Calsequestrin 2 deletion shortens the refractoriness of Ca²⁺ release and reduces rate-dependent Ca²⁺-alternans in intact mouse hearts

Calsequestrin (Casq2) is a low affinity Ca(2+)-binding protein located in sarcoplasmic reticulum (SR) of cardiac myocytes. Casq2 acts as a Ca(2+) buffer regulating free Ca(2+) concentration in the SR lumen and plays a significant role in the regulation of Ca(2+) release from this intracellular organelle. In addition, there is experimental evidence supporting the hypothesis that Casq2 also modulates the activity of the cardiac Ca(2+) release channels, ryanodine receptors (RyR2). In this study, Casq2 knockout mice (Casq2-/-) were used as a model to evaluate the effects of the Casq2 on the cytosolic and intra-SR Ca(2+) dynamics, and the electrical activity in the ventricular epicardial layer of intact beating hearts. Casq2-/- mice have accelerated intra-SR Ca(2+) refilling kinetics (76 ± 22 vs. 136.5 ± 15 ms) and a reduced refractoriness of Ca(2+) release (182 ± 32 ms Casq2+/+ and 111 ± 22 ms Casq2-/- ). In addition, mice display reduced Ca(2+) alternans (67% decline in the amplitude of Ca(2+) alternans at 7 Hz, 21oC) and less T-wave alternans at the electrocardiographic level. The results presented in this paper support the idea of Casq2 acting both as a buffer and a direct regulator of the Ca(2+) release process. Finally, we propose that alterations in Ca(2+) release refractoriness shown here could explain the relationship between Casq2 function and an increase in the risk for ventricular arrhythmias.     

KMUP-1 inhibits L-type Ca²⁺ channels involved the protein kinase C in rat basilar artery myocytes

This study investigated whether KMUP-1, a xanthine-based derivative, inhibits L-type Ca(2+) currents (I(Ca,L)) in rat basilar artery smooth muscle cells (RBASMCs). We used whole cell patch-clamp recording to monitor Ba(2+) currents (I(Ba)) through L-type Ca(2+) channels (LTCCs). Under voltage-clamp conditions, holding at -40 mV, KMUP-1 (1, 3, 10 μM) inhibited I(Ba) in a concentration-dependent manner and its IC(50) value was 2.27 ± 0.45 μM. A high concentration of KMUP-1 (10 μM) showed without modifying the I(Ba) current-voltage relationship. On the other hand, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM) increase I(Ba) was inhibited by KMUP-1. Pretreatment with the PKC inhibitor chelerythrine (5 μM) intensified KMUP-1-inhibited I(Ba). However, the Rho kinase inhibitor Y-27632 (30 μM) failed to affect the I(Ba) inhibition by KMUP-1. In light of these results, we suggest that KMUP-1 inhibition of LTCCs in concentration- and voltage-dependent manners in RBASMCs may be due, at least in part, to its modulation of the PKC pathway.     

Postnatal development of transmural gradients in expression of ion channels and Ca²⁺-handling proteins in the ventricle

Transmural gradients in myocyte action potential duration (APD) and Ca(2+)-handling proteins are argued to be important for both the normal functioning of the ventricle and arrhythmogenesis. In rabbit, the transmural gradient in APD (left ventricular wedge preparation) is minimal in the neonate. During postnatal development, APD increases both in the epicardium and the endocardium, but the prolongation is more substantial in the endocardium leading to a significant transmural gradient. We have investigated changes in the expression of ion channels and also Ca(2+)-handling proteins in the subepicardial and subendocardial layers of the left ventricular free wall in neonatal (2-7 days of age) and adult male (~6 months of age) New Zealand White rabbits using quantitative PCR and also, when possible, in situ hybridisation and immunohistochemistry. In the adult, there were significant and substantial transmural gradients in Ca(v)1.2, KChIP2, ERG, K(v)LQT1, K(ir)2.1, NCX1, SERCA2a and RyR2 at the mRNA and, in some cases, protein level-in every case the mRNA or protein was more abundant in the epicardium than the endocardium. Of the eight transmural gradients seen in the adult, only three were observed in the neonate and, in two of these cases, the gradients were smaller than those in the adult. However, in the neonate there were also transmural gradients not observed in the adult: in HCN4, Na(v)1.5, minK, K(ir)3.1 and Cx40 mRNAs - in every case the mRNA was more abundant in the endocardium than the epicardium. If the postnatal changes in ion channel mRNAs are used to predict changes in ionic conductances, mathematical modelling predicts the changes in APD observed experimentally. It is concluded that many of the well known transmural gradients in the ventricle develop postnatally.     

Different left ventricular relaxation parameters in isolated working rat and guinea pig hearts – Influence of preload, afterload, temperature, and isoprenaline

In isolated ejecting rat and guinea pig hearts, the sensitivity of the time constant of left ventricular isovolumic pressure fall, the maximum pressure fall velocity min LVdP/dt, and the relaxation time to different hemodynamic conditions, temperature, and isoprenaline were investigated. was obtained by fitting the isovolumic pressure fall three-parametrically to the exponential p(t) = p + (p0- p) exp (- t) which was found to be superior to semilogarithmic estimation. The influence of different working conditions on the relaxation parameters was tested by a rank correlation test and quantified by calculating standardized regression coefficients. Hemodynamic conditions were altered by changing left ventricular end-diastolic pressure (increasing inflow to the heart) and peak pressure (max LVP, varying aortic outflow resistance), and by atrial pacing (variation of interbeat interval). Lusitropic sensitivity was investigated by changing temperature and by applying isoprenaline. All regression parameters were only moderately sensitive to changes in end-diastolic pressure, max LVP, or heart rate, with the exception of a considerable afterload dependence of min LVdP/dt in rat hearts. This dependence, however, can be overcome to a large extent by dividing min LVdP/dt by mean aortic pressure. Isoprenaline strongly influenced all relaxation parameters, and so did temperature, except for relaxation time in guinea pig hearts. We conclude that serves as a reliable relaxation parameter, also in the hearts of small animals with heart rates up to 450 beats/min. In isolated hearts, min LVdP/dt, corrected for afterload dependence, is also suitable as a complementary index of the early relaxation phase.     

Cardiac contractility modulation increases action potential duration dispersion and decreases ventricular fibrillation threshold via β1-adrenoceptor activation in the crystalloid perfused normal rabbit heart

Background/objectives

Cardiac contractility modulation (CCM) is a new treatment being developed for heart failure (HF) involving application of electrical current during the absolute refractory period. We have previously shown that CCM increases ventricular force through β1-adrenoceptor activation in the whole heart, a potential pro-arrhythmic mechanism. This study aimed to investigate the effect of CCM on ventricular fibrillation susceptibility.

Methods

Experiments were conducted in isolated New Zealand white rabbit hearts (2.0–2.5 kg, n = 25). The effects of CCM (± 20 mA, 10 ms phase duration) on the left ventricular basal and apical monophasic action potential duration (MAPD) were assessed during constant pacing (200 bpm). Ventricular fibrillation threshold (VFT) was defined as the minimum current required to induce sustained VF with rapid pacing (30 × 30 ms). Protocols were repeated during perfusion of the β1-adrenoceptor antagonist metoprolol (1.8 μM). In separate hearts, the dynamic and spatial electrophysiological effects of CCM were assessed using optical mapping with di-4-ANEPPS.

Results

CCM significantly shortened MAPD close to the stimulation site (Basal: 102 ± 5 [CCM] vs. 131 ± 6 [Control] ms, P < 0.001). VFT was reduced during CCM (2.6 ± 0.6 [CCM] vs. 6.1 ± 0.8 [Control] mA, P < 0.01) and was correlated (r² = 0.40, P < 0.01) with increased MAPD dispersion (26 ± 4 [CCM] vs. 5 ± 1 [Control] ms, P < 0.01) (n = 8). Optical mapping revealed greater spread of CCM induced MAPD shortening during basal vs. apical stimulation. CCM effects were abolished by metoprolol and exogenous acetylcholine. No evidence for direct electrotonic modulation of APD was found, with APD adaptation occurring secondary to adrenergic stimulation.

Conclusions

CCM decreases VFT in a manner associated with increased MAPD dispersion in the crystalloid perfused normal rabbit heart.     

Chronic l-arginine treatment increases cardiac cyclic guanosine 5′-monophosphate in rats with aortic stenosis: effects on left ventricular mass and beta-adrenergic contractile reserve

Objectives. We tested the hypothesis that nitric oxide (NO) cyclic guanosine 5′-monophosphate (GMP) signaling is deficient in pressure overload hypertrophy due to ascending aortic stenosis, and that long-term l-arginine treatment will increase cardiac cyclic GMP production and modify left ventricular (LV) pressure overload hypertrophy and beta-adrenergic contractile response.Background. Nitric oxide cyclic GMP signaling is postulated to depress vascular growth, but its effects on cardiac hypertrophic growth are controversial.Methods. Forty control rats and 40 rats with aortic stenosis left ventricular hypertrophy ([LVH] group) were randomized to receive either l-arginine (0.40 g/kg/day) or no drug for 6 weeks.Results. The dose of l-arginine did not alter systemic blood pressure. Animals with LVH had similar LV constitutive nitric oxide synthase (cNOS) mRNA and protein levels, and LV cyclic GMP levels as compared with age-matched controls. In rats with LVH l-arginine treatment led to a 35% increase in cNOS protein levels (p = 0.09 vs untreated animals with LVH) and a 1.7-fold increase in LV cyclic GMP levels (p < 0.05 vs untreated animals with LVH). However, l-arginine treatment did not suppress LVH in the animals with aortic stenosis. In contrast, in vivo LV systolic pressure was depressed in l-arginine treated versus untreated rats with LVH (163 ± 16 vs 198 ± 10 mm Hg, p < 0.05). In addition, the contractile response to isoproterenol was blunted in both isolated intact hearts and isolated myocytes from l-arginine treated rats with LVH compared with untreated rats with LVH. This effect was mediated by a blunted increase in peak systolic intracellular calcium in response to beta-adrenergic stimulation.Conclusions. Left ventricular hypertrophy due to chronic mechanical systolic pressure overload is not characterized by a deficiency of LV cNOS and cyclic GMP levels. In rats with aortic stenosis, l-arginine treatment increased cardiac levels of cyclic GMP, but it did not modify cardiac mass in rats with aortic stenosis. However, long-term stimulation of NO-cyclic GMP signaling depressed in vivo LV systolic function in LVH rats and markedly blunted the contractile response to beta-adrenergic stimulation.     

Expression of the cytoplasmic domain of β1 integrin induces apoptosis in adult rat ventricular myocytes (ARVM) via the involvement of caspase-8 and mitochondrial death pathway

Abstract Stimulation of β-adrenergic receptor (β-AR) induces cardiac myocyte apoptosis. Integrins, a family of cell-surface receptors, play an important role in the regulation of cardiac myocyte apoptosis and ventricular remodeling. Cleavage of extracellular domain of β1 integrin, also called integrin shedding, is observed during cardiac hypertrophy and progression to early heart failure. Here we show that stimulation of β-AR induces β1 integrin fragmentation in mouse heart. To examine the role of intracellular domain of β1 integrin in cardiac myocyte apoptosis, a chimeric receptor consisting of the cytoplasmic tail domain of β_1A integrin and the extracellular/transmembrane domain of the interleukin-2 receptor (TAC-β1) was expressed in adult rat ventricular myocytes (ARVM) using adenoviruses. TAC-β1 increased the percentage of apoptotic ARVM as measured by TUNEL-staining assay. TAC-β1-induced apoptosis was found to be associated with increased cytosolic cytochrome c and decreased mitochondrial membrane potential. TAC-β1 increased caspase-8 activity. Z-IETD-FMK, a specific caspase-8 inhibitor, significantly inhibited TAC-β1-induced apoptosis. TAC-β1 expression also increased cleavage of Bid, a pro-apoptotic Bcl-2 family protein. These data suggest that shedding of β1 integrin may be a mechanism of induction of apoptosis during β-AR-stimulated cardiac remodeling.     

K201 (JTV-519) alters the spatiotemporal properties of diastolic Ca<Superscript>2+</Superscript> release and the associated diastolic contraction during β-adrenergic stimulation in rat ventricular cardiomyocytes

K201 has previously been shown to reduce diastolic contractions in vivo during β-adrenergic stimulation and elevated extracellular calcium concentration ([Ca²⁺]_o). The present study characterised the effect of K201 on electrically stimulated and spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca²⁺ release and contractile events in isolated rat cardiomyocytes during β-adrenergic stimulation and elevated [Ca²⁺]_o. Parallel experiments using confocal microscopy examined spontaneous diastolic Ca²⁺ release events at an enhanced spatiotemporal resolution. 1.0 μmol/L K201 in the presence of 150 nmol/L isoproterenol (ISO) and 4.75 mmol/L [Ca²⁺]_o significantly decreased the amplitude of diastolic contractions to ~16% of control levels. The stimulated free Ca²⁺ transient amplitude was significantly reduced, but stimulated cell shortening was not significantly altered. When intracellular buffering was taken into account, K201 led to an increase in action potential-induced SR Ca²⁺ release. Myofilament sensitivity to Ca²⁺ was not changed by K201. Confocal microscopy revealed diastolic events composed of multiple Ca²⁺ waves (2–3) originating at various points along the cardiomyocyte length during each diastolic period. 1.0 μmol/L K201 significantly reduced the (a) frequency of diastolic events and (b) initiation points/diastolic interval in the remaining diastolic events to 61% and 71% of control levels respectively. 1.0 μmol/L K201 can reduce the probability of spontaneous diastolic Ca²⁺ release and their associated contractions which may limit the propensity for the contractile dysfunction observed in vivo.     

Contribution of <Emphasis Type="Italic">I</Emphasis><Subscript>Kr</Subscript> and <Emphasis Type="Italic">I</Emphasis><Subscript>K1</Subscript> to ventricular repolarization in canine and human myocytes: is there any influence of action potential duration?

Background  The aim of the present work was to study the profile of the rapid delayed rectifier potassium current (I _Kr) and the inward rectifier potassium current (I _K1) during ventricular repolarization as a function of action potential duration and rate of repolarization. Methods  Whole cell configuration of the patch clamp technique was used to monitor I _Kr and I _K1 during the action potential plateau and terminal repolarization. Action potentials recorded at various cycle lengths (0.4–5 s) and repolarizing voltage ramps having various slopes (0.5–3 V/s) were used as command signals. I _Kr and I _K1 were identified as difference currents dissected by E-4031 and BaCl₂, respectively. Results  Neither peak amplitudes nor mean values of I _Kr and I _K1 recorded during the plateau of canine action potentials were influenced by action potential duration. The membrane potential where I _Kr and I _K1 peaked during the terminal repolarization was also independent of action potential duration. Similar results were obtained in undiseased human ventricular myocytes, and also in canine cells when I _Kr and I _K1 were evoked using repolarizing voltage ramps of various slopes. Action potential voltage clamp experiments revealed that the peak values of I _Kr, I _K1, and net outward current during the terminal repolarization were independent of the pacing cycle length within the range of 0.4 and 5 s. Conclusions  The results indicate that action potential configuration fails to influence the amplitude of I _Kr and I _K1 during the ventricular action potential in dogs and humans, suggesting that rate-dependent changes in action potential duration are not likely related to rate-dependent alterations in I _Kr or I _K1 kinetics in these species. Returned for 1. Revision: 5 February 2008 1. Revision received: 11 April 2008 Returned for 2. Revision: 14 May 2008 2. Revision received: 15 May 2008