Novel in vitro cardiovascular constructs composed of vascular-like networks and cardiomyocytes.

The interaction between different cardiac cells has shown to be important for critical biological properties including cell survival, proliferation, differentiation and function. The improvement of culture conditions with different cell types and to study their effects on cardiomyocyte viability and functionality is essential. For practical applications including general toxicity testing, drug development and tissue engineering it is important to study whether co-cultures have additional advantages over cardiomyocyte monoculture. Two multicellular in vitro cardiovascular constructs devoid of added biomaterial were developed in this study. In the first construct, neonatal rat cardiomyocytes (CM) were seeded on vascular-like network formed by human umbilical vein endothelial cells (HUVEC) and human adipose stromal cells (hASC). In the second construct, CMs were seeded on vascular-like network formed by HUVECs and human foreskin fibroblasts. The ability of these two vascular-like networks to support the viability and functionality of CMs was analyzed. Different culture media compositions were evaluated to support the development of optimal cardiovascular construct. Our results demonstrate that both vascular-like networks markedly improved CM viability and functionality. In the constructs, co-localization of CMs and vascular-like networks was seen. Multicellular constructs also allowed synchronized contractility of CMs. Serum-free medium supplemented with vascular endothelial growth factor and basic fibroblast growth factor was found to provide the most optimal conditions for cardiovascular construct as an entity. In conclusion, when combining a vascular-like network with CMs, the viability and functionality of CMs was markedly improved. The results suggest that the cardiovascular constructs developed provide a promising new tool for the assessment of toxicological and safety pharmacological effects of compounds in vitro.

Cell model of catecholaminergic polymorphic ventricular tachycardia reveals early and delayed afterdepolarizations.

BACKGROUND: Induced pluripotent stem cells (iPSC) provide means to study the pathophysiology of genetic disorders. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant inherited ion channel disorder predominantly caused by mutations in the cardiac ryanodine receptor (RyR2). In this study the cellular characteristics of CPVT are investigated and whether the electrophysiological features of this mutation can be mimicked using iPSC -derived cardiomyocytes (CM). METHODOLOGY/PRINCIPAL FINDINGS: Spontaneously beating CMs were differentiated from iPSCs derived from a CPVT patient carrying a P2328S mutation in RyR2 and from two healthy controls. Calcium (Ca(2+)) cycling and electrophysiological properties were studied by Ca(2+) imaging and patch-clamp techniques. Monophasic action potential (MAP) recordings and 24h-ECGs of CPVT-P2328S patients were analyzed for the presence of afterdepolarizations. We found defects in Ca(2+) cycling and electrophysiology in CPVT CMs, reflecting the cardiac phenotype observed in the patients. Catecholaminergic stress led to abnormal Ca(2+) signaling and induced arrhythmias in CPVT CMs. CPVT CMs also displayed reduced sarcoplasmic reticulum (SR) Ca(2+) content, indicating leakage of Ca(2+) from the SR. Patch-clamp recordings of CPVT CMs revealed both delayed afterdepolarizations (DADs) during spontaneous beating and in response to adrenaline and also early afterdepolarizations (EADs) during spontaneous beating, recapitulating the changes seen in MAP and 24h-ECG recordings of patients carrying the same mutation. CONCLUSIONS/SIGNIFICANCE: This cell model shows aberrant Ca(2+) cycling characteristic of CPVT and in addition to DADs it displays EADs. This cell model for CPVT provides a platform to study basic pathology, to screen drugs, and to optimize drug therapy.

Electrical Field Stimulation with a Novel Platform: Effect on Cardiomyocyte Gene Expression but not on Orientation.

Electrical field stimulation has been shown to improve cardiac cell alignment and functional properties. In this study, neonatal rat cardiomyocytes were exposed to both long-term and short-term stimulation with the goal of investigating whether it is possible to achieve cell orientation and the maturation of cardiomyocytes with a novel, microelectrode array (MEA)-compatible electrical stimulation platform. Cells were viable after electrical stimulation, but no orientation or other morphological changes were observed. However, the electrode wires in MEA dishes affected the cell orientation. Cell contractions synchronized with pacing, but settled back to their original frequency in the absence of stimulation. The expression of genes encoding a gap junction protein connexin-43 (Cx-43), and contractile cardiac protein beta myosin heavy chain 7, was stronger in stimulated cells than in controls (p<0.05). In summary, the surface topography influenced to cardiomyocyte orientation, suggesting that the micro architecture of the biomaterials should be carefully designed for cell applications. However, as electrical stimulation and its duration affected gene expression of some main cardiac proteins, the stimulation system may prove useful to enhance the cardiac differentiation of stem cells.