The beta1- and beta2-adrenergic receptors (betaARs) on the surface of cardiomyocytes mediate distinct effects on cardiac function and the development of heart failure by regulating production of the second messenger cyclic adenosine monophosphate (cAMP). The spatial localization in cardiomyocytes of these betaARs, which are coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins), and the functional implications of their localization have been unclear. We combined nanoscale live-cell scanning ion conductance and fluorescence resonance energy transfer microscopy techniques and found that, in cardiomyocytes from healthy adult rats and mice, spatially confined beta2AR-induced cAMP signals are localized exclusively to the deep transverse tubules, whereas functional beta1ARs are distributed across the entire cell surface. In cardiomyocytes derived from a rat model of chronic heart failure, beta2ARs were redistributed from the transverse tubules to the cell crest, which led to diffuse receptor-mediated cAMP signaling. Thus, the redistribution of beta(2)ARs in heart failure changes compartmentation of cAMP and might contribute to the failing myocardial phenotype.

Abstract-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 m and was characterized by length-dependent local propagation delays ranging from 11 to 68 ms (apparent local "conduction velocities" 4.6Ϯ1.8 mm/s, nϭ23). 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 m. 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.

Abstract-Structural remodeling of the myocardium associated with mechanical overload or cardiac infarction is accompanied by the appearance of myofibroblasts. These fibroblast-like cells express ␣-smooth muscle actin (␣SMA) 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 [] 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 ␣SMA-positive myofibroblasts that expressed connexin 43 and 45 both among themselves and at contact sites with cardiomyocytes. Myofibroblasts affected 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 and dV/dt max , which was followed by a progressive decline at higher ratios. On full coverage of the strands with myofibroblasts (ratio Ͼ20:100), fell Ͻ200 mm/s. This biphasic dependence of 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 Ϫ78 mV to Ϫ50 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. Key Words: electrophysiology Ⅲ slow conduction Ⅲ cardiac myofibroblasts Ⅲ fibrosis Ⅲ gap junctions T wo thirds of the cells of normal hearts are noncardiomyocytes, with fibroblasts constituting the largest fraction. At 2 months of age, fibroblasts outnumber cardiomyocytes by a factor of Ϸ2 in human hearts. [1][2][3] Under physiological conditions, fibroblasts are responsible for providing cardiomyocytes with a mechanical scaffold, which integrates the contractile activity of individual cells so as to result in the coordinated pump function of the organ. Accordingly, fibroblasts are found throughout the myocardium, where they form a 3D cellular network surrounding groups of cardiomyocytes. 4 The integrity of this structure is adversely affected by a large number of cardiac diseases ranging from volume to pressure overload and to myocardial infarction. Under these pathological conditions, complex reactions involving changes in extracellular matrix production, cell proliferation, and cell death cause structural remodeling of the ventricular wall, which compromises pump function and predisposes the heart to arrhythmias. 5 Moreover, it has been shown that these disease states are associated with the appearan...

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) S tructural remodeling of the myocardium during pressure overload and following infarction is typically accompanied by the appearance of interstitial myofibroblasts which contribute to cardiac fibrosis by excessive secretion of extracellular matrix proteins. 1 The resulting collagenous septa contribute to arrhythmogenesis in structurally remodeled hearts by inducing discontinuous slow conduction. 2 More recently, studies in vitro demonstrated that myofibroblasts can directly induce arrhythmogenic slow conduction following establishment of heterocellular gap junctional coupling with cardiomyocytes. 3 This slowing of conduction is the result of a decrease in inward currents secondary to the partial depolarization of the cardiomyocytes by the less polarized myofibroblasts. Because partial depolarization of cardiac tissue has previously been shown to induce abnormal automaticity, 4 we investigated in the present study whether heterocellular electrotonic interactions between myofibroblasts and cardiomyocytes might precipitate spontaneous ectopic activity. Materials and MethodsThe effects of myofibroblasts on cardiac excitability were investigated in patterned growth strands of neonatal rat ventricular cardiomyocytes using optical recording of transmembrane voltage, immunocytochemistry and patch clamp recording techniques. Detailed descriptions of the materials and methods used are available in the online data supplement at http://circres.ahajournals.org. ResultsThe hypothesis that myofibroblasts might generate abnormal automaticity in cardiac tissue was investigated in patterned growth strands of neonatal rat ventricular cardiomyocytes (Figure 1). Whereas control preparations were invariably quiescent (nϭ102; Figure 1A,C), coating of the strands with increasing numbers of myofibroblasts (25 to 950 cells/mm 2 ) elicited spontaneous electrical activity in 54.2% of the preparations with an average frequency of 64.4Ϯ21.7 min Ϫ1 (nϭ548; Figure 1B,C). In contrast, control cardiomyocyte strands cocultured with myofibroblast in a noncontact configuration remained quiescent indicating that induction of spontaneous activity was not dependent on conditioning of the medi...