We have combined freeze-fracture and electrophysiological methods in a study of It has been suggested that the direct diffusion of small molecules between the interiors of adjacent cells could be an important factor in cellular homeostasis and even the regulation of growth and differentiation (1-3). This exchange of molecules is believed to occur through gap junctions (4-7), which are specializations of two closely apposed membranes (8), each containing an aggregate of intramembranous particles (9, 10). The particles are presumed to bridge the narrowed extracellular space of a gap junction amd may provide an intercytoplasmic pathway for diffusing molecules (6, 10).While many ultrastructural and physiological properties of gap junctions have been elucidated (1,11,12)

Cellular specialization and interaction with other cell types in cardiac tissue is essential for the coordinated function of cell populations in the heart. The complex interplay between cardiomyocytes, endothelial cells and fibroblasts is necessary for adaptation but can also lead to pathophysiological remodeling. To understand this complex interplay, we developed 3D vascularized cardiac tissue mimetics (CTM) to study heterocellular crosstalk in hypertrophic, hypoxic and fibrogenic environments. This 3D platform responds to physiologic and pathologic stressors and mimics the microenvironment of diseased tissue. In combination with endothelial cell fluorescence reporters, these cardiac tissue mimetics can be used to precisely visualize and quantify cellular and functional responses upon stress stimulation. Utilizing this platform, we demonstrate that stimulation of α/β-adrenergic receptors with phenylephrine (PE) promotes cardiomyocyte hypertrophy, metabolic maturation and vascularization of CTMs. Increased vascularization was promoted by conditioned medium of PE-stimulated cardiomyocytes and blocked by inhibiting VEGF or upon β-adrenergic receptor antagonist treatment, demonstrating cardiomyocyte-endothelial cross-talk. Pathophysiological stressors such as severe hypoxia reduced angiogenic sprouting and increased cell death, while TGF β2 stimulation increased collagen deposition concomitant to endothelial-to-mesenchymal transition. In sum, we have developed a cardiac 3D culture system that reflects native cardiac tissue function, metabolism and morphology-and for the first time enables the tracking and analysis of cardiac vascularization dynamics in physiology and pathology.

Carter (1) has described inhibition of cytoplasmic division (cytokinesis) with unimpaired nuclear division (karyokinesis) in mammalian cells treated with cytochalasin B (CB), a metabolite from the mold, Helminthosporium dematioideum. We report a similar effect of CB on the initial cleavages of Xenopus Zaevis embryos. Our data on both the duration and magnitude of drug effect define temporal limits for the act of cytokinesis and suggest that a pool of material which will be used for subsequent cleavages is available prior to the first cleavage. The demonstration that nuclear division can be separated from cytoplasmic division has been made before with both chemical and physical agents (2-7). However, because CB seems to have minimal effects on other ceIl processes (8), we felt that it would be an ideal tool for the definition of those events surrounding cytoplasmic division.Methods. Batches of 10-1 5 eggs, fertilized within approximately 2 min of each other, were collected from X . Zaezris adults hormonally induced to mate ( 9 ) . The outer jelly coats were removed using watchmakers forceps and the eggs were exposed to 1-10 pg/mlpared by diluting, with distilled water, stock solutions of 1 mg/ml of CB in dimethyl sulfoxide (DMSO) . Although CB is only sparingly soluble in pure water, it is readily soluble in dilute solutions of DMSQ. ControIs were exposed to 1% DMSO in distilled water, the maximum concentration of DMSO used in the experimental solutions. I n our initial experiments we found that solutions of 10 pg/ml or more of CB caused pigment clumping and rotation of the eggs, both signifying death, while controls in 1% DMSO were unaffected. The remainder of our experiments were run with doses of 1-5 pglml.Further details of procedure, ie., concentration of CB, time and duration of exposure to CB, are given in the text below and in the figure legend. Results. We have focused our attention on the first two cleavages and the beginning of the third. CB produced consistent alterations in the external features of the cleavage furrow as well as in the internal structure seen in histological sections. The following external changes occurred when the eggs were placed in 5 pg/ml of CB immediately after removal of the outer jelly coat: (a) the first two cleavage furrows began at the usual time and position and with a normal appearance; the third cleavage furrow began a t the usual time but was disoriented, i.e., it was nearly meridional rather than equatorial ; (b) the progression of the first two furrows around the egg appeared normal; (c) however, about the time each furrow was completed at the vegetal pole, "pigment separation" was observed in the depths of the furrow a t the animal pole; ( d ) pigment separation became more pronounced and continued along the furrow which became shallower and finally disappeared leaving a white band of poorly pigmented cortex as the only surface indication of the cleavage plane. Events (c) and (d) are referred to below as "rever~al" of the furrow.Certain alterations in internal struct...

Endoreplication, duplication of the nuclear genome without cell division, occurs in disease to drive morphologic growth, cell fate, and function. Despite its criticality, the metabolic underpinnings of disease-induced endoreplication and its link to morphologic growth are unknown. Heart disease is characterized by endoreplication preceding cardiac hypertrophy. We identify ATP synthase as a central control node and determinant of cardiac endoreplication and hypertrophy by rechanneling free mitochondrial ADP to methylenetetrahydrofolate dehydrogenase 1 L (MTHFD1L), a mitochondrial localized rate-limiting enzyme of formate and de novo nucleotide biosynthesis. Concomitant activation of the adenosine monophosphate-activated protein kinase (AMPK)-retinoblastoma protein (Rb)-E2F axis co-opts metabolic products of MTHFD1L function to support DNA endoreplication and pathologic growth. Gain-and loss-of-function studies in genetic and surgical mouse heart disease models and correlation in individuals confirm direct coupling of deregulated energetics with endoreplication and pathologic overgrowth. Together, we identify cardiometabolic endoreplication as a hitherto unknown mechanism dictating pathologic growth progression in the failing myocardium.

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