To characterize the role of the gap junction protein connexin43 (Cx43) in ventricular conduction, we studied hearts of mice with targeted deletion of the Cx43 gene. Mice homozygous for the Cx43 null mutation (Cx43 Ϫ / Ϫ ) die shortly after birth. Attempts to record electrical activity in neonatal Cx43 Ϫ / Ϫ hearts ( n ϭ 5) were unsuccessful. Ventricular epicardial conduction of paced beats, however, was 30% slower in heterozygous (Cx43 Ϫ / ϩ ) neonatal hearts (0.14 Ϯ 0.04 m/s, n ϭ 27) than in wild-type (Cx43 ϩ / ϩ ) hearts (0.20 Ϯ 0.07 m/s, n ϭ 32; P Ͻ 0.001). This phenotype was even more severe in adult mice; ventricular epicardial conduction was 44% slower in 6-9 mo-old Cx43 Ϫ / ϩ hearts (0.18 Ϯ 0.03 m/s, n ϭ 5) than in wild-type hearts (0.32 Ϯ 0.07 m/s, n ϭ 7, P Ͻ 0.001). Electrocardiograms revealed significant prolongation of the QRS complex in adult Cx43 Ϫ / ϩ mice (13.4 Ϯ 1.8 ms, n ϭ 13) compared with Cx43 ϩ / ϩ mice (11.5 Ϯ 1.4 ms, n ϭ 12, P Ͻ 0.01). Whole-cell recordings of action potential parameters in cultured disaggregated neonatal ventricular myocytes from Cx43 Ϫ / ϩ and ϩ / ϩ hearts showed no differences. Thus, reduction in the abundance of a major cardiac gap junction protein through targeted deletion of a Cx43 allele directly leads to slowed ventricular conduction. ( J. Clin. Invest. 99:1991-1998.)

Most vertebrate internal organs show a distinctive left/right asymmetry. The inv (inversion of embryonic turning) mutation in mice was created previously by random insertional mutagenesis; it produces both a constant reversal of left/right polarity (situs inversus) and cyst formation in the kidneys. Asymmetric expression patterns of the genes nodal and lefty are reversed in the inv mutant, indicating that inv may act early in left/right determination. Here we identify a new gene located at the inv locus. The encoded protein contains 15 consecutive repeats of an Ank/Swi6 motif at its amino terminus. Expression of the gene is the highest in the kidneys and liver among adult tissues, and is seen in presomite-stage embryos. Analysis of the transgenic genome and the structure of the candidate gene indicate that the candidate gene is the only gene that is disrupted in inv mutants. Transgenic introduction of a minigene encoding the candidate protein restores normal left/right asymmetry and kidney development in the inv mutant, confirming the identity of the candidate gene.

Background— Atrial fibrillation (AF) is common after cardiac surgery. Abnormal conduction is an important substrate for AF. We hypothesized that atrial inflammation alters atrial conduction properties. Methods and Results— Normal mongrel canines (n=24) were divided into 4 groups consisting of anesthesia alone (control group); pericardiotomy (pericardiotomy group); lateral right atriotomy (atriotomy group); and lateral right atriotomy with antiinflammatory therapy (methylprednisolone 2 mg/kg per day) (antiinflammatory group). Right atrial activation was examined 3 days after surgery. Inhomogeneity of conduction was quantified by the variation of maximum local activation phase difference. To initiate AF, burst pacing was performed. Myeloperoxidase activity and neutrophil cell infiltration in the atrial myocardium were measured to quantify the degree of inflammation. The inhomogeneity of atrial conduction of the atriotomy and pericardiotomy groups was higher than that of the control group (2.02±0.10, 1.51±0.03 versus 0.96±0.08, respectively; P <0.005). Antiinflammatory therapy decreased the inhomogeneity of atrial conduction after atriotomy (1.16±0.10; P <0.001). AF duration was longer in the atriotomy and pericardiotomy groups than in the control and antiinflammatory groups ( P =0.012). There also were significant differences in myeloperoxidase activity between the atriotomy and pericardiotomy groups and the control group (0.72±0.09, 0.41±0.08 versus 0.18±0.03 ΔOD/min per milligram protein, respectively; P <0.001). Myeloperoxidase activity of the antiinflammatory group was lower than that of the atriotomy group (0.17±0.02; P <0.001). Inhomogeneity of conduction correlated with myeloperoxidase activity ( r =0.851, P <0.001). Conclusions— The degree of atrial inflammation was associated with a proportional increase in the inhomogeneity of atrial conduction and AF duration. This may be a factor in the pathogenesis of early postoperative AF. Antiinflammatory therapy has the potential to decrease the incidence of AF after cardiac surgery.

Abstract-To define mechanisms regulating expression of cell-cell junction proteins, we have developed an in vitro system in which neonatal rat ventricular myocytes were subjected to pulsatile stretch. Previously, we showed that expression of the gap junction protein, connexin (Cx) 43, is increased by Ϸ2-fold after 1 hour of stretch, and this response is mediated by stretch-induced secretion of vascular endothelial growth factor (VEGF). Here, we report that the mechanical junction proteins plakoglobin, desmoplakin, and N-cadherin are also upregulated by pulsatile stretch but by a mechanism independent of VEGF or other secreted chemical signals. Stretch-induced upregulation of mechanical junction proteins was blocked by anti-␤ 1 and anti-␤ 3 integrin antibodies. Transfection of cells with adenovirus expressing GFP-FRNK, a dominant-negative inhibitor of focal adhesion kinase (FAK)-dependent signaling, blocked stretch-induced upregulation of Cx43 and mechanical junction proteins but did not block the ability of exogenous VEGF to upregulate Cx43 expression. Conditioned medium removed from uninfected cells after stretch increased Cx43 expression when added to nonstretched cells, and this effect was blocked by anti-VEGF antibodies, but stretchconditioned medium from GFP-FRNK cells had no effect on Cx43 expression. The src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolol

Abstract-To elucidate mechanisms responsible for mechanotransduction in the heart and define the effects of remodeling of the extracellular matrix, we cultured neonatal rat ventricular myocytes on native type I collagen, fibronectin, or denatured collagen and subjected them to uniaxial, pulsatile stretch. Changes in expression of the cardiac gap junction protein, Cx43, were measured by confocal microscopy and immunoblotting. Cells grown on fibronectin or denatured collagen exhibited significantly greater Cx43 expression than cells grown on native collagen. Stretch induced a Ϸ2-fold increase in Cx43 expression in cells grown on native collagen but no increase in cells grown on fibronectin or denatured collagen. Incubation of cells on native collagen with a peptide containing the arginine-glycine-aspartate (RGD) motif upregulated Cx43 expression equivalent to that induced by stretch.

These results illustrate how a defect in a protein conventionally thought to fulfill a mechanical function in the heart can also lead to electrophysiological alterations that may contribute to arrhythmogenesis.

Inorganic polyphosphate [Poly(P)] is especially prevalent in osteoblasts. We tested the hypothesis that Poly(P) stimulates osteoblastic differentiation and polyphosphate metabolism for bone formation. The osteoblast-like cell line, MC 3T3-E1, was cultured with Poly(P), and gene expression was evaluated by real-time reverse-transcription polymerase chain-reaction. Phosphatase activity and extracellular matrix mineralization were also determined. The role of Poly(P) was assessed in a beagle dog alveolar bone regeneration model. Poly(P) increased osteocalcin, osterix, bone sialoprotein, and tissue non-specific alkaline phosphatase gene expression, with a high level of end-polyphosphatase activity, resulting in low-chain-length Poly(P), inorganic pyrophosphate, and inorganic phosphate production. MC3T3-E1 cells differentiated into mature osteoblasts and showed expression of ectonucleotide pyrophosphatase phosphodiesterase 1, while mouse progressive ankylosis gene expression remained unchanged. Promotion of alveolar bone regeneration was observed in Poly(P)-treated beagle dogs. These findings suggest that Poly(P) induces osteoblastic differentiation and bone mineralization, and acts as a resource for mineralization.

A new cell line (KT21‐MG1) has been established from a human malignant meningioma transplanted into nude mice. The cultured cells showed epithelial cell‐like morphology and were positive immunohis‐tochemically for vimentin as the original tumor. They have been grown continuously in vitro for more than 2 years. The population doubling time was about 24 hours at the 30th passage. The cells are capable of proliferating in soft agar medium and produced tumors in nude mice, the histologies of which were similar to the original patient‐derived tumor. Analysis of cellular oncogenes showed that myc and fps were amplified approximately tenfold and threefold, respectively, in this cell line, whereas N‐myc, L,‐myc, N‐ras, K‐ras, H‐ras, abl, erbB2, Blym, src, raf‐1, myb, and sis were not changed significantly. The amplification of myc was accompanied by an enhanced expression. Chromosome studies of cultured cells showed the monosomy of chromosome 22 that has been reported to be a specific abnormality in meningiomas.