Ca Spark Frequency Research Articles

NO-dependent CaMKII activation during β-adrenergic stimulation of cardiac muscle

During β-adrenergic receptor (β-AR) stimulation, phosphorylation of cardiomyocyte ryanodine receptors by protein kinases may contribute to an increased diastolic Ca(2+) spark frequency. Regardless of prompt activation of protein kinase A during β-AR stimulation, this appears to rely more on activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), by a not yet identified signalling pathway. The goal of the present study was to identify and characterize the mechanisms which lead to CaMKII activation and elevated Ca(2+) spark frequencies during β-AR stimulation in single cardiomyocytes in diastolic conditions. Confocal imaging revealed that β-AR stimulation increases endogenous NO production in cardiomyocytes, resulting in NO-dependent activation of CaMKII and a subsequent increase in diastolic Ca(2+) spark frequency. These changes of spark frequency could be mimicked by exposure to the NO donor GSNO and were sensitive to the CaMKII inhibitors KN-93 and AIP. In vitro, CaMKII became nitrosated and its activity remained increased independent of Ca(2+) in the presence of GSNO, as assessed with biochemical assays. β-AR stimulation of cardiomyocytes may activate CaMKII by a novel direct pathway involving NO, without requiring Ca(2+) transients. This crosstalk between two established signalling pathways may contribute to arrhythmogenic diastolic Ca(2+) release and Ca(2+) waves during adrenergic stress, particularly in combination with cardiac diseases. In addition, NO-dependent activation of CaMKII is likely to have repercussions in many cellular signalling systems and cell types.

An elevated late INa increases the diastolic SR-Ca-leak in atrial cardiomyocytes via a CaMKII-dependent mechanism

The late Na current (late INa) is elevated in heart failure (HF) and atrial fibrillation (AF) and may induce cardiac arrhythmias. Similarly, SR-Ca-leak is increased in both pathologies. The aim of the study was to determine a crosstalk between late INa, Ca/calmodulin-dependent kinase II (CaMKII) and the SR-Ca-leak in atrial myocytes. We hypothesized that an increased late INa leads to diastolic Ca-overload (via Na/Ca-exchanger, NCX), which activates CaMKII in AF and triggers CaMKII-dependent phosophorylation of ryanodine-receptor type 2 (RyR2). Furthermore, CaMKII was found to phosphorylate cardiac Na-channles thereby elevating late INa. Thus, a vitious circle could take place maintaining arrhythmogenity in AF. We isolated atrial cardiomyocytes (CM) from mice, paced them at 3 Hz for 20 beats and scanned for diastolic Ca-sparks (confocal microscopy). The low basal Ca-spark-frequency (CaSpF) of atrial CM (22±8/s/10-4 m) could be increased by the late INa enhancer ATX-II (0.5 nM) to 169±28/s/10-4m (by 668±155%, n=43 vs. 43, P<0.001). Simultaneous CaMKII-inhibition by AIP (1 μM) completely prevented the ATX-II-induced 11-fold increase of the total calculated SR-Ca-leak (n=48 vs. 43, p<0.001) suggesting a CaMKII-dependent mechanism. Late INa inhibition using ranolazine (Ran 10 μM, n=32 vs. 43) as well as the specific Na+-channel blocker tetrodotoxin (TTX, n=28 vs. 26) also normalized the total SR-Ca-leak in ATX-II-treated atrial cardiomyocytes (P=0.001). The ATX-II mediated induction of the SR-Ca2+-leak could further neither be detected when the Na+-Ca2+-exchanger (NCX) was inhibited with KBr (n=35 vs. 20) nor in CaMKIIδc-knockout mice (n=34 vs. 27). To validate these data for human AF we freshly isolated human atrial CM from patients in sinus rhythm (SR) and permanent AF. There was a strong tendency towards a higher calculated SR-Ca-leak in AF compared to SR (338.4±88.9 vs. 148.6±31.5, n =31 vs. 38, p=0.07). Indeed, superfusion of atrial CM from AF patients with Ran (10 μM) could significantly reduce the diastolic SR-Ca-leak compared to untreated control (by 78±9%, n=27 vs. 30, p<0.05). Additionally, treatment with AIP (1 μM) significantly reduced Ca-spark-frequency by 63±12% (p<0.05, n =28 vs. 41). These data suggest that an increased late INa induces diastolic SR-Ca-leak in murine and human atrial CM via CaMKII-dependent ryanodine receptor regulation. This mechanism might constitute a trigger for the induction and perpetuation of atrial arrhythmias. Ran seems to exert antiarrhythmic effects by reducing the SR-Ca-leak and is currently evaluated for antiarrhythmic therapy in patients with AF.

Enhanced Ca2+ influx through cardiac L-type Ca2+ channels maintains the systolic Ca2+ transient in early cardiac atrophy induced by mechanical unloading

Cardiac atrophy as a consequence of mechanical unloading develops following exposure to microgravity or prolonged bed rest. It also plays a central role in the reverse remodelling induced by left ventricular unloading in patients with heart failure. Surprisingly, the intracellular Ca2+ transients which are pivotal to electromechanical coupling and to cardiac plasticity were repeatedly found to remain unaffected in early cardiac atrophy. To elucidate the mechanisms underlying the preservation of the Ca2+ transients, we investigated Ca2+ cycling in cardiomyocytes from mechanically unloaded (heterotopic abdominal heart transplantation) and control (orthotopic) hearts in syngeneic Lewis rats. Following 2 weeks of unloading, sarcoplasmic reticulum (SR) Ca2+ content was reduced by ~55 %. Atrophic cardiac myocytes also showed a much lower frequency of spontaneous diastolic Ca2+ sparks and a diminished systolic Ca2+ release, even though the expression of ryanodine receptors was increased by ~30 %. In contrast, current clamp recordings revealed prolonged action potentials in endocardial as well as epicardial myocytes which were associated with a two to fourfold higher sarcolemmal Ca2+ influx under action potential clamp. In addition, Cav1.2 subunits which form the pore of L-type Ca2+ channels (LTCC) were upregulated in atrophic myocardium. These data suggest that in early cardiac atrophy induced by mechanical unloading, an augmented sarcolemmal Ca2+ influx through LTCC fully compensates for a reduced systolic SR Ca2+ release to preserve the Ca2+ transient. This interplay involves an electrophysiological remodelling as well as changes in the expression of cardiac ion channels.

Targeted ablation of the histidine-rich Ca2+-binding protein (HRC) gene is associated with abnormal SR Ca2+-cycling and severe pathology under pressure-overload stress

The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild-type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca(2+) transients, and maximal SR Ca(2+) uptake rates in the heart. However, after-contractions were developed in 50 % of HRC-KO cardiomyocytes, compared to 11 % in WT mice under stress conditions of high-frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca(2+) spontaneous SR Ca(2+) release and delayed afterdepolarizations with ISO in HRC-KO, compared to WT cells. The frequency of Ca(2+) sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca(2+) load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca(2+)-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC-null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca(2+)-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.

Adiponectin vasodilates small arteries by increasing BK activity

We have previously shown that human perivascular adipose tissue (PVAT) vasodilates adjacent arteries, in part by release of adiponectin. This study was designed to further elucidate this adipose‐coupling at a cellular level. Adiponectin caused a small vasodilation of 3rd order mesenteric arteries constricted with pressure‐induced myogenic tone. The vasodilation was substantially reduced by pre‐incubation of arteries with ryanodine (Apn: 5.5%±0.7 vs Apn&amp;Ryr: 2.2%±1, p=0.03). Confocal microscopy of Fluo‐4 loaded mesenteric arteries showed that the presence of intact PVAT increased vascular smooth muscle cell (VSMC) calcium (Ca) spark frequency in mesenteric arteries. However, there was no effect on VSMC Ca spark frequency following incubation with adiponectin. Using patch clamp of mesenteric VSMC, we found that adiponectin significantly increased the BK current when cells were incubated with ryanodine (20μM). Consistent with this, adiponectin increased amplitude but not frequency of spontaneous transient outward currents in untreated cells. Adiponectin had no effect on the Kv component of the whole cell current. It is concluded that adiponectin vasodilates small arteries predominantly by modulating VSMC BK activity, independently of Ca sparks. Intact PVAT appears to have an additive effect by increasing Ca spark frequency, suggesting the contribution of alternative adipose derived vasodilators.

Abnormal Ca2+ Spark/STOC Coupling in Cerebral Artery Smooth Muscle Cells of Obese Type 2 Diabetic Mice

Diabetes is a major risk factor for stroke. However, the molecular mechanisms involved in cerebral artery dysfunction found in the diabetic patients are not completely elucidated. In cerebral artery smooth muscle cells (CASMCs), spontaneous and local increases of intracellular Ca2+ due to the opening of ryanodine receptors (Ca2+ sparks) activate large conductance Ca2+-activated K+ (BK) channels that generate spontaneous transient outward currents (STOCs). STOCs have a key participation in the control of vascular myogenic tone and blood pressure. Our goal was to investigate whether alterations in Ca2+ spark and STOC activities, measured by confocal microscopy and patch-clamp technique, respectively, occur in isolated CASMCs of an experimental model of type-2 diabetes (db/db mouse). We found that mean Ca2+ spark amplitude, duration, size and rate-of-rise were significantly smaller in Fluo-3 loaded db/db compared to control CASMCs, with a subsequent decrease in the total amount of Ca2+ released through Ca2+ sparks in db/db CASMCs, though Ca2+ spark frequency remained. Interestingly, the frequency of large-amplitude Ca2+ sparks was also significantly reduced in db/db cells. In addition, the frequency and amplitude of STOCs were markedly reduced at all voltages tested (from −50 to 0 mV) in db/db CASMCs. The latter correlates with decreased BK channel β1/α subunit ratio found in db/db vascular tissues. Taken together, Ca2+ spark alterations lead to inappropriate BK channels activation in CASMCs of db/db mice and this condition is aggravated by the decrease in the BK β1 subunit/α subunit ratio which underlies the significant reduction of Ca2+ spark/STOC coupling in CASMCs of diabetic animals.

Remodeling of Atrial Ca Handling in Canine Model of Chronic Heart Failure Points to Non-Triggered Mechanisms of Atrial Fibrillation

Contractile dysfunction of atria and atrial arrhythmias are common comorbidities in heart failure (HF). Although abnormal Ca handling has been recognized to underlie both reduced contractility and increased propensity to arrhythmias in failing left ventricle, its role in HF-related atrial pathogenesis is not well established. We studied Ca handling in control and HF myocytes isolated from left atrial appendage using canine model of chronic heart failure. In this model, chronic HF was associated with reduced left atrial contractility, increased inducibility of atrial fibrillation, and shortened atrial action potential. Amplitude of Ca transients and cellular shortening was significantly reduced in field-stimulated myocytes from HF compared to control group. Diminished Ca transients recorded in voltage-clamped HF myocytes were associated with reduced amplitude of the L-type Ca currents and decreased gain of excitation-contraction coupling. HF myocytes displayed spatial segregation of Ca signaling manifested in impaired propagation of Ca transient from surface to the center. Frequency of Ca sparks in permeabilzed myocytes was higher in HF, while sarcoplasmic reticulum (SR) Ca content was similar in both control and HF groups. Despite of the increased functional activity of ryanodine receptors, rate of occurrence of diastolic Ca waves and corresponding delayed after depolarizations recorded in paced intact myocytes in a presence of beta-adrenergic receptor agonist isoproterenol was significantly lower in HF than in controls. We conclude that reduced atrial contractility observed in canine model of chronic HF can be attributed, at least in part, to the decreased efficacy of Ca current to trigger SR Ca release. Furthermore, our cellular data do not support Ca-dependent triggered activity in left atrial appendage as a mechanism for atrial arrhythmia in chronic HF.

TRPC3 Channels in Angiotensin II-Induced Calcium- Dependent Arrhythmias in Mouse and Human Cardiomyocytes

Angiotensin II (ATII) is associated with cardiac remodeling, heart failure and arrhythmias. In cardiomyocytes (CM), ATII signaling involves IP3- and DAG-dependent pathways and results in increased diastolic Ca release from the sarcoplasmic reticulum (SR). Recent evidence suggests that TRPC3 channels and NCX may also be regulated by ATII contributing to arrhythmogenity. We investigated the mechanisms of Ca-mediated cellular arrhythmias induced by ATII. In CM from murine or human non-failing left ventricle, [Ca] transients (Fluo4-AM) during 1 Hz, as well as SR Ca load (caffeine), SR Ca leak (Ca spark frequency,SparkF) and arrhythmic action potentials (arrAP, as non-stimulated synchronized Ca release) were visualized confocally. Action potentials (AP) were recorded in a subset of cells. ATII (100 nM,20 min), IP3-inhibitor 2-ABP, TRPC3-inhibitor Pyr3, or Na/K-pump inhibitor ouabain (100 nM) were used in parallel experiments. In mouse CM ATII increased the [Ca] transient amplitude (F/F0: 4.1±0.3 vs. 2.7±0.2 in CTRL). ATII also induced Ca sparks (SparkF (s-1∗pL-1): 277±28 vs. 48±20) and arrhAP (0.77±0.12 vs. 0.03±0.01 p<0.05) despite unchanged SR [Ca]. Interestingly, ATII induced significantly more arrhAP than ouabain at comparable SparkF. ATII increased AP duration from 53±6 to 92±34 ms (APD90; P=0.1). 2-APB significantly reduced ATII -induced Ca sparks and arrhAP. In CM matched for SparkF, ATII-induced arrAP were significantly reduced with Pyr3.ATII induced redistribution of TRPC3 to the sarcolemmal membrane. In human non-failing CM, Pyr3 also reduced ATII-induced arrhythmogeneity.Conclusion: ATII facilitates Ca-dependent arrhythmias by mechanisms beyond an increase in SR Ca leak in mouse and human CM. Sarcolemmal TRPC3-channels may modulate the arrhythmogenity of diastolic SR Ca release in the presence of ATII.

Effects of Redox Environment on Calcium Alternans in Isolated Rabbit Cardiomyocytes

Cardiac alternans is a multifactorial phenomenon linked to cardiac arrythmias. At the cellular level cardiac alternans is defined by beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (electrical or action potential duration alternans) and Ca transient amplitude (Ca alternans) at constant stimulation frequency. The aim of this project was to characterize the effect of changes in the cellular redox environment on Ca alternans in cardiac myocytes. Single myocytes (from New Zealand White rabbits) were isolated enzymatically by retrograde Langendorff perfusion. Ca alternans were induced by incrementally increasing the pacing frequency (electrical field stimulation) until stable Ca alternans occurred. The frequency at which stable Ca alternans were observed varied from cell to cell and ranged from >1 to 2.5 Hz at room temperature. Global cytosolic Ca transients were measured with Indo-1. In some experiments, cytosolic Ca alternans and intra-SR Ca alternans were simultaneously measured with the fluorescent Ca indicators Rhod-2 and Fluo-5N, respectively. Confocal microscopy was used to measure Ca sparks with Fluo-4.Reducing agents dithiothreitol and reduced glutathione partially abolished Ca and mechanical alternans by restoring diastolic Ca and Ca transient amplitudes. A decreased sarcoplasmic reticulum (SR) Ca release flux but not Ca content, together with a decreased Ca spark frequency, suggest that reducing agents normalized alternans through effects on the SR Ca release channel (ryanodine receptor type-2). Addition of a membrane permeant superoxide dismutase mimetic, Tempol, had little effect on Ca alternans, suggesting the possible role of dithiotreitol directly acting on the ryanodine receptor. These data highlight that the redox state of the cell may be important in the generation of Ca and mechanical alternans during oxidative stress.

Spatio-Temporal Properties of IP3 Receptor-Mediated Ca Release in Cardiac Myocytes

In cardiac myocytes cytosolic Ca increase and subsequent cell contraction are mainly determined by ryanodine receptor (RyR) mediated Ca release. However atrial cells are also equipped with IP3 receptors (IP3Rs), a second type of Ca release channels. We investigated IP3R-mediated Ca release events and their subcellular spatio-temporal properties in single membrane-permeabilized rabbit atrial myocytes. Local Ca release events were detected by confocal microscopy (fluo-4, longitudinal line scan mode). Local IP3R-mediated Ca release events were evoked by exposure to inositol-1,4,5-triphosphate (IP3) in the presence of tetracaine to inhibit RyR-mediated Ca spark activity, and could be inhibited by the IP3R blocker 2-APB. We classified IP3R-mediated Ca release events as subsarcolemmal, perinuclear (events 5μm) or nuclear. Nuclear events were considered membrane associated when occurring at a distance <1μm from the nuclear envelope or membranes of the nucleoplasmic reticulum. With tetracaine the Ca spark frequency decreased to 0.7±0.3 (from 8.8±1.2 in control), whereas the frequency of events detected after subsequent addition of IP3 increased to 2.9±1.3 (events/100μm/s). Subsequent exposure to 2-APB reduced the frequency to 1.8±0.8. Compared to Ca sparks, local Ca release events in the presence of IP3 were lower in amplitude, prolonged in duration and had a slower rise time. The increase in frequency of local Ca release events was particularly pronounced in the perinucler regions compared to the cytosol or the subsarcolemmal space. Furthermore, IP3-mediated Ca release events could also be detected within the nucleus. These nuclear events occurred at a higher incidence at locations that were closely associated with membrane structures of the nuclear envelope or the nucleoplasmic reticulum. In conclusion, in atrial myocytes IP3R-mediated Ca release is spatially inhomogeneous and preferentially occurs in the nuclear and perinuclear regions.

Cardiomyocyte Ca2+ handling and structure is regulated by degree and duration of mechanical load variation

Cardiac transverse (t)-tubules are altered during disease and may be regulated by stretch-sensitive molecules. The relationship between variations in the degree and duration of load and t-tubule structure remains unknown, as well as its implications for local Ca2+-induced Ca2+ release (CICR). Rat hearts were studied after 4 or 8 weeks of moderate mechanical unloading [using heterotopic abdominal heart–lung transplantation (HAHLT)] and 6 or 10 weeks of pressure overloading using thoracic aortic constriction. CICR, cell and t-tubule structure were assessed using confocal-microscopy, patch-clamping and scanning ion conductance microscopy. Moderate unloading was compared with severe unloading [using heart-only transplantation (HAHT)]. Mechanical unloading reduced cardiomyocyte volume in a time-dependent manner. Ca2+ release synchronicity was reduced at 8 weeks moderate unloading only. Ca2+ sparks increased in frequency and duration at 8 weeks of moderate unloading, which also induced t-tubule disorganization. Overloading increased cardiomyocyte volume and disrupted t-tubule morphology at 10 weeks but not 6 weeks. Moderate mechanical unloading for 4 weeks had milder effects compared with severe mechanical unloading (37% reduction in cell volume at 4 weeks compared to 56% reduction after severe mechanical unloading) and did not cause depression and delay of the Ca2+ transient, increased Ca2+ spark frequency or impaired t-tubule and cell surface structure. These data suggest that variations in chronic mechanical load influence local CICR and t-tubule structure in a time- and degree-dependent manner, and that physiological states of increased and reduced cell size, without pathological changes are possible.

Abstract 19257: TRPC3 and IP3 Regulate Angiotensin II Induced Ca2+ Dependent Arrhythmias in Mouse and Human Cardiomyocytes

Angiotensin II (ATII) is associated with cardiac remodeling, heart failure and arrhythmia. In cardiomyocytes (CM), ATII signals primarily via the Gq-coupled ATII receptor type 1 (AT1), phospholipase C signaling pathway including IP3-mediated Ca release and diacylglycerol activation of protein kinase C. Diastolic Ca release from the sarcomplasmic reticulum (SR) triggers arrhythmogenic inward current via the sarcolemmal Na-Ca-exchanger (NCX). Recent evidence suggests that TRPC3 channels and NCX may also be regulated by ATII contributing to arrhythmogenity. We investigated the mechanisms of Ca-mediated cellular arrhythmias induced by ATII. In CM from murine or human non-failing left ventricle, [Ca] transients (Fluo4-AM) during 1 Hz, as well as SR Ca load (caffeine), SR Ca leak (Ca spark frequency,SparkF) and arrhythmic (arrh) action potentials (as synchronized diastolic Ca release) during prolonged diastole were quantified in confocal images. Action potentials (AP) were recorded (current clamp) in a subset of cells. ATII (100 nM,20 min) in combination with IP3-inhibitor 2-ABP (3 µM) or TRPC3-inhibitor Pyr3 (10 µM), or ouabain (100 nM) were used in parallel experiments. In mouse CM ATII increased [Ca] transient amplitude (F/F0: 4.1±0.3 vs. 2.7±0.2 in CTRL). ATII also induced Ca sparks (SparkF (s-1*pL-1): 277±28 vs. 48±20) and arrhAP (0.77±0.12 vs. 0.03±0.01arrhAP*s-1) despite unchanged SR [Ca]. ATII induced significantly more arrhAP than ouabain (0.06±0.02) despite similarly increased SparkF (ouabain: 284±44). ATII tended to increase AP duration from 53±6 to 92±34 ms (APD90; P=0.1). 2-APB significantly reduced ATII -induced Ca sparks and arrhAP. In CM matched for SR Ca leak (SparkF), 2-APB significantly reduced - and Pyr3 abolished ATII-induced arrhAP. ATII was also arrhythmogenic in human non-failing CM (SparkF: 407±93 vs. 90±23 in CTRL;cells with arrhAP: 6/10 vs. 2/11; p&lt;0.05). Pyr3 also effectively reduced AT-II induced arrhythmogenity in human CM (SparkF: 101±21; 0/7 cells with arrhAP; both P&lt;0.05). Conclusion: ATII facilitates Ca-dependent arrhythmias by mechanisms beyond an increase in SR Ca leak in mouse and human CM. TRPC3-channels promote the arrhythmogenity of diastolic SR Ca release in the presence of ATII.

A critical role for Telethonin in regulating t-tubule structure and function in the mammalian heart

The transverse (t)-tubule system plays an essential role in healthy and diseased heart muscle, particularly in Ca(2+)-induced Ca(2+) release (CICR), and its structural disruption is an early event in heart failure. Both mechanical overload and unloading alter t-tubule structure, but the mechanisms mediating the normally tight regulation of the t-tubules in response to load variation are poorly understood. Telethonin (Tcap) is a stretch-sensitive Z-disc protein that binds to proteins in the t-tubule membrane. To assess its role in regulating t-tubule structure and function, we used Tcap knockout (KO) mice and investigated cardiomyocyte t-tubule and cell structure and CICR over time and following mechanical overload. In cardiomyocytes from 3-month-old KO (3mKO), there were isolated t-tubule defects and Ca(2+) transient dysynchrony without whole heart and cellular dysfunction. Ca(2+) spark frequency more than doubled in 3mKO. At 8 months of age (8mKO), cardiomyocytes showed progressive loss of t-tubules and remodelling of the cell surface, with prolonged and dysynchronous Ca(2+) transients. Ca(2+) spark frequency was elevated and the L-type Ca(2+) channel was depressed at 8 months only. After mechanical overload obtained by aortic banding constriction, the Ca(2+) transient was prolonged in both wild type and KO. Mechanical overload increased the Ca(2+) spark frequency in KO alone, where there was also significantly more t-tubule loss, with a greater deterioration in t-tubule regularity. In conjunction, Tcap KO showed severe loss of cell surface ultrastructure. These data suggest that Tcap is a critical, load-sensitive regulator of t-tubule structure and function.

Enhanced binding of calmodulin to the ryanodine receptor corrects contractile dysfunction in failing hearts

The channel function of the cardiac ryanodine receptor (RyR2) is modulated by calmodulin (CaM). However, the involvement of CaM in aberrant Ca(2+) release in diseased hearts remains unclear. Here, we investigated the pathogenic role of defective CaM binding to the RyR2 in the channel dysfunction associated with heart failure. The involvement of CaM in aberrant Ca(2+) release was assessed in normal and pacing-induced failing canine hearts. The apparent affinity of CaM for RyR2 was considerably lower in failing sarcoplasmic reticulum (SR) compared with normal SR. Thus, the amount of CaM bound to RyR2 was markedly decreased in failing myocytes. Expression of the CaM isoform Gly-Ser-His-CaM (GSH-CaM), which has much higher binding affinity than wild-type CaM for RyR1, restored normal CaM binding to RyR2 in both SR and myocytes of failing hearts. The Ca(2+) spark frequency (SpF) was markedly higher and the SR Ca(2+) content was lower in failing myocytes compared with normal myocytes. The incorporation of GSH-CaM into the failing myocytes corrected the aberrant SpF and SR Ca(2+) content to normal levels. Reduced CaM binding to RyR2 seems to play a critical role in the pathogenesis of aberrant Ca(2+) release in failing hearts. Correction of the reduced CaM binding to RyR2 stabilizes the RyR2 channel function and thereby restores normal Ca(2+) handling and contractile function to failing hearts.

Mechanical unloading reverses transverse tubule remodelling and normalizes local Ca2+‐induced Ca2+release in a rodent model of heart failure

AimsCa2+-induced Ca2+ release (CICR) is critical for contraction in cardiomyocytes. The transverse (t)-tubule system guarantees the proximity of the triggers for Ca2+ release [L-type Ca2+ channel, dihydropyridine receptors (DHPRs)] and the sarcoplasmic reticulum Ca2+ release channels [ryanodine receptors (RyRs)]. Transverse tubule disruption occurs early in heart failure (HF). Clinical studies of left ventricular assist devices in HF indicate that mechanical unloading induces reverse remodelling. We hypothesize that unloading of failing hearts normalizes t-tubule structure and improves CICR.Methods and resultsHeart failure was induced in Lewis rats by left coronary artery ligation for 12 weeks; sham-operated animals were used as controls. Failing hearts were mechanically unloaded for 4 weeks by heterotopic abdominal heart transplantation (HF-UN). HF reduced the t-tubule density measured by di-8-ANEPPS staining in isolated left ventricular myocytes, and this was reversed by unloading. The deterioration in the regularity of the t-tubule system in HF was also reversed in HF-UN. Scanning ion conductance microscopy showed the reappearance of normal surface striations in HF-UN. Electron microscopy revealed recovery of normal t-tubule microarchitecture in HF-UN. L-type Ca2+ current density, measured using whole-cell patch clamping, was reduced in HF but unaffected by unloading. The variance of the time-to-peak of the Ca2+ transient, an index of CICR dyssynchrony, was increased in HF and normalized by unloading. The increased Ca2+ spark frequency observed in HF was reduced in HF-UN. These results could be explained by the recoupling of orphaned RyRs in HF, as indicated by immunofluorescence.ConclusionsOur data show that mechanical unloading of the failing heart reverses the pathological remodelling of the t-tubule system and improves CICR.

Functional evidence for an active role of B-type natriuretic peptide in cardiac remodelling and pro-arrhythmogenicity

During heart failure (HF), the left ventricle (LV) releases B-type natriuretic peptide (BNP), possibly contributing to adverse cardiovascular events including ventricular arrhythmias (VAs) and LV remodelling. We investigated the cardiac effects of chronic BNP elevation in healthy mice and compared the results with a model of HF after myocardial infarction (PMI mice). Healthy mice were exposed to circulating BNP levels (BNP-Sham) similar to those measured in PMI mice. Telemetric surface electrocardiograms showed that in contrast with fibrotic PMI mice, electrical conduction was not affected in BNP-Sham mice. VAs were observed in both BNP-Sham and PMI but not in Sham mice. Analysis of heart rate variability indicated that chronic BNP infusion increased cardiac sympathetic tone. At the cellular level, BNP reduced Ca(2+) transients and impaired Ca(2+) reuptake in the sarcoplasmic reticulum, in line with blunted SR Ca(2+) ATPase 2a and S100A1 expression. BNP increased Ca(2+) spark frequency, reflecting Ca(2+) leak through ryanodine receptors, elevated diastolic Ca(2+), and promoted spontaneous Ca(2+) waves. Similar effects were observed in PMI mice. Most of these effects were reduced in BNP-Sham and PMI mice by the selective β1-adrenergic blocker metoprolol. Elevated BNP levels, by inducing sympathetic overdrive and altering Ca(2+) handling, promote adverse cardiac remodelling and VAs, which could account in part for the progression of HF after MI. The early use of β-blockers to prevent the deleterious effects of chronic BNP exposure may be beneficial in HF.

Refractoriness of sarcoplasmic reticulum Ca2+ release determines Ca2+ alternans in atrial myocytes.

Cardiac alternans is a recognized risk factor for cardiac arrhythmia and sudden cardiac death. At the cellular level, Ca(2+) alternans appears as cytosolic Ca(2+) transients of alternating amplitude at regular beating frequency. Cardiac alternans is a multifactorial process but has been linked to disturbances in intracellular Ca(2+) regulation. In atrial myocytes, we tested the role of voltage-gated Ca(2+) current, sarcoplasmic reticulum (SR) Ca(2+) load, and restitution properties of SR Ca(2+) release for the occurrence of pacing-induced Ca(2+) alternans. Voltage-clamp experiments revealed that peak Ca(2+) current was not affected during alternans, and alternans of end-diastolic SR Ca(2+) load, evaluated by application of caffeine or measured directly with an intra-SR fluorescent Ca(2+) indicator (fluo-5N), were not a requirement for cytosolic Ca(2+) alternans. Restitution properties and kinetics of refractoriness of Ca(2+) release after activation during alternans were evaluated by four different approaches: measurements of 1) the delay (latency) of occurrence of spontaneous global Ca(2+) releases and 2) Ca(2+) spark frequency, both during rest after a large and small alternans Ca(2+) transient; 3) the magnitude of premature action potential-induced Ca(2+) transients after a large and small beat; and 4) the efficacy of a photolytically induced Ca(2+) signal (Ca(2+) uncaging from DM-nitrophen) to trigger additional Ca(2+) release during alternans. The results showed that the latency of global spontaneous Ca(2+) release was prolonged and Ca(2+) spark frequency was decreased after the large Ca(2+) transient during alternans. Furthermore, the restitution curve of the Ca(2+) transient elicited by premature action potentials or by photolysis-induced Ca(2+) release from the SR lagged behind after a large-amplitude transient during alternans compared with the small-amplitude transient. The data demonstrate that beat-to-beat alternation of the time-dependent restitution properties and refractory kinetics of the SR Ca(2+) release mechanism represents a key mechanism underlying cardiac alternans.

Cardiac FKBP12.6 overexpression protects against triggered ventricular tachycardia in pressure overloaded mouse hearts

Alterations in RyR2 function have been proposed as a major pathophysiological mechanism of arrhythmias and heart failure (HF). Cardiac FKBP12.6 overexpression protects against myocardial infarction-induced HF and catecholamine-promoted ventricular arrhythmias. We tested the hypothesis that FKBP12.6 overexpression protects against maladaptive LVH and triggered ventricular arrhythmias following transverse aorta constriction (TAC) in the mouse. The TAC-associated mortality rate was significantly lower in male transgenic (DT) than in Ctr mice (p<0.05). TAC-associated maladaptive hypertrophy was blunted in DT mice especially 1month post-TAC and their SERCA2a/PLB ratio remained unchanged 1 and 2months post-TAC. Two months after TAC, trains of 30 stimuli (burst pacing) performed following isoproterenol injection (0.2mg/kg, ip), induced VT in 50% of the TAC-Ctr and in none of the TAC-DT mice (p=0.022). The increase in myocyte shortening and Ca(2+) spark frequency observed in sham-operated Ctr mice in response to 50 nM isoproterenol was reduced in DT mice, and abolished in TAC-DT mice. NCX1 function was reduced in Sham-DT and TAC-DT compared with Sham-Ctr and TAC-Ctr mice, respectively (p<0.05 for the 2 comparisons). In mice killed after isoproterenol injection and burst pacing, RyR2 S2814 phosphorylation was decreased by 50% in TAC-DT versus TAC-Ctr mice (p<0.05), with no change in RyR2 S2808 and PLB S16 and T17 phosphorylation. Cardiac FKBP12.6 overexpression in the mouse blunts pressure overload-induced maladaptive LV remodelling and protects against catecholamine-promoted burst pacing-induced ventricular tachycardia by decreasing cardiac sensitivity to adrenergic stress and RyR2 S2814 phosphorylation, and decreasing NCX1 activity.

Effects of Cytosolic Ca Buffering on Sarcoplasmic Reticulum Ca Leak in Permeabilized Rabbit Ventricular Myocytes

In this study we tested the hypothesis that local Ca-induced Ca release (CICR) within ryanodine receptor (RyR2) release clusters plays an important role in defining spark-mediated sarcoplasmic reticulum (SR) Ca leak. We studied to what degree changes of cytosolic Ca buffer capacity affect single RyR activity in lipid bilayers, Ca spark properties, and SR Ca leak in permeabilized ventricular myocytes. No substantial changes in single RyR2 function were observed if BAPTA (a fast Ca buffer) was added (holding free Ca constant) to the EGTA-buffered cytosolic solution. However, the same cytosolic BAPTA addition almost completely eliminated Ca sparks in cells. The elimination of Ca sparks by BAPTA was not due to a decrease in SR Ca load. The addition of the fast cytosolic Ca buffer significantly decreased SR Ca leak, particularly at high [Ca]SR (>400 μM) where Ca sparks significantly contribute to SR Ca leak. In contrast, lowering cytosolic Ca buffer capacity (by decreasing EGTA from 0.36 to 0.1 mM; keeping free Ca constant) drastically increased Ca spark frequency and width, leading to generation of multifocal (or propagating) Ca sparks. This decrease in cytosolic Ca buffer capacity also significantly accelerated SR Ca leak as a result of augmentation of Ca spark-mediated component of SR Ca leak. In conclusion, by changing cytosolic Ca buffering (capacity or affinity) we were able to experimentally separate Ca spark- and non-spark- mediated component of the overall SR Ca leak in ventricular myocytes. These data also suggest that changes in the dynamics of local cytosolic Ca handling (buffering, extrusion, etc.) are physiologically important in determining the degree of spark and non-spark mediated SR Ca leak.

Differential impact of mitochondrial positioning on mitochondrial Ca(2+) uptake and Ca(2+) spark suppression in skeletal muscle.

Muscle contraction requires ATP and Ca(2+) and, thus, is under direct control of mitochondria and the sarcoplasmic reticulum. During postnatal skeletal muscle maturation, the mitochondrial network exhibits a shift from a longitudinal ("longitudinal mitochondria") to a mostly transversal orientation as a result of a progressive increase in mitochondrial association with Ca(2+) release units (CRUs) or triads ("triadic mitochondria"). To determine the physiological implications of this shift in mitochondrial disposition, we used confocal microscopy to monitor activity-dependent changes in myoplasmic (fluo 4) and mitochondrial (rhod 2) Ca(2+) in single flexor digitorum brevis (FDB) fibers from 1- to 4-mo-old mice. A robust and sustained Ca(2+) accumulation in triadic mitochondria was triggered by repetitive tetanic stimulation (500 ms, 100 Hz, every 2.5 s) in FDB fibers from 4-mo-old mice. Specifically, mitochondrial rhod 2 fluorescence increased 272 ± 39% after a single tetanus and 412 ± 45% after five tetani and decayed slowly over 10 min following the final tetanus. Similar results were observed in fibers expressing mitochondrial pericam, a mitochondrial-targeted ratiometric Ca(2+) indicator. Interestingly, sustained mitochondrial Ca(2+) uptake following repetitive tetanic stimulation was similar for triadic and longitudinal mitochondria in FDB fibers from 1-mo-old mice, and both mitochondrial populations were found by electron microscopy to be continuous and structurally tethered to the sarcoplasmic reticulum. Conversely, the frequency of osmotic shock-induced Ca(2+) sparks per CRU density decreased threefold (from 3.6 ± 0.2 to 1.2 ± 0.1 events·CRU(-1)·min(-1)·100 μm(-2)) during postnatal development in direct linear correspondence (r(2) = 0.95) to an increase in mitochondrion-CRU pairing. Together, these results indicate that mitochondrion-CRU association promotes Ca(2+) spark suppression but does not significantly impact mitochondrial Ca(2+) uptake.