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GLUT4 expression and glucose transport in human induced pluripotent stem cell-derived cardiomyocytes.

ABSTRACT: Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) have the potential to transform regenerative cardiac medicine and the modelling of cardiac disease. This is of particular importance in the context of diabetic cardiomyopathy where diabetic individuals exhibit reduced cardiac diastolic contractile performance in the absence of vascular disease, significantly contributing towards high cardiovascular morbidity. In this study, the capacity of iPSC-CM to act as a novel cellular model of cardiomyocytes was assessed. The diabetic phenotype is characterised by insulin resistance, therefore there was a specific focus upon metabolic parameters. Despite expressing crucial insulin signalling intermediates and relevant trafficking proteins, it was identified that iPSC-CM do not exhibit insulin-stimulated glucose uptake. iPSC-CM are spontaneously contractile however contraction mediated uptake was not found to mask any insulin response. The fundamental limitation identified in these cells was a critical lack of expression of the insulin sensitive glucose transporter GLUT4. Using comparative immunoblot analysis and the GLUT-selective inhibitor BAY-876 to quantify expression of these transporters, we show that iPSC-CM express high levels of GLUT1 and low levels of GLUT4 compared to primary cardiomyocytes and cultured adipocytes. Interventions to overcome this limitation were unsuccessful. We suggest that the utility of iPSC-CMs to study cardiac metabolic disorders may be limited by their apparent foetal-like phenotype.

SUBMITTER: Bowman PRT

PROVIDER: S-EPMC6657831 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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GLUT4 expression and glucose transport in human induced pluripotent stem cell-derived cardiomyocytes.

Bowman Peter R T PRT Smith Godfrey L GL Gould Gwyn W GW

PloS one 20190725 7

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) have the potential to transform regenerative cardiac medicine and the modelling of cardiac disease. This is of particular importance in the context of diabetic cardiomyopathy where diabetic individuals exhibit reduced cardiac diastolic contractile performance in the absence of vascular disease, significantly contributing towards high cardiovascular morbidity. In this study, the capacity of iPSC-CM to act as a novel cellular model of ...[more]

PMID: 31344028

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Project description:RationaleCardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis, the key process underlying excitation-contraction coupling.ObjectiveWe investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs).Methods and resultsWe differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes, spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore, in contrast with hESC-derived cardiomyocytes, ryanodine did not reduce the amplitudes, maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition, spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2), sacroplasmic recticulum calcium-ATPase (SERCA), junction (Jun) and triadin (TRDN), were significantly lower in hiPSC than in hESCs.ConclusionsThe results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts.

Project description:Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alteration has been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes (CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells (iPSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since iPSC preserve the entire patients' genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with iPSC.

Dataset Information

GLUT4 expression and glucose transport in human induced pluripotent stem cell-derived cardiomyocytes.

Publications

GLUT4 expression and glucose transport in human induced pluripotent stem cell-derived cardiomyocytes.

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