ABSTRACT
Ion channels activity is regulated through soluble guanylate cyclase (sGC) and adenylate cyclase (AC) pathways, while phosphodiesterases (PDE) control the intracellular levels of cAMP and cGMP. Here we applied RNA transcriptome sequencing to study changes in the gene expression of the sGC, AC, and PDE isoforms in isolated rat ventricular cardiomyocytes under conditions of microgravity and hypergravity. Our results demonstrate that microgravity reduces the expression of sGC isoform genes, while hypergravity increases their expression. For a subset of AC isoforms, gene expression either increased or decreased under both microgravity and hypergravity conditions. The expression of genes encoding 10 PDE isoforms decreased under microgravity, but increased under hypergravity. However, under both microgravity and hypergravity, the gene expression increased for 7 PDE isoforms and decreased for 3 PDE isoforms. Overall, our findings indicate specific gravity-dependent changes in the expression of genes of isoforms associated with the studied enzymes.
Subject(s)
Hypergravity , Weightlessness , Rats , Animals , Phosphoric Diester Hydrolases/metabolism , Soluble Guanylyl Cyclase , Adenylyl Cyclases/genetics , Myocytes, Cardiac/metabolism , Protein Isoforms/genetics , Guanylate Cyclase/genetics , Guanylate Cyclase/metabolism , Cyclic GMP/metabolismABSTRACT
Since hypergravity changes the morphological and physiological properties of the heart, it was assumed that the expression of ion channels that respond to cell stretching or compressing, mechanically gated channels (MGC) and mechanosensitive channels (MSC), would be affected. Using RNA transcriptome sequencing, the change in the number of transcripts for MGC and MSC genes was studied in isolated rat ventricular cardiomyocytes under 4g hypergravity for 5 days. It was shown for the first time that hypergravity induces changes in the number of transcripts of MGC genes: an increase for TRPC1, TRPC3, TRPM7, TRPP1 (PKD1), TRPP2 (PKD2), TMEM63A, TMEM63B, but a decrease for TRPV2, Piezo1, Piezo2. The number of MSC gene transcripts increases: TREK-1, Kir6.2, Nav1.5, Cav1.2, Cav1.3, Kv7.1, and Kv1.2. This potentially leads to an increase in the expression of MGC and MSC proteins leading to an increase in the net current and, as a result, pathological changes in the heart function.
Subject(s)
Hypergravity , Myocytes, Cardiac , Rats , Animals , RNA , Base SequenceABSTRACT
The effect of small G-proteins of the Rho family on sodium current conducted by cardiac isoform NaV1.5 of voltage-gated sodium channels was studied in heterologous expression system, CHO-K1 cell line transfected with a plasmid containing the NaV1.5 gene. The influence of cotransfection with genes of wild-type, constitutively-active, and dominant-negative small G-proteins RhoA, Rac1, and Cdc2 on the parameters of sodium current and its noninactivating component (INa,late) was estimated. Among three studied small G-proteins, only RhoA (wild-type and constitutively-active type) strongly affected sodium current reducing its peak amplitude, but not the value of INa,late. Cotransfection with wild-type Rac1 resulted in a minor decrease in sodium current. Thus, small G-protein RhoA has potential capability for suppression of sodium current, although physiological relevance of this property has to be verified.
Subject(s)
Gene Expression Regulation , Membrane Potentials/physiology , NAV1.5 Voltage-Gated Sodium Channel/genetics , cdc42 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/genetics , Animals , CHO Cells , Cnidarian Venoms/pharmacology , Cricetulus , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Membrane Potentials/drug effects , NAV1.5 Voltage-Gated Sodium Channel/metabolism , Neurotoxins/pharmacology , Patch-Clamp Techniques , Plasmids/chemistry , Plasmids/metabolism , Transfection , Transgenes , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
The whole-cell patch-clamp technique was used to examine the effect of gadolinium Gd3+ (a non-specific blocker of mechanically gated current IMGCh, a component of late current IL) on ionic currents in insolated rat ventricular cardiomyocytes alone and in combination with the blockers of L-type calcium currents (ICaL) nifedipine (10 µM) or verapamil (1 µM). In K+in/K+out or Cs+in/Cs+out media, blockade of ICaL produced no effect on IL at negative potentials, but inhibited IL at positive ones. In K+in/K+out medium, Gd3+ (5 µM) decreased the net persistent current (Inp) at -45 mV from 198.6±6.4 to 96.7±9.5 pA over 15 min. Gd3+ alone or in combination with ICaL blockers shifted the reversal potential of IL to more negative values. At negative potentials, Gd3+ decreased IK1 and inward current including IMGCh. At positive potentials, Gd3+ alone or in combination with ICaL blockers decreased IL. When applied for 15 min in Cs+in/Cs+out medium at -45 mV, Gd3+ produced no effect on net current and inward and outward components of IL. Thus, Gd3+ can be viewed as a specific blocker of IMGCh only in Cs+ medium.
