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The sustained release of alprenolol, a ß-adrenergic antagonist, could be beneficial for the treatment of various heart diseases while reducing the side effects resulting from its continuous use. The novel and branched copolymers uniquely composed of biodegradable components (lactide and glycolide) have been synthesized using natural and therapeutically-efficient ß-aescin-initiator, and consequently characterized to determine their structures and physicochemical properties. The obtained matrices were not cyto- and genotoxic towards bacterial luminescence, protozoan, and Salmonella typhimurium TA1535. The copolymers release the drug in vitro in a sustained manner and without burst release. The value of the drug released was strongly dependent on the copolymer composition and highly correlated with the hydrolytic matrices’ degradation results.

We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of alphaENaC using the serine protease subtilisin. Trimeric channels formed with alphaFM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an approximately 10-fold decrease in amiloride-sensitive whole cell conductance as compared with alphaWT (2.2 versus 21.2 microsiemens (microS)). Both alphaWT and alphaFM were activated to similar levels by subtilisin cleavage. Channels formed with alphaFD, a construct that deleted the segment between the two furin sites (Delta175-204), exhibited an intermediate conductance of 13.2 microS. More importantly, alphaFD retained the ability to be activated by subtilisin to 108.8 +/- 20.9 microS, a level not significantly different from that of subtilisin activated alphaWT (125.6 +/- 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of alpha was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with alphaLD, a construct that extended the deleted sequence of alphaFD by 17 amino acids (Delta175-221). Channels with alphaLD were uncleaved, exhibited low baseline activity (4.1 microS), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.

Recent studies have characterized cardiac ionic currents in cardiocytes derived from human induced pluripotent stem cells. However, direct comparison with human ventricular myocytes is hampered by differences in experimental conditions. Similarly, stem cell derived cardiocytes are different and potentially more variable than native myocytes. We examined commercially prepared cells (iCells) and scale production cells (hIPS-CMs) using electronic expression of IK1 to distinguish ventricular cells, compared with myocytes (VM) from biopsy samples.APs were recorded in VM, iCells and hiPSC-CMs. iCells and hiPSC-CMs had depolarized membrane potentials, slower upstroke velocities, and prolonged AP relative to VM. These differences were minimized by electronic expression of IK1. Capacitances were larger in VM (298.97 ± 27.5 pF (4 hearts, n=44)) than in iCells (73.21 ± 5.4 (n=32), p

Human induced pluripotent stem cell derived cardiac myocytes (h-iPSC-CMs) are a major advance in drug safety testing and research into human cardiac electrophysiology. However, these cells have potential limitations when used for quantitative action potential (AP) analysis. These cells are a mixture of atrial and ventricular cell types, which can be distinguished, to some extent, by action potential (AP) morphology. However, the spontaneous activity of h-iPSC-CMs results in parameter variability and anomalous pharmacological responses, leading to cell misidentification. The spontaneous behavior is largely due to the absence of an IK1 inwardly rectifying potassium channel (IK1).We examined the effect of using an in silico interface to electronically express this missing IK1. An in silico interface was developed to express synthetic IK1 in cells under whole cell voltage clamp using a variant of the dynamic clamp approach. Electronic IK1 expression resulted in a stable physiological resting potential, eliminated spontaneous activity, reduced spontaneous early and delayed after depolarizations, and decreased AP variability. Stimulated APs had a rapid upstroke and spike and dome morphology, and the readily recognizable repolarization attributes of ventricular and atrial cells. When examining the late components of outward current, we found that APs classified on the basis of AP morphology as “atrial-like” had a significantly larger sustained outward Kv1.5-like component at +50 mV than “ventricular-like” APs (in pA/pF: 1.76 ± 0.47 (n=6) vs. 0.74 ± 0.07 (n=7), p 0.4). These data indicate that h-IPSC-CM myocytes with synthetic expression of IK1 can easily be differentiated into ventricular-like and atrial-like myocytes by AP morphology, and that atrial-like cells exhibit an atrial-specific current.

Human-induced pluripotent stem cell (h-iPSC)-derived cardiac myocytes are a unique model in which human myocyte function and dysfunction are studied, especially those from patients with genetic disorders. They are also considered a major advance for drug safety testing. However, these cells have considerable unexplored potential limitations when applied to quantitative action potential (AP) analysis. One major factor is spontaneous activity and resulting variability and potentially anomalous behavior of AP parameters.To demonstrate the effect of using an in silico interface on electronically expressed I(K1), a major component lacking in h-iPSC-derived cardiac myocytes.An in silico interface was developed to express synthetic I(K1) in cells under whole-cell voltage clamp.Electronic I(K1) expression established a physiological resting potential, eliminated spontaneous activity, reduced spontaneous early and delayed afterdepolarizations, and decreased AP variability. The initiated APs had the classic rapid upstroke and spike and dome morphology consistent with data obtained with freshly isolated human myocytes as well as the readily recognizable repolarization attributes of ventricular and atrial cells. The application of 1 µM of BayK-8644 resulted in anomalous AP shortening in h-iPSC-derived cardiac myocytes. When I(K1) was electronically expressed, BayK-8644 prolonged the AP, which is consistent with the existing results on native cardiac myocytes.The electronic expression of I(K1) is a simple and robust method to significantly improve the physiological behavior of the AP and electrical profile of h-iPSC-derived cardiac myocytes. Increased stability enables the use of this preparation for a controlled quantitative analysis of AP parameters, for example, drug responsiveness, genetic disorders, and dynamic behavior restitution profiles.

The delayed rectifier, IKr, is formed by homo/heterotetramers of the 2 HERG splice variants HERG1a and HERG1b. The only difference between isoforms is that HERG1b has a shorter N-terminal than HERG1a. Despite their similarities, HERG1a and 1b have profoundly different gating kinetics: HERG1b has faster activation and deactivation kinetics than HERG1a. HERG activation is a complex multi-step process involving voltage-dependent and voltage independent transitions.We used an envelope of tails protocol to isolate activation from the fast overlapping inactivation for both HERG1a and 1b. The activation rate of both HERG1a and 1b reaches an identical saturation value at very positive potentials, which suggests that the voltage-independent step is rate limiting at these potentials for both isoforms. This suggests that the voltage-insensitive step is not affected by isoform, i.e., the presence or absence of the N-terminal. At lower potentials, activation was strongly voltage dependent and was significantly different between isoforms. This indicates that the N-terminal modifies at least one voltage-sensitive step in the activation pathway. Deactivation is also strongly affected by the presence or absence of the N-terminal. This suggests that the N-terminal affects voltage-dependent transitions near the open state. Sensitivity analysis of our HERG model suggests that the effects may be more broad. Manipulation of only the early states failed to optimize the rate of deactivation, but optimization of only the final voltage dependent steps fails to recapitulate the combination of shifted steady-state activation and slope factor for the HERG1b isoform. In conclusion, our data indicate that the N-terminal interacts primarily through modification of voltage-sensitive transitions.

The channels underlying native human IKr are thought to be formed by homo/heterotetramers of the 2 HERG splice variants HERG1a and HERG1b. HERG1b has a shorter and highly basic 36 amino acid N-terminal, but is otherwise identical to HERG1a. This minor structural difference is sufficient to produce a significant change in gating kinetics: HERG1b has faster activation and deactivation kinetics than HERG1a. HERG activation is a multi-step process involving voltage-dependent and voltage independent transitions. It is unclear whether the difference in activation gating between HERG1a and 1b reflects modification of the voltage sensitive or insensitive steps by the N-terminal domain.We used an envelope of tails protocol to isolate activation from the fast overlapping inactivation for both HERG1a and 1b. Both of these isoforms showed saturation of activation rate at very positive potentials. This suggests that the voltage-independent step is rate limiting at these potentials for both isoforms. At +140 mV the rate of activation of HERG1a was 13.09 ± 0.66 ms (n=3), and HERG1b was 14.25 ± 0.37 ms (n=3). This suggests that the voltage-insensitive step is not affected by the presence or absence of the N-terminal. In contrast, activation at lower potentials was strongly dependent on voltage and was significantly different between isoforms. This indicates that the N-terminal modifies a voltage-sensitive step in the activation pathway. Deactivation is also strongly affected by the presence or absence of the N-terminal. This suggests that the N-terminal affects voltage-dependent transitions near the open state. In conclusion, our data indicate that the HERG N-terminal interacts primarily through modification of voltage-sensitive transitions.

Several voltage-gated channels share a proline-valine-proline (proline hinge) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels, which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine or alanine: P558A, P558G. These mutations were studied in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N- and C-type inactivation. Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed by more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement play a significant role in inactivation and recovery. Computer modeling suggests that both P558G and P558A mutations modify early voltage-dependent steps and make a final voltage-insensitive step that is rate limiting at positive potentials.

C-type inactivation is sensitive to mutation on extra- and intracellular side of the channel, indicating it may involve conformational changes at both extracellular and intracellular mouth of the channel. This movement is critical in determining the allosteric coupling between N-type and C-type inactivation. X-ray crystallography has provided insights into the structure of low-energy stable states in inactivation. In order to investigate the path connecting two stable end states and the sequence of movement over the energy landscape of the critical domains involved in C-type inactivation of Kv1.4 channel, we applied ϕ value analysis to this channel. We chose the V561 at intracellular side of S6. Mutations:[V561A], [V561C], [V561Q], [V561S] and [V561T] were made in N-terminal deleted and N-terminal intact channels. In the N-terminal deleted constructs a ϕ of 0.49 was observed for C-type inactivation. With N-terminal binding, the inactivation process became more complex, but analysis of kinetic components revealed a ϕ value of 0.88 for N-terminal binding, indicating an early interaction with the open pore and a ϕ value of 0.64 was obtained C-type inactivation, indicating facilitation of this step to earlier in the total process. Extracellular function was probed by titration of H508. The slopes of Hammond energy plot for pH titration of H508 from all different mutants are consistent with an average ϕ value of 0.218 (range from 0.20 to 0.23), which indicates the movement of extracellular region near selectivity filter is after the movement of intracellular side of S6. Our results reveal that C-type inactivation can be accelerated by binding of N-terminal to the intracellular mouth of the pore, followed by conformational change at intracellular portion of S6 which is transduced to the extracellular mouth region through an allosteric mechanism.

Cardiac myocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are a useful and renewable human myocyte model. Despite their promise, these cells have unexplored limitations when applied to action potential (AP) analysis. APs occur spontaneously and are associated with variability due to a small/missing inwardly rectifying potassium channel (IK1). We used whole cell voltage clamp with dynamic clamp to express IK1, which significantly improved the physiological behavior of the AP and electrical profile of hiPSC-CMs.hiPSC-CMs have a negligible peak IK1 at −120 mV (−0.81 ± 0.4 pA/pF) which results in depolarized resting membrane potentials (RMP) (−60.0 ± 1.7 mV, n=17). “Electronic transfection” of IK1 into hiPSC-CMs results in reestablishing a physiological RMP (−84.0 ± 0.2 mV), increases the maximal upstroke velocity (from 82.1 ± 2.4 to 161.6 ± 11.5 mV/ms), reduces AP duration, and increases the rate of repolarization (from 0.35 ± 0.03 to 1.1 ± 0.1 mV/ms, n=17). Despite a detectable transient outward potassium current in hiPSC-CMs, “spike and dome” morphology is generally absent in spontaneously active cells; addition of electronic IK1 restored this morphology in 12 out of 17 ventricular cells. It also restored the relationship between maximum upstroke velocity and sodium current density. The stabilized membrane potential allowed systematic measurement of dynamic parameters. The rate dependence of the AP duration was measured in at different pacing rates from 4000 to 500 ms in 12 electronic IK1 expressing hiPSC-CMs and showed a classical monotonic restitution curve, with AP increasing with increased cycle length. By removing sodium channel inactivation, electronic expression of IK1 improves hiPSC-CMs utility in assess mechanisms involving sodium channels and phase 1 repolarization such as LQT3 and Brugada Syndrome.