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Stem cell transplantation has been successfully used for amelioration of cardiomyopathic injury using adult cardiac progenitor cells (CPC). Engineering of mouse CPC with the human serine/threonine kinase Pim-1 (CPCeP) enhances regeneration and cell survival in vivo, but it is unknown if such apparent lineage commitment is associated with maturation of electrophysiological properties and excitation-contraction coupling. This study aims to determine electrophysiology and Ca(2+)-handling properties of CPCeP using neonatal rat cardiomyocyte (NRCM) co-culture to promote cardiomyocyte lineage commitment. Measurements of membrane capacitance, dye transfer, expression of connexin 43 (Cx43), and transmission of ionic currents (I(Ca), I(Na)) from one cell to the next suggest that a subset of co-cultured CPCeP and NRCM becomes connected via gap junctions. Unlike NRCM, CPCeP had no significant I(Na), but expressed nifedipine-sensitive I(Ca) that could be measured more consistently with Ba(2+) as permeant ion using ramp-clamp protocols than with Ca(2+) and step-depolarization protocols. The magnitude of I(Ca) in CPCeP increased during culture (4-7 days vs. 1-3 days) and was larger in co-cultures with NRCM and with NRCM-conditioned medium, than in mono-cultured CPCeP. I(Ca) was virtually absent in CPC without engineered expression of Pim-1. Caffeine and KCl-activated Ca(2+)-transients were significantly present in co-cultured CPCeP, but smaller than in NRCM. Conversely, ATP-induced (IP(3)-mediated) Ca(2+) transients were larger in CPCeP than in NRCM. I(NCX) and I(ATP) were expressed in equivalent densities in CPCeP and NRCM. These in vitro studies suggest that CPCeP in co-culture with NRCM: a) develop I(Ca) current and Ca(2+) signaling consistent with cardiac lineage, b) form electrical connections via Cx43 gap junctions, and c) respond to paracrine signals from NRCM. These properties may be essential for durable and functional myocardial regeneration under in vivo conditions.

How chronic pressure overload affects the Purkinje fibers of the ventricular peripheral conduction system (PCS) is not known. Here, we used a connexin (Cx)40 knockout/enhanced green fluorescent protein knockin transgenic mouse model to specifically label the PCS. We hypothesized that the subendocardially located PCS would remodel after chronic pressure overload and therefore analyzed cell size, markers of hypertrophy, and PCS-specific Cx and ion channel expression patterns. Left ventricular hypertrophy with preserved systolic function was induced by 30 days of surgical transaortic constriction. After transaortic constriction, we observed that PCS cardiomyocytes hypertrophied by 23% ( P < 0.05) and that microdissected PCS tissue exhibited upregulated markers of hypertrophy. PCS cardiomyocytes showed a 98% increase in the number of Cx40-positive gap junction particles, with an associated twofold increase in gene expression ( P < 0.05). We also identified a 50% reduction in Cx43 gap junction particles located at the interface between PCS cardiomyocytes and the working cardiomyocyte. In addition, we measured a fourfold increase of an ion channel, hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, throughout the PCS ( P < 0.05). As a direct consequence of PCS remodeling, we found that pressure-overloaded hearts exhibited marked changes in ventricular activation patterns during normal sinus rhythm. These novel findings characterize PCS cardiomyocyte remodeling after chronic pressure overload. We identified significant hypertrophic growth accompanied by modified expression of Cx40, Cx43, and HCN4 within PCS cardiomyocytes. We found that a functional outcome of these changes is a failure of the PCS to activate the ventricular myocardium normally. Our findings provide a proof of concept that pressure overload induces specific cellular changes, not just within the working myocardium but also within the specialized PCS.

Five strains representing a novel family within theGammaproteobacteriawere isolated from the estuarine grassesSpartina alternifloraandJuncus roemerianus. All strains were facultatively anaerobic, Gram-negative, short, motile, polar monotrichous rods that were mesophilic, oxidase-negative, catalase-positive, had DNA G+C contents of 41.5–44.4 mol% and required seawater salts or NaCl. Growth was observed at pH 3.5–8.0. Polar lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, aminophospholipid, phospholipids and unidentified aminolipids were found in the representative strain S-G2-2T. The major menaquinone and ubiquinone were MK-8 (100 %) and Q-8 (93 %), respectively. Predominant fatty acids present were C12 : 0aldehyde and/or unknown fatty acid 10.9525 (MIDI designation) and/or iso-C16 : 1I/C14 : 03-OH, C16 : 1ω7c/C16 : 1ω6c, C16 : 0, C17 : 0cyclo and C18 : 1ω7cand/or C18 : 1ω6c. The nearly full-length 16S rRNA gene sequences of the strains were very similar (99–100 % similarity), and the strains were identified as members of the same species by DNA–DNA relatedness measurements. 16S rRNA gene sequence analysis revealed that the strains formed a monophyletic lineage within the orderAlteromonadales. All five strains fixed N2. Analysis of partialnifHgene sequences also revealed a monophyletic lineage within theGammaproteobacteria, and the sequences were dissimilar to those of any previously described diazotroph. Differences between the novel strains and other members of theAlteromonadalesinclude the inability to produce cytochrome oxidase. The novel strains were metabolically versatile. On the basis of the information described above, the new genus and speciesCelerinatantimonas diazotrophicagen. nov., sp. nov. are proposed to accommodate the five strains within a new family,Celerinatantimonadaceaefam. nov. The type strain ofCelerinatantimonas diazotrophicais S-G2-2T( = ATCC BAA-1368T = DSM 18577T).