Faculty Bibliography

GJA1 (also known and referred to here as connexin 43 and abbreviated CX43) is the predominant testicular gap junction protein, and CX43 may regulate Sertoli cell maturation and spermatogenesis. We hypothesized that lack of CX43 would inhibit Sertoli cell differentiation and extend proliferation. To test this, a Sertoli cell-specific Cx43 knockout (SC-Cx43 KO) mouse was generated using Cre-lox technology. Immunohistochemistry indicated that CX43 was not expressed in the Sertoli cells of SC-Cx43 KO mice, but was normal in organs such as the heart. Testicular weight was reduced by 41% and 76% in SC-Cx43 KO mice at 20 and 60 days, respectively, vs. wild-type (wt) mice. Seminiferous tubules of SC-Cx43 KO mice contained only Sertoli cells and actively proliferating early spermatogonia. Sertoli cells normally cease proliferation at 2 wk of age in mice and become terminally differentiated. However, proliferating Sertoli cells were present in SC-Cx43 KO but not wt mice at 20 and 60 days of age. Thyroid hormone receptor alpha (THRA) is high in proliferating Sertoli cells, then declines sharply in adulthood. Thra mRNA expression was increased in 20-day SC-Cx43 KO vs. wt mice, and it showed a trend toward an increase in 60-day mice, indicating that loss of CX43 in Sertoli cells inhibited their maturation. In conclusion, we have generated mice lacking CX43 in Sertoli cells but not other tissues. Our data indicate that CX43 in Sertoli cells is essential for spermatogenesis but not spermatogonial maintenance/proliferation. SC-Cx43 KO mice showed continued Sertoli cell proliferation and delayed maturation in adulthood, indicating that CX43 plays key roles in Sertoli cell development

The specialized cardiac conduction system (CCS) consists of the sinoatrial node (SAN) and the atrioventricular (AV) conduction system (AVCS), which includes proximal (AV node, bundle of His and bundle branches) and distal (Purkinje fibers) components. In four CCS marker mice [two transgenic (cGATA6|lacZ, CCS|lacZ) and two targeted gene knock-in (minK|lacZ, Hop|lacZ)] the expression of the lacZ gene (beta-gal) has been reported to mark portions of the proximal and distal AVCS; the expression of this marker in the adult SAN is unknown. The primary objective of this study was to analyze the utility of these marker mice in the identification of the SAN. Intercaval and interventricular septal regions, containing all the components of the CCS, were freshly dissected from adult mice based on the anatomical landmarks and sectioned. Immunohistochemical characterization was performed with SAN markers (Cx45, HCN4), compared to the reporter expression (beta-gal) and markers of the working myocardium (Cx40 and Cx43). In all four of the CCS marker mice, we found that beta-gal expression is consistently observed in the proximal and distal AVCS. However, the presence of lacZ gene expression in the working myocardium outside the CCS and/or the absence of this reporter expression in the SAN prevent the effective use of these mice to identify the SAN, leading us to conclude that none of the four CCS marker mice we studied specifically mark the SAN.

Sorcin is a Ca2+ binding protein implicated in the regulation of intracellular Ca2+ cycling and cardiac excitation-contraction coupling. Structural and human genetic studies suggest that a naturally occurring sequence variant encoding L112-sorcin disrupts an E-F hand Ca2+ binding domain and may be responsible for a heritable form of hypertension and hypertrophic heart disease. We generated transgenic mice overexpressing L112-sorcin in the heart and characterized the effects on Ca2+ regulation and cardiac function both in vivo and in dissociated cardiomyocytes. Hearts of sorcin(F112L) transgenic mice were mildly dilated but ventricular function was preserved and systemic blood pressure was normal. Sorcin(F112L) myocytes were smaller than control cells and displayed complex alterations in Ca2+ regulation and contractility, including a slowed inactivation of L-type Ca2+ current, enhanced Ca2+ spark width, duration, and frequency, and increased Na+-Ca2+ exchange activity. In contrast, mice with cardiac-specific overexpression of wild-type sorcin displayed directionally opposite effects on L-type Ca2+ channel function and Ca2+ spark behavior. These data further define the role of sorcin in cardiac excitation-contraction coupling and highlight its negative regulation of SR calcium release. Our results also suggest that additional factors may be responsible for the development of cardiac hypertrophy and hypertension in humans expressing the L112-sorcin sequence variant.

The cardiac conduction system (CCS)-lacZ insertional mouse mutant strain genetically labels the developing and mature CCS. This pattern of expression is presumed to reflect the site of transgene integration rather than regulatory elements within the transgene proper. We sought to characterize the genomic structure of the integration locus and identify nearby gene(s) that might potentially confer the observed CCS-specific transcription. We found rearrangement of chromosome 7 between regions D1 and E1 with altered transcription of multiple genes in the D1 region. Several lines of evidence suggested that regulatory elements from at least one gene, Slco3A1, influenced CCS-restricted reporter gene expression. In embryonic hearts, Slco3A1 was expressed in a spatial pattern similar to the CCS-lacZ transgene and was similarly neuregulin-responsive. At later stages, however, expression patterns of the transgene and Slco3A1 diverged, suggesting that the Slco3A1 locus may be necessary, but not sufficient to confer CCS-specific transgene expression in the CCS-lacZ line

Cardiac KATP channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice which express dominant negative Kir6.x pore-forming subunits (Kir6.1-AAA or Kir6.2-AAA) in cardiac myocytes by driving their expression with the alpha-myosin heavy chain promoter. Weight gain and development after birth of these mice were similar to wild-type mice, but an increased mortality was noted after the age of 4-5 months. Transgenic mice lacked cardiac KATP channel activity as assessed with patch clamp techniques. Consistent with a decreased current density observed at positive voltages, the action potential duration was increased in these mice. Some myocytes developed early afterdepolarizations following isoproterenol treatment. Hemodynamic measurements revealed no significant effects on ventricular function (apart from a slightly elevated heart rate) whereas in-vivo electrophysiological recordings revealed a prolonged ventricular effective refractory period in transgenic mice. The transgenic mice tolerated stress less well as evident from treadmill stress tests. The pro-arrhythmogenic features and lack of adaptation to a stress response in transgenic mice suggests that these features are intrinsic to the myocardium and that KATP channels in the myocardium have an important role in protecting the heart from lethal arrhythmias and adaptation to stress situations

A number of human congenital disorders present with both heart and limb defects, consistent with common genetic pathways. We have recently shown that the LIM homeodomain transcription factor islet 1 (Isl1) marks a subset of cardiac progenitors. Here, we perform lineage studies with an Isl1Cre mouse line to demonstrate that Isl1 also marks a subset of limb progenitors. In both cardiac and limb progenitors, Isl1 expression is downregulated as progenitors migrate in to form either heart or limb. To investigate common heart-limb pathways in Isl1-expressing progenitors, we ablated the Type I Bmp receptor, Bmpr1a utilizing Isl1Cre/+. Analysis of consequent heart and limb phenotypes has revealed novel requirements for Bmp signaling. Additionally, we find that Bmp signaling in Isl1-expressing progenitors is required for expression of T-box transcription factors Tbx2 and Tbx3 in heart and limb. Tbx3 is required for heart and limb formation, and is mutated in ulnar-mammary syndrome. We provide evidence that the Tbx3 promoter is directly regulated by Bmp Smads in vivo

Vertebrate gap junction channels are formed by a family of more than 20 connexin proteins. These gap junction proteins are expressed with overlapping cellular and tissue specificity, and coding region mutations can cause human hereditary diseases. Here we present a summary of what has been learned from voltage clamp studies performed on cell pairs either endogenously expressing gap junctions or in which connexins are exogenously expressed. General protocols presented here are currently used to transfect mammalian cells with connexins and to study the biophysical properties of the heterologously expressed connexin channels. Transient transfection is accomplished overnight with maximal expression occurring at about 36 h; stable transfectants normally can be generated within three or four weeks through colony selection. Electrophysiological protocols are presented for analysis of voltage dependence and single-channel conductance of gap junction channels as well as for studies of chemical gating of these channels

Sarcomere protein gene mutations cause hypertrophic cardiomyopathy (HCM), a disease with distinctive histopathology and increased susceptibility to cardiac arrhythmias and risk for sudden death. Myocyte disarray (disorganized cell-cell contact) and cardiac fibrosis, the prototypic but protean features of HCM histopathology, are presumed triggers for ventricular arrhythmias that precipitate sudden death events. To assess relationships between arrhythmias and HCM pathology without confounding human variables, such as genetic heterogeneity of disease-causing mutations, background genotypes, and lifestyles, we studied cardiac electrophysiology, hypertrophy, and histopathology in mice engineered to carry an HCM mutation. Both genetically outbred and inbred HCM mice had variable susceptibility to arrhythmias, differences in ventricular hypertrophy, and variable amounts and distribution of histopathology. Among inbred HCM mice, neither the extent nor location of myocyte disarray or cardiac fibrosis correlated with ex vivo signal conduction properties or in vivo electrophysiologically stimulated arrhythmias. In contrast, the amount of ventricular hypertrophy was significantly associated with increased arrhythmia susceptibility. These data demonstrate that distinct somatic events contribute to variable HCM pathology and that cardiac hypertrophy, more than fibrosis or disarray, correlates with arrhythmic risk. We suggest that a shared pathway triggered by sarcomere gene mutations links cardiac hypertrophy and arrhythmias in HCM

Endocardial cushions are precursors of mature atrioventricular (AV) valves. Their formation is induced by signaling molecules originating from the AV myocardium, including bone morphogenetic proteins (BMPs). Here, we hypothesized that BMP signaling plays an important role in the AV myocardium during the maturation of AV valves from the cushions. To test our hypothesis, we used a unique Cre/lox system to target the deletion of a floxed Alk3 allele, the type IA receptor for BMPs, to cardiac myocytes of the AV canal (AVC). Lineage analysis indicated that cardiac myocytes of the AVC contributed to the tricuspid mural and posterior leaflets, the mitral septal leaflet, and the atrial border of the annulus fibrosus. When Alk3 was deleted in these cells, defects were seen in the same leaflets, ie, the tricuspid mural leaflet and mitral septal leaflet were longer, the tricuspid posterior leaflet was displaced and adherent to the ventricular wall, and the annulus fibrosus was disrupted resulting in ventricular preexcitation. The defects seen in mice with AVC-targeted deletion of Alk3 provide strong support for a role of Alk3 in human congenital heart diseases, such as Ebstein's anomaly. In conclusion, our mouse model demonstrated critical roles for Alk3 signaling in the AV myocardium during the development of AV valves and the annulus fibrosus