Heart Cell Signaling in 3D: A healthy heart relies on the proper transduction of cellular signals through the β₁- and β₂-adrenergic receptors (βARs), which are located on the surface of the heart's muscle cells (cardiomyocytes). The surface of these cells resembles a highly organized series of hills and valleys and it has been unclear whether this topography plays a role in the βAR signaling events that are critical to cell function.Nikolaevet al.(p.1653, published online 25 February; see Perspective byDorn) monitored the cyclic adenosine monophosphate (cAMP) signals generated by the βARs in living cardiomyocytes. In cells from healthy rats and from rats with heart failure, the β₁ARs were localized across the entire cell surface. In contrast, the spatial localization of the β₂ARs differed in healthy and failing cells. In healthy cardiomyocytes, the β₂ARs resided exclusively within surface invaginations called transverse tubules, thereby producing spatially confined cAMP signals, whereas in failing cardiomyocytes, the β₂ARs redistributed to other cell surface areas, thereby producing diffuse cAMP signals. Thus, changes in the spatial localization of β₂AR-induced cAMP signaling may contribute to heart failure.

Roughly half of the cells of the heart consist of nonmyocardial cells, with fibroblasts representing the predominant cell type. It is well established that individual cardiomyocytes and fibroblasts in culture establish gap junctional communication at the single cell level (short-range interaction). However, it is not known whether such coupling permits activation of cardiac tissue over extended distances (long-range interaction). Long-range interactions may be responsible for electrical synchronization of donor and recipient tissue after heart transplantation and may play a role in arrhythmogenesis. This question was investigated using a novel heterocellular culture model with strands of cardiomyocytes interrupted by cardiac fibroblasts over defined distances. With use of optical recording techniques, it could be shown that impulse propagation along fibroblast inserts was successful over distances up to 300 μm and was characterized by length-dependent local propagation delays ranging from 11 to 68 ms (apparent local “conduction velocities” 4.6±1.8 mm/s, n=23). Involvement of mechanical stretch in this phenomenon was excluded by showing that inserts consisting of communication-deficient HeLa cells were incapable of supporting propagation. In contrast, HeLa cells expressing connexin43 permitted impulse conduction over distances as long as 600 μm. Immunocytochemistry showed that fibroblasts and cardiomyocytes expressed connexin43 and connexin45, whereas connexin40 was absent. These results illustrate that fibroblasts of cardiac origin are capable of synchronizing electrical activity of multicellular cardiac tissue over extended distances through electrotonic interactions. This synchronization is accompanied by extremely large local conduction delays, which might contribute to the generation of arrhythmias in fibrotic hearts.

Structural remodeling of the myocardium associated with mechanical overload or cardiac infarction is accompanied by the appearance of myofibroblasts. These fibroblast-like cells express α-smooth muscle actin (αSMA) and have been shown to express connexins in tissues other than heart. The present study examined whether myofibroblasts of cardiac origin establish heterocellular gap junctional coupling with cardiomyocytes and whether ensuing electrotonic interactions affect impulse propagation. For this purpose, impulse conduction characteristics (conduction velocity [θ] and maximal upstroke velocity [dV/dt _max ]) were assessed optically in cultured strands of cardiomyocytes, which were coated with fibroblasts of cardiac origin. Immunocytochemistry showed that cultured fibroblasts underwent a phenotype switch to αSMA-positive myofibroblasts that expressed connexin 43 and 45 both among themselves and at contact sites with cardiomyocytes. Myofibroblasts affected θ and dV/dt _max in a cell density-dependent manner; a gradual increase of myofibroblast-to-cardiomyocyte ratios up to 7:100 caused an increase of both θ and dV/dt _max , which was followed by a progressive decline at higher ratios. On full coverage of the strands with myofibroblasts (ratio >20:100), θ fell <200 mm/s. This biphasic dependence of θ and dV/dt _max on myofibroblast density is reminiscent of “supernormal conduction” and is explained by a myofibroblast density-dependent gradual depolarization of the cardiomyocyte strands from −78 mV to −50 mV as measured using microelectrode recordings. These findings suggest that myofibroblasts, apart from their role in structural remodeling, might contribute to arrhythmogenesis by direct electrotonic modulation of conduction and that prevention of their appearance might represent an antiarrhythmic therapeutic target.

Focal ectopic activity in cardiac tissue is a key factor in the initiation and perpetuation of tachyarrhythmias. Because myofibroblasts as present in fibrotic remodeled myocardia and infarct scars depolarize cardiomyocytes by heterocellular electrotonic interactions via gap junctions in vitro, we investigated using strands of cultured ventricular cardiomyocytes coated with myofibroblasts, whether this interaction might give rise to depolarization-induced abnormal automaticity. Whereas uncoated cardiomyocyte strands were invariably quiescent, myofibroblasts induced synchronized spontaneous activity in a density dependent manner. Activations appeared at spatial myofibroblast densities >15.7% and involved more than 80% of the preparations at myofibroblast densities of 50%. Spontaneous activity was based on depolarization-induced automaticity as evidenced by: (1) suppression of activity by the sarcolemmal K _ATP channel opener P-1075; (2) induction of activity in current-clamped single cardiomyocytes undergoing depolarization to potentials similar to those induced by myofibroblasts in cardiomyocyte strands; and (3) induction of spontaneous activity in cardiomyocyte strands coated with connexin 43 transfected Hela cells but not with communication deficient HeLa wild-type cells. Apart from unveiling the mechanism underlying the hallmark of monolayer cultures of cardiomyocytes, ie, spontaneous electromechanical activity, these findings open the perspective that myofibroblasts present in structurally remodeled myocardia following pressure overload and infarction might contribute to arrhythmogenesis by induction of ectopic activity.

Rationale:: The miR-143/145 cluster is highly expressed in smooth muscle cells (SMCs), where it regulates phenotypic switch and vascular homeostasis. Whether it plays a role in neighboring endothelial cells (ECs) is still unknown. Objective:: To determine whether SMCs control EC functions through passage of miR-143 and miR-145. Methods and Results:: We used cocultures of SMCs and ECs under different conditions, as well as intact vessels to assess the transfer of miR-143 and miR-145 from one cell type to another. Imaging of cocultured cells transduced with fluorescent miRNAs suggested that miRNA transfer involves membrane protrusions known as tunneling nanotubes. Furthermore, we show that miRNA passage is modulated by the transforming growth factor (TGF) β pathway because both a specific transforming growth factor-β (TGFβ) inhibitor (SB431542) and an shRNA against TGFβRII suppressed the passage of miR-143/145 from SMCs to ECs. Moreover, miR-143 and miR-145 modulated angiogenesis by reducing the proliferation index of ECs and their capacity to form vessel-like structures when cultured on matrigel. We also identified hexokinase II ( HKII ) and integrin β 8 ( ITGβ8 )—2 genes essential for the angiogenic potential of ECs—as targets of miR-143 and miR-145, respectively. The inhibition of these genes modulated EC phenotype, similarly to miR-143 and miR-145 overexpression in ECs. These findings were confirmed by ex vivo and in vivo approaches, in which it was shown that TGFβ and vessel stress, respectively, triggered miR-143/145 transfer from SMCs to ECs. Conclusions:: Our results demonstrate that miR-143 and miR-145 act as communication molecules between SMCs and ECs to modulate the angiogenic and vessel stabilization properties of ECs.

Inhalation delivers drug-loaded nanoparticles to the heart, improving cardiac function in murine and porcine models.