Eef Dries

Selective Modulation of Coupled Ryanodine Receptors During Microdomain Activation of Calcium/Calmodulin-Dependent Kinase II in the Dyadic Cleft

Rationale: In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs. Objective: Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation. Methods and Results: We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca2+ transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca2+ indicator). Ca2+ sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca2+/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca2+/calmodulin-dependent kinase II blockers autocamtide-2–related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca2+/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca2+ transients, frequency-dependent increase of the rate of Ca2+ rise was seen in coupled RyR regions only and abolished by autocamtide-2–related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost. Conclusions: Coupled RyRs have a distinct modulation by Ca2+/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca2+/reactive oxygen species microdomain, and subject to modification with disease.

Selective Modulation of Coupled Ryanodine Receptors During Microdomain Activation of CaMKII in the Dyadic Cleft

Rationale: In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs. Objective: Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation. Methods and Results: We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca2+ transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca2+ indicator). Ca2+ sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca2+/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca2+/calmodulin-dependent kinase II blockers autocamtide-2–related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca2+/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca2+ transients, frequency-dependent increase of the rate of Ca2+ rise was seen in coupled RyR regions only and abolished by autocamtide-2–related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost. Conclusions: Coupled RyRs have a distinct modulation by Ca2+/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca2+/reactive oxygen species microdomain, and subject to modification with disease.

Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1-dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft.

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 β‐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 β‐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.

Calcium release near L-type calcium channels promotes beat-to-beat variability in ventricular myocytes from the chronic AV block dog.

Beat-to-beat variability of ventricular repolarization (BVR) has been proposed as a strong predictor of Torsades de Pointes (TdP). BVR is also observed at the myocyte level, and a number of studies have shown the importance of calcium handling in influencing this parameter. The chronic AV block (CAVB) dog is a model of TdP arrhythmia in cardiac hypertrophy, and myocytes from these animals show extensive remodeling, including of Ca(2+) handling. This remodeling process also leads to increased BVR. We aimed to determine the role that (local) Ca(2+) handling plays in BVR. In isolated LV myocytes an exponential relationship was observed between BVR magnitude and action potential duration (APD) at baseline. Inhibition of Ca(2+) release from sarcoplasmic reticulum (SR) with thapsigargin resulted in a reduction of [Ca(2+)]i, and of both BVR and APD. Increasing ICaL in the presence of thapsigargin restored APD but BVR remained low. In contrast, increasing ICaL with preserved Ca(2+) release increased both APD and BVR. Inhibition of Ca(2+) release with caffeine, as with thapsigargin, reduced BVR despite maintained APD. Simultaneous inhibition of Na(+)/Ca(2+) exchange and ICaL decreased APD and BVR to similar degrees, whilst increasing diastolic Ca(2+). Buffering of Ca(2+) transients with BAPTA reduced BVR for a given APD to a greater extent than buffering with EGTA, suggesting subsarcolemmal Ca(2+) transients modulated BVR to a larger extent than the cytosolic Ca(2+) transient. In conclusion, BVR in hypertrophied dog myocytes, at any APD, is strongly dependent on SR Ca(2+) release, which may act through modulation of the l-type Ca(2+) current in a subsarcolemmal microdomain.

Reduced mitochondrial respiration in the ischemic as well as in the remote nonischemic region in postmyocardial infarction remodeling

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.

Hyperactive ryanodine receptors in human heart failure and ischaemic cardiomyopathy reside outside of couplons

Abstract 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.