Mechanical stretch is thought to play an important role in remodeling atrial and ventricular myocardium and may produce substrates that promote arrhythmogenesis. In the present work, neonatal rat ventricular myocytes were cultured for 4 days as confluent monolayers on thin silicone membranes and then subjected to linear pulsatile stretch for up to 6 hours. Action potential upstrokes and propagation velocity (theta) were measured with multisite optical recording of transmembrane voltage of the cells stained with the voltage-sensitive dye RH237. Expression of the gap junction protein connexin43 (Cx43) and the fascia adherens junction protein N-cadherin was measured immunohistochemically in the same preparations. Pulsatile stretch caused dramatic upregulation of intercellular junction proteins after only 1 hour and a further increase after 6 hours (Cx43 signal increased from 0.73 to 1.86 and 2.02% cell area, and N-cadherin signal increased from 1.21 to 2.11 and 2.74% cell area after 1 and 6 hours, respectively). This was paralleled by an increase in theta from 27 to 35 cm/s after 1 hour and 37 cm/s after 6 hours. No significant change in the upstroke velocity of the action potential or cell size was observed. Increased theta and protein expression were not reversible after 24 hours of relaxation. Nonpulsatile (static) stretch produced qualitatively similar but significantly smaller changes than pulsatile stretch. Thus, pulsatile linear stretch in vitro causes marked upregulation of proteins that form electrical and mechanical junctions, as well as a concomitant increase in propagation velocity. These changes may contribute to arrhythmogenesis in myocardium exposed to acute stretch.

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Abstract-The aim of the present study was to morphologically and electrically characterize synthetic strands of mouse ventricular myocytes. Linear strands of mouse ventricular myocytes with widths of 34.7Ϯ4.4 m (W 1 ), 57.9Ϯ2.5 m (W 2 ), and 86.4Ϯ3.6 m (W 3 ) and a length of 10 mm were produced on glass coverslips with a photolithographic technique. Action potentials (APs) were measured from individual cells within the strands with cell-attached microelectrodes. Impulse propagation and AP upstrokes were measured with multisite optical mapping (RH237).Immunostaining was performed to assess cell-cell connections and myofibril arrangement with polyclonal antisera against connexin43 and N-cadherins and monoclonal antibodies against cardiac myosin. Light microscopy and myosin staining showed dense growth of well-developed elongated myocytes with lengths of 34.2Ϯ4.2 m (W 1 ), 36.9Ϯ5.8 m (W 2 ), and 43.7Ϯ6.9 m (W 3 ), and length/width ratios of 3.9Ϯ0.2. Gap junctions were distributed around the cell borders (3 to 4 junctions/m 2 cell area). Each cell was connected by gap junctions to 6.5Ϯ1.1 neighboring cells. AP duration shortened with time in culture (action potential duration at 50% repolarization: day 4, 103Ϯ34 ms; day 8, 16Ϯ3 ms; PϽ0.01). Minimum diastolic potential and AP amplitude were 71Ϯ5 and 97.2Ϯ7.6 mV, respectively. Conduction velocity and the maximum dV/dt of the AP upstroke were 43.9Ϯ13.6 cm/s and 196Ϯ67 V/s, respectively. Thus, neonatal ventricular mouse myocytes can be grown in continuous synthetic strands. Gap junction distribution is similar to the neonatal pattern observed in the hearts of larger mammals. Conduction velocity is in the range observed in adult mice and in the higher range for mammalian species probably due to the higher dV/dt max . This technique will permit the study of propagation, AP, and structure-function relations at cellular resolution in genetically modified mice. (Circ Res. 2000;87:467-473.)

Purified major outer membrane protein of Campylobacter jejuni exhibited different classes of molecules by SDS/PAGE and immunoblotting. A high-molecular-masc product (120-140 kDa) was observed under mild conditions of solubilization, a folded monomeric form of 35 kDa was seen when treated at high SDS concentrations and finally, a single band around 45 kDa occurred when the sample was heated to 96°C [Bolla, J. M., Loret, E., Zalewski, M. & Pages, J. M. (1995) J. Bucteriol. 177, 4266-42711. The high-molecular-mass product was reconstituted into two-dimensional crystals in the presence of phospholipids and Mg". The C. jejuni porin required different conditions for successful reconstitution into twodimensional crystals than the Escherichia coli porin OmpF. Electron microscopy and digital image processing of negatively stained specimens revealed a rectangular lattice with a unit cells size of a = 8.9 nm, b = 14.9 nm, an oblique lattice with a = 8.9 nm, b = 30.1 nm, y = 98", and a trigonal lattice with a = b = 9.6 nm. Projection maps were calculated to a resolution of 2 nm, and exhibited a trimeric protein with three stain-filled indentations.Keywords: Campylobacter jejuni porin; two-dimensional crystallization.Campylohacter jejuni, a gram-negative, microaerophilic bacterium is a inajor cause of acute enteritis and diarrhea in humans throughout the world (Fauchkre and Rosenau, 1991 ;Walker et al., 1988). As with other gram-negative bacteria, Campylobacter species contain outer membrane proteins which facilitate the diffusion of solutes across the outer membrane. The major outer membrane protein (MOMP) of Campylobacter has been identified by Blaser et al. (1983) and Logan and Trust (1982). The MOMP has been shown to possess the characteristic properties of porins (Huyer et al., 1986). More recently, the stability of this porin has been investigated (Bolla et al., 1995). In addition to the denatured monomeric form, two folded species were identified: a stable monomeric conformation and the native trimer, both containing a high level of P-sheet secondary structure characteristic for bacterial porins (Cowan et a]., 1992; Weiss et al., 1991).Here we present three different crystallographic packing arrangements obtained by reconstituting the purified c. jejuni (strain 85H) porin into two-dimensional crystals in the presence of lipids. Negative stain electron microscopy and digital image processing revealed a highly ordered rectangular lattice with unit cell dimensions a = 8.9 C 0.2 nm, b = 14.9 C 0.2 nm, and an oblique lattice a = 8.9 5 0 . 2 nm, h = 30.1 2 0 . 2 nm, y = 98". In addition, a trigonal lattice was observed that had a unit cell size of a = b = 9.620.5 nm, similar to lattices found in native C. jejuni outer membranes (Amako et al., 1996). Correlation averaging was used to calculate projection maps to a resolution of 2 nm. A trimeric protein with three prominent stain-filled indentations was revealed, suggesting that the C. jejuni MOMP is structurally related to the family of the trimeric bacterial porins. MATERIA...

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