Advanced maturation of human cardiac tissue grown from pluripotent stem cells

Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease1-6. However, the predictive power of these models is presently limited by the immature state of the cells1, 2, 5, 6. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force-frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease.The authors acknowledge funding support from the National Institutes of Health of the USA (NIBIB and NCATS grant EB17103 (G.V.-N.); NIBIB, NCATS, NIAMS, NIDCR and NIEHS grant EB025765 (G.V.-N.); NHLBI grants HL076485 (G.V.-N.) and HL138486 (M.Y.); Columbia University MD/PhD program (S.P.M., T.C.); University of Minho MD/PhD program (D.T.); Japan Society for the Promotion of Science fellowship (K.M.); and Columbia University Stem Cell Initiative (D.S., L.S., M.Y.). We thank S. Duncan and B. Conklin for providing human iPSCs, M.B. Bouchard for assistance with image and video analysis, and L. Cohen-Gould for transmission electron microscopy services.info:eu-repo/semantics/publishedVersio

Role of physiological ClC-1 Cl- ion channel regulation for the excitability and function of working skeletal muscle.

Electrical membrane properties of skeletal muscle fibers have been thoroughly studied over the last five to six decades. This has shown that muscle fibers from a wide range of species, including fish, amphibians, reptiles, birds, and mammals, are all characterized by high resting membrane permeability for Cl(-) ions. Thus, in resting human muscle, ClC-1 Cl(-) ion channels account for ∼80% of the membrane conductance, and because active Cl(-) transport is limited in muscle fibers, the equilibrium potential for Cl(-) lies close to the resting membrane potential. These conditions-high membrane conductance and passive distribution-enable ClC-1 to conduct membrane current that inhibits muscle excitability. This depressing effect of ClC-1 current on muscle excitability has mostly been associated with skeletal muscle hyperexcitability in myotonia congenita, which arises from loss-of-function mutations in the CLCN1 gene. However, given that ClC-1 must be drastically inhibited (∼80%) before myotonia develops, more recent studies have explored whether acute and more subtle ClC-1 regulation contributes to controlling the excitability of working muscle. Methods were developed to measure ClC-1 function with subsecond temporal resolution in action potential firing muscle fibers. These and other techniques have revealed that ClC-1 function is controlled by multiple cellular signals during muscle activity. Thus, onset of muscle activity triggers ClC-1 inhibition via protein kinase C, intracellular acidosis, and lactate ions. This inhibition is important for preserving excitability of working muscle in the face of activity-induced elevation of extracellular K(+) and accumulating inactivation of voltage-gated sodium channels. Furthermore, during prolonged activity, a marked ClC-1 activation can develop that compromises muscle excitability. Data from ClC-1 expression systems suggest that this ClC-1 activation may arise from loss of regulation by adenosine nucleotides and/or oxidation. The present review summarizes the current knowledge of the physiological factors that control ClC-1 function in active muscle

Pro-arrhythmic Effects of Low Plasma [K+] in Human Ventricle: An Illustrated Review

[EN] Potassium levels in the plasma, [Kþ]o, are regulated precisely under physiological conditions. However, increases (from approx. 4.5 to 8.0 mM) can occur as a consequence of, e.g., endurance exercise, ischemic insult or kidney failure. This hyperkalemic modulation of ventricular electrophysiology has been studied extensively. Hypokalemia is also common. It can occur in response to diuretic therapy, following renal dialysis, or during recovery from endurance exercise. In the human ventricle, clinical hypokalemia (e.g., [Kþ]o levels of approx. 3.0 mM) can cause marked changes in both the resting potential and the action potential waveform, and these may promote arrhythmias. Here, we provide essential background information concerning the main Kþ-sensitive ion channel mechanisms that act in concert to produce prominent short-term ventricular electrophysiological changes, and illustrate these by implementing recent mathematical models of the human ventricular action potential. Even small changes (~1 mM) in [Kþ]o result in significant alterations in two different Kþ currents, IK1 and HERG. These changes can markedly alter in resting membrane potential and/or action potential waveform in human ventricle. Specifically, a reduction in net outward transmembrane Kþ currents (repolarization reserve) and an increased substrate input resistance contribute to electrophysiological instability during the plateau of the action potential and may promote pro-arrhythmic early after-depolarizations (EADs). Translational settings where these insights apply include: optimal diuretic therapy, and the interpretation of data from Phase II and III trials for anti-arrhythmic drug candidates.In Valencia, this work was supported by: (i) the “Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016” from the Ministerio de Economía, Industria y Competitividad of Spain (DPI2016-75799-R) and AEI/FEDER, UE, and by the “Programa Prometeu (PROMETEU/2016/088) de la Conselleria d'Educació, Formació I Ocupació, Generalitat Valenciana”. and (v) GileadSciences, Ltd. Wayne Giles acknowledges receipt of financial support in the form of a salary award (Medical Scientist) from Alberta Innovates-Health Solutions, and operating funding from the Canadian Institutes for Health Research and the Heart and Stroke Foundation of Alberta.Trénor Gomis, BA.; Cardona-Urrego, KE.; Romero Pérez, L.; Gómez García, JF.; Saiz Rodríguez, FJ.; Rajamani, S.; Belardinelli, L.... (2018). Pro-arrhythmic Effects of Low Plasma [K+] in Human Ventricle: An Illustrated Review. Trends in Cardiovascular Medicine. 28(4):233-242. https://doi.org/10.1016/j.tcm.2017.11.002S23324228

The effects of progressive heart failure on the Na/K ATPase and intracellular calcium regulation

The aim of this thesis is to investigate the effects of progressive heart failure on the function of the Na/K ATPase and intracellular calcium regulation. Progressive heart failure was induced in male guinea pigs by constriction of the ascending aorta. Sham operations were carried out on the control group. Hearts were harvested and used for various experiments at 30, 60, and 150 days after the aortic constriction operation. Echocardiography was performed on unanaesthetized guinea pigs to monitor in vivo changes of contractile function. Significantly smaller fractional shortening was noted after aortic constriction although this remained stable during the progression of disease until the 150 day end point when fractional shortening decreased greatly and circulatory insufficiency presented. Heart weight : body weight ratio increased 60 days after the operation, but the lung weight : body weight ratio only increased at the 150 day end point. Electrophysiological measurements of action potential duration (APD), Ca transients, SR Ca content, and Na/K ATPase current were performed to investigate alterations of Ca regulation during the progression of heart failure using enzymatically isolated ventricular cardiac myocytes. In end-stage heart failure, APD remained prolonged and the function of other mechanisms all declined. These results supported the view that compensated hypertrophy developed at 60 days after aortic constriction, and decompensation with end stage heart failure occurred about 150 days after the operation. At the compensated hypertrophy stage, only Na/K ATPase pump function declined but other mechanisms were enhanced or were the same as the situation prior to the constriction. These results suggested that alteration of Na/K ATPase function might assist the compensation phase and may trigger the decompensatory progression. Since t-tubule remodelling was also observed early in the heart failure progression in other study, we used membrane staining with wheat germ agglutinin (WGA) to investigate the change of t-tubule density and regularity in guinea pigs with heart failure. Our results showed a decrease in density and disorganization with regular but narrower intervals of t-tubule arrangement. Immunocytochemical staining of the α1 and α2 isoforms of the Na/K ATPase was further carried out to study their distribution in the sarcolemma when the heart fails. The results showed decreased expression of both α1 and α2 isoforms, which is compatible with our functional findings of Na/K ATPase pump current decline. Furthermore, corresponding to WGA membrane staining, α2 isoforms were found to colocalize with t-tubules. These finding suggested that alteration of Na/K ATPase pump function occurs early in the progression of hypertrophy toward heart failure, and α2 isoforms may play a more prominent role in regulating Ca handling.Open Acces

Sex differences in the progression from cardiac hypertrophy towards heart failure

This thesis aims to investigate differential changes in Ca2+ and Na+ regulation during the development from cardiac hypertrophy to heart failure (HF) between sexes. Clinical evidences show females are more resistant to the development of cardiac hypertrophy and have better survival in HF than males. Oestrogen is postulated to provide cardioprotection although this is still under debate. This work used guinea pigs (GPs), a species with electrophysiology akin to human, that were subjected to aortic constriction (AC) to study the progression from pressure-overload cardiac hypertrophy to HF between sexes. Selected female animals underwent ovariectomy (OVx), mimicking postmenopausal status, to examine the effects of long-term deprivation of ovarian hormones. The effect of oestradiol supplementation was also investigated. Ventricular myocytes isolated from hearts at cardiac hypertrophy had prolonged action potential duration (APD), increased Ca2+ transient amplitudes and SR Ca2+ content, reduced Na+/K+ ATPase (NKA) function and increased late sodium current (INa,L). Fractional shortening (FS) remained unchanged in these hearts. Compromised FS with detrimental Ca2+ handling, more reduced NKA function and enhanced INa,L were noted at HF. Males showed earlier declined NKA function, more compromised FS and more detrimental Ca2+ handling than females at HF. Ventricular myocytes from OVx animals showed increased L-type Ca2+ channel current with gating shifts and larger window current, larger Ca2+ transient amplitudes, greater SR Ca2+ content, and increased Ca2+ sparks and waves. OVx myocytes showed more early and delayed afterdepolarisations (EADs and DADs) with DAD-induced extrasystoles following β-adrenergic stimulation. AC with OVx GPs showed more reduced FS, more dysregulated Ca2+ handling, more reduced NKA function and larger INa,L than AC females. In conclusion, females were more resistant to pressure-overload. Long-term deprivation of ovarian hormones abolishes the slower onset of HF in females, and provides pro-arrhythmic substrates to females. Oestradiol supplementation offered protective effects on OVx GPs.Open Acces

Changes in cardiomyocyte structure and cAMP/cGMP signalling during heart failure

The contractile function of the heart depends on efficient β adrenergic receptor (βAR) signalling which involves cycling nucleotides as second messengers. Correct secondary messenger signalling is only possible in healthy, well structured cardiac myocytes. Of the three βAR subtypes present in human cardiomyocytes β1AR and β2AR classically signal via 3'-5' cyclic adenosine monophosphate (cAMP) to regulate contraction after catecholamine administration, whereby the second isoform may also be cardioprotective. The far less characterised β3AR has been controversially associated to both increasing contraction through cAMP and protecting the heart through 3'-5' cyclic guanosine monophosphate (cGMP) signalling. During the progression of heart failure following myocardial infarction (MI) both the normal cell structure and the regulation of cAMP and cGMP signalling are changed. This happens in part due to changes in catecholaminergic stimulation of the βARs and in mechanical load, as well as due to a progressive development of hypertrophy. Some of the alterations initially appear to be of a compensatory nature but escalate into HF by worsening cardiomyocyte function and cell survival. The work presented here (1) investigates the structural integrity of healthy, isolated, single cardiomyocytes by looking at the surface topography via Scanning Ion Conductance Microscopy (SICM) imaging and by examining the internal Transverse Axial Tubule (TAT) network via confocal imaging; (2) elucidates the cyclic nucleotide response to catecholamine stimulation following either global (in the solution) or local (in the SICM pipette) stimulation of either β2ARs or β3ARs and measuring either cAMP or cGMP levels via Förster Resonance Energy Transfer (FRET) sensors in a combined FRET/SICM imaging setup; (3) determines how both the structure and β2AR and β3AR dependent second messenger signalling change in a progressive rat model of HF 4, 8 and 16 weeks after the induction of chronic MI. The major findings of the presented work are as follows: In control cardiomyocytes the structure is highly intricate with regular Z-grooves and crest areas. In MI cells the normal suface topography progressively deteriorates, with the eventual disappearance of Z-grooves by week 16, which correlates with the disorganisation of the cardiomyocyte’s internal transverse axial tubule (TAT) network of T-tubules emanating from the cell surface and traversing into the cell centre. This is accompanied by the gradual redistribution of β2ARs from their normal position inside the T-tubules to the unstructured areas on the cardiomyocyte membrane. The regularity and density of the TAT network is already severely compromised at 4 weeks post MI; at the same time a significant drop in the expression of the structural protein Junctophilin 2 (JPH2) occurs. At 4 and 8 weeks post MI a potentially compensatory increase in the number of longitudinal elements takes place which was no longer detectable at 16 weeks. The production of cAMP following local stimulation of β2ARs in the T-tubule openings was already suppressed at 4 weeks post MI and a β2AR response becomes detectable after local stimulation at the cell crests (areas between Z-grooves) at 8 weeks post MI. At 16 weeks post MI the β2AR-dependent cAMP level following both global and local stimulations was reduced due to an overall decrease in the adenylate cyclase (AC) activity. The production of the second cyclic nucleotide, cGMP, following β3AR stimulation is evident in control cells and to a significantly lesser extent in myocytes isolated from hearts at the end stage of HF. These β3AR-cGMP levels were degraded mainly by phosphodiesterases (PDE) 2 and 5. Local stimulation through the SICM pipette reveals that functional β3ARs are primarily localized inside T-tubules in control cells but redistribute equally in between T-tubules and crests in cells isolated from failing hearts. To improve the accuracy and reliability of local application of agonists via the SICM nanopipette voltage was applied to the pipette, as opposed to previously employed displacement of the liquid in the pipette via air pressure. Mathematical modelling served to determine the correct settings for this voltage driven application. It shows that the SICM nanopipette can reliably and precisely unload the βAR agonist ISO onto the nanoscale structure of cardiomyocytes via voltage.Open Acces