ABSTRACT
miR-1, the most abundant miRNA in the heart, modulates expression of several transcription factors and ion channels. Conditions affecting the heart rate, such as endurance training and cardiac diseases, show a concomitant miR-1 up- or down-regulation. Here, we investigated the role of miR-1 overexpression in the development and function of sinoatrial (SAN) cells using murine embryonic stem cells (mESC). We generated mESCs either overexpressing miR-1 and EGFP (miR1OE) or EGFP only (EM). SAN-like cells were selected from differentiating mESC using the CD166 marker. Gene expression and electrophysiological analysis were carried out on both early mES-derived cardiac progenitors and SAN-like cells and on beating neonatal rat ventricular cardiomyocytes (NRVC) over-expressing miR-1. miR1OE cells increased significantly the proportion of CD166+ SAN precursors compared to EM cells (23% vs 12%) and the levels of the transcription factors TBX5 and TBX18, both involved in SAN development. miR1OE SAN-like cells were bradycardic (1,3 vs 2 Hz) compared to EM cells. In agreement with data on native SAN cells, EM SAN-like cardiomyocytes show two populations of cells expressing either slow- or fast-activating If currents; miR1OE SAN-like cells instead have only fast-activating If with a significantly reduced conductance. Western Blot and immunofluorescence analysis showed a reduced HCN4 signal in miR-1OE vs EM CD166+ precursors. Together these data point out to a specific down-regulation of the slow-activating HCN4 subunit by miR-1. Importantly, the rate and If alterations were independent of the developmental effects of miR-1, being similar in NRVC transiently overexpressing miR-1. In conclusion, we demonstrated a dual role of miR-1, during development it controls the proper development of sinoatrial-precursor, while in mature SAN-like cells it modulates the HCN4 pacemaker channel translation and thus the beating rate.
Subject(s)
Gene Expression Regulation , MicroRNAs/genetics , Sinoatrial Node/cytology , Sinoatrial Node/metabolism , Action Potentials , Activated-Leukocyte Cell Adhesion Molecule/metabolism , Animals , Biomarkers , Cell Differentiation/genetics , Electrophysiological Phenomena , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression , Immunophenotyping , Mice , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , RatsABSTRACT
Ivabradine is a 'heart rate-reducing' agent able to slow heart rate, without complicating side-effects. Its action results from a selective and specific block of pacemaker f-channels of the cardiac sinoatrial node (SAN). Investigation has shown that block by ivabradine requires open f-channels, is use dependent, and is affected by the direction of current flow. The constitutive elements of native pacemaker channels are the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, of which four isoforms (HCN1-4) are known; in rabbit SAN tissue HCN4 is expressed strongly, and HCN1 weakly. In this study we have investigated the blocking action of ivabradine on mouse (m) HCN1 and human (h) HCN4 channels heterologously expressed in HEK 293 cells. Ivabradine blocked both channels in a dose-dependent way with half-block concentrations of 0.94 microm for mHCN1 and 2.0 microm for hHCN4. Properties of block changed substantially for the two channels. Block of hHCN4 required open channels, was strengthened by depolarization and was relieved by hyperpolarization. Block of mHCN1 did not occur, nor was it relieved, when channels were in the open state during hyperpolarization; block required channels to be either closed, or in a transitional state between open and closed configurations. The dependence of block upon current flow was limited for hHCN4, and not significant for mHCN1 channels. In summary our results indicate that ivabradine is an 'open-channel' blocker of hHCN4, and a 'closed-channel' blocker of mHCN1 channels. The mode of action of ivabradine on the two channels is discussed by implementing a simplified version of a previously developed model of f-channel kinetics.
Subject(s)
Benzazepines/pharmacology , Biological Clocks/drug effects , Ion Channels/drug effects , Muscle Proteins/drug effects , Nerve Tissue Proteins/drug effects , Action Potentials/physiology , Cell Line , Cyclic Nucleotide-Gated Cation Channels , Dose-Response Relationship, Drug , Electrophysiology , Heart Rate/physiology , Humans , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Ion Channels/antagonists & inhibitors , Ion Channels/physiology , Ivabradine , Kidney/cytology , Muscle Proteins/antagonists & inhibitors , Muscle Proteins/physiology , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/physiology , Patch-Clamp Techniques , Potassium Channels , Protein Isoforms , Sinoatrial Node/drug effects , Sinoatrial Node/physiologyABSTRACT
A survey was taken of the amount of radionuclides, acquired, utilized, and released in one year's time, in the Adige river by the USL (Local Division of the National Public Health System) of Verona. The critical pathways of environmental pollution were analyzed and the individual and collective doses of some critical population groups and the population as a whole were compiled. Some suggestions to reduce the collective doses both from radioactive releases and diagnostic use of radionuclides are given.
