Downregulation of RyR and NCX in the neonatal rat ventricular myocyte modulates cytosolic [Ca2+]

Calcium (Ca2+) is necessary for cardiac muscle contraction. RyR, NCX, and SERCA are key regulatory protein channels for cytosolic Ca2+ in the cardiomyocyte. Expression levels of these proteins are a function of development and disease. We investigated how downregulation by siRNAs of RyR and NCX affected expression levels of complimentary proteins their corresponding intracellular Ca2+ transients. We compared the experimentally observed Ca2+ transients to those predicted by mathematical models. Experimental results show RyR downregulation decreased SERCA and increased NCX protein levels. The associated Ca2+ transient had decreased amplitude, increased time-to-peak (TTP), time-to-50% (T50), and time-to-90% (T90) Ca2+ removal with respect to the control population. NCX downregulation increased SERCA production without significant changes in RyR expression levels. The corresponding Ca2+ transient increased amplitude, no change in TTP and T50 Ca2+ removal, but increased T90 Ca2+ removal with respect to the control population. Computational models that accurately predict the observed experimental data suggest compensatory changes occurring in the expression levels as well as biochemical activity of the regulatory proteins.

The effects of rosiglitazone on the neonatal rat cardiomyocyte transcriptome: a temporal analysis

Aim: The objective was to determine via high-throughput RNA sequencing the temporal effects of rosiglitazone (Avandia®) on the neonatal rat ventricular myocyte transcriptome. Materials & methods: Neonatal rat ventricular myocytes (NRVMs) were exposed to rosiglitazone in vitro. Meta analyses utilized temporal comparisons of 0.5 h control versus 0.5 h treatment, 0.5 h treatment versus 24 h treatment and 24 h treatment versus 48 h treatment. Results: Time dependent responses were observed. At 0.5 h, the PI3K-AKT signaling pathway was impacted. At 24 h endoplasmic reticulum activity and protein degradation were altered. At 48 h, oxytocin signaling was perturbed. Conclusion: The effects of rosiglitazone occured early and increased in magnitude over time. A protective molecular response was triggered at 24 h and maintained until 48 h. In parallel, a response that can cause cardiac damage was activated. Our findings suggest that rosiglitazone has deleterious effects.

Manipulation of KCNE2 expression modulates action potential duration and Ito and IK in rat and mouse ventricular myocytes

In heterologous expression systems, KCNE2 has been demonstrated to interact with multiple α-subunits of voltage-dependent cation channels and modulate their functions. However, the physiological and pathological roles of KCNE2 in cardiomyocytes are poorly understood. The present study aimed to investigate the effects of bidirectional modulation of KCNE2 expression on action potential (AP) duration (APD) and voltage-dependent K+ channels in cardiomyocytes. Adenoviral gene delivery and RNA interference were used to either increase or decrease KCNE2 expression in cultured neonatal and adult rat or neonatal mouse ventricular myocytes. Knockdown of KCNE2 prolonged APD in both neonatal and adult myocytes, whereas overexpression of KCNE2 shortened APD in neonatal but not adult myocytes. Consistent with the alterations in APD, KCNE2 knockdown decreased transient outward K+ current ( Ito) densities in neonatal and adult myocytes, whereas KCNE2 overexpression increased Ito densities in neonatal but not adult myocytes. Furthermore, KCNE2 knockdown accelerated the rates of Ito activation and inactivation, whereas KCNE2 overexpression slowed Ito gating kinetics in neonatal but not adult myocytes. Delayed rectifier K+ current densities were remarkably affected by manipulation of KCNE2 expression in mouse but not rat cardiomyocytes. Simulation of the AP of a rat ventricular myocyte with a mathematical model showed that alterations in Ito densities and gating properties can result in similar APD alterations in KCNE2 overexpression and knockdown cells. In conclusion, endogenous KCNE2 in cardiomyocytes is important in maintaining cardiac electrical stability mainly by regulating Ito and APD. Perturbation of KCNE2 expression may predispose the heart to ventricular arrhythmia by prolonging APD.