The developmental rehearsal for the debut of hearing is marked by massive changes in the membrane properties of hair cells (HCs) and spiral ganglion neurons (SGNs). Whereas the underlying mechanisms for the developing HC transition to mature stage are understood in detail, the maturation of SGNs from hyperexcitable prehearing to quiescent posthearing neurons with broad dynamic range is unknown. Here, we demonstrated using pharmacological approaches, caged-Ca(2+) photolysis, and gramicidin patch recordings that the prehearing SGN uses Ca(2+)-activated Cl(-) conductance to depolarize the resting membrane potential and to prime the neurons in a hyperexcitable state. Immunostaining of the cochlea preparation revealed the identity and expression of the Ca(2+)-activated Cl(-) channel transmembrane member 16A (TMEM16A) in SGNs. Moreover, null deletion of TMEM16A reduced the Ca(2+)-activated Cl(-) currents and action potential firing in SGNs. To determine whether Cl(-) ions and TMEM16A are involved in the transition between pre- and posthearing features of SGNs we measured the intracellular Cl(-) concentration [Cl(-)]i in SGNs. Surprisingly, [Cl(-)]i in SGNs from prehearing mice was ∼90 mM, which was significantly higher than posthearing neurons, ∼20 mM, demonstrating discernible altered roles of Cl(-) channels in the developing neuron. The switch in [Cl(-)]i stems from delayed expression of the development of intracellular Cl(-) regulating mechanisms. Because the Cl(-) channel is the only active ion-selective conductance with a reversal potential that lies within the dynamic range of SGN action potentials, developmental alteration of [Cl(-)]i, and hence the equilibrium potential for Cl(-) (ECl), transforms pre- to posthearing phenotype.

Small-conductance Ca²⁺-activated K⁺ (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca²⁺ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca²⁺ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca²⁺ microdomains necessary for SK channel activation. SK currents coupled with Ca²⁺ influx via L-type Ca²⁺ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca²⁺ store, suggesting that LTCCs provide the immediate Ca²⁺ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Ca_v1.2 Ca²⁺ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Ca_v1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca²⁺ signaling.

Cardiac cells generate and amplify force in the context of cardiac load, yet the membranous sheath enclosing the muscle fibers-the sarcolemma-does not experience displacement. That the sarcolemma sustains beat-to-beat pressure changes without experiencing significant distortion is a muscle-contraction paradox. Here, we report that an elastic element-the motor protein prestin (Slc26a5)-serves to amplify actin-myosin force generation in mouse and human cardiac myocytes, accounting partly for the nonlinear capacitance of cardiomyocytes. The functional significance of prestin is underpinned by significant alterations of cardiac contractility in Prestin-knockout mice. Prestin was previously considered exclusive to the inner ear's outer hair cells; however, our results show that prestin serves a broader cellular motor function.

Purpose

The delivery of transgenes into human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) represents an important tool in cardiac regeneration with potential for clinical applications. Gene transfection is more difficult, however, for hiPSCs and hiPSC-CMs than for somatic cells. Despite improvements in transfection and transduction, the efficiency, cytotoxicity, safety, and cost of these methods remain unsatisfactory. The objective of this study is to examine gene transfection in hiPSCs and hiPSC-CMs using magnetic nanoparticles (NPs).

Methods

Magnetic NPs are unique transfection reagents that form complexes with nucleic acids by ionic interaction. The particles, loaded with nucleic acids, can be guided by a magnetic field to allow their concentration onto the surface of the cell membrane. Subsequent uptake of the loaded particles by the cells allows for high efficiency transfection of the cells with nucleic acids. We developed a new method using magnetic NPs to transfect hiPSCs and hiPSC-CMs. HiPSCs and hiPSC-CMs were cultured and analyzed using confocal microscopy, flow cytometry, and patch clamp recordings to quantify the transfection efficiency and cellular function.

Results

We compared the transfection efficiency of hiPSCs with that of human embryonic kidney (HEK 293) cells. We observed that the average efficiency in hiPSCs was 43%±2% compared to 62%±4% in HEK 293 cells. Further analysis of the transfected hiPSCs showed that the differentiation of hiPSCs to hiPSC-CMs was not altered by NPs. Finally, robust transfection of hiPSC-CMs with an efficiency of 18%±2% was obtained.

Conclusion

The difficult-to-transfect hiPSCs and hiPSC-CMs were efficiently transfected using magnetic NPs. Our study offers a novel approach for transfection of hiPSCs and hiPSC-CMs without the need for viral vector generation.

Background

Intracellular pH (pH_i) is critical to cardiac excitation and contraction; uncompensated changes in pH_i impair cardiac function and trigger arrhythmia. Several ion transporters participate in cardiac pH_i regulation. Our previous studies identified several isoforms of a solute carrier Slc26a6 to be highly expressed in cardiomyocytes. We show that Slc26a6 mediates electrogenic Cl^-/HCO₃^- exchange activities in cardiomyocytes, suggesting the potential role of Slc26a6 in regulation of not only pH_i, but also cardiac excitability.

Methods and results

To test the mechanistic role of Slc26a6 in the heart, we took advantage of Slc26a6 knockout (Slc26a6^-/- ) mice using both in vivo and in vitro analyses. Consistent with our prediction of its electrogenic activities, ablation of Slc26a6 results in action potential shortening. There are reduced Ca²⁺ transient and sarcoplasmic reticulum Ca²⁺ load, together with decreased sarcomere shortening in Slc26a6^-/- cardiomyocytes. These abnormalities translate into reduced fractional shortening and cardiac contractility at the in vivo level. Additionally, pH_i is elevated in Slc26a6^-/- cardiomyocytes with slower recovery kinetics from intracellular alkalization, consistent with the Cl^-/HCO₃^- exchange activities of Slc26a6. Moreover, Slc26a6^-/- mice show evidence of sinus bradycardia and fragmented QRS complex, supporting the critical role of Slc26a6 in cardiac conduction system.

Conclusions

Our study provides mechanistic insights into Slc26a6, a unique cardiac electrogenic Cl^-/HCO₃^- transporter in ventricular myocytes, linking the critical roles of Slc26a6 in regulation of pH_i, excitability, and contractility. pH_i is a critical regulator of other membrane and contractile proteins. Future studies are needed to investigate possible changes in these proteins in Slc26a6^-/- mice.