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Pompe disease results in a Golgi-based glycosylation deficit in human induced pluripotent stem cell-derived cardiomyocytes.

ABSTRACT: Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid ?-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were differentiated to cardiomyocytes (iPSC-CM). Pompe iPSC-CMs had undetectable GAA activity and pathognomonic glycogen-filled lysosomes. Nonetheless, Pompe and control iPSC-CMs exhibited comparable contractile properties in engineered cardiac tissue. Impaired autophagy has been implicated in Pompe skeletal muscle; however, control and Pompe iPSC-CMs had comparable clearance rates of LC3-II-detected autophagosomes. Unexpectedly, the lysosome-associated membrane proteins, LAMP1 and LAMP2, from Pompe iPSC-CMs demonstrated higher electrophoretic mobility compared with control iPSC-CMs. Brefeldin A induced disruption of the Golgi in control iPSC-CMs reproduced the higher mobility forms of the LAMPs, suggesting that Pompe iPSC-CMs produce LAMPs lacking appropriate glycosylation. Isoelectric focusing studies revealed that LAMP2 has a more alkaline pI in Pompe compared with control iPSC-CMs due largely to hyposialylation. MALDI-TOF-MS analysis of N-linked glycans demonstrated reduced diversity of multiantennary structures and the major presence of a trimannose complex glycan precursor in Pompe iPSC-CMs. These data suggest that Pompe cardiomyopathy has a glycan processing abnormality and thus shares features with hypertrophic cardiomyopathies observed in the congenital disorders of glycosylation.

SUBMITTER: Raval KK

PROVIDER: S-EPMC4317045 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Pompe disease results in a Golgi-based glycosylation deficit in human induced pluripotent stem cell-derived cardiomyocytes.

Raval Kunil K KK Tao Ran R White Brent E BE De Lange Willem J WJ Koonce Chad H CH Yu Junying J Kishnani Priya S PS Thomson James A JA Mosher Deane F DF Ralphe John C JC Kamp Timothy J TJ

The Journal of biological chemistry 20141208 5

Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were diffe ...[more]

PMID: 25488666

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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

Pompe disease results in a Golgi-based glycosylation deficit in human induced pluripotent stem cell-derived cardiomyocytes.

Publications

Pompe disease results in a Golgi-based glycosylation deficit in human induced pluripotent stem cell-derived cardiomyocytes.

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