Electrical Modulation of Cardiac Contractility: Clinical Aspects in Congestive Heart Failure

Heart failure is a highly prevalent disease in western society. Drug therapies aimed at increasing myocardial contractility have been associated with decreased survival. Several short and mid term clinical studies have suggested adjuvant or alternative therapies to congestive heart failure using modified pacing techniques that were aimed to increase contractility (e.g. Paired pacing) or restore synchrony of contraction (biventricular pacing). While delivery of paired pacing was abandoned during the early 70's, biventricular pacing has recently emerged as an adjuvant treatment to limited group of congestive heart failure patients with aberrant left ventricular conduction. In this brief review, we describe our initial safety and efficacy experience in patients with heart failure using a novel non-stimulatory electrical approach to the delivery of positive inotropic therapy to the failing myocardium. The study suggests that unlike modified pacing techniques, delivery of the signal to the left ventricle during the refractory period resulted in a rapid increase in myocardial contractility and improved hemodynamic performance. The near instantaneous contractility improvement achieved by this type of stimulus was shown to be safe and effective independently of the primary cause of heart failure or the function of the conduction system. Unlike pharmacologic treatments, which have a relatively constant effect, use of electrical stimuli may prove useful as a new therapeutic modality in the treatment of heart failure with which contractility can be improved when and as needed.     

短时刺激对心力衰竭心室肌电生理特性及钠钙交换体的影响

观察短时刺激对阿霉素心力衰竭 (简称心衰 )模型心室肌的电生理特性的影响 ,并研究钠钙交换体在其中的作用。 2 4只纯种日本大耳白兔随机分为对照组和阿霉素心衰组。心衰模型通过耳缘静脉每周注射一次 0 .2 %的阿霉素 ( 2mg/kg) ,持续 8周获得 ;对照组每周注射生理盐水 ( 2ml/kg)持续 8周。从第 10周开始进行实验 ,经兔耳缘静脉推注阿托品 ( 0 .0 4mg/kg) ,美多心安 ( 0 .2mg/kg)阻断神经支配。于心房快速刺激 30min前后 ,应用心脏电刺激及单相动作电位 (MAP)记录技术测定心电生理参数。左心室心肌组织钠钙交换体的蛋白量用免疫印迹方法定量。结果 :快速刺激后 ,心衰组和对照组心室肌的 90 %MAP时程 (MAPD90 )分别延长 14.1± 8.8ms和 8.7± 5 .6ms(P <0 .0 1) ,心室有效不应期 (ERP)分别延长 17.8± 8.4ms和 11.3± 6 .1ms(P <0 .0 1)。钠钙交换体的蛋白量没有明显的差异 (P >0 .0 5 )。结论 :短时快速刺激可引起阿霉素兔心衰模型心室肌的电生理特性发生改变 ,钠钙交换体的表达没有明显的改变。     

Role of Cardiac Contractility Modulation in Heart Failure With a Higher Ejection Fraction

Cardiac contractility modulation (also known as CCM) is a novel device therapy that delivers nonexcitatory electric stimulation to cardiac myocytes during the absolute refractory period, and it has been shown to improve functional status and clinical outcomes in patients with heart failure (HF) with reduced ejection fraction (HFrEF). CCM therapy is currently recommended for a subset of patients with advanced HFrEF who are not candidates for cardiac resynchronization therapy. A growing body of evidence demonstrates the benefit of CCM therapy in patients with HFrEF and with ejection fraction at the upper end of the spectrum and in patients with HF and with mildly reduced ejection fraction (HFmrEF). Experimental studies have also observed reversal of pathological biomolecular intracellular changes with CCM therapy in HF with preserved ejection fraction (HFpEF), indicating the potential for clinically meaningful benefits of CCM therapy in these patients. In this review, we sought to discuss the basis of CCM therapy and its potential for management of patients with HF with higher ejection fractions.     

Cardiac Contractility Modulation With the Impulse Dynamics Signal: Studies in Dogs With Chronic Heart Failure

The intravenous use of positive inotropic agents, such as sympathomimetics and phosphodiesterase inhibitors, in heart failure is limited by pro-arrhythmic and positive chronotropic effects. Chronic use of these agents, while eliciting an improvement in the quality of life of patients with advanced heart failure, has been abandoned because of marked increase in mortality when compared to placebo. Nevertheless, patients with advanced heart failure can benefit from long-term positive inotropic support if the therapy can be delivered on demand and in a manner that is both safe and effective. In this review, we will examine the use of a novel, non-stimulatory electrical signal that can acutely modulate left ventricular (LV) contractility in dogs with chronic heart failure in such a way as to elicit a positive inotropic support. Cardiac contractility modulation (CCM) with the Impulse Dynamic signal was examined in dogs with chronic heart failure produced by intracoronary microembolizations. Delivery of the CCM signal from a lead placed in the great coronary vein for periods up to 10 minutes resulted in significant improvements in cardiac output, LV peak+dP/dt, LV fractional area of shortening and LV ejection fraction measured angiographically. Discontinuation of the signal resulted in a return of all functional parameters to baseline values. In cardiomyocytes isolated from dogs with chronic heart failure, application of the CCM signal resulted in improved shortening, rate of change of shortening and rate of change of relengthening suggesting that CCM application is associated with intrinsic improvement of cardiomyocyte function. The improvement in isolated cardiomyocyte function after application of the CCM signal was accompanied by an increase in the peak and integral of the Ca²⁺ transient suggesting modulation of calcium cycling by CCM application. In a limited number of normal dogs, intermittent chronic delivery of the CCM signal for up to 7 days showed chronic maintenance of LV functional improvement. In conclusion, pre-clinical results to date with the Impulse Dynamics CCM signal indicate that this non-pharmacologic therapeutic modality can provide short-term positive inotropic support to the failing heart and as such, may be a useful adjunct in the treatment of advanced heart failure. Additional, long-term studies in dogs with heart failure are needed to establish the safety and efficacy of this therapeutic modality for the chronic treatment of this disease syndrome.     

Improved Physical Function After Cardiac Contractility Modulation Therapy in 10 Patients With Chronic Heart Failure

BackgroundA new generation of therapeutic devices has expanded the options for managing advanced heart failure. We examined the outcomes of cardiac contractility therapy in a series of 10 patients with chronic heart failure.MethodsTen patients with chronic heart failure were nonrandomly selected to receive cardiac contractility modulation therapy. Hemodynamics, left ventricular ejection fraction, functional capacity, and clinical outcomes were evaluated at baseline and after 6 months of therapy.ResultsEight male and 2 female patients (mean [SD] age, 63.4 [9.4] years) received cardiac contractility modulation therapy. Between baseline and 6-month follow-up, mean (SD) left ventricular ejection fraction improved from 27.1% (4.18%) to 35.1% (9.89%), New York Heart Association class declined from 3.9 (0.32) to 2.44 (0.52), and 6-minute walk test distance increased from 159.2 (93.79) m to 212.4 (87.24) m. Furthermore, the mean (SD) number of hospital admissions within the 6 months before cardiac contractility modulation therapy was 2.4 (2.27) compared with 1 (1.52) during the 6 months after therapy.ConclusionCardiac contractility modulation therapy improved physical functioning and reduced hospital admissions in these patients.     

Acute Effects of Intravenous Nesiritide on Cardiac Contractility in Heart Failure

BackgroundAlthough nesiritide is a potent vasodilator, studies using myocytes and isolated muscle strips have shown that recombinant B-type natriuretic peptide (BNP; nesiritide) decreases contractility. We sought to determine whether nesiritide decreases contractility in heart failure patients.Methods and ResultsTwenty-five heart failure patients underwent left heart catheterization (using a pressure-volume conductance catheter) and echocardiography at baseline and after a 2 mcg/kg bolus and 30-minute nesiritide infusion (0.01 mcg·kg·min). From invasive and noninvasive measurements, left ventricular (LV) systolic function indices were calculated, including ejection fraction, end-systolic elastance (E_es; single-beat invasive and noninvasive methods) and preload-recruitable stroke work (PRSW; noninvasive, single-beat method). The mean age was 60 ± 11 years, 48% were male, 56% had coronary disease, and 64% had hypertension. Although nesiritide did not change LV ejection fraction, it did decrease contractility on pressure-volume analysis. Noninvasive E_es decreased from 2.6 ± 1.6 to 2.0 ± 1.4 mm Hg/mL (P = .02). For those with reduced ejection fraction, E_es decreased by invasive (P = .006) and noninvasive (P = .02) methods. PRSW decreased from 76 ± 37 to 62 ± 28 g/cm² (P = .003). On tissue Doppler imaging, nesiritide reduced the systolic annular tissue velocity of the mitral annulus from 8.0 ± 1.9 to 6.9 ± 1.3 cm/s (P = .04).ConclusionsNesiritide infusion acutely decreases derived measures of contractility and systolic function in patients with chronic heart failure.     

Alterations in membrane Na+-Ca2+ exchange in the aging myocardium

Heart failure is a common event in the elderly. Although it is generally believed that age-induced biochemical alterations of the myocardium are directly involved in the heart failure usually encountered in the elderly, several gaps still exist in our understanding of the mechanisms involved in the contractile failure of the aged myocardium. Virtually nothing is known, for example, about the integrity of the sarcolemmal (SL) Na⁺-Ca²⁺ exchange mechanism in the aging head. In this study we have examined the status of Na⁺-Ca²⁺. exchange in SL membranes from the aging myocardium. Male Sprague Dawley rats were used and were divided into 3 groups: young (4–6 months old); middle aged (14–17 months) and old age (24–27 months). Purified SL membranes were isolated from ventricular tissues. Ca²⁺-influx of Na⁺-loaded vesicles from old hearts was depressed relative to the middle-aged group, which in turn was lower than the Ca²⁺ accumulated by vesicles from young hearts. These changes were observed at different concentrations of Ca²⁺ and at different times of incubation. The results suggest that Ca²⁺ transport by SL Na⁺-Ca²⁺ exchange mechanism is attenuated in the aging myocardium and might therefore be involved in age-induced heart failure.     

Alterations of sarcolemmal Na+-Ca2+ exchange in catecholamine-induced cardiomyopathy

Rats were injected intraperitoneally with 40 mg/kg body weight isoproterenol and the heart sarcolemma was isolated 3, 9 and 24 hours later. The heart/body weight ratio increased and varying degrees of change in cardiac ultrastructure were apparent at 9 and 24 hours after isoproterenol injection. Na+-dependent Ca2+ uptake activities of heart sarcolemma were depressed at 3, 9 and 24 hours; such alterations in 24 hour preparations were evident at different times of incubation and at different concentrations of Ca2+. No differences in Na+-induced Ca2+ release or Na+-K+ ATPase activities were observed between the control and experimental membranes. The control and isoproterenol-treated heart sarcolemmal preparations were minimally but equally contaminated by other subcellular organelles. Although there was no significant change in the phospholipid composition, the protein pattern as determined by gel electrophoresis was altered in sarcolemma at 24 hours of isoproterenol treatment. These results indicate an abnormality of heart sarcolemmal Na+-dependent Ca+ uptake during the development of catecholamine-induced cardiotoxicity. It is suggested that a depression in the ability of the cell to remove Ca2+ through the Na+-Ca2+ exchange in sarcolemma may contribute to the development of intracellular Ca2+ overload in catecholamine induced cardiomyopathy.     

Involvement of mitochondrial Na＋-Ca2＋ exchange in intestinal pacemaking activity

AIM: Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. We have aimed to investigate the involvement of mitochondrial Na+-Ca2+ exchange in intestinal pacemaking activity in cultured interstitial cells of Cajal. METHODS: Enzymatic digestions were used to dissociate ICCs from the small intestine of a mouse. The whole-cell patch-clamp configuration was used to record membrane currents (voltage clamp) and potentials (current clamp) from cultured ICCs. RESULTS: Clonazepam and CGP37157 inhibited the pacemaking activity of ICCs in a dose-dependent manner. Clonazepam from 20 to 60 micromol/L and CGP37157 from 10 to 30 micromol/L effectively inhibited Ca2+ efflux from mitochondria in pacemaking activity of ICCs. The IC50s of clonazepam and CGP37157 were 37.1 and 18.2 micromol/L, respectively. The addition of 20 micromol/L NiCl2 to the internal solution caused a "wax and wane" phenomenon of pacemaking activity of ICCs. CONCLUSION: These results suggest that mitochondrial Na+-Ca2+ exchange has an important role in intestinal pacemaking activity.     

Isolation, purification, and reconstitution of the Na+ gradient-dependent Ca2+ transporter (Na+-Ca2+ exchanger) from brain synaptic plasma membranes.

A [Na+]-gradient-dependent Ca2+ transporter from brain synaptic plasma membranes has been isolated, purified, and reconstituted into brain phospholipid vesicles. The purification was achieved by sucrose-gradient centrifugation after solubilization of the synaptic membranes in cholate in the presence of a 30-fold excess (by weight) of added brain phospholipids and [Na+]-gradient-dependent Ca2+ loading of the reconstituted vesicles. A 128-fold increase in specific activity of [Na+]-gradient-dependent Ca2+ uptake per mg of protein has been obtained. The purified and reconstituted vesicles took up Ca2+ only in response to an outward-oriented [Na+] gradient. The Ca2+ uptake could be inhibited by dissipation of the [Na+] gradient with nigericin. Successful purification was based on the initial [Na+]-gradient dependency of the Ca2+-transport process, the magnitude of the [Na+]-gradient-dependent uptake, and the presence of purified brain phospholipids. Analysis of the sucrose-gradient-purified reconstituted vesicles on NaDodSO4/polyacrylamide gels showed that the activity coincided with enriched appearance of a 70,000-Da protein.     

Adverse bioenergetic consequences of Na+-Ca2+ exchanger-mediated Ca2+ influx in cardiac myocytes

BACKGROUND: In heart failure, the Na+-Ca2+ exchanger (NCX) is upregulated and mediates Ca2+ influx (instead of efflux) during the cardiac action potential. Although this partly compensates for impaired sarcoplasmic reticulum Ca2+ release and supports inotropy, the energetic consequences have never been considered. Because NCX-mediated Ca2+ influx is rather slow and mitochondrial Ca2+ uptake (which stimulates NADH production by the Krebs cycle) is thought to be facilitated by high Ca2+ gradients in a "mitochondrial Ca2+ microdomain," we speculated that NCX-mediated Ca2+ influx negatively affects the bioenergetic feedback response. Methods and Results- With the use of a patch-clamp-based approach in guinea-pig myocytes, cytosolic and mitochondrial Ca2+ ([Ca2+](c) and [Ca2+](m), respectively) was determined within the same cell after varying Ca2+ influx via L-type Ca2+ channels (I(Ca,L)) or the NCX. The efficiency of mitochondrial Ca2+ uptake, indexed by the slope of plotting [Ca2+](m) against [Ca2+](c) during each Ca2+ transient, was maximal during I(Ca,L)-triggered sarcoplasmic reticulum Ca2+ release. Depletion of sarcoplasmic reticulum Ca2+ load and increased contribution of the NCX to cytosolic Ca2+ influx independently reduced the efficiency of mitochondrial Ca2+ uptake. The upstroke velocity of cytosolic Ca2+ transients closely correlated with the efficiency of mitochondrial Ca2+ uptake. Despite comparable [Ca2+](c), sarcoplasmic reticulum Ca2+ release, but not NCX-mediated Ca2+ influx, led to stimulation of Ca2+-sensitive dehydrogenases of the Krebs cycle. Conclusions- Increased contribution of the NCX to cytosolic Ca2+ transients, which occurs in cardiac myocytes from failing hearts, impairs mitochondrial Ca2+ uptake and the bioenergetic feedback response. This mechanism could contribute to energy starvation of failing hearts.     

Modification of cardiac sarcolemmal Na+-Ca2+ exchange by diltiazem and verapamil

Cardiac sarcolemmal membranes were isolated from the rat heart and their ability for Na+-Ca2+ exchange in the absence or presence of diltiazem and verapamil was examined. Maximal Ca2+ influx activity of membranes due to Na+-dependent reaction occurred within 3 min and was about 5 nmol Ca2+/mg protein. Diltiazem (0.1 to 10 microM) depressed the Ca2+ influx activity significantly whereas verapamil (0.1 to 10 microM) had no effect at initial stages of the reaction (10 to 20 sec). The inhibitory effect of diltiazem on Ca2+ influx was found to be of an uncompetitive nature. Sodium was found to cause a rapid Ca2+ efflux from the calcium loaded membrane vesicles; about 70% of the Ca2+ efflux activity was increased by 0.1 to 10 microM of verapamil and 10 microM of diltiazem significantly. The stimulatory effect of these agents on Ca2+ efflux was associated with a change in Ka value from 16 to 5 mM Na+. Both diltiazem (0.1-3 microM) and verapamil (0.1-10 microM) did not affect the membrane Na+-K+ ATPase activity, but diltiazem in high concentrations (10-30 microM) had an inhibitory action. Specific calcium channel blocking agents, nitrendipine and nifedipine, depressed sodium-dependent Ca2+-efflux activity. A beta-adrenoreceptor antagonist, propranolol, unlike acebutolol, increased sodium-induced Ca2+-influx at high concentrations (10-100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in sarcolemmal Na+-Ca2+ exchange and ATP-dependent Ca2+-binding in hypertrophied heart

In order to examine changes in Ca2+ transport in heart sarcolemma, cardiac hypertrophy was induced in rabbits by stenosis of the abdominal aorta and hearts were removed 18-20 weeks later; sham-operated normal rabbits were used as controls. Sarcolemmal vesicles were isolated from the left ventricular tissue by a sucrose density gradient method and the membrane composition as well as activities of certain marker enzymes were monitored to determine the purity of control and experimental fractions; Na+-Ca2+ exchange and Ca2+-pump activities were assessed by the Millipore filtration technique. No changes in Ca2+-influx were observed in Na+-loaded vesicles from the hypertrophied hearts when studied in the presence of different concentrations of calcium as well as at different times of incubation. In contrast, Na+-induced Ca2+-efflux from Ca2+-loaded vesicles was enhanced in the hypertrophied heart at different times of incubation and at different concentrations of sodium. ATP-dependent Ca2+-binding activity of sarcolemma from hypertrophied heart, when measured at different times of incubation and at several concentrations of calcium, was more than the control. Minimal but an equal amount of cross contamination was seen in both control and experimental preparations; however, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid contents were increased in sarcolemma from hypertrophied hearts. These results suggest that the sarcolemmal Ca2+-transport systems may become adapted during the development of hypertrophy for augmenting Ca2+-efflux from the hypertrophied myocardial cell and this may prevent the occurrence of intracellular Ca2+ overload in a stable form of cardiac hypertrophy.     

Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle.

Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.     

Charge movements during the Na+-Ca2+ exchange in heart sarcolemmal vesicles.

The Na+-Ca2+ exchange was studied in a highly purified vesicular preparation derived from heart sarcolemma. The initial velocity of the Na+-driven Ca2+ influx, which was monitored continuously with a specific electrode, was 15 nmol/mg of protein per sec; the total Ca2+-accumulation capacity was 80 nmol/mg of protein. The Na+-Ca2+ exchange generated a current that was compensated for by the uptake of tetraphenylphosphonium in (Ph4P+) (when the latter was present in the medium), the influx of K+, and the efflux of Cl-. The movements of Ph4P were followed with a specific electrode. Ca2+ in the concentration range 3-50 microM induced an increase in the permeability of the sarcolemmal membrane to K+. Under conditions of optimal charge neutralization by K+ (i.e., in the presence of valinomycin), the Km (Ca2+) of the exchanger was 1.5 microM. The Na+-Ca2+ exchange was inhibited by chlorpromazine and was not inhibited by vanadate.     

Regulation of Cardiac Sarcolemmal Na+/H+ Exchanger Activity: Potential Pathophysiological Significance of Endogenous Mediators and Oxidant Stress

The cardiac sarcolemmal Na⁺/H⁺ exchanger (NHE) extrudes one H⁺ in exchange for one Na⁺ entering the myocyte, utilizing for its driving force the inwardly directed Na⁺ gradient maintained by the Na⁺, K⁺-ATPase. The exchanger is quiescent at physiological values of intracellular pH but becomes activated in response to intracellular acidosis. Recent evidence suggests that a variety of extracellular signals (e.g., adrenergic agonists, thrombin, endothelin, and oxidant stress) also modulate sarcolemmal NHE activity by altering its sensitivity to intracellular H⁺. Because sarcolemmal NHE activity is believed to be an important determinant of the extent of myocardial injury during ischemia and reperfusion, regulation of exchanger activity by factors that are associated with ischemia is likely to be pathophysiological importance.     

Role of the Na+-Ca2+ exchange in the calcium paradox in frog auricular trabeculae

After a Ca2+-free ouabain perfusion of 8-10 min duration, reperfusion of isolated stimulated frog auricular trabeculae with Ca2+ ouabain containing medium resulted in a large and transient contracture. The contracture was weaker in a quiescent preparation or in the absence of ouabain. In sodium-free (Li+ substitute) ouabain containing medium, the amplitude of the contracture was largely decreased while it disappeared completely in Na+-free medium without ouabain. Moreover this contracture was suppressed by 15 mM Mn2+. Although the approach was only indirect, these results suggest that the contracture is due to an entry of Ca2+ ions through the Na+-Ca2+ exchange mechanism and that this could be the unique route of calcium entry during calcium repletion.     

Na+-Ca2+ exchange activity is localized in the T-tubules of rat ventricular myocytes

Detubulation of rat ventricular myocytes has been used to investigate the role of the t-tubules in Ca2+ cycling during excitation-contraction coupling in rat ventricular myocytes. Ca2+ was monitored using fluo-3 and confocal microscopy. In control myocytes, electrical stimulation caused a spatially uniform increase in intracellular [Ca2+] across the cell width. After detubulation, [Ca2+] rose initially at the cell periphery and then propagated into the center of the cell. Application of caffeine to control myocytes resulted in a rapid and uniform increase of intracellular [Ca2+]; the distribution and amplitude of this increase was the same in detubulated myocytes, although its decline was slower. On application of caffeine to control cells, there was a large, rapid, and transient rise in extracellular [Ca2+] as Ca2+ was extruded from the cell; this rise was significantly smaller in detubulated cells, and the remaining increase was blocked by the sarcolemmal Ca2+ ATPase inhibitor carboxyeosin. The treatment used to produce detubulation had no significant effect on Ca2+ efflux in atrial cells, which lack t-tubules. Detubulation of ventricular myocytes also resulted in loss of Na+-Ca2+ exchange current, although the density of the fast Na+ current was unaltered. It is concluded that Na+-Ca2+ exchange function, and hence Ca2+ efflux by this mechanism, is concentrated in the t-tubules, and that the concentration of Ca2+ flux pathways in the t-tubules is important in producing a uniform increase in intracellular Ca2+ on stimulation.