Faculty Bibliography

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21(cip1.) and p16(INK4a), and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

Sudden cardiac death is responsible for several hundred thousand deaths each year in the United States. Multiple lines of evidence suggest that perturbation of gap junction expression and function in the heart, or what has come to be known as cardiac gap junction remodeling, plays a key mechanistic role in the pathophysiology of clinically significant cardiac arrhythmias. Here we review recent studies from our laboratory using genetically engineered murine models to explore mechanisms implicated in pathologic gap junction remodeling and their proarrhythmic consequences, with a particular focus on aberrant posttranslational phosphorylation of connexin43.

Oculodentodigital dysplasia (ODDD) is a rare developmental disease resulting from germline mutations in the GJA1 gene that encodes the gap junction protein connexin43 (Cx43). In addition to the classical ODDD symptoms that affect the eyes, teeth, bone and digits, in some cases ODDD patients have reported bladder impairments. Thus, we chose to characterize the bladder in mutant mouse models of ODDD that harbor two distinct Cx43 mutations, G60S and I130T. Histological assessment revealed no difference in bladder detrusor wall thickness in mutant compared to littermate control mice. The overall localization of Cx43 in the lamina propria and detrusor also appeared to be similar in the bladders of mutant mice with the exception that the G60S mice had more instances of intracellular Cx43. However, both mutant mouse lines exhibited a significant reduction in the phosphorylated P1 and P2 isoforms of Cx43, while only the I130T mice exhibited a reduction in total Cx43 levels. Interestingly, Cx26 levels and distribution were not altered in mutant mice as it was localized to intracellular compartments and restricted to the basal cell layers of the urothelium. Our studies suggest that these two distinct genetically modified mouse models of ODDD probably mimic patients who lack bladder defects or other factors, such as aging or co-morbidities, are necessary to reveal a bladder phenotype.

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals.

Recently, we had the opportunity to review the progress that has been made in the field of cardiovascular disease over the past century in The FASEB Journal and, based on those thoughts, in this article we predict what may transpire inthis 'century of biology'. Although it is true that 'the best way to predict the future is to invent it', we gaze through the prism of modern biomolecular science for a vision of a possible future and see cardiology practice that is transformed. In the second half of the 20th century, we developed a more fundamental understanding of atherosclerotic vascular disorders and invented life-saving therapeutics. We saw a similar development of mechanism-based pharmacotherapy to address heart failure, primarily through agents that antagonize the excessive concentration of circulating neurohumoral agents. Now we are in the midst of the device era, from stents to cardiac resynchronization therapy to transcatheter valves.The next wave of treatments will build on an increasingly sophisticated understanding of the molecular determinants of cardiovascular disorders and engineering feats that are barely perceptible now. Genomic profiling, molecular prescriptions for prevention and personalized therapeutics, regenerative medicine and the new field of cardiovascular tissue bioengineering will transform cardiovascular medicine. If the human species can survive threats of our own doing, such as the related epidemics of obesity and diabetes, by the turn of the next century, treatment of cardiovascular disease will not resemble the present in almost any way. Touch Medical Media 2012

Skeletal muscle development involves the differentiation of myoblasts into myotubes; likely involving connexins (Cx) and the gap junctional exchange of secondary messengers and metabolites. Some patients with oculodentodigital dysplasia (ODDD), a rare primarily autosomal dominant disease caused by mutations in the gene encoding Cx43, become less ambulatory with aging and exhibit symptoms that may reflect defects in skeletal muscle development, maintenance, and repair. In this study, the role of Cx43 in skeletal muscle development was examined in differentiation-competent L6 myoblasts and two ODDD-linked Cx43-mutant mouse lines. Undifferentiated L6 myoblasts exhibited high levels of Cx43-based gap junctions which were inhibited by the co-expression of I130T and G60S mutants. Upon myotube formation, Cx43 was rapidly down-regulated and gap junctions were lost but myotubes continued to enlarge. Cx43-mutant mice heterozygous for the I130T mutation exhibited similar body weight, tibial length and muscle fiber size as littermate controls. While mutant mice heterozygous for the G60S mutation also had similar tibial length their overall size and muscle fibre diameter were significantly smaller. Overall, Cx43 regulation may be important for initial myoblast differentiation but not linked to myotube growth and while our mutant mouse studies suggest that some ODDD patients are predicted to have smaller muscle fibres, this appears to be dependent on the site of the Cx43 mutation or other unknown etiologies

BACKGROUND: Sodium channel alpha-subunits in ventricular myocytes (VMs) segregate either to the intercalated disc, or to lateral membranes, where they associate with region-specific molecules. OBJECTIVE: To determine the functional properties of sodium channels as a function of their location in the cell. METHODS: Local sodium currents were recorded from adult rodent VMs and Purkinje cells using the cell-attached macropatch configuration. Electrodes were placed either in the cell midsection (M), or cell end (area originally occupied by the intercalated disc; ID). Channels were identified as TTX-sensitive (TTX-S) or TTX-resistant (TTX-R) by application of 100 nM TTX. RESULTS: Average peak-current amplitude was larger in ID than M, and largest at site of contact between attached cells. TTX-S channels were found only in M region of VMs, and not in Purkinje myocytes. TTX-R channels were found in M and ID, but their biophysical properties differed depending on recording location. Sodium current in rat VMs was upregulated by TNF-alpha. The magnitude of current increase was largest in M, but this difference was abolished by 100 nM TTX. CONCLUSIONS: Our data suggest that: a) a large fraction of TTX-R (likely Na(v)1.5) channels in the M region of VMs are inactivated at normal resting potential, leaving most of the burden of excitation to TTX-R channels in the ID; b) cell-cell adhesion increases functional channel density at ID. c) TTX-S (likely non-Na(v)1.5) channels make a minimal contribution to sodium current under control conditions, but represent a functional reserve that can be upregulated by exogenous factors

Background- The specialized cardiac conduction system (CCS) expresses a unique complement of ion channels that confer a specific electrophysiological profile. ATP-sensitive potassium (K(ATP)) channels in these myocytes have not been systemically investigated. Methods and Results- We recorded K(ATP) channels in isolated CCS myocytes using Cntn2-EGFP reporter mice. The CCS K(ATP) channels were less sensitive to inhibitory cytosolic ATP compared with ventricular channels and more strongly activated by MgADP. They also had a smaller slope conductance. The 2 types of channels had similar intraburst open and closed times, but the CCS K(ATP) channel had a prolonged interburst closed time. CCS K(ATP) channels were strongly activated by diazoxide and less by levcromakalim, whereas the ventricular K(ATP) channel had a reverse pharmacological profile. CCS myocytes express elevated levels of Kir6.1 but reduced Kir6.2 and SUR2A mRNA compared with ventricular myocytes (SUR1 expression was negligible). SUR2B mRNA expression was higher in CCS myocytes relative to SUR2A. Canine Purkinje fibers expressed higher levels of Kir6.1 and SUR2B protein relative to the ventricle. Numeric simulation predicts a high sensitivity of the Purkinje action potential to changes in ATP:ADP ratio. Cardiac conduction time was prolonged by low-flow ischemia in isolated, perfused mouse hearts, which was prevented by glibenclamide. Conclusions- These data imply a differential electrophysiological response (and possible contribution to arrhythmias) of the ventricular CCS to K(ATP) channel opening during periods of ischemia