Cees A. Schumacher

Potassium accumulation in the globally ischemic mammalian heart. A role for the ATP-sensitive potassium channel.

We investigated the contribution of opening of the ATP-sensitive K+ channel to extracellular accumulation of K+ during ischemia with the use of glibenclamide, a specific blocker of this K+ channel. To characterize the electrophysiological effects of glibenclamide during metabolic inhibition (by either application of dinitrophenol or hypoxia) we performed patch-clamp studies in isolated membrane patches of guinea pig myocytes and in intact guinea pig myocytes and studied action potential parameters in isolated superfused guinea pig papillary muscle. We studied the effect of glibenclamide on extracellular accumulation of K+ and H+ in isolated retrogradely perfused globally ischemic hearts of rat, guinea pig, and rabbit. Experimental evidence is presented that supports the conclusions that glibenclamide 1) effectively blocks open K+ATP channels, 2) reverses the dinitrophenol-induced increase of the outward current and prevents the hypoxia-induced shortening of the action potential, 3) decreases the rate of K+ accumulation during the first minutes of ischemia in stimulated hearts, an effect which was entirely absent in quiescent hearts, and 4) does not influence the rate and extent of ischemia-induced extracellular acidification.

Increased Na+/H+-exchange activity is the cause of increased [Na+]i and underlies disturbed calcium handling in the rabbit pressure and volume overload heart failure model

OBJECTIVE Cytosolic sodium ([Na+]i) is increased in heart failure (HF). We hypothesize that up-regulation of Na+/H+-exchanger (NHE) in heart failure is causal to the increase of [Na+]i and underlies disturbance of cytosolic calcium ([Ca2+]i) handling. METHODS Heart failure was induced in rabbits by combined volume and pressure overload. Age-matched animals served as control. [Na+]i, cytosolic calcium [Ca2+]i and cytosolic pH (pH(i)) were measured in isolated left ventricular midmural myocytes with SBFI, indo-1 and SNARF. SR calcium content was measured as the response of [Ca2+]i to rapid cooling (RC). Calcium after-transients were elicited by cessation of rapid stimulation (3 Hz) in the presence of 100 nmol/l noradrenalin. NHE and Na+/K+-ATPase activity were inhibited with 10 micromol/l cariporide and 100 micromol/l ouabain, respectively. RESULTS At all stimulation rates (0-3 Hz) [Na+]i and diastolic [Ca2+]i were significantly higher in HF than in control. With increasing frequency [Na+]i and diastolic [Ca2+]i progressively increased in HF and control, and the calcium transient amplitude (measured as total calcium released from SR) decreased in HF and increased in control. In HF (at 2 Hz), SR calcium content was reduced by 40% and the calcium gradient across the SR membrane by 60%. Fractional systolic SR calcium release was 90% in HF and 60% in control. In HF the rate of pH(i) recovery following acid loading was much faster at all pH(i) and NHE dependent sodium influx was almost twice as high as in control. In HF cariporide (10 micromol/l, 5 min) reduced [Na+]i and end diastolic [Ca2+]i to almost control values, and reversed the relation between calcium transient amplitude and stimulation rate from negative to positive. It increased SR calcium content and SR membrane gradient and decreased fractional systolic SR depletion to 60%. Cariporide greatly reduced the susceptibility to develop calcium after-transients. In control animals, cariporide had only minor effects on all these parameters. Increase of [Na+]i with ouabain in control myocytes induced abnormal calcium handling as found in HF. CONCLUSIONS In HF up-regulation of NHE activity is causal to increased [Na+]i and secondarily to disturbed diastolic, systolic and SR calcium handling. Specific inhibition of NHE partly normalized [Na+]i, end diastolic [Ca2+]i, and SR calcium handling and reduced the incidence of calcium after-transients. Chronic treatment with specific NHE inhibitors may provide a useful future therapeutic option in treatment of developing hypertrophy and heart failure.

Acute administration of fish oil inhibits triggered activity in isolated myocytes from rabbits and patients with heart failure

Background— Fish oil reduces sudden death in patients with prior myocardial infarction. Sudden death in heart failure may be due to triggered activity based on disturbed calcium handling. We hypothesized that superfusion with &ohgr;3-polyunsaturated fatty acids (&ohgr;3-PUFAs) from fish inhibits triggered activity in heart failure. Methods and Results— Ventricular myocytes were isolated from explanted hearts of rabbits with volume- and pressure-overload–induced heart failure and of patients with end-stage heart failure. Membrane potentials (patch-clamp technique) and intracellular calcium (indo-1 fluorescence) were recorded after 5 minutes of superfusion with Tyrode’s solution (control), &ohgr;-9 monounsaturated fatty acid oleic acid (20 &mgr;mol/L), or &ohgr;3-PUFAs (docosahexaenoic acid or eicosapentaenoic acid 20 &mgr;mol/L). &ohgr;3-PUFAs shortened the action potential at low stimulation frequencies and caused an ≈25% decrease in diastolic and systolic calcium (all P<0.05). Subsequently, noradrenalin and rapid pacing were used to evoke triggered activity, delayed afterdepolarizations, and calcium aftertransients. &ohgr;3-PUFAs abolished triggered activity and reduced the number of delayed afterdepolarizations and calcium aftertransients compared with control and oleic acid. &ohgr;3-PUFAs reduced action potential shortening and intracellular calcium elevation in response to noradrenalin. Results from human myocytes were in accordance with the findings obtained in rabbit myocytes. Conclusion— Superfusion with &ohgr;3-PUFAs from fish inhibits triggered arrhythmias in myocytes from rabbits and patients with heart failure by lowering intracellular calcium and reducing the response to noradrenalin.

Functional NaV1.8 Channels in Intracardiac Neurons The Link Between SCN10A and Cardiac Electrophysiology

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A/Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 &mgr;mol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A/NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A /Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity. # Novelty and Significance {#article-title-33}

[Na+](i) and the driving force of the Na+/Ca2+-exchanger in heart failure

OBJECTIVE Diastolic calcium is increased in myocytes from failing hearts despite up-regulation of the principal calcium extruding mechanism the Na+/Ca2+-exchanger (NCX). We hypothesize that increased diastolic calcium ([Ca2+]i) is secondary to increased cytosolic sodium ([Na+]i) and decreased driving force of NCX (DeltaG(exch)). METHODS The stimulation rate dependence of simultaneously measured cytosolic sodium ([Na+]i), calcium transients ([Ca2+]i) and action potentials were determined with SBFI, indo-1 and the perforated patch technique in midmural left ventricular myocytes isolated from rabbits with pressure and volume overload induced heart failure (HF) and in age matched controls. Dynamic changes of DeltaG(exch) were calculated. RESULTS With increasing stimulation frequency, 0.2-3 Hz (all data HF versus control): [Na+]i increased (6.4 to 10.8 versus 3.8 to 6.4 mmol/l), diastolic [Ca2+]i increased (142 to 219 versus 47 to 98 nmol/l), calcium transient amplitude decreased in HF (300 to 250 nmol/l) but increased in control (201 to 479 nmol/l), action potential duration (APD90) decreased (380 to 260 versus 325 to 205 ms) and time averaged DeltaG(exch) decreased (6.8 to 2.8 versus 8.7 to 6.4 kJ/mol. With increasing stimulation rate the forward mode time integral of DeltaG(exch) decreased in HF by about 30%, the reversed mode time integral increased about ninefold and the duration of reversed mode operation more than sixfold relative to control. CONCLUSIONS [Na+]i is increased in HF and the driving force of NCX is decreased. NCX exerts thermodynamic control over diastolic calcium. Disturbed diastolic calcium handling in HF is due to decreased forward mode DeltaG(exch) secondary to increased [Na+]i and prolongation of the action potential. Enhanced reversed mode DeltaG(exch) may account for increased contribution of NCX to e-c coupling in HF.

Mitochondrial oxygen tension within the heart

By using a newly developed optical technique which enables non-invasive measurement of mitochondrial oxygenation (mitoPO(2)) in the intact heart, we addressed three long-standing oxygenation questions in cardiac physiology: 1) what is mitoPO(2) within the in vivo heart?, 2) is mitoPO(2) heterogeneously distributed?, and 3) how does mitoPO(2) of the isolated Langendorff-perfused heart compare with that in the in vivo working heart? Following calibration and validation studies of the optical technique in isolated cardiomyocytes, mitochondria and intact hearts, we show that in the in vivo condition mean mitoPO(2) was 35+/-5 mm Hg. The mitoPO(2) was highly heterogeneous, with the largest fraction (26%) of mitochondria having a mitoPO(2) between 10 and 20 mm Hg, and 10% between 0 and 10 mm Hg. Hypoxic ventilation (10% oxygen) increased the fraction of mitochondria in the 0-10 mm Hg range to 45%, whereas hyperoxic ventilation (100% oxygen) had no major effect on mitoPO(2). For Langendorff-perfused rat hearts, mean mitoPO(2) was 29+/-5 mm Hg with the largest fraction of mitochondria (30%) having a mitoPO(2) between 0 and 10 mm Hg. Only in the maximally vasodilated condition, did the isolated heart compare with the in vivo heart (11% of mitochondria between 0 and 10 mm Hg). These data indicate 1) that the mean oxygen tension at the level of the mitochondria within the heart in vivo is higher than generally considered, 2) that mitoPO(2) is considerably heterogeneous, and 3) that mitoPO(2) of the classic buffer-perfused Langendorff heart is shifted to lower values as compared to the in vivo heart.

Functional NaV1.8 Channels in Intracardiac NeuronsNovelty and Significance: The Link Between SCN10A and Cardiac Electrophysiology

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A/Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 &mgr;mol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A/NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A /Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity. # Novelty and Significance {#article-title-33}

Transmural dispersion of refractoriness and conduction velocity is associated with heterogeneously reduced connexin43 in a rabbit model of heart failure

BACKGROUND Heterogeneity of repolarization and conduction is a potential source of arrhythmogenesis. In heart failure (HF), intercellular coupling is reduced and heterogeneities may become evident because of reduced intercellular coupling. OBJECTIVE This study sought to investigate connexin43 (Cx43) expression, conduction velocity (CV), refractoriness and inducibility of arrhythmias at multiple sites of the left ventricle during HF. METHODS HF was induced by pressure-volume overload in rabbits. Epicardial and intramural mapping was performed in isolated perfused hearts following programmed stimulation. Myocytes were enzymatically dissociated and studied using D-4-ANEPPS fluorescence. Western blotting and immunohistochemistry was performed to quantify heterogeneity of Cx43 expression. RESULTS Cx43 was heterogeneously reduced in the midmyocardial, but not in the sub epicardium layer of the left ventricular free wall in HF compared to control rabbits. In HF, subepicardial and midmyocardial refractory periods (RPs) were increased compared to control rabbits (148 +/- 3 ms and 143 +/- 3 versus 131 +/- 2 and 129 +/- 2 ms, respectively, both P < 0.001). Also, transmural dispersion of RPs was larger in HF (30 +/- 4 ms) than in control rabbits (24 +/- 3 ms, P < 0.05). Intrinsic dispersion of action potential duration in isolated myocytes was similar in HF and control rabbits. Transmural CV was heterogeneous, although the mean CV was not different between groups. Arrhythmias were more easily inducible in HF, especially from midmyocardium. CONCLUSION In HF, midmyocardial Cx43 expression is heterogeneously reduced. This is associated with increased transmural dispersion in refractoriness and conduction, and with increased arrhythmia inducibility.

Chronic inhibition of the Na+/H+‐ exchanger causes regression of hypertrophy, heart failure, and ionic and electrophysiological remodelling

Increased activity of the Na+/H+‐exchanger (NHE‐1) in heart failure underlies raised [Na+]i causing disturbances of calcium handling. Inhibition of NHE‐1, initiated at the onset of pressure/volume overload, prevents development of hypertrophy, heart failure and remodelling. We hypothesized that chronic inhibition of NHE‐1, initiated at a later stage, would induce regression of hypertrophy, heart failure, and ionic and electrophysiological remodelling.

PGC-1α and Reactive Oxygen Species Regulate Human Embryonic Stem Cell-Derived Cardiomyocyte Function

Summary Diminished mitochondrial function is causally related to some heart diseases. Here, we developed a human disease model based on cardiomyocytes from human embryonic stem cells (hESCs), in which an important pathway of mitochondrial gene expression was inactivated. Repression of PGC-1α, which is normally induced during development of cardiomyocytes, decreased mitochondrial content and activity and decreased the capacity for coping with energetic stress. Yet, concurrently, reactive oxygen species (ROS) levels were lowered, and the amplitude of the action potential and the maximum amplitude of the calcium transient were in fact increased. Importantly, in control cardiomyocytes, lowering ROS levels emulated this beneficial effect of PGC-1α knockdown and similarly increased the calcium transient amplitude. Our results suggest that controlling ROS levels may be of key physiological importance for recapitulating mature cardiomyocyte phenotypes, and the combination of bioassays used in this study may have broad application in the analysis of cardiac physiology pertaining to disease.