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Background: The sympathetic nervous system is critical in maintaining the normal physiological function of the heart. Its dysfunction in pathological states may exacerbate the substrate for arrhythmias. Obviously, knowledge of its three-dimensional (3D) structure is important, however, it has been revealed by conventional methods only to a limited extent. In this study, a new method of tissue clearance in combination with immunostaining unravels the 3D structure of the sympathetic cardiac network as well as its changes after myocardial infarction., Methods and Results: Hearts isolated from adult male mice were optically cleared using the CUBIC-perfusion protocol. After making the hearts transparent, sympathetic nerves and coronary vessels were immunofluorescently labeled, and then images were acquired. The spatial distribution of sympathetic nerves was visualized not only along the epicardial surface, but also transmurally. They were distributed over the epicardial surface and penetrated into the myocardium to twist around coronary vessels, but also independent from the coronary vasculature. At 2 weeks after myocardial infarction, we were able to quantify both denervation distal from the site of infarction and nerve sprouting (hyperinnervation) at the ischemic border zone of the hearts in a 3D manner. The nerve density at the ischemic border zone was more than doubled in hearts with myocardial infarction compared to intact mice hearts (3D analyses; n = 5, p<0.05)., Conclusions: There is both sympathetic hyperinnervation and denervation after myocardial infarction. Both can be visualized and quantified by a new imaging technique in transparent hearts and thereby become a useful tool in elucidating the role of the sympathetic nervous system in arrhythmias associated with myocardial infarction.

Cardiac fibrosis is a pathological feature of myocardium of failing heart and plays causative roles in arrhythmia and cardiac dysfunction, but its regulatory mechanisms remain largely elusive. In this study, we investigated the effects of the novel EP4 receptor agonist ONO-0260164 on cardiac fibrosis in hypertrophied heart and explored the regulatory mechanisms in cardiac fibroblasts.In a mouse model of cardiac hypertrophy generated by transverse aortic constriction (TAC), ONO-0260164 treatment significantly prevented systolic dysfunction and progression of myocardial fibrosis at 5 weeks after TAC. In cultured neonatal rat cardiac fibroblasts, transforming growth factor-β1 (TGF-β1) induced upregulation of collagen type 1, alpha 1 (Col1a1) and type 3, alpha 1 (Col3a1), which was inhibited by ONO-0260164 treatment. ONO-0260164 activated protein kinase A (PKA) in the presence of TGF-β1 in the cardiac fibroblasts. PKA activation suppressed an increase in collagen expression induced by TGF-β1, indicating the important inhibitory roles of PKA activation in TGF-β1mediated collagen induction.We have demonstrated for the first time the antifibrotic effects of the novel EP4 agonist ONO-0260164 in vivo and in vitro, and the important role of PKA activation in the effects.

Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step in the salvage pathway for nicotinamide adenine dinucleotide (NAD(+)) biosynthesis, and thereby regulates the deacetylase activity of sirtuins. Here we show accommodative regulation of myocardial NAD(+) by monocyte-derived extracellular Nampt (eNampt), which is essential for hemodynamic compensation to pressure overload. Although intracellular Nampt (iNampt) expression was decreased in pressure-overloaded hearts, myocardial NAD(+) concentration and Sirt1 activity were preserved. In contrast, iNampt was up-regulated in spleen and monocytes, and circulating eNampt protein and nicotinamide mononucleotide (NMN), a key precursor of NAD(+), were significantly increased. Pharmacological inhibition of Nampt by FK866 or depletion of monocytes/macrophages by clodronate liposomes disrupted the homeostatic mechanism of myocardial NAD(+) levels and NAD(+)-dependent Sirt1 activity, leading to susceptibility to cardiomyocyte apoptosis and cardiac decompensation in pressure-overloaded mice. These biochemical and hemodynamic defects were prevented by systemic administration of NMN. Our studies uncover a crucial role of monocyte-derived eNampt in myocardial adaptation to pressure overload, and highlight a potential intervention controlling myocardial NAD(+) against heart failure.

Engineering of three-dimensional (3D) cardiac tissues using decellularized extracellular matrix could be a new technique to create an "organ-like" structure of the heart. To engineer artificial hearts functionally comparable to native hearts, however, much remain to be solved including stable excitation-propagation. To elucidate the points, we examined conduction properties of engineered tissues. We repopulated the decellularized hearts with neonatal rat cardiac cells and then, we observed excitation-propagation of spontaneous beatings using high resolution cameras. We also conducted immunofluorescence staining to examine morphological aspects. Live tissue imaging revealed that GFP-labeled-isolated cardiac cells were migrated into interstitial spaces through extravasation from coronary arteries. Engineered hearts repopulated with Ca(2+)-indicating protein (GCaMP2)-expressing cardiac cells were subjected to optical imaging experiments. Although the engineered hearts generally showed well-organized stable excitation-propagation, the hearts also demonstrated arrhythmogenic propensity such as disorganized propagation. Immunofluorescence study revealed randomly-mixed alignment of cardiomyocytes, endothelial cells and smooth muscle cells. The recellularized hearts also showed disarray of cardiomyocytes and markedly decreased expression of connexin43. In conclusion, we successfully demonstrated that the recellularized hearts showed dynamic excitation-propagation as a "whole organ". Our strategy could provide prerequisite information to construct a 3D-engineered heart, functionally comparable to the native heart., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The aim of myocardial tissue engineering is to repair or regenerate damaged myocardium with engineered cardiac tissue. However, this strategy has been hampered by lack of functional integration of grafts with native myocardium. Autonomic innervation may be crucial for grafts to function properly with host myocardium. In this study, we explored the feasibility of in vivo induction of autonomic innervation to engineered myocardial tissue using genetic modulation by adenovirus encoding glial cell line derived neurotrophic factor (GDNF). GFP-transgene (control group) or GDNF overexpressing (GDNF group) engineered cardiomyocyte sheets were transplanted on cryoinjured hearts in rats. Nerve fibers in the grafts were examined by immunohistochemistry at 1, 2, and 4 weeks postoperatively. Growth associated protein-43 positive growing nerves and tyrosine hydroxylase positive sympathetic nerves were first detected in the grafts at 2 weeks postoperatively in control group and 1 week in GDNF group. The densities of growing nerve and sympathetic nerve in grafts were significantly increased in GDNF group. No choline acetyltransferase immunopositive parasympathetic nerves were observed in grafts. In conclusion, sympathetic innervation could be effectively induced into engrafted engineered cardiomyocyte sheets using GDNF.

Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose of this study was to investigate the effect of neurotrophic factors on sympathetic neurite growth towards cardiomyocytes. Cardiomyocytes (CMs) and sympathetic neurons (SNs) were isolated from neonatal rat hearts and superior cervical ganglia, and were co-cultured, either in a random or localized way. Neurite growth from SNs toward CMs was assessed by immunohistochemistry for neurofilament M and α-actinin in response to neurotrophic factors-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and a chemical repellent, semaphorin 3A. As a result, GDNF as well as NGF and BDNF stimulated neurite growth. GDNF enhanced neurite outgrowth even under the NGF-depleted culture condition, excluding an indirect effect of GDNF via NGF. Quantification of mRNA and protein by real-time PCR and immunohistochemistry at different developmental stages revealed that GDNF is abundantly expressed in the hearts of embryos and neonates, but not in adult hearts. GDNF plays an important role in inducing cardiac sympathetic innervation at the early developmental stages. A possible role in (re)innervation of injured or transplanted or cultured and transplanted myocardium may deserve investigation.

The long-term cardiac effects of amiodarone resemble many aspects of hypothyroidism. The anti-arrhythmic potential of amiodarone may therefore be the result of a drug-induced, local hypothyroid-like condition. To investigate this controversial issue, we compared gene expression profiles in the hearts of rats treated with amiodarone with those of rats with hypothyroidism. Wistar male rats were assigned to 3 groups (n=6-8): Control, systemic hypothyroidism (hypothyroidism) and amiodarone treatment (amiodarone, 150 mg/kg/day, p.o., 4 weeks). Electrocardiogram (ECG) recordings, gene profiling by DNA microarray and Northern blotting were carried out. Amiodarone, like hypothyroidism, caused significant prolongation of RR and QT intervals in ECGs. Microarray analysis of 8435 genes in the left ventricular myocardium revealed a significant similarity in expression profiles between hypothyroidism and amiodarone (R=0.63, p<0.00001). The gene expression profiles of hypothyroidism and amiodarone showed closer correlation when top 100 up-regulated and 100 down-regulated genes in hypothyroidism (total 200 genes) were analyzed (R=0.78, p<0.00001). Northern blots of left ventricular myocardium showed a parallel decrease in mRNAs for myosin heavy chain (MHC)-alpha and a parallel increase for myosin heavy chain (MHC)-beta in hypothyroidism and amiodarone. In the liver and pituitary, in contrast, Northern blots showed quite different changes in the transcripts of the representative T3-responsive genes in the hypothyroidism and amiodarone. In conclusion, long-term treatment with amiodarone causes cardioselective hypothyroid-like alterations in gene expression profiles. The potent anti-arrhythmic activity of amiodarone may be attributable, in part at least, to this unique transcriptional remodeling.

Sympathetic innervation is critical for effective cardiac function. However, the developmental and regulatory mechanisms determining the density and patterning of cardiac sympathetic innervation remain unclear, as does the role of this innervation in arrhythmogenesis. Here we show that a neural chemorepellent, Sema3a, establishes cardiac sympathetic innervation patterning. Sema3a is abundantly expressed in the trabecular layer in early-stage embryos but is restricted to Purkinje fibers after birth, forming an epicardial-to-endocardial transmural sympathetic innervation patterning. Sema3a(-/-) mice lacked a cardiac sympathetic innervation gradient and exhibited stellate ganglia malformation, which led to marked sinus bradycardia due to sympathetic dysfunction. Cardiac-specific overexpression of Sema3a in transgenic mice (SemaTG) was associated with reduced sympathetic innervation and attenuation of the epicardial-to-endocardial innervation gradient. SemaTG mice demonstrated sudden death and susceptibility to ventricular tachycardia, due to catecholamine supersensitivity and prolongation of the action potential duration. We conclude that appropriate cardiac Sema3a expression is needed for sympathetic innervation patterning and is critical for heart rate control.

Background: Spiral re-entry plays the principal role in the genesis of ventricular tachycardia and ventricular fibrillation (VT/VF). The specific I(Kr) blocker, nifekakant (NIF) has, often in combination with lidocaine (LID), recently been used in Japan to prevent recurrent VT/VF, but the combined effects of these drugs on spiral re-entry had never been investigated., Methods and Results: A ventricular epicardial sheet was obtained from 13 Langendorff-perfused rabbit hearts by means of a cryoprocedure, and epicardial excitations were analyzed with a high-resolution optical mapping system. Nifekakant (0.5 micromol/L) caused significant prolongation of action potential duration (APD) and LID (3 micromol/L) attenuated the APD prolongation without affecting the conduction velocity. VT were induced in 6 hearts by cross-field stimulation, and single- or double-loop spirals circulating around variable functional block lines were visualized during the VT. Nifekakant reduced VT cycle length and caused early termination in association with destabilization of the spiral dynamics (prolongation of functional block line, frequent local conduction block, and extensive meandering). These modifications of spiral-type re-entrant VT by NIF were prevented by addition of LID., Conclusions: The effects of NIF on the spiral excitations are reversed by LID. This interaction should be taken into account when these drugs are used in combination to treat VT/VF.

Background: It is controversial which subtypes of T type Ca(2+) channels are implicated in automaticity of cardiac cells during the embryonic period., Method and Results: The effect of Ni(2+) on the automaticity of Nkx2.5-positive cardiac precursor cells sorted from embryonic stem cells during their differentiation was examined using patch clamp techniques. Although 40 micromol/L Ni(2+), which is enough to block Ni(2+)sensitive T type-Ca(2+) channels, decreased the spontaneous beating rate in all cells in the early and intermediate stage, Ni(2+) did not show any effects on the automaticity of 50% of the cells in the late stage., Conclusion: These results indicate that Ni(2+)-sensitive T-type Ca(2+) channels expressed in the Nkx2.5-positive cardiac precursor cells are developmentally regulated.

T-type Ca2+ channels are implicated in cardiac automaticity, cell growth, and cardiovascular remodeling. Two voltage-gated Ca2+ subtypes (Ca(v)3.1 and Ca(v)3.2) have been cloned for the pore-forming alpha(1)-subunit of the T-type Ca2+ channel in cardiac muscle, but their differential roles remain to be clarified. The aim of this study was to elucidate the relative contribution of the two subtypes in the normal development of mouse hearts. A whole cell patch clamp was used to record ionic currents from ventricular myocytes isolated from mice of early (E9.5) and late embryonic days (E18) and from adult 10-wk-old mice. Large T-type Ca2+ current (I(Ca,T)) was observed at both E9.5 and E18, displaying similar voltage-dependence and kinetics of activation and inactivation. The current was inhibited by Ni2+ at relatively low concentrations (IC(50) 26-31 microM). I(Ca,T) was undetectable in adult myocytes. Quantitative PCR analysis revealed that Ca(v)3.2 mRNA is the predominant subtype encoding T-type Ca2+ channels at both E9.5 and E18. Ca(v)3.1 mRNA increased from E9.5 to E18, but remained low compared with Ca(v)3.2 mRNA during the whole embryonic period. In the adulthood, in contrast, Ca(v)3.1 mRNA is greater than Ca(v)3.2 mRNA. These results indicate that Ca(v)3.2 underlies the functional T-type Ca2+ channels in the embryonic murine heart, and there is a subtype switching of transcripts from Ca(v)3.2 to Ca(v)3.1 in the perinatal period.