Daniël A. Pijnappels

Forced Alignment of Mesenchymal Stem Cells Undergoing Cardiomyogenic Differentiation Affects Functional Integration With Cardiomyocyte Cultures

Alignment of cardiomyocytes (CMCs) contributes to the anisotropic (direction-related) tissue structure of the heart, thereby facilitating efficient electrical and mechanical activation of the ventricles. This study aimed to investigate the effects of forced alignment of stem cells during cardiomyogenic differentiation on their functional integration with CMC cultures. Labeled neonatal rat (nr) mesenchymal stem cells (nrMSCs) were allowed to differentiate into functional heart muscle cells in different cell-alignment patterns during 10 days of coculture with nrCMCs. Development of functional cellular properties was assessed by measuring impulse transmission across these stem cells between 2 adjacent nrCMC fields, cultured onto microelectrode arrays and previously separated by a laser-dissected channel (230±10 &mgr;m) for nrMSC transplantation. Coatings in these channels were microabraded in a direction (1) parallel or (2) perpendicular to the channel or were (3) left unabraded to establish different cell patterns. Application of cells onto microabraded coatings resulted in anisotropic cell alignment within the channel. Application on unabraded coatings resulted in isotropic (random) alignment. After coculture, conduction across seeded nrMSCs occurred from day 1 (perpendicular and isotropic) or day 6 (parallel) onward. Conduction velocity across nrMSCs at day 10 was highest in the perpendicular (11±0.9 cm/sec; n=12), intermediate in the isotropic (7.1±1 cm/sec; n=11) and lowest in the parallel configuration (4.9±1 cm/sec; n=11) (P<0.01). nrCMCs and fibroblasts served as positive and negative control, respectively. Also, immunocytochemical analysis showed alignment-dependent increases in connexin 43 expression. In conclusion, forced alignment of nrMSCs undergoing cardiomyogenic differentiation affects the time course and degree of functional integration with surrounding cardiac tissue.

Resynchronization of Separated Rat Cardiomyocyte Fields With Genetically Modified Human Ventricular Scar Fibroblasts

Background— Nonresponse to cardiac resynchronization therapy is associated with the presence of slow or nonconducting scar tissue. Genetic modification of scar tissue, aimed at improving conduction, may be a novel approach to achieve effective resynchronization. Therefore, the feasibility of resynchronization with genetically modified human ventricular scar fibroblasts was studied in a coculture model. Methods and Results— An in vitro model was used to study the effects of forced expression of the myocardin (MyoC) gene in human ventricular scar fibroblasts (hVSFs) on resynchronization of 2 rat cardiomyocyte fields separated by a strip of hVSFs. Furthermore, the effects of MyoC expression on the capacity of hVSFs to serve as pacing sites were studied. MyoC-dependent gene activation in hVSFs was examined by gene and immunocytochemical analysis. Forced MyoC expression in hVSFs decreased dyssynchrony, expressed as the activation delay between 2 cardiomyocyte fields (control hVSFs 27.6±0.2 ms [n=11] versus MyoC-hVSFs 3.6±0.3 ms [n=11] at day 8, P<0.01). Also, MyoC-hVSFs could be stimulated electrically, which resulted in simultaneous activation of the 2 adjacent cardiomyocyte fields. Forced MyoC expression in hVSFs led to the expression of various connexin and cardiac ion channel genes. Intracellular measurements of MyoC-hVSFs coupled to surrounding cardiomyocytes showed strongly improved action potential conduction. Conclusions— Forced MyoC gene expression in hVSFs allowed electrical stimulation of these cells and conferred the ability to conduct an electrical impulse at high velocity, which resulted in resynchronization of 2 separated cardiomyocyte fields. Both phenomena appear mediated mainly by MyoC-dependent activation of genes that encode connexins, strongly enforcing intercellular electrical coupling.

Response to the Letter by Rose et al

We would like to reply to the letter by Drs Rose, Keating, and Backx,1 in which they gave their response to our recent publication in Circulation Research .2 In this study, we introduced alignment of transplanted stem cells as a novel determinant of functional integration of these cells with native cardiac tissue. In this study, we used neonatal rat mesenchymal stem cells (MSCs), which differentiated into functional cardiac cells after coculture with neonatal rat cardiomyocytes (CMCs). In their letter, Rose et al raise the important question of whether MSCs can differentiate into functional CMCs.1 However, we demonstrated that neonatal rat MSCs do differentiate into functional CMCs. Although we were one of the first to address the issue of cell alignment and stem cell transplantation, cardiomyogenic differentiation of MSCs …