RBM20 Mutations Induce an Arrhythmogenic Dilated Cardiomyopathy Related to Disturbed Calcium Handling.

BACKGROUND: Mutations in RBM20 (RNA-binding motif protein 20) cause a clinically aggressive form of dilated cardiomyopathy, with an increased risk of malignant ventricular arrhythmias. RBM20 is a splicing factor that targets multiple pivotal cardiac genes, such as Titin (TTN) and CAMK2D (calcium/calmodulin-dependent kinase II delta). Aberrant TTN splicing is thought to be the main determinant of RBM20-induced dilated cardiomyopathy, but is not likely to explain the increased risk of arrhythmias. Here, we investigated the extent to which RBM20 mutation carriers have an increased risk of arrhythmias and explore the underlying molecular mechanism. METHODS: We compared clinical characteristics of RBM20 and TTN mutation carriers and used our previously generated Rbm20 knockout (KO) mice to investigate downstream effects of Rbm20-dependent splicing. Cellular electrophysiology and Ca2+ measurements were performed on isolated cardiomyocytes from Rbm20 KO mice to determine the intracellular consequences of reduced Rbm20 levels. RESULTS: Sustained ventricular arrhythmias were more frequent in human RBM20 mutation carriers than in TTN mutation carriers (44% versus 5%, respectively, P=0.006). Splicing events that affected Ca2+- and ion-handling genes were enriched in Rbm20 KO mice, most notably in the genes CamkIIδ and RyR2. Aberrant splicing of CamkIIδ in Rbm20 KO mice resulted in a remarkable shift of CamkIIδ toward the δ-A isoform that is known to activate the L-type Ca2+ current ( ICa,L). In line with this, we found an increased ICa,L, intracellular Ca2+ overload and increased sarcoplasmic reticulum Ca2+ content in Rbm20 KO myocytes. In addition, not only complete loss of Rbm20, but also heterozygous loss of Rbm20 increased spontaneous sarcoplasmic reticulum Ca2+ releases, which could be attenuated by treatment with the ICa,L antagonist verapamil. CONCLUSIONS: We show that loss of Rbm20 disturbs Ca2+ handling and leads to more proarrhythmic Ca2+ releases from the sarcoplasmic reticulum. Patients that carry a pathogenic RBM20 mutation have more ventricular arrhythmias despite a similar left ventricular function, in comparison with patients with a TTN mutation. Our experimental data suggest that RBM20 mutation carriers may benefit from treatment with an ICa,L blocker to reduce their arrhythmia burden.

Class effects of SGLT2 inhibitors in mouse cardiomyocytes and hearts: inhibition of Na+/H+ exchanger, lowering of cytosolic Na+ and vasodilation.

AIMS/HYPOTHESIS: Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i) constitute a novel class of glucose-lowering (type 2) kidney-targeted agents. We recently reported that the SGLT2i empagliflozin (EMPA) reduced cardiac cytosolic Na+ ([Na+]c) and cytosolic Ca2+ ([Ca2+]c) concentrations through inhibition of Na+/H+ exchanger (NHE). Here, we examine (1) whether the SGLT2i dapagliflozin (DAPA) and canagliflozin (CANA) also inhibit NHE and reduce [Na+]c; (2) a structural model for the interaction of SGLT2i to NHE; (3) to what extent SGLT2i affect the haemodynamic and metabolic performance of isolated hearts of healthy mice. METHODS: Cardiac NHE activity and [Na+]c in mouse cardiomyocytes were measured in the presence of clinically relevant concentrations of EMPA (1 µmol/l), DAPA (1 µmol/l), CANA (3 µmol/l) or vehicle. NHE docking simulation studies were applied to explore potential binding sites for SGTL2i. Constant-flow Langendorff-perfused mouse hearts were subjected to SGLT2i for 30 min, and cardiovascular function, O2 consumption and energetics (phosphocreatine (PCr)/ATP) were determined. RESULTS: EMPA, DAPA and CANA inhibited NHE activity (measured through low pH recovery after NH4+ pulse: EMPA 6.69 ± 0.09, DAPA 6.77 ± 0.12 and CANA 6.80 ± 0.18 vs vehicle 7.09 ± 0.09; p < 0.001 for all three comparisons) and reduced [Na+]c (in mmol/l: EMPA 10.0 ± 0.5, DAPA 10.7 ± 0.7 and CANA 11.0 ± 0.9 vs vehicle 12.7 ± 0.7; p < 0.001). Docking studies provided high binding affinity of all three SGLT2i with the extracellular Na+-binding site of NHE. EMPA and CANA, but not DAPA, induced coronary vasodilation of the intact heart. PCr/ATP remained unaffected. CONCLUSIONS/INTERPRETATION: EMPA, DAPA and CANA directly inhibit cardiac NHE flux and reduce [Na+]c, possibly by binding with the Na+-binding site of NHE-1. Furthermore, EMPA and CANA affect the healthy heart by inducing vasodilation. The [Na+]c-lowering class effect of SGLT2i is a potential approach to combat elevated [Na+]c that is known to occur in heart failure and diabetes.

Empagliflozin decreases myocardial cytoplasmic Na+ through inhibition of the cardiac Na+/H+ exchanger in rats and rabbits.

AIMS/HYPOTHESIS: Empagliflozin (EMPA), an inhibitor of the renal sodium-glucose cotransporter (SGLT) 2, reduces the risk of cardiovascular death in patients with type 2 diabetes. The underlying mechanism of this effect is unknown. Elevated cardiac cytoplasmic Na+ ([Na+]c) and Ca2+ ([Ca2+]c) concentrations and decreased mitochondrial Ca2+ concentration ([Ca2+]m) are drivers of heart failure and cardiac death. We therefore hypothesised that EMPA would directly modify [Na+]c, [Ca2+]c and [Ca2+]m in cardiomyocytes. METHODS: [Na+]c, [Ca2+]c, [Ca 2+]m and Na+/H+ exchanger (NHE) activity were measured fluorometrically in isolated ventricular myocytes from rabbits and rats. RESULTS: An increase in extracellular glucose, from 5.5 mmol/l to 11 mmol/l, resulted in increased [Na+]c and [Ca2+]c levels. EMPA treatment directly inhibited NHE flux, caused a reduction in [Na+]c and [Ca2+]c and increased [Ca2+]m. After pretreatment with the NHE inhibitor, Cariporide, these effects of EMPA were strongly reduced. EMPA also affected [Na+]c and NHE flux in the absence of extracellular glucose. CONCLUSIONS/INTERPRETATION: The glucose lowering kidney-targeted agent, EMPA, demonstrates direct cardiac effects by lowering myocardial [Na+]c and [Ca2+]c and enhancing [Ca2+]m, through impairment of myocardial NHE flux, independent of SGLT2 activity.

In vivo mouse myocardial (31)P MRS using three-dimensional image-selected in vivo spectroscopy (3D ISIS): technical considerations and biochemical validations.

(31)P MRS provides a unique non-invasive window into myocardial energy homeostasis. Mouse models of cardiac disease are widely used in preclinical studies, but the application of (31)P MRS in the in vivo mouse heart has been limited. The small-sized, fast-beating mouse heart imposes challenges regarding localized signal acquisition devoid of contamination with signal originating from surrounding tissues. Here, we report the implementation and validation of three-dimensional image-selected in vivo spectroscopy (3D ISIS) for localized (31)P MRS of the in vivo mouse heart at 9.4 T. Cardiac (31)P MR spectra were acquired in vivo in healthy mice (n = 9) and in transverse aortic constricted (TAC) mice (n = 8) using respiratory-gated, cardiac-triggered 3D ISIS. Localization and potential signal contamination were assessed with (31)P MRS experiments in the anterior myocardial wall, liver, skeletal muscle and blood. For healthy hearts, results were validated against ex vivo biochemical assays. Effects of isoflurane anesthesia were assessed by measuring in vivo hemodynamics and blood gases. The myocardial energy status, assessed via the phosphocreatine (PCr) to adenosine 5'-triphosphate (ATP) ratio, was approximately 25% lower in TAC mice compared with controls (0.76 ± 0.13 versus 1.00 ± 0.15; P < 0.01). Localization with one-dimensional (1D) ISIS resulted in two-fold higher PCr/ATP ratios than measured with 3D ISIS, because of the high PCr levels of chest skeletal muscle that contaminate the 1D ISIS measurements. Ex vivo determinations of the myocardial PCr/ATP ratio (0.94 ± 0.24; n = 8) confirmed the in vivo observations in control mice. Heart rate (497 ± 76 beats/min), mean arterial pressure (90 ± 3.3 mmHg) and blood oxygen saturation (96.2 ± 0.6%) during the experimental conditions of in vivo (31)P MRS were within the normal physiological range. Our results show that respiratory-gated, cardiac-triggered 3D ISIS allows for non-invasive assessments of in vivo mouse myocardial energy homeostasis with (31)P MRS under physiological conditions.

Functional Nav1.8 channels in intracardiac neurons: the link between SCN10A and cardiac electrophysiology.

RATIONALE: The SCN10A gene encodes the neuronal sodium channel isoform Na(V)1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for Na(V)1.8 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 Na(V)1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest Na(V)1.8 expression within the myocardium. Immunocytochemistry further revealed substantial Na(V)1.8 staining in isolated neurons from murine intracardiac ganglia but no Na(V)1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the Na(V)1.8 blocker A-803467 (0.5-2 µmol/L) had no effect on either mean sodium current (I(Na)) density or I(Na) gating kinetics in isolated myocytes but significantly reduced I(Na) 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 Na(V)1.8-based I(Na). 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 Na(V)1.8 in cardiac neural activity. CONCLUSIONS: Our findings demonstrate the functional presence of SCN10A/Na(V)1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.

Protease XIV abolishes NHE inhibition by empagliflozin in cardiac cells.

Background: SGLT2i directly inhibit the cardiac sodium-hydrogen exchanger-1 (NHE1) in isolated ventricular cardiomyocytes (CMs). However, other studies with SGLT2i have yielded conflicting results. This may be explained by methodological factors including cell isolation techniques, cell types and ambient pH. In this study, we tested whether the use of protease XIV (PXIV) may abrogate inhibition of SGLT2i on cardiac NHE1 activity in isolated rabbit CMs or rat cardiomyoblast cells (H9c2), in a pH dependent manner. Methods: Rabbit ventricular CMs were enzymatically isolated from Langendorff-perfused hearts during a 30-min perfusion period followed by a 25-min after-dissociation period, using a collagenase mixture without or with a low dose PXIV (0.009 mg/mL) present for different periods. Empagliflozin (EMPA) inhibition on NHE activity was then assessed at pH of 7.0, 7.2 and 7.4. In addition, effects of 10 min PXIV treatment were also evaluated in H9c2 cells for EMPA and cariporide NHE inhibition. Results: EMPA reduced NHE activity in rabbit CMs that were not exposed to PXIV treatment or undergoing a 35-min PXIV treatment, independent of pH levels. However, when exposure time to PXIV was extended to 55 min, NHE inhibition by Empa was completely abolished at all three pH levels. In H9c2 cells, NHE inhibition by EMPA was evident in non-treated cells but lost after 10-min incubation with PXIV. NHE inhibition by cariporide was unaffected by PXIV. Conclusion: The use of protease XIV in cardiac cell isolation procedures obliterates the inhibitory effects of SGLT2i on NHE1 activity in isolated cardiac cells, independent of pH.

Ventricular fibrillation hampers the restoration of creatine-phosphate levels during simulated cardiopulmonary resuscitations.

AIMS: Recurrences of ventricular fibrillation (VF) during cardiopulmonary resuscitation (CPR) are associated with a reduced chance of survival. The effect of VF during CPR on the myocardium is unknown. We tested the hypothesis that VF during simulated CPR reduces the restoration of the myocardial energy state and contractile function. METHODS AND RESULTS: Twelve porcine hearts were isolated and perfused with the pig's own blood. First, cardiac oxygen consumption was measured by blood gas analysis. Secondly, we simulated sudden cardiac arrest by VF (7 min VF, zero flow) followed by simulated CPR (7 min, 0.3 mL/g/min perfusion rate) in the absence and presence of VF [six hearts were maintained in VF (VF-group), six were defibrillated (defib-group)]. The VF increased the cardiac oxygen consumption by 71% (0.87 ± 0.12 vs. 1.49 ± 0.14 µmol O2/g/min; mean ± SEM, P< 0.001) compared with a ventricular rhythm of 62 beats/min. The presence of VF during simulated CPR after 7 min of cardiac arrest hampered restoration of myocardial creatine-phosphate levels compared with defibrillated hearts (61 ± 9 vs. 87 ± 7% of baseline values, respectively; P< 0.05). The cardiac contractile function was significantly higher in the defib- than in the VF-group (area under the pressure curve 2.29 ± 0.22 vs. 1.72 ± 0.14 s×mm Hg respectively; P< 0.05). CONCLUSIONS: These data demonstrate that the cardiac oxygen consumption is increased by VF and that the presence of VF during CPR hampers the restoration of the myocardial energy state and contractility. Strategies that reduce VF duration without disrupting chest compressions will benefit the restoration of the cardiac energy state during resuscitations.

Empagliflozin reduces oxidative stress through inhibition of the novel inflammation/NHE/[Na+]c/ROS-pathway in human endothelial cells.

Inflammation causing oxidative stress in endothelial cells contributes to heart failure development. Sodium/glucose cotransporter 2 inhibitors (SGLT2i's) were shown to reduce heart failure hospitalization and oxidative stress. However, how inflammation causes oxidative stress in endothelial cells, and how SGLT2i's can reduce this is unknown. Here we hypothesized that 1) TNF-α activates the Na+/H+ exchanger (NHE) and raises cytoplasmatic Na+ ([Na+]c), 2) increased [Na+]c causes reactive oxygen species (ROS) production, and 3) empagliflozin (EMPA) reduces inflammation-induced ROS through NHE inhibition and lowering of [Na+]c in human endothelial cells. Human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) were incubated with vehicle (V), 10 ng/ml TNF-α, 1 µM EMPA or the NHE inhibitor Cariporide (CARI, 10 µM) and NHE activity, intracellular [Na+]c and ROS were analyzed. TNF-α enhanced NHE activity in HCAECs and HUVECs by 92% (p < 0.01) and 51% (p < 0.05), respectively, and increased [Na+]c from 8.2 ± 1.6 to 11.2 ± 0.1 mM (p < 0.05) in HCAECs. Increasing [Na+]c by ouabain elevated ROS generation in both HCAECs and HUVECs. EMPA inhibited NHE activity in HCAECs and in HUVECs. EMPA concomitantly lowered [Na+]c in both cell types. In both cell types, TNF α-induced ROS was lowered by EMPA or CARI, with no further ROS lowering by EMPA in the presence of CARI, indicating EMPA attenuated ROS through NHE inhibition. In conclusion, inflammation induces oxidative stress in human endothelial cells through NHE activation causing elevations in [Na+]c, a process that is inhibited by EMPA through NHE inhibition.

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.

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 omega3-polyunsaturated fatty acids (omega3-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), omega-9 monounsaturated fatty acid oleic acid (20 micromol/L), or omega3-PUFAs (docosahexaenoic acid or eicosapentaenoic acid 20 micromol/L). omega3-PUFAs shortened the action potential at low stimulation frequencies and caused an approximately 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. omega3-PUFAs abolished triggered activity and reduced the number of delayed afterdepolarizations and calcium aftertransients compared with control and oleic acid. Omega3-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. CONCLUSIONS: Superfusion with omega3-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.

Dietary n-3 fatty acids promote arrhythmias during acute regional myocardial ischemia in isolated pig hearts.

OBJECTIVE: Dietary supplementation with fish oil-derived n-3 fatty acids reduces mortality in patients with myocardial infarction, but may have adverse effects in angina patients. The underlying electrophysiologic mechanisms are poorly understood. We studied the arrhythmias and the electrophysiologic changes during regional ischemia in hearts from pigs fed a diet rich in fish oil. METHODS: Pigs received diets rich in fish oil, in sunflower oil, or a control diet for 8 weeks. Hearts were isolated and perfused. Ischemia was created by occluding the left anterior descending artery. Diastolic stimulation threshold, refractory period, conduction velocity, activation recovery intervals and the maximum downstroke velocity of 176 electrograms were measured in the ischemic zone. Spontaneous arrhythmias during 75 min of regional ischemia were counted. RESULTS: More episodes of spontaneous ischemia-induced sustained ventricular tachycardia and ventricular fibrillation occurred in the fish oil and sunflower oil group than in the control group. More inexcitable myocardium was present in the ischemic zone in the group fed fish oil or sunflower oil than in the control group after 20 min of ischemia. After 40 min of ischemia, more block occurred in the control group than in the other groups. The downstroke velocity of the electrograms in the ischemic border zone was lower in the fish oil group and sunflower oil group than in the control after 20 min. CONCLUSIONS: A diet rich in fish oil results in proarrhythmia compared to a control diet during regional ischemia in pigs. Myocardial excitability is reduced in the fish oil and sunflower oil group during the early phase of arrhythmogenesis. In the late phase of arrhythmogenesis, excitability is more reduced in the control group than in the fish oil and sunflower oil group.

Direct Cardiac Actions of Sodium Glucose Cotransporter 2 Inhibitors Target Pathogenic Mechanisms Underlying Heart Failure in Diabetic Patients.

Sodium glucose cotransporter 2 inhibitors (SGLT2i) are the first antidiabetic compounds that effectively reduce heart failure hospitalization and cardiovascular death in type 2 diabetics. Being explicitly designed to inhibit SGLT2 in the kidney, SGLT2i have lately been investigated for their off-target cardiac actions. Here, we review the direct effects of SGLT2i Empagliflozin (Empa), Dapagliflozin (Dapa), and Canagliflozin (Cana) on various cardiac cell types and cardiac function, and how these may contribute to the cardiovascular benefits observed in large clinical trials. SGLT2i impaired the Na+/H+ exchanger 1 (NHE-1), reduced cytosolic [Ca2+] and [Na+] and increased mitochondrial [Ca2+] in healthy cardiomyocytes. Empa, one of the best studied SGLT2i, maintained cell viability and ATP content following hypoxia/reoxygenation in cardiomyocytes and endothelial cells. SGLT2i recovered vasoreactivity of hyperglycemic and TNF-α-stimulated aortic rings and of hyperglycemic endothelial cells. Anti-inflammatory actions of Cana in IL-1ß-treated HUVEC and of Dapa in LPS-treated cardiofibroblast were mediated by AMPK activation. In isolated mouse hearts, Empa and Cana, but not Dapa, induced vasodilation. In ischemia-reperfusion studies of the isolated heart, Empa delayed contracture development during ischemia and increased mitochondrial respiration post-ischemia. Direct cardiac effects of SGLT2i target well-known drivers of diabetes and heart failure (elevated cardiac cytosolic [Ca2+] and [Na+], activated NHE-1, elevated inflammation, impaired vasorelaxation, and reduced AMPK activity). These cardiac effects may contribute to the large beneficial clinical effects of these antidiabetic drugs.

Neurokinin-3 receptor activation selectively prolongs atrial refractoriness by inhibition of a background K+ channel.

The cardiac autonomic nervous system (ANS) controls normal atrial electrical function. The cardiac ANS produces various neuropeptides, among which the neurokinins, whose actions on atrial electrophysiology are largely unknown. We here demonstrate that the neurokinin substance-P (Sub-P) activates a neurokinin-3 receptor (NK-3R) in rabbit, prolonging action potential (AP) duration through inhibition of a background potassium current. In contrast, ventricular AP duration was unaffected by NK-3R activation. NK-3R stimulation lengthened atrial repolarization in intact rabbit hearts and consequently suppressed arrhythmia duration and occurrence in a rabbit isolated heart model of atrial fibrillation (AF). In human atrial appendages, the phenomenon of NK-3R mediated lengthening of atrial repolarization was also observed. Our findings thus uncover a pathway to selectively modulate atrial AP duration by activation of a hitherto unidentified neurokinin-3 receptor in the membrane of atrial myocytes. NK-3R stimulation may therefore represent an anti-arrhythmic concept to suppress re-entry-based atrial tachyarrhythmias, including AF.

Dietary fish oil reduces the incidence of triggered arrhythmias in pig ventricular myocytes.

BACKGROUND: Fish oil reduces the incidence of sudden cardiac death in postmyocardial infarction patients. Triggered activity is the principal mechanism of arrhythmogenesis under these conditions. OBJECTIVE: The purpose of this study was to test whether dietary fish oil in pigs inhibits Ca2+ overload-induced triggered activity. METHODS: Pigs were fed a diet of fish oil or sunflower oil for 8 weeks. Ventricular myocytes (omega3: fish oil, n = 11; control: sunflower oil, n = 8) were isolated by enzymatic dissociation and used for patch clamp studies and intracellular Ca2+ recordings. Triggered activity was induced by rapid pacing in the presence of norepinephrine. RESULTS: Dietary fish oil reduced the incidence of triggered action potentials and delayed afterdepolarizations compared to control (9.1% in omega3 and 84.6% in control, P <.05), concomitant with a reduction in spontaneous Ca2+ release. Dietary fish oil prevented Ca2+ overload and reduced action potential prolongation in response to norepinephrine (DeltaAPD(90): 23.2 +/- 8.5 ms in omega3 and 107.4 +/- 15.9 in control, P <.05). omega3 myocytes displayed decreased sarcoplasmic reticulum Ca2+ content, reduced L-type Ca2+ current (I(Ca,L)), and less recruitment of the Na+/Ca2+ exchange current (I(NCX)) in response to norepinephrine compared to control. In the absence of norepinephrine, the slow component of the delayed rectifier current (I(Ks)) was larger in omega3 myocytes. In the presence of norepinephrine, I(Ks) increased to the same level in omega3 and control myocytes. CONCLUSION: Dietary fish oil reduces the incidence of triggered activity and prevents Ca2+ overload and AP prolongation in response to norepinephrine. Fish oil may prevent arrhythmias in patients with heart failure.

Incorporated sarcolemmal fish oil fatty acids shorten pig ventricular action potentials.

BACKGROUND: Omega-3 polyunsaturated fatty acids (omega3-PUFAs) from fish oil reduce the risk of sudden death presumably by preventing life-threatening arrhythmias. Acutely administered omega3-PUFAs modulate the activity of several cardiac ion channels, but the chronic effects of a diet enriched with fish oil leading to omega3-PUFA-incorporation into the sarcolemma on membrane currents are unknown. METHODS: Pigs received a diet either rich in omega3-PUFAs or in omega9-fatty acids for 8 weeks. Ventricular myocytes (VMs) were isolated and used for patch-clamp studies. RESULTS: omega3-VMs contained higher amounts of omega3-PUFAs and had a shorter action potential (AP) with a more negative plateau than control VM. In omega3 VMs, L-type Ca(2+) current (I(Ca,L)) and Na(+)-Ca(2+) exchange current (I(NCX)) were reduced by approximately 20% and 60%, respectively, and inward rectifier K(+) current (I(K1)) and slow delayed rectifier K(+) current (I(Ks)) were increased by approximately 50% and 70%, respectively, compared to control. Densities of rapid delayed rectifier K(+) current, Ca(2+)-activated Cl(-) current, and Na(+) current (I(Na)) were unchanged, although voltage-dependence of I(Na) inactivation was more negative in omega3 VMs. CONCLUSIONS: A fish oil diet increases omega3-PUFA content in the ventricular sarcolemma, decreases I(Ca,L) and I(NCX), and increases I(K1) and I(Ks), resulting in AP shortening. Incorporation of omega3-PUFAs in the sarcolemma may have consequences for arrhythmias independent of circulating omega3-PUFAs.

Chronic inhibition of Na+/H+-exchanger attenuates cardiac hypertrophy and prevents cellular remodeling in heart failure.

OBJECTIVE: In patients with heart disease, the transition from compensatory hypertrophy to heart failure (HF) is associated with altered calcium handling. Up-regulated Na(+)/H(+)-exchanger (NHE-1) activity underlies increased [Na(+)](i) and disturbance of cellular calcium handling in HF. We hypothesize that chronic inhibition of NHE-1 activity prevents the hypertrophic response, cellular remodeling, and development of HF. METHODS: Rabbits received a control or cariporide (inhibitor of NHE-1) diet for 3 months, starting after induction of combined volume and pressure overload. Age-matched animals served as control. Development of HF was examined echocardiographically and electrocardiographically after 3 months. [Na(+)](i), [Ca(2+)](i), pH(i), and action potentials were measured in left ventricular midmural myocytes with SBFI, indo-1, SNARF, and di-4-anepps. Sarcoplasmic reticulum calcium content was calculated from the response of [Ca(2+)](i) to rapid cooling. Calcium after-transients were elicited by cessation of rapid stimulation (3 Hz) in the presence of 100 nmol/l noradrenalin. RESULTS: Chronic treatment of rabbits with the specific Na(+)/H(+)-exchanger activity inhibitor cariporide greatly attenuated development of hypertrophy and entirely abolished development of HF; the heart/body weight ratio increased only little, no change in lung weight occurred, left ventricular dimensions and fractional shortening changed mildly, ascites was not present, QT duration did not increase, and sudden death did not occur. Chronic cariporide treatment also prevented cellular electrical and ionic remodeling. Myocyte dimensions were unaltered, action potentials were not prolonged, cytoplasmic sodium and NHE-1 activity did not increase, cytoplasmic and SR calcium handling remained undisturbed, and no increase of the incidence of calcium after-transient dependent delayed after depolarizations (DADs) occurred. CONCLUSION: We conclude that enhanced activity of NHE-1 underlies cardiac cellular electrical and ionic remodeling in experimental heart failure, and that chronic dietary treatment with cariporide attenuates hypertrophy, development of HF, and cellular remodeling.

Reducing mitochondrial bound hexokinase II mediates transition from non-injurious into injurious ischemia/reperfusion of the intact heart.

Ischemia/reperfusion (I/R) of the heart becomes injurious when duration of the ischemic insult exceeds a certain threshold (approximately ≥20 min). Mitochondrial bound hexokinase II (mtHKII) protects against I/R injury, with the amount of mtHKII correlating with injury. Here, we examine whether mtHKII can induce the transition from non-injurious to injurious I/R, by detaching HKII from mitochondria during a non-injurious I/R interval. Additionally, we examine possible underlying mechanisms (increased reactive oxygen species (ROS), increased oxygen consumption (MVO2) and decreased cardiac energetics) associated with this transition. Langendorff perfused rat hearts were treated for 20 min with saline, TAT-only or 200 nM TAT-HKII, a peptide that translocates HKII from mitochondria. Then, hearts were exposed to non-injurious 15-min ischemia, followed by 30-min reperfusion. I/R injury was determined by necrosis (LDH release) and cardiac mechanical recovery. ROS were measured by DHE fluorescence. Changes in cardiac respiratory activity (cardiac MVO2 and efficiency and mitochondrial oxygen tension (mitoPO2) using protoporphyrin IX) and cardiac energetics (ATP, PCr, ∆GATP) were determined following peptide treatment. When exposed to 15-min ischemia, control hearts had no necrosis and 85% recovery of function. Conversely, TAT-HKII treatment resulted in significant LDH release and reduced cardiac recovery (25%), indicating injurious I/R. This was associated with increased ROS during ischemia and reperfusion. TAT-HKII treatment reduced MVO2 and improved energetics (increased PCr) before ischemia, without affecting MVO2/RPP ratio or mitoPO2. In conclusion, a reduction in mtHKII turns non-injurious I/R into injurious I/R. Loss of mtHKII was associated with increased ROS during ischemia and reperfusion, but not with increased MVO2 or decreased cardiac energetics before damage occurs.

SR calcium handling and calcium after-transients in a rabbit model of heart failure.

OBJECTIVE: After-depolarization associated arrhythmias are frequently observed in heart failure and associated with spontaneous calcium release from sarcoplasmic reticulum (SR), calcium after-transients. We hypothesize that disturbed SR calcium handling underlies calcium after-transients in heart failure (HF). METHODS: We measured the stimulation rate dependence (0.2-3 Hz) of diastolic calcium, calcium transient amplitude and SR calcium content in left ventricular myocytes isolated from hearts of rabbits with pressure and volume overload-induced HF and age-matched control animals. Cytosolic calcium was measured with indo-1. In some experiments, delayed after-depolarizations (DADs) were monitored with the voltage sensitive dye di-4-Annepps. SR calcium content was estimated from the response to rapid cooling (RC). After-transients were elicited in the presence of norepinephrine (100 nmol/l) after cessation of burst pacing. RESULTS: With increasing stimulation rate (0.2-3.0 Hz): (1) steady state diastolic [Ca](i) increased from 102 to 174 nmol/l in HF and from 44 to 103 nmol/l in control, (2) calcium transient amplitudes decreased from 310 to 254 nmol/l in HF and increased from 186 to 429 nmol/l in control, (3) SR calcium content decreased from 1.25 to 1.09 mmol/l in HF and increased from 1.51 to 2.48 mmol/l in control, (4) in HF and control, the end diastolic SR membrane calcium gradient decreased by about 30%; at any stimulation rate, the magnitude of gradient in HF was one-third of control, (5) systolic depletion of SR was 85% in HF and 60% in control. In HF, noradrenaline (100 nmol/l) increased SR calcium content and SR membrane gradient by 40% versus about 7% in control. Calcium after-transients were observed in 14 out of 18 HF rabbits, and none in eight control animals and were associated with DADs. Calcium after-transients were associated with a 35% decrease in SR calcium content. The frequency of occurrence of calcium after-transients was related to diastolic calcium. CONCLUSIONS: in HF, diastolic calcium is increased and both SR calcium content and SR membrane calcium gradient are decreased in a stimulation rate-dependent manner. In HF, beta-adrenergic stimulation can partly restore the SR calcium content and SR membrane gradient at higher stimulation rates in a meta-stable condition; upon transition to low stimulation rates, the SR membrane can no longer maintain this high unbalanced SR calcium load at increased diastolic calcium, the magnitude of which is causally related to the occurrence of calcium after-transients.

Intrinsic heterogeneity in repolarization is increased in isolated failing rabbit cardiomyocytes during simulated ischemia.

OBJECTIVE: Myocardial ischemia and ventricular arrhythmias often complicate congestive heart failure. Ischemia-induced dispersion in repolarization is an important arrhythmogenic factor that might be caused by intrinsic cellular differences in response to simulated ischemia (SI) or by changed coupling of myocytes. We hypothesized that intrinsic heterogeneity in action potential duration (APD) or the occurrence of rigor is larger in failing than in normal rabbit myocytes during SI. METHODS: Heart failure (HF) was induced with volume and pressure overload. Left ventricular myocytes from apex, free wall and base were enzymatically isolated and exposed to SI with NaCN. RESULTS: There were no baseline differences in APD before SI. During SI no differences in time to inexcitability occurred but the range in APD increased more in HF than in normal cells. Rigor occurred after 16.8+/-3.5 and 23.0+/-7.5 min (P<0.05) in normal and HF myocytes, with no differences between apical, free wall or base cells. Variance in time to rigor was larger in HF than in normal cells (55.7 versus 12.4 min(2)). Blockade of anaerobic reserve decreased variance in time to rigor, also when normalized to mean, in HF and normal myocytes. In coupled normal and HF cell pairs, no delay in action potential propagation or differences in APD occurred during SI, and time to rigor was synchronized (P<0.05 vs. single cells). CONCLUSIONS: Intercellular differences in APD and in time of rigor arise in normal and HF myocytes subjected to SI, and are inhibited by blockade of anaerobic glycolysis. Dispersion in APD and tolerance to SI is increased in HF cells. APD and time to rigor are completely synchronized in coupled cell pairs.