Engineered Cardiac Tissues Generated in the Biowire II: A Platform for Human-Based Drug Discovery

Recent advances in techniques to differentiate human induced pluripotent stem cells (hiPSCs) hold the promise of an unlimited supply of human derived cardiac cells from both healthy and disease populations. That promise has been tempered by the observation that hiPSC-derived cardiomyocytes (hiPSC-CMs) typically retain a fetal-like phenotype, raising concern about the translatability of the in vitro data obtained to drug safety, discovery, and development studies. The Biowire II platform was used to generate 3D engineered cardiac tissues (ECTs) from hiPSC-CMs and cardiac fibroblasts. Long term electrical stimulation was employed to obtain ECTs that possess a phenotype like that of adult human myocardium including a lack of spontaneous beating, the presence of a positive force-frequency response from 1 to 4 Hz and prominent postrest potentiation. Pharmacology studies were performed in the ECTs to confirm the presence and functionality of pathways that modulate cardiac contractility in humans. Canonical responses were observed for compounds that act via the β-adrenergic/cAMP-mediated pathway, eg, isoproterenol and milrinone; the L-type calcium channel, eg, FPL64176 and nifedipine; and indirectly effect intracellular Ca2+ concentrations, eg, digoxin. Expected positive inotropic responses were observed for compounds that modulate proteins of the cardiac sarcomere, eg, omecamtiv mecarbil and levosimendan. ECTs generated in the Biowire II platform display adult-like properties and have canonical responses to cardiotherapeutic and cardiotoxic agents that affect contractility in humans via a variety of mechanisms. These data demonstrate that this human-based model can be used to assess the effects of novel compounds on contractility early in the drug discovery and development process.

Effects of increased preload on the force-frequency response and contractile kinetics in early stages of cardiac muscle hypertrophy

Numerous studies have aimed to elucidate markers for the onset of decompensatory hypertrophy and heart failure in vivo and in vitro. Alterations in the force-frequency relationship are commonly used as markers for heart failure with a negative staircase being a hallmark of decompensated cardiac function. Here we aim to determine the functional and molecular alterations in the very early stages of compensatory hypertrophy through analysis of the force-frequency relationship, using a novel isolated muscle culture system that allows assessment of force-frequency relationship during the development of hypertrophy. New Zealand white male rabbit trabeculae excised from the right ventricular free wall were utilized for all experiments. Briefly, muscles held at constant preload and contracting isometrically were stimulated to contract in culture for 24 h, and in a subset up to 48 h. We found that, upon an increase in the preload and maintaining the muscles in culture for up to 24 h, there was an increase in baseline force produced by isolated trabeculae over time. This suggests a gradual compensatory response to the impact of increased preload. Temporal analysis of the force-frequency response during this progression revealed a significant blunting (at 12 h) and then reversal of the positive staircase as culture time increased (at 24 h). Phosphorylation analysis revealed a significant decrease in desmin and troponin (Tn)I phosphorylation from 12 to 24 h in culture. These results show that even very early on in the compensatory hypertrophy state, the force-frequency relationship is already affected. This effect on force-frequency relationship may, in addition to protein expression changes, be partially attributed to the alterations in myofilament protein phosphorylation.

The cellular force-frequency response in ventricular myocytes from the varanid lizard, Varanus exanthematicus

To investigate the cellular mechanisms underlying the negative force-frequency relationship (FFR) in the ventricle of the varanid lizard, Varanus exanthematicus , we measured sarcomere and cell shortening, intracellular Ca2+ ([Ca2+]i), action potentials (APs), and K+ currents in isolated ventricular myocytes. Experiments were conducted between 0.2 and 1.0 Hz, which spans the physiological range of in vivo heart rates at 20–22°C for this species. As stimulation frequency increased, diastolic length, percent change in sarcomere length, and relaxation time all decreased significantly. Shortening velocity was unaffected. These changes corresponded to a faster rate of rise of [Ca2+]i, a decrease in [Ca2+]i transient amplitude, and a seven-fold increase in diastolic [Ca2+]i. The time constant for the decay of the Ca2+ transient (τ) decreased at higher frequencies, indicating a frequency-dependent acceleration of relaxation (FDAR) but then reached a plateau at moderate frequencies and did not change above 0.5 Hz. The rate of rise of the AP was unaffected, but the AP duration (APD) decreased with increasing frequency. Peak depolarization tended to decrease, but it was only significant at 1.0 Hz. The decrease in APD was not due to frequency-dependent changes in the delayed inward rectifier ( IKr) or the transient outward ( Ito) current, as neither appeared to be present in varanid ventricular myocytes. Our results suggest that a negative FFR relationship in varanid lizard ventricle is caused by decreased amplitude of the Ca2+ transient coupled with an increase in diastolic Ca2+, which leads to incomplete relaxation between beats at high frequencies. This coincides with shortened APD at higher frequencies.

Abstract 5408: PKA and PKC Phosphorylation of Troponin I Canonical Sites Regulate Calcium Sensitivity During Force Frequency Response

In failing hearts, the force-frequency response (FFR) is blunted, flat or negative. A positive FFR is crucial for healthy myocardium to respond to an increased working demand. There is no consensus in weather a positive FFR relies on myofilament Ca 2+ sensitization or desensitization and weather this is modulated by cTnI phosphorylation. In the present work we aimed to address the FFR and Ca 2+ cycling in intact mouse trabeculae loaded with Fura-2. To achieve this we used two transgenic models with pseudo phosphorylation mutants of troponin I (TnI), TnIDD 22,23 mice, which mimic increased phosphorylation at PKA sites of TnI at Ser 22 and 23 and TnI PKA/PKC mice, which mimic dephosphorylation at same PKA sites and increased phosphorylation at PKC sites of TnI at Ser 42 and 44. We hypothesized that controlling for cTnI phosphorylation will clarify the contribution of cTnI to the differences in force and Ca 2+ dynamics during FFR. When we examined the isometric contraction and Ca 2+ dynamics in each of these lines (TnIDD 22,23 , n= 8; TnI PKA/PKC, n=6) and non transgenic controls (NTG, n=7) we found that all three groups showed a positive FFR, although peak Ca 2+ increased with frequency rate in all three a less steep Ca 2+ transient increase (myofilament Ca 2+ sensitization) was observed in both transgenic lines compared to NTG (TnIDD 22,23 , p= 0.001; TnI PKA/PKC, p=0.03). Additionally, the peak force during the FFR was greater in the TnIDD 22,23 mice compared to NTG (p < 0.0001), suggesting that TnIDD 22,23 mice posses an enhanced frequency rate-related myofilament Ca 2+ sensitivity. WB analysis of Ca 2+ handling proteins including PLB, pPLB, SERCA2a and Ryanodine receptor normalized levels showed no major differences among all three groups, suggesting the differences observed in TnIDD 22,23 mice were not due to altered Ca 2+ handling but rather to myofilament Ca 2+ sensitivity. We conclude that a positive systolic peak FFR is followed by increasing myofilament Ca 2+ esensitization but mimicking increased phosphorylation at PKA sites of TnI Ser 22,23 enhances FFR and Ca 2+ responsiveness. Overall, our results support the concept that myofilament alterations feedback onto Ca 2+ handling mechanisms and these findings have important implications for human heart failure.

Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban

Studies have shown that neuronal nitric oxide synthase (nNOS, NOS1) knockout mice (NOS1−/−) have increased or decreased contractility, but consistently have found a slowed rate of intracellular Ca2+ ([Ca2+]i) decline and relengthening. Contraction and [Ca2+]i decline are determined by many factors, one of which is phospholamban (PLB). The purpose of this study is to determine the involvement of PLB in the NOS1-mediated effects. Force-frequency experiments were performed in trabeculae isolated from NOS1−/− and wild-type (WT) mice. We also simultaneously measured Ca2+ transients (Fluo-4) and cell shortening (edge detection) in myocytes isolated from WT, NOS1−/−, and PLB−/− mice. NOS1−/− trabeculae had a blunted force-frequency response and prolonged relaxation. We observed similar effects in myocytes with NOS1 knockout or specific NOS1 inhibition with S-methyl-l-thiocitrulline (SMLT) in WT myocytes (i.e., decreased Ca2+ transient and cell shortening amplitudes and prolonged decline of [Ca2+]i). Alternatively, NOS1 inhibition with SMLT in PLB−/− myocytes had no effect. Acute inhibition of NOS1 with SMLT in WT myocytes also decreased basal PLB serine16 phosphorylation. Furthermore, there was a decreased SR Ca2+ load with NOS1 knockout or inhibition, which is consistent with the negative contractile effects. Perfusion with FeTPPS (peroxynitrite decomposition catalyst) mimicked the effects of NOS1 knockout or inhibition. β-Adrenergic stimulation restored the slowed [Ca2+]i decline in NOS1−/− myocytes, but a blunted contraction remained, suggesting additional protein target(s). In summary, NOS1 inhibition or knockout leads to decreased contraction and slowed [Ca2+]i decline, and this effect is absent in PLB−/− myocytes. Thus NOS1 signaling modulates PLB serine16 phosphorylation, in part, via peroxynitrite.