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Role of Heme-Oxygenase-1 in Biology of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

ABSTRACT: Heme oxygenase-1 (HO-1, encoded by HMOX1) is a cytoprotective enzyme degrading heme into CO, Fe2+, and biliverdin. HO-1 was demonstrated to affect cardiac differentiation of murine pluripotent stem cells (PSCs), regulate the metabolism of murine adult cardiomyocytes, and influence regeneration of infarcted myocardium in mice. However, the enzyme's effect on human cardiogenesis and human cardiomyocytes' electromechanical properties has not been described so far. Thus, this study aimed to investigate the role of HO-1 in the differentiation of human induced pluripotent stem cells (hiPSCs) into hiPSC-derived cardiomyocytes (hiPSC-CMs). hiPSCs were generated from human fibroblasts and peripheral blood mononuclear cells using Sendai vectors and subjected to CRISPR/Cas9-mediated HMOX1 knock-out. After confirming lack of HO-1 expression on the protein level, isogenic control and HO-1-deficient hiPSCs were differentiated into hiPSC-CMs. No differences in differentiation efficiency and hiPSC-CMs metabolism were observed in both cell types. The global transcriptomic analysis revealed, on the other hand, alterations in electrophysiological pathways in hiPSC-CMs devoid of HO-1, which also demonstrated increased size. Functional consequences in changes in expression of ion channels genes were then confirmed by patch-clamp analysis. To the best of our knowledge, this is the first report demonstrating the link between HO-1 and electrophysiology in human cardiomyocytes.

SUBMITTER: Jez M

PROVIDER: S-EPMC8000937 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Role of Heme-Oxygenase-1 in Biology of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

Jeż Mateusz M Martyniak Alicja A Andrysiak Kalina K Mucha Olga O Szade Krzysztof K Kania Alan A Chrobok Łukasz Ł Palus-Chramiec Katarzyna K Sanetra Anna M AM Lewandowski Marian H MH Pośpiech Ewelina E Stępniewski Jacek J Dulak Józef J

Cells 20210301 3

Heme oxygenase-1 (HO-1, encoded by <i>HMOX1</i>) is a cytoprotective enzyme degrading heme into CO, Fe<sup>2+</sup>, and biliverdin. HO-1 was demonstrated to affect cardiac differentiation of murine pluripotent stem cells (PSCs), regulate the metabolism of murine adult cardiomyocytes, and influence regeneration of infarcted myocardium in mice. However, the enzyme's effect on human cardiogenesis and human cardiomyocytes' electromechanical properties has not been described so far. Thus, this study ...[more]

PMID: 33804563

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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

Role of Heme-Oxygenase-1 in Biology of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

Publications

Role of Heme-Oxygenase-1 in Biology of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

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