Investigation in the Role of Lamin A (LMNA) Deficiency in Heart Block and Dilated Cardiomyopathy (DCM) Using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes
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1
The University of Hong Kong, Cardiology Division, Department of Medicine, China
Motivation Ð Background and Aims
Mutations in LMNA gene that encodes two major intermediate filament protein lamin A and C isoforms, cause a large range of laminopathies adversely affect striated tissue and vascular system. Lamin A/C is an essential component in nuclear matrix for maintenance of nuclear architecture. Clinical manifestations indicate that cardiac laminopathy was associated with prognosis and an early onset of atrial fibrillation (AF), AV blocks (AVB) and dilated cardiomyopathy (DCM). Nevertheless, there is a lack of study dissecting the direct role of LMNA in cardiac electrophysiology. In current study, we aimed to investigate the role of LMNA in disease progression from cardiac arrhythmia, electrical signal propagation defeats and subsequently development of DCM using human induced pluripotent stem cell (hiPSC)-cardiomyocytes (CMs).
Material and Methods
To recapitulate arrhythmic and DCM phenotypes mediated by laminopathy, the current study relied on our previously generated hiPSC of a patient bearing LMNAR225X/WT 1 and another lentiviral shRNA LMNA knockdown (shLMNA)-hiPSC cell line replicating the haploinsufficient phenotype of LMNAR225X/W. The role of LMNA on the cardiac arrthymic and hypertrophic phenotypes were investigated in 1) cytoskeletal organization that affects force generation; 2) transcriptional level of junction protein connexin; and 3) electrical-contraction coupling events in terms of calcium handling and contractility; and 4) adrenergic responses and rate dependency of QTc to evaluate rate adaptiation. Hypertrophic stress was induced by treatment of the differentiated CMs with 1 mM angiotensin-II (Ang-II) and 10 µM endothelin-1 (Et-1) for 48 hours in serum free medium. Multielectrode array (MEA) analysis was carried out to perform electrophysiological studies on the hiPSC-CMs. Calcium transient was recorded by fluorescence imaging of CMC stained with FURA-2 while video edge detector was used for measurement of the contractile force simultaneously. Conduction system gene expressions were also studied to determine tight junction abundance during heart development as reflected by Day 30 of cardiac differentiation.
Results
Pathological microstructure analysis by electronic microscopy revealed that the shLMNA-hiPSC-CMCs showed disorganized sarcomeric structure with a condensed Z-band, thus affecting force generation. The loss of nulcear integrity was also observed in shLMNA cell as a typical phenotype of laminopathy (Figure A). Relative gene expression of connexin 40 (CX40) /troponin-T (cTnT) in shLMNA was significantly reduced which indicated the reduced abundance of conduction system-specific tight junction (n=4; ****p<0.0001; Figure B). Regarding electrical-contraction coupling properties, the diastolic intracellular calcium was remarkedly elevated, together with the significantly depleted upstroke and decay velocity of calcium transients in the shLMNA (n=8; *p<0.05). A corresponding impaired contraction force generation as reflected by the significant suppression for 2.7-fold in % of cell shortening (n=14; ****p<0.0001) (Figure C). Hypertrophic stimulus by Ang-II and Et-1 to further overload the calcium content of iPSC-CMs that remarkedly impaired the calcium transients propagation. MEA analysis indicated the shLMNA-hiPSC-CMC were refractory to beta-adrenergic stimulation by Isoproterenol (ISO). The beat rate of shLMNA cells to ISO treatment ranged from 100-200 nM was significantly lower than GFP cells by 20-28% (n=3; *p<0.05; **p<0.001). A further significant loss in rate dependency of field potential duration (FPD) shortening was observed with a failure in field potential duration (FPD) shortening upon ISO treatment from the minimal dose (Figure D).
Discussion
With regard to structural basis of cardiac cells bearing mutated LMNA, the formation of weak lamina diminishes cardiac mechanical stability which is critical during contraction2. Loss of lamina integrity would destroy cytoskeletal structure connecting nucleus to cell membrane surface that might affect proper ion channel function. The Z band is composed of desmin which act as an intermediate filament to interconnect nuclear lamina to sarcomere and cell surface. Visualization of F-actin as revealed by immunofluorescence stain indicated such intermediate filament interacting with lamin and desmin was also disorganized, thus further reduced nuclear integrity in the contracting striated tissue and imparied force generation. In fact, the high diastolic calcium in shLMNA-hiPSC-CMC could account for the heart block since resting membrane potential elevated far from the depolarization threshold, thus reducing the excitability of cells to propagate action potential that caused earlier block when intercellular coupling was reduced. This could be explained by intracellular calcium concentrationÊaffected conduction via calcium-dependent inactivation of L-type calcium channel (ICa(L)) 3 or maybe due to defeats in (SR)-mediated calcium handling. Our results indicated blunted response to ISO was probably due to such reason when the cells are under stress condition that required rapid response of cells excitability.
Conclusion
The loss of nuclear integrity due to lamin haploinsufficiency, accelerate DCM progression due to premature cell aging and cell death associated with mechanical stress to nuclear structure in cardiac cell. Heart block phenotype of cardiolaminopathy is probably due to impaired SR (probably those linked with nuclear laminar) calcium handling properties and elevated diastolic calcium that contributes to the reduced cell excitability as reflected in irresponsive to beta-adrenergic stimulation.
References
1. Siu CW, Lee YK, Ho JC, Lai WH, Chan YC, Ng KM, Wong LY, Au KW, Lau YM, Zhang J, Lay KW, Colman A, Tse HF. Modeling of lamin A/C mutation premature cardiac aging using patient-specific induced pluripotent stem cells. Aging (Albany NY). Nov 2012;4(11):803-822.
2. Vaikhanskaya T, Sivitskaya L, Danilenko N, Davydenko O, Kurushka T, Sidorenko I. LMNA-related dilated cardiomyopathy. Oxf Med Case Reports. Sep 2014;2014(6):102-104.
3. Shaw RM, Rudy Y. Ionic mechanisms of propagation in cardiac tissue. Roles of the sodium and L-type calcium currents during reduced excitability and decreased gap junction coupling. Circ Res. Nov 1997;81(5):727-741.
Acknowledgements
This work was supported by grants from the Hong Kong Research Grant Council, Theme-Based Research Theme (T12-705/11 to Prof. Tse and Dr. Siu); Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01020106, to MAE).
Keywords:
multielectrode array,
human induced pluripotent stem cell,
Calcium transient,
cardiac laminopathy
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Lee
YK,
Ran
XR,
Jiang
Y and
Tse
HF
(2016). Investigation in the Role of Lamin A (LMNA) Deficiency in Heart Block and Dilated Cardiomyopathy (DCM) Using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00033
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Yee K Lee, The University of Hong Kong, Cardiology Division, Department of Medicine, Hong Kong SAR, China, carol801@hku.hk