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Biomedical subjects

Glenn I Fishman

Publications and source records attributed to Glenn I Fishman.

At least 19 recordsLinked to original sources
PubMed2007

Proliferation of adult sertoli cells following conditional knockout of the Gap junctional protein GJA1 (connexin 43) in mice.

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.

Animals↗
PubMed2006

Expression of a sorcin missense mutation in the heart modulates excitation-contraction coupling.

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.

Animals↗
PubMed2006

Consequences of cardiac myocyte-specific ablation of KATP channels in transgenic mice expressing dominant negative Kir6 subunits.

Cardiac ATP-sensitive K+ (K(ATP)) channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice that 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 nontransgenic mice, but an increased mortality was noted after the age of 4-5 mo. Transgenic mice lacked cardiac K(ATP) 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 EADs after 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 proarrhythmogenic features and lack of adaptation to a stress response in transgenic mice suggest that these features are intrinsic to the myocardium and that K(ATP) channels in the myocardium have an important role in protecting the heart from lethal arrhythmias and adaptation to stress situations.

ATP-Binding Cassette Transporters↗
PubMed2006

Isl1Cre reveals a common Bmp pathway in heart and limb development.

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.

Animals↗
PubMed2005

Alk3/Bmpr1a receptor is required for development of the atrioventricular canal into valves and annulus fibrosus.

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.

Animals↗
PubMed2005

Conditional mineralocorticoid receptor expression in the heart leads to life-threatening arrhythmias.

BACKGROUND: Life-threatening cardiac arrhythmia is a major source of mortality worldwide. Besides rare inherited monogenic diseases such as long-QT or Brugada syndromes, which reflect abnormalities in ion fluxes across cardiac ion channels as a final common pathway, arrhythmias are most frequently acquired and associated with heart disease. The mineralocorticoid hormone aldosterone is an important contributor to morbidity and mortality in heart failure, but its mechanisms of action are incompletely understood. METHODS AND RESULTS: To specifically assess the role of the mineralocorticoid receptor (MR) in the heart, in the absence of changes in aldosteronemia, we generated a transgenic mouse model with conditional cardiac-specific overexpression of the human MR. Mice exhibit a high rate of death prevented by spironolactone, an MR antagonist used in human therapy. Cardiac MR overexpression led to ion channel remodeling, resulting in prolonged ventricular repolarization at both the cellular and integrated levels and in severe ventricular arrhythmias. CONCLUSIONS: Our results indicate that cardiac MR triggers cardiac arrhythmias, suggesting novel opportunities for prevention of arrhythmia-related sudden death.

Animals↗
PubMed2005

Focal gap junction uncoupling and spontaneous ventricular ectopy.

Genetic studies in the mouse have demonstrated that conditional cardiac-restricted loss of connexin43 (Cx43), the major ventricular gap junction protein, is highly arrhythmogenic. However, whether more focal gap junction remodeling, as is commonly seen in acquired cardiomyopathies, influences the propensity for arrhythmogenesis is not known. We examined electrophysiological properties and the frequency of spontaneous and inducible arrhythmias in genetically engineered chimeric mice derived from injection of Cx43-deficient embryonic stem cells into wild-type recipient blastocysts. Chimeric mice had numerous well-circumscribed microscopic Cx43-negative foci in their hearts, comprising approximately 15% of the total surface area as determined by immunohistochemical analysis. Systolic function in the chimeric mice was significantly depressed as measured echocardiographically (19.0% decline in fractional shortening compared with controls, P < 0.05) and by invasive hemodynamics (17.6% reduction in change of pressure over time, P < 0.01). Chimeras had significantly more spontaneous arrhythmic events than controls (P < 0.01), including frequent runs of nonsustained ventricular tachycardia in some of the chimeric mice. However, in contrast to mice with conditional cardiac-resricted loss of Cx43 in the heart, no sustained ventricular tachyarrhythmias were observed. We conclude that focal areas of uncoupling in the myocardium increase the likelihood of arrhythmic triggers, but more widespread uncoupling is required to support sustained arrhythmias.

Animals↗
PubMed2005

Reduced intercellular coupling leads to paradoxical propagation across the Purkinje-ventricular junction and aberrant myocardial activation.

Ventricular tachycardia is a common heart rhythm disorder and a frequent cause of sudden cardiac death. Aberrant cell-cell coupling through gap junction channels, a process termed gap junction remodeling, is observed in many of the major forms of human heart disease and is associated with increased arrhythmic risk in both humans and in animal models. Genetically engineered mice with cardiac-restricted knockout of Connexin43, the major cardiac gap junctional protein, uniformly develop sudden cardiac death, although a detailed electrophysiological understanding of their profound arrhythmic propensity is unclear. Using voltage-sensitive dyes and high resolution optical mapping techniques, we found that uncoupling of the ventricular myocardium results in ectopic sites of ventricular activation. Our data indicate that this behavior reflects alterations in source-sink relationships and paradoxical conduction across normally quiescent Purkinje-ventricular muscle junctions. The aberrant activation profiles are associated with wavefront collisions, which in the setting of slow conduction may account for the highly arrhythmogenic behavior of Connexin43-deficient hearts. Thus, the extent of gap junction remodeling in diseased myocardium is a critical determinant of cardiac excitation patterns and arrhythmia susceptibility.

Animals↗
PubMed2004

Left atrial tachycardia originating from the mitral annulus-aorta junction.

BACKGROUND: At the mitral annulus-aorta (MA-Ao) junction, the left atrium is continuous through the subaortic curtain with the musculature of the anterior mitral leaflet. Under experimental conditions, this region can generate abnormal electrical activity. In patients with left atrial tachycardia, we investigated whether this region could be the source of this arrhythmia. METHODS AND RESULTS: In 10 (28%) of 35 consecutive patients with left atrial tachycardia, the arrhythmia originated from the MA-Ao junction. Sustained, self-limited episodes of atrial tachycardia (cycle length, 340+/-56 ms; duration, 125+/-69 seconds) were repeatedly induced. Prematurity of the extrastimulus and time to first atrial tachycardia complex were directly correlated (R=0.66; P<0.001). During tachycardia, bipolar electrograms at the earliest site preceded onset of the P wave by 44+/-14 ms and were of longer duration and lower amplitude than those recorded from nearby left atrial sites (52+/-8 versus 24+/-4 ms, P<0.001; and 0.53+/-0.08 versus 3.45+/-0.96 mV, respectively; P<0.001). Ablation eliminated the tachycardia with no recurrence after a mean follow-up of 24+/-19 months. A comparative study in mouse embryos demonstrated the presence of the developing specialized conduction system in the MA-Ao region starting at embryonic age 11.5. CONCLUSIONS: The MA-Ao junction can be a frequent source of left atrial tachycardia. This previously unrecognized site of origin may explain why catheter ablation has been less successful in eliminating left versus right atrial tachycardias. Remnants of the developing specialized conduction system could be the underlying substrate of this arrhythmia.

Adrenergic beta-Agonists↗
PubMed2004

Modulation of cardiac gap junction expression and arrhythmic susceptibility.

Connexin43 (Cx43), the predominant ventricular gap junction protein, is critical for maintaining normal cardiac electrical conduction, and its absence in the mouse heart results in sudden arrhythmic death. The mechanisms linking reduced Cx43 abundance in the heart and inducibility of malignant ventricular arrhythmias have yet to be established. In this report, we investigate arrhythmic susceptibility in a murine model genetically engineered to express progressively decreasing levels of Cx43. Progressively older cardiac-restricted Cx43 conditional knockout (CKO) mice were selectively bred to produce a heart-specific Cx43-deficient subline ("O-CKO" mice) in which the loss of Cx43 in the heart occurs more gradually. O-CKO mice lived significantly longer than the initial series of CKO mice but still died suddenly and prematurely. At 25 days of age, cardiac Cx43 protein levels decreased to 59% of control values (P<0.01), but conduction velocity was not significantly decreased and no O-CKO mice were inducible into sustained ventricular tachyarrhythmias. By 45 days of age, cardiac Cx43 abundance had decreased in a heterogeneous fashion to 18% of control levels, conduction velocity had slowed to half of that observed in control hearts, and 80% of O-CKO mice were inducible into lethal tachyarrhythmias. Enhanced susceptibility to induced arrhythmias was not associated with altered invasive hemodynamic measurements or changes in ventricular effective refractory period. Thus, moderately severe reductions in Cx43 abundance are associated with slowing of impulse propagation and a dramatic increase in the susceptibility to inducible ventricular arrhythmias.

Age Factors↗
PubMed2004

The neural crest is contiguous with the cardiac conduction system in the mouse embryo: a role in induction?

In this study we present data on the spatial relationship between neural crest-derived cells (NCC) and the specialized cardiac conduction system (CCS) in the developing murine heart. Using Wnt1-Cre/R26R conditional reporter mice that express beta-galactosidase from ROSA26 upon Cre-mediated recombination, two populations of NCC are seen: one migrates through the arterial pole and contributes to the bundle branches, whereas the second population enters by way of the venous pole and provides cells to the sinoatrial and atrioventricular node areas. The CCS/ lacZ construct is found in the myocardium of the early embryonic heart and afterward only persists in the definitive CCS and is acknowledged as a reporter for the developing conduction system. The contiguous expression of both reporters is suggestive for a potential role of cardiac NCC in the induction of the final differentiation of the CCS.

Animals↗
PubMed2004

Alterations of intercellular communication in neonatal cardiac myocytes from connexin43 null mice.

OBJECTIVE: To compare gap junction expression and intercellular coupling in wildtype neonatal cardiac myocytes to those from mice lacking the most abundant cardiac gap junction protein (connexin43, Cx43). METHODS: Northern and Western blots compared connexin mRNA and protein levels, immunocytochemistry evaluated connexin distribution in neonatal Cx43 null(-/-), heterozygous(+/-) and wildtype(+/+) mouse hearts. Ca(2+) imaging, dye coupling and electrophysiological methods evaluated intercellular communication. RESULTS: Similar levels of Cx40 and Cx45 were detected in all genotypes, although in adult cardiac tissue from wildtype mice, Cx43 expression was higher than in heterozygotes. After culturing dissociated cells for 3-4 days, cardiocytes beat spontaneously; in Cx43(+/+) and (+/-) cultures, the beating was generally quite synchronous. In Cx43(-/-) mice, interbeat intervals were on average twice as long and more variable than in Cx43(+/+) or Cx43(+/-) cultures. Junctional conductance was lower by about 60% in Cx43(-/-) as compared to Cx43(+/-) and (+/+) littermates; Lucifer Yellow dye coupling was virtually absent in Cx43(-/-) cardiomyocytes but was comparably strong in wildtype and heterozygous siblings. Macroscopic junctional conductance measurements on Cx43(-/-) cardiocytes showed slightly stronger voltage sensitivity in these cells than in Cx43(+/+) cardiocytes. Unitary junctional conductance measurements revealed distinct populations of channels contributing to macroscopic conductance for Cx43(+/+) and Cx43(-/-) genotypes. CONCLUSIONS: In Cx43-deficient cardiac myocytes, the expression of other connexins only partially compensates for the functional loss, with dye coupling and spontaneous beating being strongly impaired.

Animals↗
PubMed2004

Toward an understanding of the genetics of murine cardiac pacemaking and conduction system development.

We distinguish the cardiac pacemaking and conduction system (CPCS) from neighboring working cardiomyocytes by its function to generate and deliver electrical impulses within the heart. Yet the CPCS is a series of integrated but distinct components. The components must act in a coordinated fashion, but they are also functionally, molecularly, and electrophysiologically unique. Understanding the differentiation and function of this elegant and complex system is an exciting challenge. Knowledge of genes and signaling pathways that direct CPCS development is at present minimal, but the use of transgenic mice represents an enormous opportunity for elucidating the unknown. Transgenic marker lines have enabled us to image and manipulate the CPCS in new ways. These tools are now being used to examine the CPCS in mutants where its formation and function is altered, generating new information and directions for study of the genetics of CPCS development.

Animals↗
PubMed2004

Embryonic conduction tissue: a spatial correlation with adult arrhythmogenic areas.

INTRODUCTION: The occurrence of arrhythmias in adult patients may arise preferentially in anatomic regions derived from the specialized cardiac conduction system. To examine this hypothesis, we performed a detailed analysis of the developing cardiac conduction system using the recently described CCS-lacZ transgenic mouse strain. METHODS AND RESULTS: Transgenic embryos (E9.5-15.5) were stained for beta-galactosidase activity and co-stained with the myocardial marker HHF35. Results were reconstructed three dimensionally. CCS-lacZ expression was observed in the sinoatrial node, left and right venous valves, septum spurium, right and left atrioventricular ring, His bundle, bundle branches, and right ventricular moderator band. Furthermore, lacZ-positive cells could be demonstrated for the first time in the left atrium, in the posterior wall surrounding the pulmonary venous orifice. and, in later stages, surrounding the pulmonary venous wall. These cells were continuous with the left venous valve in the right atrium. LacZ-positive tissue also could be identified in Bachmann's bundle, running retro-aortically between the right atrium and left atrium. CONCLUSION: Known arrhythmogenic areas including Bachmann's bundle, the pulmonary veins, and sinus venosus derived internodal structures, demonstrate lacZ expression. These data support the hypothesis that areas derived from the developing cardiac conduction system may contribute to the arrhythmogenic substrate in adult hearts.

Animals↗
PubMed2003

Cell coupling between ventricular myocyte pairs from connexin43-deficient murine hearts.

Mice with cardiac-restricted inactivation of the connexin43 gene (CKO mice) have moderate slowing of ventricular conduction and lethal arrhythmias. Mechanisms through which propagation is maintained in the absence of Cx43 are unknown. We evaluated gap junctional conductance in CKO ventricular pairs using dual patch clamp methods. Junctional coupling was reduced to 4+/-2 nS (side-to-side) and 11+/-2 nS (end-to-end), including 21% of cell-pairs with no detectable coupling, compared with 588+/-104 nS (side-to-side) and 558+/-92 nS (end-to-end) in control cell-pairs. Voltage dependence of control gap junctions was characteristic of Cx43. CKO conductance showed increased voltage dependence, suggesting low-level expression of other connexin isoforms. From theoretical models, this degree of CKO coupling is not expected to support levels of conduction persisting in vivo, suggesting the possibility that there are additional mechanisms for maintained propagation when gap junctional conductance is severely reduced.

Animals↗
PubMed2003

Sorcin regulates excitation-contraction coupling in the heart.

Sorcin is a penta-EF hand Ca2+-binding protein that associates with both cardiac ryanodine receptors and L-type Ca2+ channels and has been implicated in the regulation of intracellular Ca2+ cycling. To better define the function of sorcin, we characterized transgenic mice in which sorcin was overexpressed in the heart. Transgenic mice developed normally with no evidence of cardiac hypertrophy and no change in expression of other calcium regulatory proteins. In vivo hemodynamics revealed significant reductions in global indices of contraction and relaxation. Contractile abnormalities were also observed in isolated adult transgenic myocytes, along with significant depression of Ca2+ transient amplitudes. Whole cell ICa density and the time course of activation were normal in transgenic myocytes, but the rate of inactivation was significantly accelerated. These effects of sorcin on L-type Ca2+ currents were confirmed in Xenopus oocyte expression studies. Finally, we examined the expression of sorcin in normal and failing hearts from spontaneous hypertensive heart failure rats. In normal myocardium, sorcin extensively co-localized with ryanodine receptors at the Z-lines, whereas in myopathic hearts the degree of co-localization was markedly disrupted. Together, these data indicate that sorcin modulates intracellular Ca2+ cycling and Ca2+ influx pathways in the heart.

Animals↗