Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O₂) environment, whereas, their in-vitro expansion is typically performed under normoxia (20–21% O₂). The comparative information about the effects of low and normal O₂ levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O₂) and normoxia (21% O₂). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p?<?0.05) improved, thus, yielding a significantly (p?<?0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O₂ conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p?<?0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

     

Repolarisation and vulnerability to re-entry in the human heart with short QT syndrome arising from KCNQ1 mutation--a simulation study

Idiopathic short QT syndrome (SQTS) is a recently identified, genetically heterogeneous condition characterised by abbreviated QT intervals and an increased susceptibility to arrhythmia and sudden death. This simulation study identifies mechanisms by which cellular electrophysiological changes in the SQT2 (slow delayed rectifier, I_Ks, -linked) SQTS variant increases arrhythmia risk. The channel kinetics of the V307L mutation of the KCNQ1 subunit of the I_Ks channel were incorporated into human ventricular action potential (AP) models and into 1D and 2D transmural tissue simulations. Incorporating the V307L mutation into simulations reproduced defining features of the SQTS: abbreviation of the QT interval, and increases in T wave amplitude and T_peak–T_end duration. In the single-cell model, the V307L mutation abbreviated ventricular cell AP duration at 90% repolarisation (APD₉₀) and increased the maximal transmural voltage heterogeneity (δV) during APs; this resulted in augmented transmural heterogeneity of APD₉₀ and of the effective refractory period (ERP). In the intact tissue model, the vulnerable window for unidirectional conduction block was also increased. In 2D tissue the V307L mutation facilitated and maintained reentrant excitation. Thus, in SQT2 increases in transmural heterogeneity of APD, δV, ERP and an increased vulnerable window for unidirectional conduction block generate an electrical substrate favourable to reentrant arrhythmia.     

Modelling changes in transmural propagation and susceptibility to arrhythmia induced by volatile anaesthetics in ventricular tissue

Volatile anaesthetics such as halothane, isoflurane and sevoflurane inhibit membrane currents contributing to the ventricular action potential. Transmural variation in the extent of current blockade induces differential effects on action potential duration (APD) in the endocardium and epicardium which may be pro-arrhythmic. Biophysical modelling techniques were used to simulate the functional impact of anaesthetic-induced blockade of membrane currents on APD and effective refractory period (ERP) in rat endocardial and epicardial cell models. Additionally, the transmural conduction of excitation waves in 1-dimensional cell arrays, the tissue's vulnerability to arrhythmogenesis and dynamic behaviour of re-entrant excitation in 2-dimensional cell arrays were studied. Simulated anaesthetic exposure reduced APD and ERP in both epicardial and endocardial cell models. The reduction in APD was greater in endocardial than epicardial cells, reducing transmural APD dispersion consistent with experimental data. However, the transmural ERP dispersion was augmented. All three anaesthetics increased the width of the tissue's vulnerable window during which a premature stimulus could induce unidirectional conduction block but only halothane reduced the critical size of ventricular substrates necessary to initiate and sustain re-entrant excitation. All three anaesthetics accelerated the rate of re-entrant excitation waves, but only halothane prolonged the lifespan of re-entry. These data illustrate in silico, that modest changes in ion channel conductance abbreviate rat ventricular APD and ERP, reduce transmural APD dispersion, but augment transmural ERP dispersion. These changes collectively enhance the propensity for arrhythmia generation and provide a substrate for re-entry circuits with a longer half life than in control conditions.     

Mechanisms underlying adaptation of action potential duration by pacing rate in rat myocytes

Heart rate is an essential determinant of cardiac performance. In rat ventricular myocytes, a sudden increase in rate yields to a prolongation of the action potential duration (APD). The mechanism underlying this prolongation is controversial: it has been proposed that the longer APD is due to either: (1) a decrease in K⁺ currents only or (2) an increase in Ca²⁺ current only. The aim of this study was to quantitatively investigate the contribution of Ca²⁺ and K⁺ currents in the adaptation of APD to pacing rate. Simulation using a mathematical model of ventricular rat cardiac cell model [Pandit, S.V., Clark, R.B., Giles, W.R., Demir, S.S., 2001. A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes. Biophys. J. 81, 3029–3051] predicted a role in the prolongation of APD for K⁺ currents only. In patch clamp experiments, increasing the pacing rate leads to a significant increase in APD in both control and detubulated myocytes, although it was more marked in control than detubulated myocytes. Supporting the model prediction, we observed that increasing stimulation frequency leads to a decrease in K⁺ currents in voltage clamped rat ventricular myocytes (square and action potential waveforms), and to a similar extent in both cell types. We have also observed that frequency-dependent facilitation of Ca²⁺ current occurred in control cells but not in detubulated cells (square and action potential waveforms). From these experiments, we calculated that the relative contribution of Ca²⁺ and K⁺ currents to the longer APD following an increase in pacing rate is 65% and 35%, respectively. Therefore, in contrast to the model prediction, Ca²⁺ current has a significant role in the adaptation of APD to pacing rate. Finally, we have introduced a simplistic modification to the Pandit's model to account for the frequency-dependent facilitation of Ca²⁺ current.     

Effects of streptozotocin-induced diabetes on connexin43 mRNA and protein expression in ventricular muscle

Abnormal QT prolongation with the associated arrhythmias is a significant predictor of mortality in diabetic patients. Gap junctional intercellular communication allows electrical coupling between heart muscle cells. The effects of streptozotocin (STZ)-induced diabetes mellitus on the expression and distribution of connexin 43 (Cx43) in ventricular muscle have been investigated. Cx43 mRNA expression was measured in ventricular muscle by quantitative PCR. The distribution of total Cx43, phosphorylated Cx43 (at serine 368) and non-phosphorylated Cx43 was measured in ventricular myocytes and ventricular muscle by immunocytochemistry and confocal microscopy. There was no significant difference in Cx43 mRNA between diabetic rat ventricle and controls. Total and phosphorylated Cx43 were significantly increased in ventricular myocytes and ventricular muscle and dephosphorylated Cx43 was not significantly altered in ventricular muscle from diabetic rat hearts compared to controls. Disturbances in gap junctional intercellular communication, which in turn may be attributed to alterations in balance between total, phosphorylated and dephosporylated Cx43, might partly underlie prolongation of QRS and QT intervals in diabetic heart.     

A mathematical model of action potentials of mouse sinoatrial node cells with molecular bases

Genetically modified mice are popular experimental models for studying the molecular bases and mechanisms of cardiac arrhythmia. A postgenome challenge is to classify the functional roles of genes in cardiac function. To unveil the functional role of various genetic isoforms of ion channels in generating cardiac pacemaking action potentials (APs), a mathematical model for spontaneous APs of mouse sinoatrial node (SAN) cells was developed. The model takes into account the biophysical properties of membrane ionic currents and intracellular mechanisms contributing to spontaneous mouse SAN APs. The model was validated by its ability to reproduce the physiological exceptionally short APs and high pacing rates of mouse SAN cells. The functional roles of individual membrane currents were evaluated by blocking their coding channels. The roles of intracellular Ca(2+)-handling mechanisms on cardiac pacemaking were also investigated in the model. The robustness of model pacemaking behavior was evaluated by means of one- and two-parameter analyses in wide parameter value ranges. This model provides a predictive tool for cellular level outcomes of electrophysiological experiments. It forms the basis for future model development and further studies into complex pacemaking mechanisms as more quantitative experimental data become available.     

Atrial proarrhythmia due to increased inward rectifier current (IK1) arising from KCNJ2 mutation – A simulation study

Atrial fibrillation (AF) has been linked to increased inward rectifier potassium current, I_K1, either due to AF-induced electrical remodelling, or from functional changes due to the Kir2.1 V93I mutation. The aim of this simulation study was to identify at cell and tissue levels' mechanisms by which increased I_K1 facilitates and perpetuates AF. The Courtemanche et al. human atrial cell action potential (AP) model was modified to incorporate reported changes in I_K1 induced by the Kir2.1 V93I mutation in both heterozygous (Het) and homozygous (Hom) mutant forms. The modified models for wild type (WT), Het and Hom conditions were incorporated into homogeneous 1D, 2D and 3D tissue models. Restitution curves of AP duration (APD), effective refractory period (ERP) and conduction velocity (CV) were computed and both the temporal and the spatial vulnerability of atrial tissue to re-entry were measured. The lifespan and tip meandering pattern of re-entry were also characterised. For comparison, parallel simulations were performed by incorporating into the Courtmanche et al. model a linear increase in maximal I_K1 conductance. It was found that the gain-in-function of V93I ‘mutant’ I_K1 led to abbreviated atrial APs and flattened APD, ERP and CV restitution curves. It also hyperpolarised atrial resting membrane potential and slowed down intra-atrial conduction. V93I ‘mutant’ I_K1 reduced the tissue's temporal vulnerability but increased spatial vulnerability to initiate and sustain re-entry, resulting in an increased overall susceptibility of atrial tissue to arrhythmogenesis. In the 2D model, spiral waves self-terminated for WT (lifespan < 3.3 s) tissue, but persisted in Het and Hom tissues for the whole simulation period (lifespan > 10 s). The tip of the spiral wave meandered more in WT tissue than in Het and Hom tissues. Increased I_K1 due to augmented maximal conductance produced similar results to those of Het and Hom Kir2.1 V93I mutant conditions. In the 3D model the dynamic behaviour of scroll waves was stabilized by increased I_K1. In conclusion, increased I_K1 current, either by the Kir2.1 V93I mutation or by augmented maximal conductance, increases atrial susceptibility to arrhythmia by increasing the lifespan of re-entrant spiral waves and the stability of scroll waves in 3D tissue, thereby facilitating initiation and maintenance of re-entrant circuits.