From Hair to Heart: Hair-Follicle-Associated Pluripotent (HAP) Stem Cells Can Simultaneously Differentiate Into Mature Cardiomyocytes and Atrial Myocytes

Hair-follicle-associated pluripotent (HAP) stem cells express nestin, and are located in the bulge area of hair follicles and can differentiate to numerous types of cells. In the present study, we demonstrate that rat HAP stem cells simultaneously differentiated to mature cardiomyocytes and atrial myocytes. The addition of isoproterenol, activin A, bone morphogenetic protein 4 (BMP 4), basic fibroblast growth factor (bFGF), and cyclosporin A (CSA), induced simultaneously differentiation of HAP stem cells to c-kit-positive cardiomyocytes and MLC-2a-expressing atrial myocytes. The results of the present study suggest that HAP stem cells differentiating to cardiomyocytes and atrial myocytes have future clinical potential for heart regeneration.

Fibroblast Growth Factor 21 Protects Against Atrial Remodeling via Reducing Oxidative Stress

Aim: The structural and electrical changes in the atrium, also known as atrial remodeling, are the main characteristics of atrial fibrillation (AF). Fibroblast growth factor 21 (Fgf21) is an important endocrine factor, which has been shown to play an important role in cardiovascular diseases. However, the effects of Fgf21 on atrial remodeling have not been addressed yet. The purpose of the present study is to evaluate the effects of Fgf21 on atrial remodeling.Methods and Results: Adult mice were treated with Ang II, and randomly administrated with or without Fgf21 for 2 weeks. The susceptibility to AF was assessed by electrical stimulation and optical mapping techniques. Here, we found that Fgf21 administration attenuated the inducibility of atrial fibrillation/atrial tachycardia (AF/AT), improved epicardial conduction velocity in the mice atria. Mechanistically, Fgf21 protected against atrial fibrosis and reduced oxidative stress of the atria. Consistently, in vitro study also demonstrated that Fgf21 blocked the upregulation of collagen by Tgf-β in fibroblasts and attenuated tachypacing-induced oxidative stress including reactive oxygen species (ROS), Tgf-β, and ox-CaMKII in atrial myocytes. We further found that Fgf21 attenuated oxidative stress by inducing antioxidant genes, such as SOD2 and UCP3. Fgf21 also improved tachypacing-induced myofibril degradation, downregulation of L-type calcium channel, and upregulation of p-RyR2, which implicated protective effects of Fgf21 on structural and electrical remodeling in the atria. Moreover, Nrf2 was identified as a downstream of Fgf21 and partly mediated Fgf21-induced antioxidant gene expression in atrial myocytes.Conclusion: Fgf21 administration effectively suppressed atrial remodeling by reducing oxidative stress, which provides a novel therapeutic insight for AF.

Transcriptome analysis of conditionally immortalized atrial myocytes: identification of a novel atrium-enriched protein involved in sarcomere assembly and maintenance

Background Heart development relies on the tight spatiotemporal control of cardiac gene expression. Genes involved in these processes have been identified using mainly (transgenic) animals models and pluripotent stem cell-derived cardiomyocytes (CMs). Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal cell line of conditionally immortalized neonatal rat atrial myocytes (NRAMs) which allows toggling between proliferative and differentiated (i.e. excitable and contractile) phenotypes in a synchronized and homogenous manner. Purpose To identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation, dedifferentiation and proliferation by exploiting the unique properties of conditionally immortalized NRAMs (iAMs). Methods and results RNA sequencing was performed during a full cycle of iAM-1 differentiation and subsequent dedifferentiation, identifying ±13,000 transcripts, of which the dynamic expressional changes during cardiomyogenic differentiation in most cases opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many genes with a known (lineage-specific) role in cardiac muscle formation, thereby confirming the relevance of iAMs as cardiomyogenic differentiation model. Filtering for cardiomyocyte-enriched low abundancy transcripts, resulted in the identification of an uncharacterized protein, which is highly conserved among Nephrozoa and up- and downregulated during cardiomyocyte differentiation and dedifferentiation, respectively. In neonatal and adult rats, this protein is muscle-specific, highly atrium-enriched and localized around the C-zone of cardiac sarcomeres. Lentiviral shRNA-mediated knockdown resulted in loss of sarcomeric organization in both NRAMs and iAMs. Neither knockdown nor overexpression of this protein affected the electrophysiological properties of differentiated iAM monolayers. Conclusions iAM-1 cells offer a relevant model to identify and characterize novel (low abundancy) genes involved in cardiomyocyte (de)differentiation as exemplified by the identification a novel uncharacterized protein that is muscle-specific, highly atrium-enriched, localized around the C-zone of cardiac sarcomeres and plays a specific role in atrial sarcomerigenesis. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Netherlands Organisation for Health Research and Development (ZonMw) Leiden Regenerative Medicine Platform Holding project with number (LRMPH) Figure 1. (A) Experimental setup. At the indicated timepoints iAM-1 cells were fixed for immunostaining and RNA extraction for transcriptome analysis. (B) Immunochemical staining of iAM-1 cells for the proliferation marker Ki-67 and the Z-line marker sarcomeric α-actinin. (C & D) Immunohistological double stainings of longitudinal sections of neonatal rat hearts for the uncharacterized protein (GOI 1) and the sarcomeric protein cardiac troponin I (TNNI3). LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. Scale bar, 250 μm.

Abstract P413: Stretch-induced Caveolae-mediated Disruption Of Cyclic AMP Microdomains Leads To Arrhythmogenic Ca 2+ Mishandling In Mouse Atrial Myocytes

Background: Atrial fibrillation (AF) often occurs during hypertension and is associated with an increase in cardiomyocyte stretch. Mechanism of ectopic beats, that trigger AF, has been linked to Ca 2+ mishandling and leaky hyperphosphorylated ryanodine receptors (RyRs), while the underlying mechanisms remain elusive. Caveolae membrane structures are involved in cell mechanosensing processes and control the cAMP signaling pathway. We hypothesized that mechanical stretch disrupts caveolae, promoting cAMP production and sarcoplasmic reticulum Ca 2+ leak via augmentation of RyRs phosphorylation. Methods and Results: Cell size analysis and Ca 2+ dynamics measurements were performed by confocal imaging of isolated mouse atrial myocytes. Cell stretch was modeled by hypoosmotic swelling (from 310 mOsM to 220 mOsM to flatten caveolae structures) resulting in a ~30% increase in cell width (p<0.05) with no changes in cell length. Swelling resulted in a biphasic effect on Ca 2+ spark activity: a fast (<10 min of exposure) ~50% increase (p<0.001) followed by a slow decrease to the level observed in isotonic conditions (>30 min of exposure). Similarly, caveolae disruption via cholesterol depletion by 10 mM methyl-β-cyclodextrin (MβCD) led to 2-fold increase in Ca 2+ sparks frequency (p<0.001). Swelling- and MβCD-induced increases in atrial Ca 2+ spark activity were prevented via inhibition of cAMP production by adenylyl cyclases by 0.1mM SQ22536 or cAMP-dependent protein kinase A (PKA) by 1μM H-89. Then, we tested if this mechanism is present in atrial myocytes from pressure-overloaded (4-weeks transaortic constriction, TAC) mice. Atrial myocytes from TAC mice showed a 1.6 times higher Ca 2+ sparks frequency than wild-type myocytes (p<0.01), which was significantly reduced (p<0.01) to wild-type level after incubation with SQ22536. Conclusions: Our findings suggest that cell stretch increases spontaneous Ca 2+ spark activity through the disruption of caveolae and cAMP-mediated augmentation of PKA activity. This mechanism could be involved in the Ca 2+ mishandling and AF in pressure overloaded hearts.

Immediate and Delayed Response of Simulated Human Atrial Myocytes to Clinically-Relevant Hypokalemia

Although plasma electrolyte levels are quickly and precisely regulated in the mammalian cardiovascular system, even small transient changes in K+, Na+, Ca2+, and/or Mg2+ can significantly alter physiological responses in the heart, blood vessels, and intrinsic (intracardiac) autonomic nervous system. We have used mathematical models of the human atrial action potential (AP) to explore the electrophysiological mechanisms that underlie changes in resting potential (Vr) and the AP following decreases in plasma K+, [K+]o, that were selected to mimic clinical hypokalemia. Such changes may be associated with arrhythmias and are commonly encountered in patients (i) in therapy for hypertension and heart failure; (ii) undergoing renal dialysis; (iii) with any disease with acid-base imbalance; or (iv) post-operatively. Our study emphasizes clinically-relevant hypokalemic conditions, corresponding to [K+]o reductions of approximately 1.5 mM from the normal value of 4 to 4.5 mM. We show how the resulting electrophysiological responses in human atrial myocytes progress within two distinct time frames:(i) Immediately after [K+]o is reduced, the K+-sensing mechanism of the background inward rectifier current (IK1) responds. Specifically, its highly non-linear current-voltage relationship changes significantly as judged by the voltage dependence of its region of outward current. This rapidly alters, and sometimes even depolarizes, Vr and can also markedly prolong the final repolarization phase of the AP, thus modulating excitability and refractoriness.(ii) A second much slower electrophysiological response (developing 5–10 minutes after [K+]o is reduced) results from alterations in the intracellular electrolyte balance. A progressive shift in intracellular [Na+]i causes a change in the outward electrogenic current generated by the Na+/K+ pump, thereby modifying Vr and AP repolarization and changing the human atrial electrophysiological substrate.In this study, these two effects were investigated quantitatively, using seven published models of the human atrial AP. This highlighted the important role of IK1 rectification when analyzing both the mechanisms by which [K+]o regulates Vr and how the AP waveform may contribute to “trigger” mechanisms within the proarrhythmic substrate. Our simulations complement and extend previous studies aimed at understanding key factors by which decreases in [K+]o can produce effects that are known to promote atrial arrhythmias in human hearts.

Autonomous initiation of pacemaking by L-type Cav1.3 calcium channels in mouse sino-atrial myocytes

Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Ecole doctorale Background Cardiac pacemaking relies on the spontaneous electrical activity in the right atrium of sino-atrial myocytes (SANCs). Automaticity in SANCs results from a robust interplay of membrane ion channels activity and intracellular calcium dynamics. However, only a fraction of isolated SANCs exhibit rhythmic firing, whereas most SANCs show irregular (dysrhythmic) firing or remain dormant. Purpose To study the capability of L-type Cav1.3 calcium channels to initiate automaticity in dormant SANCs under β-adrenergic stimulation, we used a knock-in mouse strain in which the sensitivity of Cav1.2 α1 subunits to dihydropyridines (DHP) was inactivated (Cav1.2DHP-/-). Methods We performed current and voltage-clamp recordings on isolated SANCs under isoprenaline (ISO, 100 nM) and in the absence or presence of the DHP blocker Nifedipine (Nife, 3 µM). Results Nife significantly reduced the spontaneous firing under ISO perfusion in all rhythmic SANCs (ISO: 447 ± 12, ISO + Nife: 233 ± 25 bpm) and 60% of dysrhythmic SANCs (ISO: 386 ± 12, ISO + Nife: 188 ± 47 bpm) whereas it completely stopped it in the remaining 40% (295 ± 29 bpm to 0). On 25 dormant SANCs, 50% started firing after ISO perfusion (0 to 320 ± 46 bpm). Strikingly, in 75% of them, Nife totally blocked this ISO-induced firing. Interestingly, these cells exhibited a significantly slower rate and a slower slope of the diastolic depolarization under ISO perfusion compared to the remaining 25% dormant SANCs in which Nife only reduced the ISO-induced firing. Moreover, dormant SANCs showed a statistically significant increase in action potential (AP) threshold under ISO compared to dysrhythmic and rhythmic SANCs (dormant: -30.1 ± 2.5, dysrhythmic: -43.3 ± 2.3, rhythmic: -41.2 ± 2.1 mV). No significant difference was observed in the other AP parameters between dormant, dysrhythmic and rhythmic SANCs under ISO. Conclusion Our results seem to point at a difference of expression in ionic channels (Cav1.3, HCN4) within isolated SANCs. Preliminary results on If density support this hypothesis with a lesser density in dormant SANCs compared to dysrhythmic SANCs. These results also tend to indicate that Cav1.3 channels can generate pacemaker activity autonomously, at least in a particular subpopulation of SANCs.