ABSTRACT
Nanomedicine offers great potential for the treatment of cardiovascular disease and particulate systems have the capacity to markedly improve bioavailability of therapeutics. The delivery of pro-angiogenic hepatocyte growth factor (HGF) and pro-survival and pro-myogenic insulin-like growth factor (IGF-1) encapsulated in Alginate-Sulfate nanoparticles (AlgS-NP) might improve left ventricular (LV) functional recovery after myocardial infarction (MI). In a porcine ischemia-reperfusion model, MI is induced by 75 min balloon occlusion of the mid-left anterior descending coronary artery followed by reperfusion. After 1 week, pigs (n = 12) with marked LV-dysfunction (LV ejection fraction, LVEF < 45%) are randomized to fusion imaging-guided intramyocardial injections of 8 mg AlgS-NP prepared with 200 µg HGF and IGF-1 (HGF/IGF1-NP) or PBS (Control). Intramyocardial injection is safe and pharmacokinetic studies of Cy5-labeled NP confirm superior cardiac retention compared to intracoronary infusion. Seven weeks after intramyocardial-injection of HGF/IGF1-NP, infarct size, measured using magnetic resonance imaging, is significantly smaller than in controls and is associated with increased coronary flow reserve. Importantly, HGF/IGF1-NP-treated pigs show significantly increased LVEF accompanied by improved myocardial remodeling. These findings demonstrate the feasibility and efficacy of using AlgS-NP as a delivery system for growth factors and offer the prospect of innovative treatment for refractory ischemic cardiomyopathy.
Subject(s)
Myocardial Infarction , Myocardial Reperfusion Injury , Nanoparticles , Animals , Disease Models, Animal , Hepatocyte Growth Factor , Insulin-Like Growth Factor I , Sulfates , SwineABSTRACT
Aims: In ventricular myocytes from humans and large mammals, the transverse and axial tubular system (TATS) network is less extensive than in rodents with consequently a greater proportion of ryanodine receptors (RyRs) not coupled to this membrane system. TATS remodelling in heart failure (HF) and after myocardial infarction (MI) increases the fraction of non-coupled RyRs. Here we investigate whether this remodelling alters the activity of coupled and non-coupled RyR sub-populations through changes in local signalling. We study myocytes from patients with end-stage HF, compared with non-failing (non-HF), and myocytes from pigs with MI and reduced left ventricular (LV) function, compared with sham intervention (SHAM). Methods and results: Single LV myocytes for functional studies were isolated according to standard protocols. Immunofluorescent staining visualized organization of TATS and RyRs. Ca2+ was measured by confocal imaging (fluo-4 as indicator) and using whole-cell patch-clamp (37°C). Spontaneous Ca2+ release events, Ca2+ sparks, as a readout for RyR activity were recorded during a 15 s period following conditioning stimulation at 2 Hz. Sparks were assigned to cell regions categorized as coupled or non-coupled sites according to a previously developed method. Human HF myocytes had more non-coupled sites and these had more spontaneous activity than in non-HF. Hyperactivity of these non-coupled RyRs was reduced by Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition. Myocytes from MI pigs had similar changes compared with SHAM controls as seen in human HF myocytes. As well as by CaMKII inhibition, in MI, the increased activity of non-coupled sites was inhibited by mitochondrial reactive oxygen species (mito-ROS) scavenging. Under adrenergic stimulation, Ca2+ waves were more frequent and originated at non-coupled sites, generating larger Na+/Ca2+ exchange currents in MI than in SHAM. Inhibition of CaMKII or mito-ROS scavenging reduced spontaneous Ca2+ waves, and improved excitation-contraction coupling. Conclusions: In HF and after MI, RyR microdomain re-organization enhances spontaneous Ca2+ release at non-coupled sites in a manner dependent on CaMKII activation and mito-ROS production. This specific modulation generates a substrate for arrhythmia that appears to be responsive to selective pharmacologic modulation.
Subject(s)
Arrhythmias, Cardiac/metabolism , Calcium Signaling , Cardiomyopathies/metabolism , Heart Failure/metabolism , Myocardial Infarction/metabolism , Myocytes, Cardiac/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Aged , Animals , Arrhythmias, Cardiac/physiopathology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cardiomyopathies/physiopathology , Case-Control Studies , Disease Models, Animal , Excitation Contraction Coupling , Female , Heart Failure/physiopathology , Humans , Male , Membrane Potentials , Middle Aged , Mitochondria, Heart/metabolism , Myocardial Contraction , Myocardial Infarction/physiopathology , NADPH Oxidase 2/metabolism , Reactive Oxygen Species/metabolism , Sodium-Calcium Exchanger/metabolism , Sus scrofa , Time Factors , Ventricular Function, Left , Ventricular RemodelingABSTRACT
Scarring and remodeling of the left ventricle (LV) after myocardial infarction (MI) results in ischemic cardiomyopathy with reduced contractile function. Regional differences related to persisting ischemia may exist. We investigated the hypothesis that mitochondrial function and structure is altered in the myocardium adjacent to MI with reduced perfusion (MIadjacent) and less so in the remote, nonischemic myocardium (MIremote). We used a pig model of chronic coronary stenosis and MI (n = 13). Functional and perfusion MR imaging 6 wk after intervention showed reduced ejection fraction and increased global wall stress compared with sham-operated animals (Sham; n = 14). Regional strain in MIadjacent was reduced with reduced contractile reserve; in MIremote strain was also reduced but responsive to dobutamine and perfusion was normal compared with Sham. Capillary density was unchanged. Cardiac myocytes isolated from both regions had reduced basal and maximal oxygen consumption rate, as well as through complex I and II, but complex IV activity was unchanged. Reduced respiration was not associated with detectable reduction of mitochondrial density. There was no significant change in AMPK or glucose transporter expression levels, but glycogen content was significantly increased in both MIadjacent and MIremote Glycogen accumulation was predominantly perinuclear; mitochondria in this area were smaller but only in MIadjacent where also subsarcolemmal mitochondria were smaller. In conclusion, after MI reduction of mitochondrial respiration and glycogen accumulation occur in all LV regions suggesting that reduced perfusion does not lead to additional specific changes and that increased hemodynamic load is the major driver for changes in mitochondrial function.
Subject(s)
Cardiomyopathies/metabolism , Mitochondria, Heart/metabolism , Myocardial Infarction/metabolism , Myocytes, Cardiac/metabolism , Oxygen Consumption , Ventricular Remodeling , AMP-Activated Protein Kinases/genetics , Animals , Blotting, Western , Cardiomyopathies/diagnostic imaging , Cardiomyopathies/etiology , Cardiomyopathies/pathology , Cell Respiration , Cicatrix , Coronary Stenosis/complications , Electron Transport Complex I/metabolism , Electron Transport Complex II/metabolism , Electron Transport Complex IV/metabolism , Glucose Transport Proteins, Facilitative/genetics , Glycogen/metabolism , Magnetic Resonance Imaging , Microscopy, Electron , Microscopy, Fluorescence , Myocardial Infarction/diagnostic imaging , Myocardial Infarction/etiology , Myocardial Infarction/pathology , Myocardial Perfusion Imaging , Myocytes, Cardiac/ultrastructure , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Stroke Volume , Sus scrofa , SwineABSTRACT
KEY POINTS: The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca2+ /calmodulin-dependent kinase II (CaMKII) activation during high-frequency stimulation. Sympathetic stimulation through ß-adrenergic receptors activates an integrated signalling cascade, enhancing Ca2+ cycling and is at least partially mediated through CaMKII. Here we report that CaMKII activation during ß-adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs. In contrast, the increase in the Ca2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca2+ transient are primarily protein kinase A-dependent. The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. ABSTRACT: In cardiac myocytes, ß-adrenergic stimulation enhances Ca2+ cycling through an integrated signalling cascade modulating L-type Ca2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca2+ /calmodulin-dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole-cell voltage-clamp and confocal line-scan imaging with Fluo-4 as a [Ca2+ ]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca2+ sparks, which are assigned to coupled and non-coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces spark frequency in coupled RyRs only; NOS1 inhibition mimics the effect of CaMKII inhibition. Moreover, ISO induces the repetitive activation of coupled RyR clusters through CaMKII activation. Immunostaining shows high levels of CaMKII phosphorylation at the dyadic cleft. CaMKII inhibition reduces ICaL and local Ca2+ transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca2+ transient. In contrast, protein kinase A (PKA) inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and relaxation. In conclusion, CaMKII activation during ß-adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC-triggered local Ca2+ release as well as spontaneous diastolic Ca2+ release whilst PKA is the major pathway increasing global Ca2+ cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy.
Subject(s)
Adrenergic Agents/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium/metabolism , Calmodulin/metabolism , Nitric Oxide Synthase Type I/metabolism , Receptors, Adrenergic, beta/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Aniline Compounds/metabolism , Animals , Calcium Signaling/drug effects , Calcium Signaling/physiology , Calcium-Binding Proteins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Swine , Xanthenes/metabolismABSTRACT
AIMS: In atrial fibrillation (AF), abnormalities in Ca(2+) release contribute to arrhythmia generation and contractile dysfunction. We explore whether ryanodine receptor (RyR) cluster ultrastructure is altered and is associated with functional abnormalities in AF. METHODS AND RESULTS: Using high-resolution confocal microscopy (STED), we examined RyR cluster morphology in fixed atrial myocytes from sheep with persistent AF (N = 6) and control (Ctrl; N = 6) animals. RyR clusters on average contained 15 contiguous RyRs; this did not differ between AF and Ctrl. However, the distance between clusters was significantly reduced in AF (288 ± 12 vs. 376 ± 17 nm). When RyR clusters were grouped into Ca(2+) release units (CRUs), i.e. clusters separated by <150 nm, CRUs in AF had more clusters (3.43 ± 0.10 vs. 2.95 ± 0.02 in Ctrl), which were more dispersed. Furthermore, in AF cells, more RyR clusters were found between Z lines. In parallel experiments, Ca(2+) sparks were monitored in live permeabilized myocytes. In AF, myocytes had >50% higher spark frequency with increased spark time to peak (TTP) and duration, and a higher incidence of macrosparks. A computational model of the CRU was used to simulate the morphological alterations observed in AF cells. Increasing cluster fragmentation to the level observed in AF cells caused the observed changes, i.e. higher spark frequency, increased TTP and duration; RyR clusters dispersed between Z-lines increased the occurrence of macrosparks. CONCLUSION: In persistent AF, ultrastructural reorganization of RyR clusters within CRUs is associated with overactive Ca(2+) release, increasing the likelihood of propagating Ca(2+) release.
Subject(s)
Atrial Fibrillation/metabolism , Calcium Signaling , Myocytes, Cardiac/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Animals , Atrial Fibrillation/physiopathology , Computer Simulation , Disease Models, Animal , Heart Atria/metabolism , Heart Atria/physiopathology , Heart Atria/ultrastructure , Kinetics , Microscopy, Confocal , Models, Cardiovascular , Models, Molecular , Myocytes, Cardiac/ultrastructure , Protein Conformation , Ryanodine Receptor Calcium Release Channel/ultrastructure , Sheep , Structure-Activity RelationshipABSTRACT
BACKGROUND: An acute increase in blood pressure is associated with the occurrence of premature ventricular complexes (PVCs). OBJECTIVE: We aimed to study the timing of these PVCs with respect to afterload-induced changes in myocardial deformation in a controlled, preclinically relevant, novel closed-chest pig model. METHODS: An acute left ventricular (LV) afterload challenge was induced by partial balloon inflation in the descending aorta, lasting 5-10 heartbeats (8 pigs; 396 inflations). RESULTS: Balloon inflation enhanced the reflected wave (augmentation index 30% ± 8% vs 59% ± 6%; P < .001), increasing systolic central blood pressure by 35% ± 4%. This challenge resulted in a more abrupt LV pressure decline, which was delayed beyond ventricular repolarization (rate of pressure decline 0.16 ± 0.01 mm Hg/s vs 0.27 ± 0.04 mm Hg/ms; P < .001 and interval T-wave to peak pressure 1 ± 12 ms vs 36 ± 9 ms; P = .008), during which the velocity of myocardial shortening at the basal septum increased abruptly (ie, postsystolic shortening) (peak strain rate -0.6 ± 0.5 s(-1) vs -2.5 ± 0.8 s(-1); P < .001). It is exactly at this time of LV pressure decline, with increased postsystolic shortening, and not at peak pressure, that PVCs occur (22% of inflations). These PVCs preferentially occurred at the basal and apical segments. In the same regions, monophasic action potentials demonstrated the appearance of delayed afterdepolarization-like transient depolarizations as origin of PVCs. CONCLUSION: An acute blood pressure increase results in a more abrupt LV pressure decline, which is delayed after ventricular repolarization. This has a profound effect on myocardial mechanics with enhanced postsystolic shortening. Coincidence with induced transient depolarizations and PVCs provides support for the mechanoelectrical origin of pressure-induced premature beats.
Subject(s)
Body Surface Potential Mapping , Hypertension/complications , Myocardial Contraction/physiology , Ventricular Function, Left/physiology , Ventricular Premature Complexes/etiology , Animals , Cardiac Complexes, Premature/diagnosis , Cardiac Complexes, Premature/etiology , Disease Models, Animal , Echocardiography, Doppler , Female , Heart Rate/physiology , Male , Mechanoreceptors/physiology , Pressure , Random Allocation , Sensitivity and Specificity , Sus scrofa , Systole/physiology , Ventricular Premature Complexes/diagnosisABSTRACT
AIMS: Oxidative stress can modulate nitric oxide (NO) signalling pathways. Both pathways have been shown to be involved in the pathophysiology of atrial fibrillation (AF), but data are conflicting. We aimed to characterize the NO-pathway and its relation to oxidative stress in persistent AF in a sheep model. METHODS AND RESULTS: Persistent AF was induced by rapid atrial pacing for a mean of 136.5 ± 21.7 days. Non-stimulated sheep served as controls. Nicotine adenine dinucleotide phosphate (NADPH) oxidase-stimulated superoxide production was significantly increased in the AF group (+51.3 ± 23.2%, P < 0.01). Although there were no changes in mRNA expression of the different NADPH oxidase subunits, the increased activity was associated with markedly increased protein expression of the NADPH oxidase activator, Rac1 (+26 ± 4.6%, P < 0.05). No differences were seen in superoxide dismutase activity, but glutathione peroxidase activity was lower in the AF group. There was a marked accumulation of 3-nitrotyrosine, a biomarker for peroxynitrite, in atrial tissue of AF animals, as demonstrated by immunohistochemical staining and dot blot analysis (+15.6 ± 1.8%, P < 0.05). Expression of atrial NOS3 mRNA was 24.9 ± 4.4% lower in the AF group vs. control (P < 0.05), while NOS1 and 2 were unchanged. Immunoblot analysis revealed no changes in protein expression. Nitrite/nitrate levels were significantly lower during AF (-24.8 ± 5.8%, P < 0.05). CONCLUSION: In a sheep model of persistent AF, NOS3 transcript levels are attenuated and circulating NOx levels decreased. Persistent AF is associated with increased oxidative stress, probably resulting from increased NADPH oxidase activity, without major changes in anti-oxidant capacity of the atrial tissue.
Subject(s)
Atrial Fibrillation/metabolism , Nitric Oxide Synthase/metabolism , Nitric Oxide/metabolism , Oxidative Stress , Reactive Oxygen Species/metabolism , Animals , Chronic Disease , Female , SheepABSTRACT
BACKGROUND: Repolarization variability is considered to predict sudden cardiac death. T-wave alternans (TWA) has been the subject of exhaustive research, whereas beat-to-beat variability of repolarization (BVR) is a new parameter that possibly predicts proarrhythmia. How these parameters interact has not been tested. OBJECTIVE: The purpose of this study was to compare TWA and BVR as predictors of proarrhythmic substrate early after myocardial infarction (MI). METHODS: In nine pigs, MI was induced by 1-hour occlusion of the left anterior descending coronary artery. Cardiac magnetic resonance imaging was performed at day 21. Six sham pigs served as control. Spectral TWA was tested during right atrial pacing before induction of MI and after 21 days. BVR was calculated from 60 consecutive QT intervals. RESULTS: Magnetic resonance imaging showed transmural MI. TWA was negative in all pigs at clinical threshold rate and equally present in MI versus sham pigs at higher rates (170 bpm: 55% vs 50% positive TWA). In MI pigs, BVR of QT intervals increased significantly during acute ischemia (2.44 ± 0.43 ms vs 3.55 ± 0.41 ms, P <.01) and even more on day 21 (5.80 ± 1.12 ms), but it differed significantly from sham (2.14 ± 0.54 ms, P <.01). A clinical ventricular tachycardia induction protocol was positive in 2 of 8 MI pigs and in none of 6 shams. CONCLUSION: In early remodeling after MI, BVR at intrinsic heart rate was a consistent phenomenon, whereas TWA during atrial pacing or baseline QT-interval changes were not. TWA and BVR could reflect different post-MI remodeling processes. BVR may be a new technique for predicting a potentially proarrhythmic substrate in the early postinfarction period.
Subject(s)
Cardiac Resynchronization Therapy/methods , Death, Sudden, Cardiac/etiology , Heart Conduction System/physiopathology , Heart Rate/physiology , Myocardial Ischemia/complications , Tachycardia, Ventricular/physiopathology , Ventricular Remodeling , Animals , Death, Sudden, Cardiac/pathology , Death, Sudden, Cardiac/prevention & control , Disease Models, Animal , Electrocardiography , Follow-Up Studies , Magnetic Resonance Imaging, Cine , Myocardial Ischemia/physiopathology , Swine , Tachycardia, Ventricular/complications , Tachycardia, Ventricular/diagnosis , Time FactorsABSTRACT
AIMS: Rapid atrial pacing for 1 week leads to decreased expression of endocardial nitric oxide (NO)-synthase and decreased NO concentrations. We hypothesized that increasing NO bioavailability may reduce electrical remodelling induced by atrial tachycardia. METHODS AND RESULTS: We examined the effect of molsidomine, a NO donor, and N(ω)-nitro-l-arginine methylester (l-NAME), a NO-synthase inhibitor, on electrical remodelling occurring during 4 h of rapid atrial pacing in sheep. Haemodynamic and electrophysiological parameters were measured at baseline, 1 h after the start of the infusion and before the start of pacing, and 2 and 4 h after pacing. We measured the effect of molsidomine on atrial monophasic action potentials (MAPs) in non-instrumented sheep and on l-type Ca(2+) currents and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients in right atrial cells, isolated from control sheep. In control sheep, rapid atrial pacing shortened the atrial effective refractory period (AERP) by 12 ± 0.18% after 4 h, an effect that was unaffected by l-NAME. Infusion of molsidomine increased AERP at baseline (+13.4 ± 1.04%) and transiently attenuated pacing-induced AERP shortening (13.6 ± 0.1% at 2 h). Molsidomine tended to increase MAP duration by 20.7 ± 13.4 ms. Incubation of isolated atrial myocytes with NO donor 3-morpholino-sydnonimine (SIN-1) increased significantly l-type Ca(2+) current and [Ca(2+)](i) transients. CONCLUSION: Infusion of molsidomine, a NO donor, delayed shortening of the action potential during short-term rapid atrial pacing, by increasing [Ca(2+)](i). Whereas the former could be protective against repetitive short episodes of atrial fibrillation, the latter might be detrimental in the long term.
Subject(s)
Heart Conduction System/drug effects , Molsidomine/pharmacology , Nitric Oxide Donors/pharmacology , Nitric Oxide/metabolism , Tachycardia, Ectopic Atrial , Action Potentials/drug effects , Action Potentials/physiology , Animals , Blood Pressure/physiology , Calcium/metabolism , Calcium Channels, L-Type/physiology , Cardiac Pacing, Artificial , Disease Models, Animal , Female , Heart Conduction System/physiology , Refractory Period, Electrophysiological/drug effects , Refractory Period, Electrophysiological/physiology , Sheep , Tachycardia, Ectopic Atrial/drug therapy , Tachycardia, Ectopic Atrial/metabolism , Tachycardia, Ectopic Atrial/physiopathologyABSTRACT
There is ample evidence that regular exercise exerts beneficial effects on left ventricular (LV) hypertrophy, remodeling and dysfunction produced by ischemic heart disease or systemic hypertension. In contrast, the effects of exercise on pathological LV hypertrophy and dysfunction produced by LV outflow obstruction have not been studied to date. Consequently, we evaluated the effects of 8 weeks of voluntary wheel running in mice (which mitigates post-infarct LV dysfunction) on LV hypertrophy and dysfunction produced by mild (mTAC) and severe (sTAC) transverse aortic constriction. mTAC produced ~40% LV hypertrophy and increased myocardial expression of hypertrophy marker genes but did not affect LV function, SERCA2a protein levels, apoptosis or capillary density. Exercise had no effect on global LV hypertrophy and function in mTAC but increased interstitial collagen, and ANP expression. sTAC produced ~80% LV hypertrophy and further increased ANP expression and interstitial fibrosis and, in contrast with mTAC, also produced LV dilation, systolic as well as diastolic dysfunction, pulmonary congestion, apoptosis and capillary rarefaction and decreased SERCA2a and ryanodine receptor (RyR) protein levels. LV diastolic dysfunction was likely aggravated by elevated passive isometric force and Ca(2+)-sensitivity of myofilaments. Exercise training failed to mitigate the sTAC-induced LV hypertrophy and capillary rarefaction or the decreases in SERCA2a and RyR. Exercise attenuated the sTAC-induced increase in passive isometric force but did not affect myofilament Ca(2+)-sensitivity and tended to aggravate interstitial fibrosis. In conclusion, exercise had no effect on LV function in compensated and decompensated cardiac hypertrophy produced by LV outflow obstruction, suggesting that the effect of exercise on pathologic LV hypertrophy and dysfunction depends critically on the underlying cause.
Subject(s)
Aortic Valve Stenosis/complications , Aortic Valve Stenosis/physiopathology , Hypertrophy, Left Ventricular/etiology , Hypertrophy, Left Ventricular/physiopathology , Physical Conditioning, Animal , Actin Cytoskeleton/metabolism , Animals , Aortic Valve Stenosis/mortality , Calcium-Binding Proteins/metabolism , Female , Genetic Markers/genetics , Homeodomain Proteins/metabolism , Hypertrophy, Left Ventricular/mortality , Male , Mice , Mice, Inbred C57BL , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Survival AnalysisABSTRACT
AIMS: Exercise started early after myocardial infarction (MI) improves in vivo cardiac function and myofilament responsiveness to Ca(2+). We investigated whether this represents partial or complete reversal of cellular remodelling. METHODS AND RESULTS: Mice with MI following left coronary ligation were given free access to a running wheel (MI(EXE), N = 22) or housed sedentary (MI(SED), N = 18) for 8 weeks and compared with sedentary sham-operated animals (SHAM, N = 11). Myocytes were enzymatically isolated from the non-infarcted left ventricle. Myocytes in MI were significantly longer and even more so with exercise (165 +/- 3 microm in MI(EXE) vs. 148 +/- 3 microm in MI(SED) and 136 +/- 2 microm in SHAM; P < 0.05, mean +/- SEM); cell width was not different. Contraction was measured during electrical field stimulation at 1, 2, and 4 Hz. Unloaded cell shortening was significantly reduced in MI(SED) (at 1 Hz, L/L(0)=4.4 +/- 0.3% vs. 6.7 +/- 0.4% in SHAM; P < 0.05, also at 2 and 4 Hz). Exercise restored cell shortening to SHAM values (MI(EXE), L/L(0)=6.4 +/- 0.5%). Membrane currents and [Ca(2+)](i) were measured via whole-cell patch clamping, with Fluo-3 as Ca(2+) indicator, all at 30 degrees C. Ca(2+) transient amplitude, I(CaL) and sarcoplasmic reticulum Ca(2+) content were not different between the three groups. Diastolic Ca(2+) levels at 4 Hz were significantly elevated in MI(SED) only, with a trend to increased spontaneous Ca(2+) release events (sparks). Action potential duration was increased and transient outward K(+) currents significantly reduced after MI; this was unaffected by exercise. CONCLUSIONS: Early voluntary exercise training after MI restores cell contraction to normal values predominantly because of changes in the myofilament Ca(2+) response and has a beneficial effect on diastolic Ca(2+) handling. However, the beneficial effect is not a complete reversal of remodelling as hypertrophy and loss of repolarizing K(+) currents are not affected.
Subject(s)
Cardiomegaly/physiopathology , Myocardial Contraction/physiology , Myocardial Infarction/physiopathology , Physical Conditioning, Animal/physiology , Ventricular Remodeling/physiology , Actin Cytoskeleton/physiology , Animals , Calcium/metabolism , Calcium Channels, L-Type/physiology , Cardiomegaly/pathology , Cardiomegaly/therapy , Diastole/physiology , Disease Models, Animal , Exercise Therapy , Mice , Myocardial Infarction/pathology , Myocardial Infarction/therapy , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Myocytes, Cardiac/physiology , Potassium/metabolism , Sarcoplasmic Reticulum/metabolismABSTRACT
RATIONALE: Persistent atrial fibrillation (AF) has been associated with structural and electric remodeling and reduced contractile function. OBJECTIVE: To unravel mechanisms underlying reduced sarcoplasmic reticulum (SR) Ca(2+) release in persistent AF. METHODS: We studied cell shortening, membrane currents, and [Ca(2+)](i) in right atrial myocytes isolated from sheep with persistent AF (duration 129+/-39 days, N=16), compared to matched control animals (N=21). T-tubule density, ryanodine receptor (RyR) distribution, and local [Ca(2+)](i) transients were examined in confocal imaging. RESULTS: Myocyte shortening and underlying [Ca(2+)](i) transients were profoundly reduced in AF (by 54.8% and 62%, P<0.01). This reduced cell shortening could be corrected by increasing [Ca(2+)](i). SR Ca(2+) content was not different. Calculated fractional SR Ca(2+) release was reduced in AF (by 20.6%, P<0.05). Peak Ca(2+) current density was modestly decreased (by 23.9%, P<0.01). T-tubules were present in the control atrial myocytes at low density and strongly reduced in AF (by 45%, P<0.01), whereas the regular distribution of RyR was unchanged. Synchrony of SR Ca(2+) release in AF was significantly reduced with increased areas of delayed Ca(2+) release. Propagation between RyR was unaffected but Ca(2+) release at subsarcolemmal sites was reduced. Rate of Ca(2+) extrusion by Na(+)/Ca(2+) exchanger was increased. CONCLUSIONS: In persistent AF, reduced SR Ca(2+) release despite preserved SR Ca(2+) content is a major factor in contractile dysfunction. Fewer Ca(2+) channel-RyR couplings and reduced efficiency of the coupling at subsarcolemmal sites, possibly related to increased Na(+)/Ca(2+) exchanger, underlie the reduction in Ca(2+) release.
Subject(s)
Atrial Fibrillation/metabolism , Atrial Function, Right , Calcium Signaling , Myocardial Contraction , Myocytes, Cardiac/metabolism , Sarcoplasmic Reticulum/metabolism , Actin Cytoskeleton/metabolism , Animals , Atrial Fibrillation/pathology , Atrial Fibrillation/physiopathology , Calcium Channels, L-Type/metabolism , Disease Models, Animal , Electrophysiologic Techniques, Cardiac , Female , Glycogen/metabolism , Heart Atria/metabolism , Heart Atria/pathology , Heart Atria/physiopathology , Membrane Potentials , Myocytes, Cardiac/ultrastructure , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcolemma/metabolism , Sarcoplasmic Reticulum/ultrastructure , Sheep , Sodium-Calcium Exchanger/metabolism , Time FactorsABSTRACT
Block of Na/Ca exchange (NCX) has potential therapeutic applications, in particular, if a mode-selective block could be achieved, but also carries serious risks for disturbing the normal Ca2+ balance maintained by NCX. We have examined the effects of partial inhibition of NCX by SEA-0400 (1 or 0.3 micromol/L) in left ventricular myocytes from healthy pigs or mice and from mice with heart failure (MLP-/-). During voltage clamp ramps with [Ca2+](i) buffering, block of reverse mode block was slightly larger than of forward mode (by 25+/-5%, P<0.05). In the absence of [Ca2+](i) buffering and with sarcoplasmic reticulum (SR) fluxes blocked, rate constants for Ca2+ influx and Ca2+ efflux were reduced to the same extent (to 36+/-6% and 32+/-4%, respectively). With normal SR function the reduction of inward NCX current (I(NCX)) was 57+/-10% (n=10); during large caffeine-induced Ca2+ transients, it was larger (82+/-3%). [Ca2+](i) transients evoked during depolarizing steps increased (from 424+/-27 to 994+/-127 nmol/L at +10 mV, P<0.05), despite a reduction of I(CaL) by 27%. Resting [Ca2+](i) increased; there was a small decrease in the rate of decline of [Ca2+](i). SR Ca2+) content increased more than 2-fold. Contraction amplitude of field-stimulated myocytes increased in healthy myocytes but not in myocytes from MLP-/- mice, in which SR Ca2+ content remained unchanged. These data provide proof-of-principle that even partial inhibition of NCX results in a net gain of Ca2+. Further development of NCX blockers, in particular, for heart failure, must balance potential benefits of I(NCX) reduction against effects on Ca2+ handling by refining mode dependence and/or including additional targets.
Subject(s)
Aniline Compounds/pharmacology , Calcium/metabolism , Heart Failure/metabolism , Myocytes, Cardiac/drug effects , Phenyl Ethers/pharmacology , Sodium-Calcium Exchanger/antagonists & inhibitors , Animals , Calcium Channel Blockers/pharmacology , Cardiotonic Agents/pharmacology , Cells, Cultured , Disease Models, Animal , Electric Stimulation/methods , Female , Male , Mice , Mice, Knockout , Myocardial Contraction/drug effects , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Sodium-Calcium Exchanger/metabolism , SwineABSTRACT
The development of atrial fibrillation (AF) is associated with electrical and structural remodeling. The aim of this study was to assess the contribution of electrical and structural remodeling to the development of AF in a rapid atrially paced ovine model with and without His bundle ablation and to determine the role of the angiotensin pathway and matrix metalloproteinases in this process. Thirty-five sheep were rapidly paced in the atrium and were randomized to undergo His bundle ablation (HBA) (21 sheep; HBA sheep) or not (14 sheep; non-HBA sheep). After HBA the ventricles were paced at 80 bpm. Both groups were subdivided to receive active treatment (quinapril+losartan) or placebo. Sheep were followed for 15 weeks. Inducible AF was defined as a rapid irregular atrial rhythm lasting >1 min. Inducible AF was considered to be persistent if during further follow-up no sinus rhythm (SR) was documented anymore. The inducibility of AF with atrial tachypacing was not different between the 4 groups. On the other hand, non-HBA sheep developed persistent AF significantly earlier than HBA sheep (p=0.028). They had elevated ventricular rates, diminished atrial MMP-2, increased TIMP-2 expression, and more extensive atrial fibrosis. Active treatment in these sheep significantly lowered AT-II (p=0.018), prevented atrial fibrogenesis (p<0.001) and slowed the development of persistent AF (p=0.049). Electrical remodeling is sufficient to induce AF, while structural changes are needed for persistent AF. Fibrosis development in our model is the result of an increased expression of AT-II in combination with changes in MMP expression. Inhibition of the angiotensin pathway suppresses atrial fibrosis and the development of persistent AF.
Subject(s)
Atrial Fibrillation/physiopathology , Cardiac Pacing, Artificial , Disease Models, Animal , Sheep , Angiotensin II/metabolism , Animals , Bundle of His/pathology , Cardiac Output , Female , Kaplan-Meier Estimate , Matrix Metalloproteinases/metabolism , Myocytes, Cardiac/enzymology , Myocytes, Cardiac/pathology , Refractory Period, Electrophysiological , Renin-Angiotensin System , Time Factors , Tissue Inhibitor of Metalloproteinases/metabolismABSTRACT
Reduced Ca(2+) release from the sarcoplasmic reticulum (SR) and a negative force-frequency relation characterize end-stage human heart failure. The MLP(-/-) mouse with dilated cardiomyopathy is used as a model to explore novel therapeutic interventions but the alterations in Ca(2+) handling in MLP(-/-) remain incompletely understood. We studied [Ca(2+)](i) in left ventricular myocytes from MLP(-/-) and WT mice (3-4 months old; whole-cell voltage clamp, 30 degrees C). At 1 Hz stimulation, the amplitude of [Ca(2+)](i) transients was similar. However, in contrast to WT, at higher frequencies the [Ca(2+)](i) transient amplitude declined in MLP(-/-) and there was no increase in SR Ca(2+) content. Unexpectedly, the decline of [Ca(2+)](i) was faster in MLP(-/-) than in WT (at 1 Hz, tau of 80 +/- 9 vs. 174 +/- 29 ms, P < 0.001) and the frequency-dependent acceleration of the decline was abolished suggesting an enhanced basal SERCA activity. Indeed, the Ca(2+) affinity of SR Ca(2+) uptake in homogenates was higher in MLP(-/-), with the maximal uptake rate similar to WT. Phosphorylation of phospholamban in MLP(-/-) was increased (2.3-fold at Ser(16) and 2.9-fold at the Thr(17) site, P < 0.001) with similar SERCA and total phospholamban protein levels. On increasing stimulation frequency to 4 Hz, WT, but not MLP(-/-), myocytes had a net gain of Ca(2+), suggesting inadequate Ca(2+) sequestration in MLP(-/-). In conclusion, increased baseline phosphorylation of phospholamban in MLP(-/-) leads to a reduced reserve for frequency-dependent increase of Ca(2+) release. This represents a novel paradigm for altered Ca(2+) handling in heart failure, underscoring the importance of phosphorylation pathways.
Subject(s)
Calcium-Binding Proteins/metabolism , Heart Diseases/metabolism , Animals , Calcium/analysis , Calcium/metabolism , Calcium Channels, L-Type/metabolism , Electric Stimulation , Heart Ventricles/cytology , Mice , Mice, Knockout , Myocytes, Cardiac/metabolism , Phosphorylation , Sarcoplasmic Reticulum/metabolismABSTRACT
BACKGROUND: Atrial fibrillation (AF) is associated with extracellular matrix remodeling involving atrial fibrosis and atrial dilatation. Angiotensin II mediated pathways and matrix metalloproteinases (MMPs) have been implicated in these processes. Our aim was to study atrial structural remodeling and the expression of the angiotensin receptor subtypes and MMPs and their inhibitors (TIMPs) in patients with mitral valve disease with and without AF. METHODS AND RESULTS: Biopsies from right and left atrial appendages (RA and LA) were taken from patients undergoing CABG (n=9, all in sinus rhythm (SR)) or mitral valve surgery (MVS; n=19; 9 with permanent AF and 10 in SR). Patients with MVS and AF had significantly larger atria (versus MVS and SR: p=0.02; versus CABG: p<0.01). The MVS patients had significantly more fibrosis than the control CABG group. Fibrosis was increased in both the AF and SR MVS groups in the LA, but only in the MVS-AF group in the RA. These AF patients had significantly more tricuspid regurgitation than SR patients. MMP-1 was down-regulated in LA of MVS patients (p=0.02) independent of the underlying rhythm (SR or AF; p=0.95). In RA biopsies, MMP-1 was down-regulated only in the MVS and AF group. MMP-9 was down-regulated in the MVS patients compared to CABG both in the RA and LA, and without a difference between the SR and AF groups. Protein expression of AT-1, AT-2, MMP-2, TIMP-1, -2 and -4, TNF-alpha, and TNF-alpha-converting enzyme did not differ significantly between the 3 groups. CONCLUSIONS: Concordant changes between MMP-expression and fibrosis during mitral valve disease, both in LA and RA, suggest involvement of MMPs in structural atrial remodeling. AF itself did not contribute to altered fibrosis or MMP-expression in the LA. The association between AF and RA changes may be precipitated by greater hemodynamic load due to tricuspid regurgitation in these patients.
Subject(s)
Atrial Appendage/enzymology , Atrial Fibrillation/enzymology , Matrix Metalloproteinases/analysis , Mitral Valve Insufficiency/enzymology , Aged , Atrial Appendage/pathology , Atrial Fibrillation/pathology , Blotting, Western/methods , Case-Control Studies , Cell Size , Female , Fibrosis , Humans , Immunohistochemistry/methods , Male , Matrix Metalloproteinase 1/analysis , Matrix Metalloproteinase 2/analysis , Matrix Metalloproteinase 9/analysis , Mitral Valve Insufficiency/pathology , Myocytes, Cardiac/enzymology , Myocytes, Cardiac/pathology , Receptor, Angiotensin, Type 1/analysis , Receptor, Angiotensin, Type 2/analysis , Statistics, Nonparametric , Tissue Inhibitor of Metalloproteinase-1/analysis , Tissue Inhibitor of Metalloproteinase-2/analysis , Tissue Inhibitor of Metalloproteinases/analysis , Tumor Necrosis Factor-alpha/analysis , Tissue Inhibitor of Metalloproteinase-4ABSTRACT
BACKGROUND: Catheter based "maze" procedures for atrial fibrillation have been hampered by difficult creation and evaluation of continuous and transmural linear lesions. Our aim was to develop an online evaluation method for effective lesion creation based on conventional techniques and using the multipolar ablation catheter, already in place. METHODS AND RESULTS: We created 15 linear lines in right atria of 13 anesthetized sheep using three multipolar catheter designs (8 x 4 mm 7 Fr, 4 x 6 mm 7 Fr, 8 x 4 mm 3.7 Fr). The lesions were placed on the right posterolateral wall between the orifices of the superior and inferior vena cava. Radiofrequency energy was applied in the temperature-controlled mode to prespecified endpoints (electrogram amplitude decrease to < or = 50%; pacing threshold increase by > or = 100%; split potentials indicating conduction block). Macroscopically transmural and continuous lesions were achieved in only 3 experiments (29 +/- 12 mm x 5 +/- 1 mm), all created by 3.7 Fr octapolar catheters inserted through long sheaths. Preset temperature was reached in 96% of the electrodes (vs. 64% in the non-effective experiments; p < 0.01). Electrogram amplitude decrease (to < or = 50%) and pacing threshold increase (by > or = 100%) did not predict effectiveness. The only criterion that could reliably predict transmural continuous necrosis at histology was the development of split potentials (p < or = 0.05). CONCLUSIONS: Effective creation of linear lesions is difficult. Pliable catheters that conform to the endocardial contour give the best results. The only endpoint that reliably predicted histological transmural continuous necrosis was development of split potentials indicating conduction block.
Subject(s)
Atrial Fibrillation/surgery , Catheter Ablation/instrumentation , Animals , Catheter Ablation/methods , Chi-Square Distribution , Disease Models, Animal , Electrocardiography , Electrodes , Electrophysiologic Techniques, Cardiac , Feasibility Studies , Heart Block , Heart Conduction System/physiology , Probability , Sensitivity and Specificity , SheepABSTRACT
INTRODUCTION: Atrial electrical remodeling, shortening of the atrial effective refractory period (AERP) underlying atrial fibrillation (AF) has been described in different animal models. However, there remains some controversy regarding the time course of this electrical remodeling and the need for secondary factors in the development of AF. We investigated the effect of instrumentation on the inducibility of AF. We hypothesized that epicardial instrumentation could be a confounding factor that accelerates the development of AF. METHODS AND RESULTS: Thirty sheep were rapidly atrially paced at 600 beats/min for 15 weeks: 15 were endocardially instrumented and paced (endo), and 15 were both endocardially and epicardially instrumented. Six of these animals were endocardially paced (sham) and 9 were epicardially paced (epi). The underlying rhythm was determined at regular intervals, and electrophysiologic study was performed. AF developed significantly faster in the epi group. After 3 weeks of pacing, the cumulative incidence of sustained AF (>1 hour) already was 70% in this group versus only 14% and 20% in the endo and sham groups, respectively. After 15 weeks of pacing, this difference was no longer evident. Baseline AERP and minimal AERP, reached before the development of AF, were not significantly different in the three groups. Epicardial instrumentation (epi and sham) increased baseline left and right atrial pressures, but only epicardial stimulation (epi) led to early development of AF. CONCLUSION: In this sheep model of AF, the experimental setup is a major determinant of the inducibility of AF. Not epicardial instrumentation per se but epicardial stimulation accelerated the development of AF. Different animal models