Isolated Guinea Pig Ventricular Cardiomyocytes Research Articles

MiRNA Expression Profiles in Isolated Ventricular Cardiomyocytes: Insights into Doxorubicin-Induced Cardiotoxicity.

Doxorubicin (DOX), widely used as a chemotherapeutic agent for various cancers, is limited in its clinical utility by its cardiotoxic effects. Despite its widespread use, the precise mechanisms underlying DOX-induced cardiotoxicity at the cellular and molecular levels remain unclear, hindering the development of preventive and early detection strategies. To characterize the cytotoxic effects of DOX on isolated ventricular cardiomyocytes, focusing on the expression of specific microRNAs (miRNAs) and their molecular targets associated with endogenous cardioprotective mechanisms such as the ATP-sensitive potassium channel (KATP), Sirtuin 1 (SIRT1), FOXO1, and GSK3β. We isolated Guinea pig ventricular cardiomyocytes by retrograde perfusion and enzymatic dissociation. We assessed cell morphology, Reactive Oxygen Species (ROS) levels, intracellular calcium, and mitochondrial membrane potential using light microscopy and specific probes. We determined the miRNA expression profile using small RNAseq and validated it using stem-loop qRT-PCR. We quantified mRNA levels of some predicted and validated molecular targets using qRT-PCR and analyzed protein expression using Western blot. Exposure to 10 µM DOX resulted in cardiomyocyte shortening, increased ROS and intracellular calcium levels, mitochondrial membrane potential depolarization, and changes in specific miRNA expression. Additionally, we observed the differential expression of KATP subunits (ABCC9, KCNJ8, and KCNJ11), FOXO1, SIRT1, and GSK3β molecules associated with endogenous cardioprotective mechanisms. Supported by miRNA gene regulatory networks and functional enrichment analysis, these findings suggest that DOX-induced cardiotoxicity disrupts biological processes associated with cardioprotective mechanisms. Further research must clarify their specific molecular changes in DOX-induced cardiac dysfunction and investigate their diagnostic biomarkers and therapeutic potential.

Protein kinase C epsilon mediates the inhibition of angiotensin II on the slowly activating delayed-rectifier potassium current through channel phosphorylation

The slowly activating delayed rectifier K+ current (IKs) is one of the main repolarizing currents in the human heart. Evidence has shown that angiotensin II (Ang II) regulates IKs through the protein kinase C (PKC) pathway, but the related results are controversial. This study was designed to identify PKC isoenzymes involved in the regulation of IKs by Ang II and the underlying molecular mechanism. The whole-cell patch-clamp technique was used to record IKs in isolated guinea pig ventricular cardiomyocytes and in human embryonic kidney (HEK) 293 cells co-transfected with human KCNQ1/KCNE1 genes and Ang II type 1 receptor genes. Ang II inhibited IKs in a concentration-dependent manner in native cardiomyocytes. A broad PKC inhibitor Gö6983 (not inhibiting PKCε) and a selective cPKC inhibitor Gö6976 did not affect the inhibitory action of Ang II. In contrast, the inhibition was significantly attenuated by PKCε-selective peptide inhibitor εV1–2. However, direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) increased the cloned human IKs in HEK293 cells. Similarly, the cPKC peptide activator significantly enhanced the current. In contrast, the PKCε peptide activator inhibited the current. Further evidence showed that PKCε knockdown by siRNA antagonized the Ang II-induced inhibition on KCNQ1/KCNE1 current, whereas knockdown of cPKCs (PKCα and PKCβ) attenuated the potentiation of the current by PMA. Moreover, deletion of four putative phosphorylation sites in the C-terminus of KCNQ1 abolished the action of PMA. Mutation of two putative phosphorylation sites in the N-terminus of KCNQ1 and one site in KCNE1 (S102) blocked the inhibition of Ang II. Our results demonstrate that PKCε isoenzyme mediates the inhibitory action of Ang II on IKs and by phosphorylating distinct sites in KCNQ1/KCNE1, cPKC and PKCε isoenzymes produce the contrary regulatory effects on the channel. These findings have provided new insight into the molecular mechanism underlying the modulation of the KCNQ1/KCNE1 channel.

Adrenergic regulation of the rapid component of delayed rectifier K+ currents in guinea pig cardiomyocytes.

Guinea pig ventricular cardiomyocytes display the rapid component of the delayed rectifier potassium current (Ikr) that contributes to ventricular repolarization and promotes stress-induced arrhythmias. Adrenergic stimulation favors ventricular arrhythmogenesis but its effects on Ikr are poorly understood. Adrenergic modulation of Ikr was studied in isolated guinea pig ventricular cardiomyocytes using whole-cell patch clamping. We found that the Ikr amplitude was reduced to 0.66±0.02 and 0.62±0.03 in response to 0.1 µM phenylephrine (PE), an α1AR agonist, and 10 µM isoproterenol (ISO), a βAR agonist, respectively. The effect of PE can be blocked by the selective α1A-adrenoceptor antagonist 5-methylurapidil, but not by the α1B-adrenoceptor antagonist chloroethylclonidine or α1D-adrenoceptor antagonist BMY7378. Additionally, the effect of ISO can be blocked by the β1-selective AR antagonist CGP-20712A, but not by the β2-selective AR antagonist ICI-118551. Although PE and ISO was continuously added to cells, ISO did not decrease the current to a greater extent when cells were first given PE. In addition, PE's effect on Ikr was suppressed by β1AR stimulation. Ikr can by regulated by both the α1 and β ARs system, and that in addition to direct regulation by each receptor system, crosstalk may exist between the two systems.

Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X)

The human ether-a-go-go-related gene (hERG) encodes the rapid component of the cardiac repolarizing delayed rectifier potassium current, I(Kr). The direct interaction of the commonly used protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) with hERG, KvLQT1/minK, and I(Kr) currents was investigated in this study. hERG and KvLQT1/minK channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. In addition, hERG currents in stably transfected human embryonic kidney (HEK 293) cells, native I(Kr) currents and action potentials in isolated guinea pig ventricular cardiomyocytes were recorded using whole-cell patch clamp electrophysiology. Bisindolylmaleimide I blocked hERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner with IC(50) values of 1.0 and 13.2 muM, respectively. hERG channels were primarily blocked in the open state in a frequency-independent manner. Analysis of the voltage-dependence of block revealed a reduction of inhibition at positive membrane potentials. BIM I caused a shift of -20.3 mV in the voltage-dependence of inactivation. The point mutations tyrosine 652 alanine (Y652A) and phenylalanine 656 alanine (F656A) attenuated hERG current blockade, indicating that BIM I binds to a common drug receptor within the pore region. KvLQT1/minK currents were not significantly altered by BIM I. Finally, 1 muM BIM I reduced native I(Kr) currents by 69.2% and lead to action potential prolongation. In summary, PKC-independent effects have to be carefully considered when using BIM I as PKC inhibitor in experimental models involving hERG channels and I(Kr) currents.

Inhibitory actions of the selective serotonin re-uptake inhibitor citalopram on HERG and ventricular L-type calcium currents

Using whole-cell patch clamp recording of heterologous HERG-mediated currents in transfected mammalian cells, we observed that the selective serotonin re-uptake inhibitor citalopram blocks HERG with an IC 50 of 3.97 μM. This is slightly less potent than fluoxetine in our system (IC 50 of 1.50 μM). In isolated guinea pig ventricular cardiomyocytes citalopram inhibited L-type calcium current ( I Ca,L). The voltage dependence of I Ca,L inactivation in the presence of 100 μM citalopram was shifted significantly leftward. As a result, the I Ca,L ‘window’ in citalopram was found to be (a) smaller and (b) leftward-shifted compared to control. The effects of citalopram on both calcium current amplitude and the I Ca,L ‘window’ may help to explain citalopram’s good cardiac safety profile, given its propensity to block HERG at excessive dosages.

On the Cardiac Contractile, Electrophysiological and Biochemical Effects of Endothall, a Protein Phosphatase Inhibitor

Protein phosphatase inhibitors, e.g. cantharidin, exert positive inotropic effects in mammalian heart preparations. Endothall, a synthetic herbicide which is chemically related to cantharidin, inhibits protein phosphatase activities in mouse liver preparations. However, the cardiac effects of endothall have hitherto not been studied. In guinea pig papillary muscles, endothall (1–100 μmol/l) failed to affect force of contraction, whereas cantharidin (1–100 μmol/l) increased force of contraction maximally to 313.4 ± 32% of control at 10 μmol/l. In isolated guinea pig ventricular cardiomyocytes, endothall did neither change the free intracellular calcium concentration nor the amplitude of calcium current nor the phosphorylation state of regulatory phosphoproteins like phospholamban. In contrast, cantharidin (30 μmol/l) increased the free intracellular calcium concentration and the L-type calcium current to 149.6 ± 9% and to 157.6 ± 12% of control, respectively. Furthermore, cantharidin (1–100 μmol/l) augmented the phosphorylation of phospholamban maximally to 140.8 ± 7% of control. Nevertheless, in guinea pig ventricular homogenates, both endothall and cantharidin inhibited phosphatase activity with EC₅₀ values of 1.92 and 0.32 μmol/l, respectively. Thus, in contrast to cantharidin, endothall failed to increase force of contraction, though it inhibited protein phosphatase activity. Clearly, endothall is not an appropriate tool to study the function of protein phosphatases in the mammalian heart.

Inhibition of the Fast Na + Current by Taurine in Guinea Pig Ventricular Myocytes

1. Effects of taurine on the fast Na + current ( I Na ) in isolated guinea pig ventricular cardiomyocytes were examined by using the whole-cell voltage-clamp method. Experiments were performed at room temperature (22°C). 2. Test pulses were applied from −60 to +40 mV from a holding potential of −90 mV. Addition of taurine to the bath solution markedly inhibited the I Na in a concentration-dependent manner; at −30 mV, by 39.0±4.1% ( n=8, P<0.01) at 10 mM and by 56.1±4.7% ( n=9, P<0.001) at 20 mM. 3. Simultaneously, the time constant of inactivation phase for I Na decreased to 0.95±0.4 ms ( n=8, P<0.05) at 10 mM and to 1.02±0.3 ms ( n=9, P<0.05) at 20 mM, from 1.29±0.3 ms in normal Tyrode solution. 4. These results indicate that taurine inhibits the I Na current, which would play an important role in the cell functions.

Inhibition by taurine of the inwardly rectifying K + current in guinea pig ventricular cardiomyocytes

Effects of taurine on the inwardly rectifying K + current ( I K1) in isolated guinea pig ventricular cardiomyocytes were examined using patch voltage-clamp methods. All experiments were performed at 36°C. Taurine (10–20 mM) increased the action potential duration, but failed to affect the resting potential. Holding potential was maintained at −30 mV. The current was activated with an inwardly going rectification, and was completely blocked by Ba 2+ (2 mM). Taurine inhibited I K1 at −120 mV by 28.3±1.1% ( n=6, P<0.05) at 10 mM and by 36.0±2.1% ( n=6, P<0.01) at 20 mM. The reversal potential was shifted in the hyperpolarizing direction by 3.7±0.6 mV ( n=6) at 20 mM. In inside-out patch-clamp experiments, the amplitude of unitary channels was −2.7±0.3 pA ( n=21) at −90 mV. Symmetrical high-K + (150 mM) solutions in both bath and pipette were used. The channel conductance was 32±2 pS ( n=9). Taurine did not affect channel conductance, but markedly decreased the open probability at −120 mV of channel by 21.5±2.4% ( n=8, P<0.01) at 10 mM, and by 56.7±3.8% ( n=8, P<0.001) at 20 mM. These responses were almost reversible. These results suggest that taurine directly modulates the open probability of the inwardly rectifying K + current, resulting in regulation of the functions of heart cells.

Actions of taurine on the L-type Ca2+ channel current in guinea pig ventricular cardiomyocytes.

Effects of taurine on the L-type channel in isolated guinea pig ventricular cardiomyocytes were examined at different Ca2+ concentrations by using whole-cell and cell-attached voltage-clamp modes. All experiments were performed at 36 degrees C. In whole-cell voltage-clamp experiments, test pulses were applied between -20 to +60 mV from a holding potential of -40 mV. When [Ca]i was pCa 6, addition of 10 and 20 mM taurine to the bath solution reduced the Ca2+ current (I(Ca)) at 0 mV by 14.4 +/- 2.0% (n = 8; p < 0.01) and 31.5 +/- 2.2% (n = 8; p < 0.001), respectively. In contrast, when [Ca]i was pCa 8, I(Ca) at +10 mV was enhanced by 10.1 +/- 2.2% (n = 7; p < 0.05) at 10 mM taurine and by 41.7 +/- 2.1% (n = 7; p < 0.001) at 20 mM taurine. Taurine increased the time constants (tau(f) and tau(s)) of inactivation phase for I(Ca) current at both pCa 8 and 6. In cell-attached voltage-clamp experiments, taurine (20 mM) decreased the open probability of unitary Ba2+ current from 0.63 +/- 0.06 to 0.39 +/- 0.09 (n = 5; p < 0.01) at 5.4 mM [Ca]o, whereas taurine increased it from 0.21 +/- 0.04 to 0.48 +/- 0.07 (n = 4; p < 0.01) at 0.9 mM [Ca]o. Taurine did not affect the channel conductance. In addition, taurine (20 mM) increased the time constants (tau(of) and tau(os)) of the open time and decreased tau(cs) of the closed time at 0.9 mM [Ca]o. At 5.4 mM [Ca]o, the tau(os) and tau(cs) were also increased and decreased, respectively. tau(of) and tau(cf) were unaffected. These results indicate that taurine modulates the open probability of L-type Ca2+ channel dependent on [Ca]i and [Ca]o, thereby maintaining the normal [Ca]i level.

Direct inhibition by taurine of the ATP-sensitive K + channel in guinea pig ventricular cardiomyocytes

1. 1. Effects of taurine on the ATP-sensitive K + channel in isolated guinea pig ventricular cardiomyocytes were examined using an inside-out patch voltage-clamp mode. All experiments were performed at 36°C. 2. 2. The ATP-sensitive K + channel was activated outwardly. ATP-free and 140 mM K + solution in the bath and 140 mM K + in the pipette solution were used. The amplitude was 2.2 ± 0.3 pA ( n=9) at + 50 mV. The channel conductance was 31 ± 3 pS ( n=9). Glibenclamide (1 μM) blocked the channel opening. 3. 3. Taurine decreased the open probability (as a mean patch current for 30 sec) of the channel by 21.5 ± 2.4% ( n = 8, P< 0.01) at 10 mM, and almost blocked it ( n = 8, P< 0.001) at 20 mM. 4. 4. Taurine did not affect the channel conductance; these responses were reversible. 5. 5. These results suggest that taurine directly modulates the open probability of the ATP-sensitive K + channel, resulting in regulation of the cell functions.

Trypsin and forskolin decrease the sensitivity of L-type calcium current to inhibition by cytoplasmic free calcium in guinea pig heart muscle cells

A key feature of trypsin action on ionic membrane currents including L-type Ca2+ current (ICa) is the removal of inactivation upon intracellular application. Here we report that trypsin also occludes the resting cytoplasmic free Ca2+ ([Ca2+]i)-induced inhibition of peak ICa in isolated guinea pig ventricular cardiomyocytes, using the whole-cell patch clamp in combination with the Fura-2 ratio-fluorescence technique. The effectiveness of trypsin to guard ICa against [Ca2+]i-induced inhibition was compared with that of forskolin, as cAMP-dependent phosphorylation had been suggested to confer protection against [Ca2+]i-induced inactivation. Intracellular dialysis of trypsin (1 mg/ml) augmented ICa by 7.2-fold, significantly larger than the threefold increase induced by forskolin (3 microM). Forskolin application after trypsin dialysis did not further enhance ICa. An increase in [Ca2+]i from resting levels (varied by 0.2, 10, and 40 mM EGTA dialysis) to submicromolar concentrations after replacement of external Na+ (Na(o)+) with tetraethylammonium (TEA+) resulted in monotonic inhibition of control ICa, elicited from a holding potential of -40 mV at 22 degrees C. AFter trypsin dialysis, however, ICa became less sensitive to submicromolar [Ca2+]i; the [Ca2+]i of half-maximal inhibition (K0.5, normally around 60 nM) increased by approximately 20-fold. Forskolin also increased the K0.5 by approximately threefold. These and accompanying kinetic data on ICa decay are compatible with a model in which it is assumed that Ca2+ channels can exist in two modes (a high open probability "willing" and a low open probability "reluctant" mode) that are in equilibrium with one another. An increase in [Ca2+]i places a larger fraction of channels in the reluctant mode. This interconversion is hindered by cAMP-dependent phosphorylation and becomes nearly impossible after tryptic digestion.

Modulation of Calcium Current Density by Intracellular Calcium in Isolated Guinea Pig Ventricular Cardiomyocytes

This study describes quantitatively the relation between L-type calcium current (ICa) density and resting cytosolic free calcium concentration ([Ca2+]i) in isolated guinea pig ventricular cardiomyocytes. ICa was measured simultaneously with [Ca2+]i at 22 and 35°C using the whole-cell patch-clamp method in combination with the Fura-2 ratio-fluorescence technique. At 22°C, the increase in [Ca2+]i upon replacing external Na+ with tetraethylammonium resulted in a monotonic decrease in ICa density. Half-maximal inhibition of ICa density occurred at a [Ca2+]i value (K0.5) of 60 ± 17 nM (mean ± S.D., n = 59). At 35°C, with a holding potential of −80 mV, a similar relation between [Ca2+]i and ICa was observed (K0.5 of 62 ± 12 nM, n = 34). When the cells were depolarized from −40 mV, however, the initial decrease in ICa density with increasing [Ca2+]i was usually followed by transient stimulation (≤ 2-fold), which was again overcome by inhibition. Stimulation was not observed in the presence of broad spectrum protein kinase inhibitors. These data suggest that Ca2+i-dependent facilitation of cardiac ICa is mediated by a temperature-sensitive enzymatic pathway that ends in voltage-dependent phosphorylation of Ca2+ channels.

Evidence for physiological functions of protein phosphatases in the heart: evaluation with okadaic acid.

Okadaic acid exerts a positive inotropic effect in cardiac preparations. We studied whether the positive inotropic effect of okadaic acid in cardiac preparations could be due to phosphatase inhibition and whether this inhibition affects the phosphorylation of cardiac proteins. In papillary muscles from guinea pigs, 30 microM okadaic acid increased force of contraction to 175% of predrug value. In isolated guinea pig ventricular cardiomyocytes, okadaic acid augmented single Ca(2+)-channel currents by enhancing channel availability. In homogenates from ventricles, 1 microM okadaic acid completely inhibited phosphorylase a phosphatase activity. In isolated 32P-labeled ventricular cardiomyocytes, 30 microM okadaic acid increased phosphorylation of phospholamban (PLB) and troponin inhibitor (TnI) to 325 and 284% of control, respectively. Furthermore, 30 microM okadaic acid increased phosphorylation of a hitherto unknown 23-kDa protein to 352% of control. It is concluded that the effects of okadaic acid could be mediated by increasing the phosphorylation state of several proteins including PLB, a 23-kDa protein, and TnI.

Inhibition in L-type Ca 2+ channel by stimulation of protein kinase C in isolated guinea pig ventricular cardiomyocytes

1. 1. Electrophysiological effects of phorbol esters on the L-type Ca 2+ current ( I Ca(L)) in isolated single ventricular cells from guinea pig hearts were investigated. 2. 2. In whole-cell voltage-clamped myocytes, 12- O-tetradecanoyl-phorbol-13-acetate (TPA) at 10 −7 M inhibited I Ca(L). An antagonist of protein kinase C (PK-C), H-7, at 10 −5 M did not modify the TPA-induced inhibition. The time-course of inactivation process for I Ca(L) was greatly slowed. 3. 3. In cell-attached patch-clamp experiments, TPA (10 −7 M) also markedly decreased the opening of L-type Ca 2+ channels. The conductance was unaffected. 4. 4. Even H-7 (10 −5 M) alone inhibited the opening of the channels. Addition of TPA (10 −7–10 −8 M) caused further decrease in the opening. 5. 5. On the other hand, 4-α-phorbol-12,13-didecanoate (not a PK-C activator) had no effect on the Ca 2+ channels. 6. 6. These results indicate that the PK-C activation induced by TPA greatly depresses the opening of L-type Ca 2+ channels in ventricular cell membranes.