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Activation of the unfolded protein response downregulates cardiac ion channels in human induced pluripotent stem cell-derived cardiomyocytes.

ABSTRACT: RATIONALE:Heart failure is characterized by electrical remodeling that contributes to arrhythmic risk. The unfolded protein response (UPR) is active in heart failure and can decrease protein levels by increasing mRNA decay, accelerating protein degradation, and inhibiting protein translation. OBJECTIVE:Therefore, we investigated whether the UPR downregulated cardiac ion channels that may contribute to arrhythmogenic electrical remodeling. METHODS:Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to study cardiac ion channels. Action potentials (APs) and ion channel currents were measured by patch clamp recording. The mRNA and protein levels of channels and the UPR effectors were determined by quantitative RT-PCR and Western blotting. Tunicamycin (TM, 50?ng/mL and 5??g/mL), GSK2606414 (GSK, 300?nmol/L), and 4?8C (5??mol/L) were utilized to activate the UPR, inhibit protein kinase-like ER kinase (PERK) and inositol-requiring protein-1 (IRE1), respectively. RESULTS:TM-induced activation of the UPR caused significant prolongation of the AP duration (APD) and a reduction of the maximum upstroke velocity (dV/dtmax) of the AP phase 0 in both acute (20-24?h) and chronic treatment (6?days). These changes were explained by reductions in the sodium, L-type calcium, the transient outward and rapidly/slowly activating delayed rectifier potassium currents. Nav1.5, Cav1.2, Kv4.3, and KvLQT1 channels showed concomitant reductions in mRNA and protein levels under activated UPR. Inhibition of PERK or IRE1 shortened the APD and reinstated dV/dtmax. The PERK branch regulated Nav1.5, Kv4.3, hERG, and KvLQT1. The IRE1 branch regulated Nav1.5, hERG, KvLQT1, and Cav1.2. CONCLUSIONS:Activated UPR downregulates all major cardiac ion currents and results in electrical remodeling in hiPSC-CMs. Both PERK and IRE1 branches downregulate Nav1.5, hERG, and KvLQT1. The PERK branch specifically downregulates Kv4.3, while the IRE1 branch downregulates Cav1.2. Therefore, the UPR contributed to electrical remodeling, and targeting the UPR might be anti-arrhythmic.

SUBMITTER: Liu M

PROVIDER: S-EPMC5861016 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Activation of the unfolded protein response downregulates cardiac ion channels in human induced pluripotent stem cell-derived cardiomyocytes.

Liu Man M Shi Guangbin G Zhou Anyu A Rupert Cassady E CE Coulombe Kareen L K KLK Dudley Samuel C SC

Journal of molecular and cellular cardiology 20180221

<h4>Rationale</h4>Heart failure is characterized by electrical remodeling that contributes to arrhythmic risk. The unfolded protein response (UPR) is active in heart failure and can decrease protein levels by increasing mRNA decay, accelerating protein degradation, and inhibiting protein translation.<h4>Objective</h4>Therefore, we investigated whether the UPR downregulated cardiac ion channels that may contribute to arrhythmogenic electrical remodeling.<h4>Methods</h4>Human induced pluripotent s ...[more]

PMID: 29474817

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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.

Project description:Stem cell therapy has the potential to regenerate cardiac function after myocardial infarction. In this study, we sought to examine if fibrin microthread technology could be leveraged to develop a contractile fiber from human pluripotent stem cell derived cardiomyocytes (hPS-CM). hPS-CM seeded onto fibrin microthreads were able to adhere to the microthread and began to contract seven days after initial seeding. A digital speckle tracking algorithm was applied to high speed video data (>60 fps) to determine contraction behaviour including beat frequency, average and maximum contractile strain, and the principal angle of contraction of hPS-CM contracting on the microthreads over 21 days. At day 7, cells seeded on tissue culture plastic beat at 0.83 ± 0.25 beats/sec with an average contractile strain of 4.23±0.23%, which was significantly different from a beat frequency of 1.11 ± 0.45 beats/sec and an average contractile strain of 3.08±0.19% at day 21 (n = 18, p < 0.05). hPS-CM seeded on microthreads beat at 0.84 ± 0.15 beats/sec with an average contractile strain of 3.56±0.22%, which significantly increased to 1.03 ± 0.19 beats/sec and 4.47±0.29%, respectively, at 21 days (n = 18, p < 0.05). At day 7, 27% of the cells had a principle angle of contraction within 20 degrees of the microthread, whereas at day 21, 65% of hPS-CM were contracting within 20 degrees of the microthread (n = 17). Utilizing high speed calcium transient data (>300 fps) of Fluo-4AM loaded hPS-CM seeded microthreads, conduction velocities significantly increased from 3.69 ± 1.76 cm/s at day 7 to 24.26 ± 8.42 cm/s at day 21 (n = 5-6, p < 0.05). hPS-CM seeded microthreads exhibited positive expression for connexin 43, a gap junction protein, between cells. These data suggest that the fibrin microthread is a suitable scaffold for hPS-CM attachment and contraction. In addition, extended culture allows cells to contract in the direction of the thread, suggesting alignment of the cells in the microthread direction.

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Activation of the unfolded protein response downregulates cardiac ion channels in human induced pluripotent stem cell-derived cardiomyocytes.

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Activation of the unfolded protein response downregulates cardiac ion channels in human induced pluripotent stem cell-derived cardiomyocytes.

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