Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) have been widely proposed as in vitro models of myocardial physiology and disease. A significant obstacle, however, is their immature phenotype. We hypothesised that Ca2+ cycling of iPSC-CM is influenced by culture conditions and can be manipulated to obtain a more mature cellular behaviour. To test this hypothesis we seeded iPSC-CM onto fibronectin coated microgrooved polydimethylsiloxane (PDMS) scaffolds fabricated using photolithography, or onto unstructured PDMS membrane. After two weeks in culture, the structure and function of iPSC-CM were studied. PDMS microgrooved culture substrates brought about cellular alignment (p < 0.0001) and more organised sarcomere. The Ca2+ cycling properties of iPSC-CM cultured on these substrates were significantly altered with a shorter time to peak amplitude (p = 0.0002 at 1 Hz), and more organised sarcoplasmic reticulum (SR) Ca2+ release in response to caffeine (p < 0.0001), suggesting improved SR Ca2+ cycling. These changes were not associated with modifications in gene expression. Whilst structured tissue culture may make iPSC-CM more representative of adult myocardium, further construct development and characterisation is required to optimise iPSC-CM as a model of adult myocardium.

The phospholipase neutral sphingomyelinase (N-SMase) has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer's, atherosclerosis, heart failure, ischemia/reperfusion damage, or combined pituitary hormone deficiency. Although activation of N-SMase by the proinflammatory cytokine TNF was described almost two decades ago, the underlying signaling pathway is unresolved. Here, we identify the Polycomb group protein EED (embryonic ectodermal development) as an interaction partner of nSMase2. In yeast, the N terminus of EED binds to the catalytic domain of nSMase2 as well as to RACK1, a protein that modulates the activation of nSMase2 by TNF in concert with the TNF receptor 1 (TNF-R1)-associated protein FAN. In mammalian cells, TNF causes endogenous EED to translocate from the nucleus and to colocalize and physically interact with both endogenous nSMase2 and RACK1. As a consequence, EED and nSMase2 are recruited to the TNF-R1•FAN•RACK1-complex in a timeframe concurrent with activation of nSMase2. After knockdown of EED by RNA interference, the TNF-dependent activation of nSMase2 is completely abrogated, identifying EED as a protein that both physically and functionally couples TNF-R1 to nSMase2, and which therefore represents the "missing link" that completes one of the last unresolved signaling pathways of TNF-R1.embryonic ectodermal development | immune response | inflammation N eutral sphingomyelinases (N-SMases) mediate stress-induced ceramide generation and participate in inflammation, development, cellular growth, differentiation and death, heart failure, ischemia/reperfusion damage, atherosclerosis, and Alzheimer's disease (1, 2). They are acutely activated by TNF, a major mediator of inflammatory and immunoregulatory responses (3, 4). Out of the three N-SMase genes cloned in mammals, nSMase2 corresponds to the biochemically characterized N-SMase. It is a membrane-bound protein with two putative N-terminal hydrophobic membrane-anchoring domains, a collagen-like linker region, and a C-terminal catalytic domain (Fig. 1A) (5). nSMase2 has been linked to cell cycle regulation and contact inhibition (1), late embryonal and postnatal development (5, 6), exosome secretion (7), and Alzheimer's disease and combined pituitary hormone deficiency (1, 6). In response to TNF, nSMase2 is important for inflammatory signaling, cell adhesion and migration, endothelial regulation, cell death, and cutaneous barrier repair (5,(8)(9)(10)(11). Even though N-SMase activation by TNF has been reported since almost 20 years ago (3), the corresponding signaling pathway is not fully resolved. In response to TNF, nSMase2 is activated exclusively by the 55-kDa receptor TNF receptor 1 (TNF-R1) (10). We have previously defined a neutral sphingomyelinase activation domain (NSD) within TNF-R1 (12) that serves as a binding site for the protein FAN (factor associated with N-SMase activation) (13). FAN recruits the WD repeat protein...

SummaryCardiomyocytes from human embryonic stem cells (hESC-CMs) and induced pluripotent stem cells (hiPSC-CMs) represent new models for drug discovery. Although hypertrophy is a high-priority target, we found that hiPSC-CMs were systematically unresponsive to hypertrophic signals such as the α-adrenoceptor (αAR) agonist phenylephrine (PE) compared to hESC-CMs. We investigated signaling at multiple levels to understand the underlying mechanism of this differential responsiveness. The expression of the normal α1AR gene, ADRA1A, was reversibly silenced during differentiation, accompanied by ADRA1B upregulation in either cell type. ADRA1B signaling was intact in hESC-CMs, but not in hiPSC-CMs. We observed an increased tonic activity of inhibitory kinase pathways in hiPSC-CMs, and inhibition of antihypertrophic kinases revealed hypertrophic increases. There is tonic suppression of cell growth in hiPSC-CMs, but not hESC-CMs, limiting their use in investigation of hypertrophic signaling. These data raise questions regarding the hiPSC-CM as a valid model for certain aspects of cardiac disease.

No abstract

No abstract