, including: cardiac anatomy and physiology, propensity for sedentary behavior, and metabolism of dietary carbohydrates and lipids. Acute toxic effects of alloxan (hypoglycemia, hyperglycemia, nephrotoxicosis) were minimized by preventive fluid loading and by use of algorithms in which insulin, food, and fluid therapy were administered. Long-term insulin and food maintenance algorithms elicited normal body weight gain in all three diabetic groups (lean experiment) and threefold greater body weight gain in pigs of an obesity experiment. Exercise-trained pigs of both experiments manifested significantly increased work performance and did not experience medical complications. We conclude that these algorithms can be used in swine, or similar algorithms can be developed for other animal species to maintain hyperglycemia and/or dyslipidemia, while avoiding diabetes-induced wasting. Importantly, animal models of diabetes mellitus that maintain positive energy balance and poor glycemic control provide a marked improvement over other models by more closely mimicking the human presentation of diabetes mellitus.

Algorithms↗
PubMed2003

Hyperglycemia-induced insulin resistance in diabetic dyslipidemic Yucatan swine.

Hyperglycemia, dyslipidemia, and associated insulin resistance are hallmarks of diabetes mellitus. Purposes of the study reported here were to develop practical methods for assessment of in vivo insulin sensitivity and determine contributions of hyperglycemia and dyslipidemia to insulin resistance in the porcine model of alloxan-induced diabetes mellitus and dyslipidemia. Male Yucatan swine groups were treated for 20 weeks: control (C), high fat-fed (2% cholesterol) hyperlipidemic (H), alloxan-induced diabetic normolipidemic (D), diabetic high fat-fed (diabetic dyslipidemic, DD), and diabetic dyslipidemic treated with the lipid-lowering agent atorvastatin (DDA). Plasma cholesterol concentration increased sixfold in animals of groups H, DD, and DDA, whereas triglyceride concentration increased threefold in animals of group DD only. Diabetics had decreases in glucose tolerance and pancreatic immunostaining for insulin. Use of the gold standard hyperinsulinemic euglycemic clamp procedure indicated that maximal insulin-stimulated glucose uptake was similar to that in humans, but this method was not practical for use in pigs. Instead, a more convenient and valid insulin sensitivity test involving suppression of insulin secretion with somatostatin and a single insulin injection was used. Insulin sensitivity was greatly impaired by anesthesia with isoflurane, but was not affected by use of the anxiolytic agent diazepam. Insulin sensitivity decreased by 75% in diabetics (groups D, DD, DDA), compared with animals of groups C and H, and was inversely related to fasting blood glucose concentration (r = -0.72). Insulin treatment to restore blood glucose values of diabetics (> 250 mg/dl) to near control values (< 100 mg/dl) promptly restored insulin sensitivity to control values. We conclude that hyperglycemia is a major cause of insulin resistance in the porcine model of alloxan-induced diabetes mellitus and dyslipidemia.

Animals↗
PubMed2002

Intermittent increases in cytosolic Ca2+ stimulate mitochondrial biogenesis in muscle cells.

Muscle contractions cause numerous disturbances in intracellular homeostasis. This makes it impossible to use contracting muscle to identify which of the many signals generated by contractions are responsible for stimulating mitochondrial biogenesis. One purpose of this study was to evaluate the usefulness of L6 myotubes, which do not contract, for studying mitochondrial biogenesis. A second purpose was to evaluate further the possibility that increases in cytosolic Ca2+ can stimulate mitochondrial biogenesis. Continuous exposure to 1 microM ionomycin, a Ca2+ ionophore, for 5 days induced an increase in mitochondrial enzymes but also caused a loss of myotubes, as reflected in an approximately 40% decrease in protein per dish. However, intermittent (5 h/day) exposure to ionomycin, or to caffeine or W7, which release Ca2+ from the sarcoplasmic reticulum, did not cause a decrease in protein per dish. Raising cytosolic Ca2+ intermittently with these agents induced significant increases in mitochondrial enzymes. EGTA blocked most of this effect of ionomycin, whereas dantrolene, which blocks Ca2+ release from the sarcoplasmic reticulum, largely prevented the increases in mitochondrial enzymes induced by W7 and caffeine. These findings provide evidence that intermittently raising cytosolic Ca2+ stimulates mitochondrial biogenesis in muscle cells.

5-Aminolevulinate Synthetase↗
PubMed2002

Regulation of GLUT4 biogenesis in muscle: evidence for involvement of AMPK and Ca(2+).

There is evidence suggesting that adaptive increases in GLUT4 and mitochondria in skeletal muscle occur in parallel. It has been reported that raising cytosolic Ca(2+) in myocytes induces increases in mitochondrial enzymes. In this study, we tested the hypothesis that an increase in cytosolic Ca(2+) induces an increase in GLUT4. We found that raising cytosolic Ca(2+) by exposing L6 myotubes to 5 mM caffeine for 3 h/day for 5 days induced increases in GLUT4 protein and in myocyte enhancer factor (MEF)2A and MEF2D, which are transcription factors involved in regulating GLUT4 expression. The caffeine-induced increases in GLUT4 and MEF2A and MEF2D were partially blocked by dantrolene, an inhibitor of sarcoplasmic reticulum Ca(2+) release, and completely blocked by KN93, an inhibitor of Ca(2+)-calmodulin-dependent protein kinase (CAMK). Caffeine also induced increases in MEF2A, MEF2D, and GLUT4 in rat epitrochlearis muscles incubated with caffeine in culture medium. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), which activates AMP-activated protein kinase (AMPK), also induced approximately twofold increases in GLUT4, MEF2A, and MEF2D in L6 myocytes. Our results provide evidence that increases in cytosolic Ca(2+) and activation of AMPK, both of which occur in exercising muscle, increase GLUT4 protein in myocytes and skeletal muscle. The data suggest that this effect of Ca(2+) is mediated by activation of CAMK and indicate that MEF2A and MEF2D are involved in this adaptive response.

AMP-Activated Protein Kinase Kinases↗