Left Ventricular Action Potentials Research Articles

SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): This work was supported by CVie Therapeutics Limited (Taipei, Taiwan) and Windtree Therapeutics (Warrington, USA) Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the double property to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). The project aims to characterize istaroxime effects at a concentration (100 nM) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in a model of mild diabetes. Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed 1) marked DD not associated to cardiac fibrosis, 2) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, 3) reduced LV SERCA2 protein level and activity and 4) slower SR Ca2+ uptake rate, 5) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, 6) increased diastolic Ca2+, 7) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nM) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment. Abstract Figure.

SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy.

Aims Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes.Methods and results Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed (i) marked DD not associated to cardiac fibrosis, (ii) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, (iii) reduced LV SERCA2 protein level and activity and (iv) slower SR Ca2+ uptake rate, (v) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, (vi) increased diastolic Ca2+, and (vii) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent.Conclusions SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment.

High heart rate associated early repolarization causes J-waves in both zebra finch and mouse.

High heart rates are a feature of small endothermic—or warm‐blooded—mammals and birds. In small mammals, the QT interval is short, and local ventricular recordings reveal early repolarization that coincides with the J‐wave on the ECG, a positive deflection following the QRS complex. Early repolarization contributes to short QT‐intervals thereby enabling brief cardiac cycles and high heart rates. We therefore hypothesized high hearts rates associate with early repolarization and J‐waves on the ECG of endothermic birds. We tested this hypothesis by comparing isolated hearts of zebra finches and mice and recorded pseudo‐ECGs and optical action potentials (zebra finch, n = 8; mouse, n = 8). In both species, heart rate exceeded 300 beats per min, and total ventricular activation was fast (QRS < 10 ms). Ventricular activation progressed from the left to the right ventricle in zebra finch, whereas it progressed from apex‐to‐base in mouse. In both species, the early repolarization front followed the activation front, causing a positive J‐wave in the pseudo‐ECG. Inhibition of early repolarization by 4‐aminopyridine reduced J‐wave amplitude in both species. Action potential duration was similar between ventricles in zebra finch, whereas in mouse the left ventricular action potential was longer. Accordingly, late repolarization had opposite directions in zebra finch (left‐right) and mouse (right‐left). This caused a similar direction for the zebra finch J‐wave and T‐wave, whereas in the mouse they were discordant. Our findings demonstrate that early repolarization and the associated J‐wave may have evolved by convergence in association with high heart rates.

Transient Receptor Potential Vanilloid 4 channels participate in mouse ventricular action potentia

Transient Receptor Potential Vanilloid 4 (TRPV4) channels, belonging to the TRP channels superfamily, are Ca 2+ -permeable channels activated by hypo-osmotic stress. In the heart, TRPV4 channels are expressed at the protein level in mouse and rat ventricular myocytes and fibroblasts. They are involved in valve development during embryogenesis, in ischemia-reperfusion injuries and in reactive fibrosis. Whether TRPV4 channel can participate in ventricular action potential (AP) is still unknown. The aim of this study is to pharmacologically unmask the contribution of TRPV4 channels in left ventricular AP. Left Ventricular myocytes were isolated from TRPV4 + / + and TRPV4 − / − mice and AP were recorded using the patch-clamp technique. Pharmacological activator (GSK1016790A) and inhibitors (HC-067047 and GSK2193874) were perfused during the recordings to evaluate the effect on AP parameters. TRPV4 activator GSK1016790A induces a transient and dose dependent increase in AP duration at 90% of repolarization (APD90) in TRPV4 + / + but not in TRPV4 − / − myocytes (+ 19.4 ± 3.8%; n = 11 vs − 0.6 ± 1.8%; n = 7 at 500 nM respectively). The activator mediated-APD increase is abolished by TRPV4 channels inhibitors and CamKII inhibitor KN93 at 1 μM. Conversely, TRPV4 channel inhibitor (GSK2193874) at 100 nM significantly decreases APD90 in TRPV4 + / + but not in TRPV4 − / − myocytes (− 7.8 ± 2.6%; n = 11 vs 1.0 ± 1.3%; n = 7 respectively). Similarly, HC-067047 at 300 nM significantly decreases APD90 in TRPV4 + / + but not in TRPV4 − / − myocytes (− 10.9 ± 5.0%; n = 6 vs − 1.2 ± 2.4%; n = 4 respectively). At the concentrations mentioned above, activator and inhibitors have no effect on the other AP parameters. This study shows that TRPV4 channels exert a basal contribution in ventricular AP and could affect repolarization. It is thus a potential target for pharmacological approaches against ventricular arrhythmias.

56-03: Increased arrhythmia vulnerability in KChIP2 knock-out mice is explained by different spatial dispersion of ventricular repolarization and location of cardiac stimulation

Purpose of the study: Downregulation of ventricular K+ channel-interacting protein 2 (KChIP2) and loss of the transient outward K+ current (Ito), is observed in failing hearts. In this study, we investigate the impact of KChIP2 and heart failure (HF) on action potential duration (APD) and repolarization time (RT) in mouse hearts. We hypothese that a dispersion of RT will provoke arrhythmias if cardiac pacing is performed from the region with the shortest RT. Method used: HF in WT and KChIP2-/- mice was induced by transverse aortic constriction and verified by echocardiography. Regional activation time (AT), APD and RT (=AT + APD) were subsequently measured in isolated, perfused and apically paced hearts using floating microelectrodes. Finally, in-vivo intracardiac pacing of the right ventricle tested arrhythmia vulnerability. Summary of results: Within the left ventricle, APD is longer at the base in KChIP2-/- hearts (n = 6) when compared to WT (n = 6) (APD90: 52.4 ± 5.3 vs. 37.6 ± 3.8 ms, p < 0.05). However, including the AT, the apico-basal dispersion of RT in KChIP2-/- hearts is not different from WT. KChIP2-/- hearts have a larger interventricular dispersion of APD (Δ-APD90: 18.7 ± 4.8 vs. 6.1 ± 1.5 ms, p < 0.05), which translates into a larger left-to-right ventricular dispersion of RT compared to WT hearts (Δ-RT: 22.7 ± 6.7 vs. 5.0 ± 1.5 ms, p < 0.05). HF prolongs AT and APD in WT and KChIP2-/- hearts, and makes spatial dispersion of RT comparable. Pacing the right ventricle of KChIP2-/- control mice to test if pacing from a region with a shorter RT will provoke arrhythmias revealed significant increased arrhythmia vulnerability in these mice. Conclusion: Absence of KChIP2 leads to a prolongation of the left ventricular action potential and an increased interventricular dispersion of RT. HF alters the APD gradients, but reduces the differences in spatial dispersion of RT between WT and KChIP2-/- hearts. Finally, the location of cardiac pacing is crucial when evaluating arrhythmia vulnerability.

From the Purkinje fibres to the ventricle: One dimensional computer simulation for the healthy and failing heart.

This study used one-dimensional computer simulation to investigate the influence of heart failure on action potential conduction through the left Purkinje fibres to the left ventricle. The study was based on a rabbit model of left ventricular heart failure caused by volume and pressure overload. To simulate the effect of heart failure, we began with models of the healthy rabbit Purkinje fibre action potential and healthy left ventricular (endocardial) action potential. In the absence of ionic current measurements from failing rabbit Purkinje fibres, we assumed that changes in ionic currents mirrored changes in ion channel expression (measured at the messenger RNA level): ionic conductances were adjusted based on changes in expression of the relevant ion channels. Ionic currents in the left ventricle were adjusted in the same way, but in addition, changes in ionic currents measured in the failing rabbit left ventricle by Ruijter et al. and Powizd et al. were used in simulations. The simulations predict a gradient in action potential duration from the Purkinje fibres to the ventricle and this gradient is exacerbated in heart failure. The predicted changes in the Purkinje fibre and left ventricular action potential were compared to actual changes measured using sharp microelectrodes.

Loss of K+ Currents in Heart Failure Is Accentuated in KChIP2 Deficient Mice

KV 4 together with KV Channel-Interacting Protein 2 (KChIP2) mediate the fast recovering transient outward potassium current (I(to,f)) in the heart. KChIP2 is downregulated in human heart failure (HF), potentially underlying the loss of I(to,f). We investigated remodeling associated with HF hypothesizing that KChIP2 plays a central role in the modulation of outward K(+) currents in HF. HF was induced by aortic banding in wild-type (WT) and KChIP2 deficient (KChIP2(-/-)) mice, evaluated by echocardiography. Action potentials were measured by floating microelectrodes in intact hearts. Ventricular cardiomyocytes were isolated and whole-cell currents were recorded by patch clamp. Left ventricular action potentials in KChIP2(-/-) mice were prolonged in a rate dependent manner, consistent with patch-clamp data showing loss of a fast recovering outward K(+) current and upregulation of the slow recovering I(to,s) and I(Kur). HF decreased all outward K(+) currents in WT mice and did not change the relative contribution of I(to,f) in WT mice. Compared to WT HF, KChIP2(-/-) HF had a larger reduction of K(+) -current density. However, the relative APD prolongation caused by HF was shorter for KChIP2(-/-) compared with WT, and the APs of the 2 HF mouse types were indistinguishable. I(to,f) is just one of many K(+) currents being downregulated in murine HF. The downregulation of repolarizing currents in HF is accentuated in KChIP2(-/-) mice. However, the prolongation of APs associated with HF is less in KChIP2(-/-) compared to WT, suggesting other compensatory mechanism(s) in the KChIP2(-/-) mouse.

Hearts of K Channel-Interacting Protein 2 Deficient Mice have Prolonged Action Potential Duration, and Reduced Outward Potassium Currents that are further reduced by Heart Failure

Cardiac myocytes exhibit a fast recovering transient outward potassium current (Ito,f) that is conducted by the channel proteins Kv4 + KChIP2. Heart failure (HF) is associated with a reduction of outward K+-currents and a downregulation of KChIP2. In this study, we investigated the electrophysiological role of KChIP2 in mice with HF.HF induced by transverse aortic constriction was defined as an ejection fraction below 50% evaluated by ultrasound. Action potentials were recorded by floating micro electrodes from the left ventricle in ex vivo perfused hearts. Whole-cell currents were recorded from disaggregated left ventricular cardiomyocytes.We found an equal reduction in the ejection fractions of wild-type (WT) and KChIP2-/- mouse hearts by transverse aortic constriction. Left ventricular action potential durations were prolonged by 32% in KChIP2-/- vs. WT at 90% repolarization (APD90) during 200 ms pacing intervals (P<0.05). Peak K+-current density was 18% lower in KChIP2-/- vs. WT (P<0.05). Induction of HF reduced peak K+-current density by 38% in KChIP2-/- and by 24% in WT (P<0.05 for both). K+-current recovery from inactivation was delayed in KChIP2-/- vs. WT, and further delayed by HF in KChIP2-/- but remained unchanged in WT.In conclusion, HF reduced K+-currents in WT, but did not abolish the KChIP2-dependent fast recovering current component. KChIP2-/- mice lack the fast recovering K+-current (Ito,f) leading to prolonged left ventricular action potentials. HF in KChIP2-/- mice induced a further loss of K+-currents and an additional delay of recovery from inactivation, suggesting a reduction of non-Ito,f K+-currents contributing to the electrophysiological changes in mouse HF.

Effects of tetraethylammonium, 4-aminopyridine and bretylium on cardiovascular tissues from normo- and hypertensive rats.

Our objective was to test whether potassium-channel blockade is a potential positive inotropic mechanism for heart failure. Thus we studied the effects of tetraethylammonium, 4-aminopyridine and bretylium on left ventricular action potentials, left ventricular contractility in the absence and presence of hypertrophy, and on isolated blood vessels from Wistar Kyoto normotensive rats (WKY) and spontaneously hypertensive rats (SHRs). Tetraethylammonium at 10(-3)-10(-2) M, 4-aminopyridine at 10(-4)-10(-3) M and bretylium at 10(-6)-10(-4) M prolonged the action potentials of the WKY left ventricular strip. Similar concentrations of tetraethylammonium, 4-aminopyridine and bretylium augmented the peak force, prolonged the contractions, and did not cause arrhythmias in the absence or presence of isoprenaline on left ventricular strips from 12-month-old WKY. The 12-month-old SHR has hypertrophy of the left ventricle with reduced contractility and prolongation of relaxation. The effects of tetraethylammonium and bretylium were similar on WKY and SHR, whereas the effects of 4-aminopyridine were reduced on SHR left ventricular contractility, which suggests that the function of the transient outward-blocking potassium channel may be impaired in hypertrophy. Bretylium at < or = 10(-4) M had no effect on the portal vein, intralobar or mesenteric arteries. Tetraethylammonium and 4-aminopyridine at > or = 10(-5) M increased the duration or amplitude, or both, of the portal vein contractions. Tetraethylammonium at > or = 10(-2) M and 4-aminopyridine at > or = 3 x 10(-4) M contracted the mesenteric artery, and 4-aminopyridine also contracted the intralobar pulmonary artery. In summary, we have demonstrated that the action potential prolonging effects of potassium-channel blockade is associated with a positive inotropic effect on the rat left ventricle. The non-specific blockers, tetraethylammonium and 4-aminopyridine, do not have potential as positive inotropes in heart failure because of their widespread effects, including vasoconstriction. The potential of bretylium and some of the newer selective potassium-channel blockers as positive inotropes requires further evaluation.

Altered gene expression may underlie prolonged duration of the QT interval and ventricular action potential in streptozotocin-induced diabetic rat heart

Ventricular electrical conduction has been investigated in the streptozotocin (STZ)-induced diabetic rat. Diabetes was induced with a single injection of STZ (60 mg/kg bodyweight, ip). The ECG was measured continuously, in vivo, using a biotelemetry system. Left ventricular action potentials were recorded with an extracellular suction electrode. Expression of mRNA transcripts for selected ion transport proteins was measured in left ventricle with real-time RT-PCR. At 10 weeks after STZ treatment, in vivo heart rate (HR) was reduced (267 +/- 3 vs. 329 +/- 5 BPM), QRS complex duration and QT interval were prolonged in diabetic rats compared to controls. In vitro spontaneous HR was reduced and paced heart action potential repolarization was prolonged in diabetic rats compared to controls. The mRNA expression for Kcnd2 (I (to) channel) and Kcne2 (I (kr) channel) was significantly reduced in diabetic rats compared to controls. Altered gene expression and, in particular, genes that encode K(+) channel proteins may underlie delayed propagation of electrical activity in the ventricular myocardium of STZ-induced diabetic rat.

Effects of tedisamil on cardiovascular tissues isolated from normo- and hypertensive rats.

This study was undertaken to characterize the effects of tedisamil on isolated rat cardiovascular tissues, and identify actions that could be beneficial or detrimental in the treatment of cardiac disease. Tedisamil prolonged the Wistar Kyoto normotensive rat (WKY) left ventricular action potential and augmented the force of contraction of left ventricle strips. On the 12-month-old SHR model of cardiac hypertrophy, the augmenting effects of tedisamil at 10(-6) and 3 x 10(-6) M were reduced. On the 21-month-old SHR model of heart failure, the augmenting effects of tedisamil at 10(-6) and 3 x 10(-6) M were further reduced. The augmenting effect of tedisamil at 10(-5) M was reduced to 47%. The rate of the right atrium of 16- to 17-month-old WKY was reduced by tedisamil at 10(-5) and 10(-4) M, and tedisamil had a similar effect on the SHR right atrium. Tedisamil at 10(-6)--3 x 10(-5) M contracted the portal veins of WKY and aortae of 12-month-old WKY and SHR. The positive inotropic and negative chronotropic effects of tedisamil in the rat, which are partially or fully maintained in hypertrophied or failing myocardium would be beneficial in the treatment of heart failure. In contrast, the vasoconstrictor action of tedisamil will be detrimental in heart failure.

Effects of clofilium on cardiovascular tissues from normo- and hypertensive rats.

1. The overall aim was to test whether clofilium has some potential as a positive inotrope for heart failure. We used Wistar Kyoto normotensive rats (WKY) and spontaneously hypertensive rats (SHRs) and studied the effects of clofilium on isolated blood vessels, left ventricular action potentials and left ventricular contractility. 2. Clofilium at < or = 10(-6) M had no effect on WKY portal vein contractions and at < or = 3 x 10(-4) M had no effect on WKY or SHR quiescent mesenteric and intralobar pulmonary arteries. 3. Clofilium at 10(7) - 10(-5) M prolonged the WKY left ventricular action potentials and with 10(-6) and 10(-5)M this included after-depolarizations. 4. Clofilium at < or = 3 x 10(-5) M augmented the peak force, prolonged the contractions and did not cause arrhythmias in the absence and presence of isoprenaline on left ventricle strips from 12-month-old WKY. 5. The 12-month-old SHR has hypertrophy of the left ventricle with reduced peak force and prolongation of relaxation. The effects of clofilium on 12-month-old SHR left ventricle contractility were similar to those in the age-matched WKY. 6 In summary, clofilium has positive inotropic effects on the rat left ventricle that are maintained in hypertrophy. Clofilium does not have effects on blood vessels that would be detrimental in heart failure. Clofilium prolongs the rat left ventricle action potential and causes after-depolarizations. The pro-arrhythmic potential of clofilium, however, makes it unlikely that it could be used as a positive inotrope in the treatment of heart failure.

Dilation and action potential lengthening in cardiomyopathic Syrian hamster heart.

The aim of our study was to determine the main ionic mechanisms responsible for the electrophysiological alterations of ventricular action potentials associated with cardiac dilation in a strain of cardiomyopathic Syrian hamsters which does not develop hypertrophy during the first five months of life. Right and left ventricular action potentials (APs) were recorded in Langendorff perfused isolated hearts from dilated cardiomyopathic (MS 200) and normal hamsters at 60, 120, and 180 days of age. AP characteristics differed in the two ventricles and in different regions (base, apex) of the left ventricle in both strains. When recorded in a given region (apex), the plateau was always of higher amplitude and longer duration, i.e., of larger area, in diseased as compared to normal hearts. The participation of the calcium-independent 4-aminopyridine (4-AP) sensitive transient outward current, Ito1, in the left ventricular AP plateau repolarization was smaller in dilated than in control hearts at any age and AP area was the same in both strains at 60 days of age in the presence of 4-AP. The participation of the cadmium (Cd) sensitive L-type Ca current was investigated in the development of AP plateau at 120 days of age and was smaller in dilated than in control hearts. The participation of the Na-Ca exchange inward current, INa-Ca, in the development of the AP plateau was similar in both strains at 60 days of age; later on, it strongly decreased in control hearts, whereas it remained high in diseased hearts. The tetrodotoxin sensitive slowly inactivating inward current was not increased in dilated hearts compared to control hearts. Our results show that the AP lengthening observed, in dilated non-hypertrophic hamster hearts, results essentially from a reduced participation of Ito1 at 60 days of age, whereas it results from both a reduced participation of Ito1 and an increased participation of INa-Ca at 120 and 180 days of age.

The effects of lignocaine and tetrodotoxin on the action potentials and contractions of left ventricles from normo- and hypertensive rats

The objective was to test the hypothesis that the effects of the sodium channel blockers lignocaine and tetrodotoxin are modified in the presence of hypertension-induced hypertrophy. We describe the effects of lignocaine and tetrodotoxin on the action potentials and contractions of left ventricles isolated from 6-month-old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). The upstroke velocity, amplitude, and overshoot of the action potential were reduced; action potentials were prolonged; and the contractions were reduced on the hypertrophied left ventricles of the SHRs. Lignocaine and tetrodotoxin reduced the upstroke velocity, amplitude, and overshoot and prolonged the left ventricular action potentials. These effects of lignocaine and tetrodotoxin on the SHR were less than those on the WKY left ventricle, possibly because the action potential was already modified by hypertrophy. Lignocaine also reduced the left ventricular contractions and the concentrations producing this reduction were lower for the hypertrophied than those for the normal left ventricle. Tetrodotoxin at 3 × 10 −6–10 −5 M caused similar attenuation of the WKY and SHR left ventricle contractions. Our study shows that the effects of lignocaine on contraction are enhanced in the hypertrophied left ventricle of the SHR, which suggests that the binding is increased or the access of lignocaine to the receptor is enhanced in hypertrophy. In contrast, the effects of tetrodotoxin on contractions are similar, and thus the binding or access of tetrodotoxin to the receptor is not altered in the hypertrophied left ventricle of the SHR.

Developmental change of Left Ventricular Action Potential and Sympathetic Effect in SHR

Developmental change of Left Ventricular Action Potential and Sympathetic Effect in SHR

Comparative study of the left ventricular action potentials in embryonic hearts of wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR)

Comparative study of the left ventricular action potentials in embryonic hearts of wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR)