Tissue regenerative potential displays striking divergence across phylogeny and ontogeny, but the underlying mechanisms remain enigmatic. Loss of mammalian cardiac regenerative potential correlates with cardiomyocyte cell-cycle arrest and polyploidization, as well as the development of postnatal endothermy. We reveal that diploid cardiomyocyte abundance across 41 species conforms to Kleiber’s law−the ¾-power law scaling of metabolism with bodyweight−and inversely correlates with standard metabolic rate, body temperature, and serum thyroxine level. Inactivation of thyroid hormone signaling reduces mouse cardiomyocyte polyploidization, delays cell-cycle exit, and retains cardiac regenerative potential in adults. Conversely, exogenous thyroid hormones inhibit zebrafish heart regeneration. Thus, our findings suggest that loss of heart regenerative capacity in adult mammals is triggered by increasing thyroid hormones and may be a tradeoff for the acquisition of endothermy.

Background-Clinically, chronic atrial dilatation is associated with an increased incidence of atrial fibrillation (AF), but the underlying mechanism is not clear. We have investigated atrial electrophysiology and tissue structure in a canine model of chronic atrial dilatation due to mitral regurgitation (MR). Methods and Results-Thirteen control and 19 MR dogs (1 month after partial mitral valve avulsion) were studied. Dogs in the MR group were monitored using echocardiography and Holter recording. In open-chest follow-up experiments, electrode arrays were placed on the atria to investigate conduction patterns, effective refractory periods, and inducibility of AF. Alterations in tissue structure and ultrastructure were assessed in atrial tissue samples. At follow-up, left atrial length in MR dogs was 4.09Ϯ0.45 cm, compared with 3.25Ϯ0.28 at baseline (PϽ0.01), corresponding to a volume of 205Ϯ61% of baseline. At follow-up, no differences in atrial conduction pattern and conduction velocities were noted between control and MR dogs. Effective refractory periods were increased homogeneously throughout the left and right atrium. Sustained AF (Ͼ1 hour) was inducible in 10 of 19 MR dogs and none of 13 control dogs (PϽ0.01). In the dilated MR left atrium, areas of increased interstitial fibrosis and chronic inflammation were accompanied by increased glycogen ultrastructurally. Conclusions-Chronic atrial dilatation in the absence of overt heart failure leads to an increased vulnerability to AF that is not based on a decrease in wavelength.

Background-It has recently been recognized that atrial fibrillation can originate from focal sources in the pulmonary veins (PVs). However, the mechanisms of focal atrial fibrillation have not been well characterized. We assessed the electrophysiological characteristics of the PVs using high-resolution optical mapping. Methods and Results-Coronary-perfused, isolated whole-atrial preparations from 33 normal dogs were studied.Programmed electrical stimulation was performed, and a 4-cm 2 area of the PV underwent optical mapping of transmembrane voltage to obtain 256 simultaneous action potentials. Marked conduction slowing was seen at the proximal PV, compared with the rest of the vein, on both the epicardial (31.3Ϯ4.47 versus 90.2Ϯ20.7 cm/s, Pϭ0.001) and endocardial (45.8Ϯ6.90 versus 67.6Ϯ10.4 cm/s, Pϭ0.012) aspects. Pronounced repolarization heterogeneity was also noted, with action potential duration at 80% repolarization being longest at the PV endocardium. Nonsustained reentrant beats were induced with single extrastimuli, and the complete reentrant loop was visualized (cycle length, 155Ϯ30.3 ms); reentrant activity could be sustained with isoproterenol. Sustained focal discharge (cycle length, 330 to 1100 ms) was seen from the endocardial surface in the presence of isoproterenol; each focus was localized near the venous ostium. Conclusions-The normal PV seems to have the necessary substrate to support reentry as well as focal activity. Although reentry occurred more distally in the vein, focal activity seemed to occur more proximally.

Abstract. The effect of cyclic mechanical strain on growth of neonatal rat vascular smooth muscle (VSM) cells were examined. Cells were grown on silicone elastomer plates subjected to cyclic strain (60 cycle/ min) by application of a vacuum under the plates. A 48 h exposure to mechanical strain increased the basal rate of thymidine incorporation by threefold and increased cell number by 40% compared with cells grown on stationary rubber plates. Strain also increased the rate of thymidine incorporation in response to ot-thrombin (from 15-to 33-fold), but not to PDGE As determined by thymidine autoradiography, strain alone induced a fourfold increase in labeled nuclei at the periphery of dishes, where strain is maximal, and a 2-3-fold increase at the center of dishes. Strain appeared to induce the production of an autocrine growth factor(s), since conditioned medium from cells subjected to strain induced a fourfold increase in DNA synthesis in control cells. Western blots of medium conditioned on the cells subjected to strain indicate that the cells secrete both AA and BB forms of PDGF in response to strain. Northern blots of total cell RNA from cells exposed to strain for 24 h show increased steady-state level of mRNA for PDGF-A. Lastly, polyclonal antibodies to the AA form of PDGF reduced by 75 % the mitogenic effect of strain and polyclonal antibodies to AB-PDGF reduced mitogenicity by 50%. Antibodies to bFGF did not significantly reduce the strain-induced thymidine incorporation.

Cyclic mechanical strain (1 Hz) causes a mitogenic response in neonatal rat vascular smooth muscle cells due to production and secretion of PDGF. In this study, the mechanism for sensing mechanical strain was investigated. Silicone elastomer strain plates were coated at varying densities with elastin, laminin, type I collagen, fibronectin, or vitronectin. Strain was applied by cyclic application of a vacuum under the dishes. Cells adhered, spread, and proliferated on each matrix protein, but the mitogenic response to strain was matrix dependent. Strain increased DNA synthesis in cells on collagen, fibronectin, or vitronectin, but not in cells on elastin or laminin. When strain was applied on matrices containing both laminin and vitronectin, the mitogenic response to strain depended upon the vitronectin content of the matrix. Fibronectin, in soluble form (0-50 jg/ml), and the integrin binding peptide GRGDTP (100 ,ug/ml) both blocked the mitogenic response to mechanical strain in cells grown on immobilized collagen. Neither soluble laminin nor the inactive peptide GRGESP blocked the response to strain. GRGDTP did not alter the mitogenic response to exogenous PDGF or a-thrombin but did prevent the secretion of PDGF in response to strain. Furthermore, GRGDTP, but not GRGESP, prevented strain-induced expression of a PDGF-A chain promoter 890 bp-chloramphenicol acetyltransferase construct that was transiently transfected into vascular smooth muscle cells. Finally, the response to strain was abrogated by antibodies to both 83 and avJ5 integrins but not by an antibody to PBu integrins. Thus interaction between integrins and specific matrix proteins is responsible for sensing mechanical strain in vascular smooth muscle cells. (J. Clin. Invest. 1995. 96:2364-2372

Stimulation of certain receptor tyrosine kinases results in the tyrosine phosphorylation and activation of phospholipase C gamma (PLC gamma), an enzyme that catalyses the hydrolysis of phosphatidylinositol (PtdIns). This hydrolysis generates diacylglycerol and free inositol phosphate, which in turn activate protein kinase C and increase intracellular Ca2+, respectively. PLC gamma physically associates with activated receptor tyrosine kinases, suggesting that it is a substrate for direct phosphorylation by these kinases. Here we report that a fibroblast growth factor (FGF) receptor with a single point mutation at residue 766 replacing tyrosine with phenylalanine fails to associate with PLC gamma in response to FGF. This mutant receptor also failed to mediate PtdIns hydrolysis and Ca2+ mobilization after FGF stimulation. However, the mutant receptor phosphorylated itself and several other cellular proteins, and it mediated mitogenesis in response to FGF. These findings show that a point mutation in the FGF receptor selectively eliminates activation of PLC gamma and that neither Ca2+ mobilization nor PtdIns hydrolysis are required for FGF-induced mitogenesis.

Background-Atrial fibrosis is an important substrate in atrial fibrillation (AF), particularly in the setting of structural heart disease. In a canine model, congestive heart failure (CHF) produces significant atrial fibrosis and the substrate for sustained AF. This atrial remodeling is a potential therapeutic target. The objective of the present study is to evaluate the effects of the antifibrotic drug pirfenidone (PFD) on arrhythmogenic atrial remodeling in a canine CHF model. Methods and Results-We studied 15 canines, divided equally into 3 groups: control, CHF canines not treated with PFD, and CHF canines treated with PFD. CHF was induced by ventricular tachypacing (220 bpm for 3 weeks), and oral PFD was administered for the 3-week pacing period. We performed electrophysiology and AF vulnerability studies, atrial fibrosis measurements, and atrial cytokine expression studies.

The L-type calcium channel is the major calcium influx pathway in vascular smooth muscle and is regulated by integrin ligands, suggesting an important link between extracellular matrix and vascular tone regulation in tissue injury and remodeling. We examined the role of integrin-linked tyrosine kinases and focal adhesion proteins in regulation of L-type calcium current in single vascular myocytes. Soluble tyrosine kinase inhibitors blocked the increase in current produced by ␣ 5 integrin antibody or fibronectin, whereas tyrosine phosphatase inhibition enhanced the effect. Cell dialysis with an antibody to focal adhesion kinase or with FRNK, the C-terminal noncatalytic domain of focal adhesion kinase, produced moderate (24 or 18%, respectively) inhibition of basal current but much greater inhibition (63 or 68%, respectively) of integrin-enhanced current. A c-Src antibody and peptide inhibitors of the Src homology-2 domain or a putative Src tyrosine phosphorylation site on the channel produced similar inhibition. Antibodies to the cytoskeletal proteins paxillin and vinculin, but not ␣-actinin, inhibited integrin-dependent current by 65-80%. Therefore, ␣ 5 ␤ 1 integrin appears to regulate a tyrosine phosphorylation cascade involving Src and various focal adhesion proteins that control the function of the L-type calcium channel. This interaction may represent a novel mechanism for control of calcium influx in vascular smooth muscle and other cell types.Mechanical forces are known to stimulate a number of cell signaling pathways, including those initiated by or resulting in ion channel activation. Several types of ion channels exhibit mechanosensitivity, but the mechanisms underlying their gating remain unclear. Membrane tension changes induced by physiologically relevant forces may not be sufficiently large to gate channels directly in eukaryotic cells (1). Thus, it is likely that mechanically advantageous associations with cytoskeletal or other membrane-bound proteins play a key role in control of gating. However, some mechanism must exist to integrate mechanosensitive gating behavior with the primary regulatory mechanism for channel gating, which is phosphorylation of channel proteins by intracellular kinases (2).Integrins are potential force transduction proteins because they span the plasma membrane and link the extracellular matrix (ECM) 1 to the underlying actin cytoskeleton (CSK) in specialized focal contact regions. Stress applied through integrin-specific adhesion sites increases cytoskeletal stiffening (3), activates second messenger formation (4) and initiates ribosomal and mRNA recruitment to focal adhesions (5). Integrin clustering by multivalent ligands, including ECM proteins, induces recruitment of CSK proteins including vinculin, talin, paxillin and tensin to the focal contact (6, 7). Kinases such as focal adhesion kinase (FAK), Src, PLC-␥, and Rho GTPase, as well as adaptor proteins such as Grb2, Sos, and Shc, are also recruited to the ECM-integrin binding site (8, 9). Subsequently, phosphorylation...