SERCA Activity Controls the Systolic Calcium Increase in the Nucleus of Cardiac Myocytes

In cardiomyocytes, nuclear calcium is involved in regulation of transcription and, thus, remodeling. The cellular mechanisms regulating nuclear calcium, however, remain elusive. Therefore, the aim of this study was to identify and characterize the factors that regulate nuclear calcium in cardiomyocytes. We focused on the roles of (1) the cytoplasmic calcium transient (CaT), (2) the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and (3) intracellular calcium stores for nuclear calcium handling. Experiments were performed on rat ventricular myocytes loaded with Fluo-4/AM. Subcellularly resolved CaTs were visualized using confocal microscopy. The cytoplasmic CaT was varied by reducing extracellular calcium (from 1.5 to 0.3 mM) or by exposure to isoprenaline (ISO, 10 nM). SERCA was blocked by thapsigargin (5 μM). There was a strict linear dependence of the nucleoplasmic CaT on the cytoplasmic CaT over a wide range of calcium concentrations. Increasing SERCA activity impaired, whereas decreasing SERCA activity augmented the systolic calcium increase in the nucleus. Perinuclear calcium store load, on the other hand, did not change with either 0.3 mM calcium or ISO and was not a decisive factor for the nucleoplasmic CaT. The results indicate, that the nucleoplasmic CaT is determined largely by the cytoplasmic CaT via diffusion of calcium through nuclear pores. They identify perinuclear SERCA activity, which limits the systolic calcium increase in the nucleus, as a novel regulator of the nuclear CaT in cardiac myocytes

Na/K Pump-Induced [Na]i Gradients in Rat Ventricular Myocytes Measured with Two-Photon Microscopy

AbstractVia the Na/Ca and Na/H exchange, intracellular Na concentration ([Na]i) is important in regulating cardiac Ca and contractility. Functional data suggest that [Na]i might be heterogeneous in myocytes that are not in steady state, but little direct spatial information is available. Here we used two-photon microscopy of SBFI to spatially resolve [Na]i in rat ventricular myocytes. In vivo calibration yielded an apparent Kd of 27±2mM Na. Similar resting [Na]i was found using two-photon or single-photon ratiometric measurements with SBFI (10.8±0.7 vs. 11.1±0.7mM). To assess longitudinal [Na]i gradients, Na/K pumps were blocked at one end of the myocyte (locally pipette-applied K-free extracellular solution) and active in the rest of the cell. This led to a marked increase in [Na]i at sites downstream of the pipette (where Na enters the myocyte and Na/K pumps are blocked). [Na]i rise was smaller at upstream sites. This resulted in sustained [Na]i gradients (up to ∼17 mM/120μm cell length). This implies that Na diffusion in cardiac myocytes is slow with respect to trans-sarcolemmal Na transport rates, although the mechanisms responsible are unclear. A simple diffusion model indicated that such gradients require a Na diffusion coefficient of 10–12μm2/s, significantly lower than in aqueous solutions

The European Network for Translational Research in Atrial Fibrillation (EUTRAF): objectives and initial results

Atrial fibrillation (AF) is the most common sustained arrhythmia in the general population. As an age-related arrhythmia AF is becoming a huge socio-economic burden for European healthcare systems. Despite significant progress in our understanding of the pathophysiology of AF, therapeutic strategies for AF have not changed substantially and the major challenges in the management of AF are still unmet. This lack of progress may be related to the multifactorial pathogenesis of atrial remodelling and AF that hampers the identification of causative pathophysiological alterations in individual patients. Also, again new mechanisms have been identified and the relative contribution of these mechanisms still has to be established. In November 2010, the European Union launched the large collaborative project EUTRAF (European Network of Translational Research in Atrial Fibrillation) to address these challenges. The main aims of EUTRAF are to study the main mechanisms of initiation and perpetuation of AF, to identify the molecular alterations underlying atrial remodelling, to develop markers allowing to monitor this processes, and suggest strategies to treat AF based on insights in newly defined disease mechanisms. This article reports on the objectives, the structure, and initial results of this networ

Left Atrial Myocardium in Arterial Hypertension

Arterial hypertension affects ≈ 1 billion people worldwide. It is associated with increased morbidity and mortality and responsible for millions of deaths each year. Hypertension mediates damage of target organs including the heart. In addition to eliciting left ventricular hypertrophy, dysfunction and heart failure, hypertension also causes left atrial remodeling that may culminate in atrial contractile dysfunction and atrial fibrillation. Here, we will summarize data on the various aspects of left atrial remodeling in (essential) hypertension gathered from studies on patients with hypertension and from spontaneously hypertensive rats, an animal model that closely mimics cardiac remodeling in human hypertension. Analyzing the timeline of remodeling processes, i.e., distinguishing between alterations occurring in prehypertension, in early hypertension and during advanced hypertensive heart disease, we will derive the potential mechanisms underlying left atrial remodeling in (essential) hypertension. Finally, we will discuss the consequences of these remodeling processes for atrial and ventricular function. The data imply that left atrial remodeling is multifactorial, starts early in hypertension and is an important contributor to the progression of hypertensive heart disease, including the development of atrial fibrillation and heart failure

Atrial remodelling in hypertensive heart disease: role of Na+ homeostasis and contractility

Arterial hypertension causes hypertensive heart disease. Constant mechanical stress and activation of neurohormonal systems cause structural and functional changes in the myocardium termed “remodelling”. Remodelling is beneficial in the beginning of the disease development; however, with time it becomes detrimental and impairs cardiac function. Remodelling of the myocardium occurs in hypertension, atrial fibrillation and heart failure. These cardiac diseases are tightly linked by the mechanisms of pathological remodelling and induce development and maintenance of one another. Ventricular remodelling has been studied intensively in hypertensive heart disease, however, atrial remodelling has been studied much less and is only poorly understood. Physiology of cardiac myocytes relies on balanced intracellular Na+ homeostasis. Na+ is involved in many cellular processes, such as action potential initiation, Ca2+ homeostasis, intracellular pH, metabolism and contractility. In the first part of the thesis I investigated ionic (Na+ homeostasis) and functional (contractility) atrial remodelling in an animal model of hypertensive heart disease – spontaneously hypertensive rats (SHR). In early hypertension, SHR exhibited elevated blood pressure and isolated left ventricular hypertrophy. The atria were not hypertrophied. Contractility of atrial myocytes and intracellular Na+ concentration ([Na+]i) were both unaltered. Expression of most Na+-handling proteins was unaffected in the atria of SHR. In advanced hypertension, SHR exhibited further progression of left ventricular hypertrophy and signs of heart failure. Left atria were hypertrophied. The contractility of atrial myocytes was reduced. [Na+]i was significantly decreased together with increased expression of the α 1 subunit of Na+/K+-ATPase. Expression of Na+/H+-exchanger was increased, suggesting activation of pro-hypertrophic pathways. Comparison of SHR with and without signs of heart failure (i.e. increased lung weight) revealed development of right ventricular hypertrophy and progression of bi-atrial hypertrophy in SHR with heart failure. Moreover, the impairment of atrial myocyte contractility progressed. However, [Na+]i and the expression of major Na+-handling proteins were not changed during the transition to heart failure. In addition to studies on atrial myocytes, we performed measurements of [Na+]i and contractility of ventricular myocytes from old SHR. In contrast to our findings in the atria, no impairment of contractility or changes in [Na+]i were observed in the ventricular myocytes, indicating atria-specific remodelling. Taken together, the presented results indicate that in early hypertension no significant signs of atrial remodelling in terms of contractility and Na+ homeostasis were found. However, in advanced hypertensive heart disease there was atria-specific functional atrial remodelling, which might contribute to the transition from compensated left ventricular hypertrophy to heart failure. Atrial ionic remodelling is an important factor in the development and maintenance of atrial fibrillation. The role of intracellular Na+ homeostasis in these processes is not understood. In the second part of the thesis, I investigated expression of Na+-handling proteins in right atrial tissue of patients suffering from paroxysmal and chronic atrial fibrillation compared to patients with sinus rhythm. The results indicated that the expression of Na+-handling proteins, including Na+ channels, Na+/H+ exchanger, alpha subunits of Na+/K+-ATPase, phospholemman, was not altered in either paroxysmal or chronic atrial fibrillation. The expression of β 1 subunit of Na+/K+-ATPase was significantly reduced in chronic atrial fibrillation. However, the functional consequences of this change require further investigation. Endothelin-1 plays an important role in the regulation of blood pressure and cardiac physiology. Enhancement of endothelin-1 system activity contributes to cardiac maladaptive remodelling, including disturbances in Ca2+ and Na+ homeostasis in cardiac myocytes. At the age of 7 months, SHR exhibit enhanced endothelin-1 signalling and altered Ca2+ handling. Therefore, in the third part of the thesis we investigated the effect of endothelin-1 receptor blockage on blood pressure and expression and phosphorylation of Ca2+-handling proteins, as well as the expression of proteins involved in endothelin-1 signalling in the atria of SHR. The results revealed that the blockage of endothelin receptors by 8 weeks treatment with macitentan (novel dual endothelin A and endothelin B receptor antagonist) did not lower blood pressure in SHR. Expression and phosphorylation of major Ca2+-handling proteins and endothelin-1 signalling proteins were both unaffected. Thus, the blockage of endothelin receptors did not cause any major changes in atrial Ca2+ remodelling in SH

Store-Operated Calcium Entry Increases Nuclear Calcium in Adult Rat Atrial and Ventricular Cardiomyocytes

Store-operated calcium entry (SOCE) in cardiomyocytes may be involved in cardiac remodeling, but the underlying mechanisms remain elusive. We hypothesized that SOCE may increase nuclear calcium, which alters gene expression via calcium/calmodulin-dependent enzyme signaling, and elucidated the underlying cellular mechanisms. An experimental protocol was established in isolated adult rat cardiomyocytes to elicit SOCE by re-addition of calcium following complete depletion of sarcoplasmic reticulum (SR) calcium and to quantify SOCE in relation to the electrically stimulated calcium transient (CaT) measured in the same cell before SR depletion. Using confocal imaging, calcium changes were recorded simultaneously in the cytosol and in the nucleus of the cell. In ventricular myocytes, SOCE was observed in the cytosol and nucleus amounting to ≈15% and ≈25% of the respective CaT. There was a linear correlation between the SOCE-mediated calcium increase in the cytosol and nucleus. Inhibitors of TRPC or Orai channels reduced SOCE by ≈33–67%, whereas detubulation did not. In atrial myocytes, SOCE with similar characteristics was observed in the cytosol and nucleus. However, the SOCE amplitudes in atrial myocytes were ≈two-fold larger than in ventricular myocytes, and this was associated with ≈1.4- to 3.6-fold larger expression of putative SOCE proteins (TRPC1, 3, 6, and STIM1) in atrial tissue. The results indicated that SOCE in atrial and ventricular myocytes is able to cause robust calcium increases in the nucleus and that both TRPC and Orai channels may contribute to SOCE in adult cardiomyocytes

Alterations of nuclear Ca2+ regulation in ventricular myocytes during development and progression of hypertensive heart disease

Hypertension is the leading risk factor for developing hypertrophy and heart failure. Pathological changes in myocardial structure and function caused by hypertension are termed ’’hypertensive heart disease’’. In response to elevated mechanical stress in hypertension and a special neurohormonal environment (catecholamines, angiotensin II, endothelin-1), the heart grows as a means of increasing pump function and decreasing wall tension thus inducing a state of ’’compensated hypertrophy’’. Initially, hypertrophy is beneficial, but chronic activation of neurohormonal mediators and altered Ca2+ signaling ultimately lead to maladaptive alterations in gene expression and progressive cardiac remodeling, which eventually becomes detrimental and impairs cardiac function. Impairment of Ca2+ handling is critically implicated in the pathophysiology of hypertrophy and heart failure. Two important Ca2+-dependent signaling pathways involved in cardiac remodeling are the Ca2+/calmodulin-dependent protein phosphatase calcineurin (CaN)–NFAT–GATA4/6 and the Ca2+/calmodulin-dependent protein kinase II (CaMKII)–HDAC–MEF2 pathway. The mechanisms by which the cardiomyocytes distinguish between Ca2+ involved in excitation-contraction coupling in the cytoplasm and transcriptional regulation in the nucleus are poorly understood. While alterations in cytoplasmic Ca2+ regulation have been clearly implicated in the pathophysiology of hypertrophy and heart failure, nucleoplasmic Ca2+ signaling has been studied much less. Increases in nucleoplasmic [Ca2+] may activate Ca2+-dependent enzymes and transcription factors modulating gene expression and may be critical for the pathogenesis of hypertrophy and heart failure. However, there is lack of studies dealing with the regulation and alterations of nucleoplasmic Ca2+ handling under pathophysiological conditions. We used spontaneously hypertensive rats (SHR) to investigate potential alterations in nuclear Ca2+ handling in response to hypertension. During the course of hypertensive heart disease, very little is known about the onset and progression of hypertrophy and heart failure. We hypothesized that maladaptive remodeling of nuclear structure and nuclear Ca2+ signaling might occur early in hypertension triggering initiation and progression of hypertrophy, and in advanced hypertension contributing to the transition from compensated hypertrophy to heart failure. Therefore, in the first part of the thesis, we studied early hypertension-induced structural and functional (Ca2+ handling) remodeling of left ventricular (LV) myocytes and nuclei. We found that LV myocytes and nuclei from early hypertensive SHR (≈3 months of age) were already in the stage of compensatory hypertrophy. Cytoplasmic and nucleoplasmic [Ca2+]i transients (CaTs) were enlarged in SHR. The increase in nucleoplasmic Ca2+ exceeded the increase in cytoplasmic Ca2+, suggesting enhanced nuclear Ca2+ signaling in SHR. Ca2+ load of SR and perinuclear Ca2+ stores was also enlarged in SHR, while fractional release from both stores was unaltered. Intranuclear Ca2+ propagation was faster in SHR, associated with preserved density of nuclear envelope invaginations and elevated nuclear expression of nucleoporins and SR-Ca2+-ATPase. Increased nucleoplasmic Ca2+ signaling was associated with activation of the CaMKIIδ–HDAC5 pathway and increased histone acetylation, suggesting increased gene transcription. The observed remodeling of nuclear Ca2+ handling might represent an early event in hypertension that contributes to initiation and progression of pathological hypertrophy in hypertensive heart disease. In the second part of the thesis, we studied structural and functional nuclear remodeling in advanced hypertension. LV myocytes and nuclei from old SHR (15-25 months of age) were larger compared to young hypertrophic SHR, suggesting further growth of the ventricle with progression of cardiac disease. Cytoplasmic and nucleoplasmic CaTs were augmented (as in young hypertrophic SHR). SR and perinuclear Ca2+ load was increased (as in young hypertrophic SHR). There were profound alterations in the kinetics of both cytoplasmic and nucleoplasmic CaTs. Development of heart failure in old SHR was associated with increased density of nuclear envelope invaginations, faster intranuclear Ca2+ propagation, acceleration of both rise time and tau of decay of nuclear CaTs and alterations in the cytoplasmic protein levels of major Ca2+-regulating proteins. Thus, we identified distinct alterations in nuclear structure and Ca2+ handling during development of heart failure in SHR, providing new insights into mechanisms of nuclear Ca2+ regulation under pathophysiological conditions. Normalization of nucleoplasmic Ca2+ handling may represent a novel target for the treatment of hypertrophy and heart failure in hypertensive heart disease

Comparison of ouabain-sensitive and -insensitive Na/K pumps in HEK293 cells

AbstractThe Na/K pump current Ip of single HEK293 cells either untransfected (endogenous Ip) or transfected with the α1 subunit of the rat Na/K pump (exogenous Ip) was investigated in Na-containing solution by means of whole-cell recording at 30°C. The endogenous Ip was irreversibly blocked by 10−4 M ouabain or 2·10−4 M dihydro-ouabain (DHO). Its density amounted to 0.33 pA pF−1 at 0 mV and 5.4 mM Ko. It was half maximally activated at 1.5 mM Ko and increased linearly with depolarization over the entire voltage range studied (−80 to +60 mV). In contrast, HEK293 cells stably transfected with cDNA for the cardiac glycoside-resistant α1 subunit of the rat Na/K pump showed an Ip in the presence of 10−4 M ouabain and 2·10−4 M DHO, respectively. This exogenous Ip was reversibly blocked by 10−2 M ouabain. Half maximal activation of the exogenous Ip occurred at 1.7 mM Ko. Its amplitude increased linearly with depolarization at negative voltages but remained almost constant at positive membrane potentials. Comparison with the Ip of isolated rat cardiac ventricular myocytes strongly suggests that the exogenous Ip in HEK293 cells is generated by the α1 subunit of the rat Na/K pump since it displays identical properties. Therefore, HEK293 cells represent an expression system well suited for the electrophysiological analysis of recombinant, cardiac glycoside-resistant Na/K pumps by means of whole-cell recording

Activation of the cAMP–protein kinase A pathway facilitates Na+ translocation by the Na+–K+ pump in guinea-pig ventricular myocytes

The effects of the adenylyl cyclase activator forskolin on steady-state and transient currents generated by the Na+-K+ pump were studied in guinea-pig ventricular myocytes by means of whole-cell voltage clamp at 30 °C.In external solution containing 144 mM Na+ (Nao+) and 10 mM K+ (Ko+), steady-state Na+-K+ pump current (Ip) activated by 5 mM pipette Na+ ( Napip+) at -20 mV was reversibly augmented by forskolin (4 μM) to 133 ± 4 % of the control current (n = 15). The forskolin analogue 1,9-dideoxyforskolin (10 μM), which does not activate adenylyl cyclases, did not increase Ip (n = 2). Application of the protein kinase A (PKA) inhibitor H-89 (10 μM) in the continued presence of forskolin reversed the forskolin-induced elevation of Ip (n = 3).The forskolin effect on Ip persisted in the presence of 50 mM Napip+ which ensured that the internal Na+-binding sites of the Na+-K+ pump were nearly saturated. Under these conditions, the drug increased Ip to 142 ± 3 % of the control Ip when the pipette free Ca2+ concentration ([Ca2+]pip) was 0·013 nM (n = 5) and to 138 ± 4 % of the control Ip when free [Ca2+]pip was 15 nM (n = 9).In Na+-free external solution, Ip activated by 50 mM Napip+ and 1·5 mM Ko+ was likewise increased by forskolin but to a lesser extent than in Na+-containing medium (116 ± 3 % of control, n = 10).In order to investigate exclusively partial reactions in the Na+ limb of the pump cycle, transient pump currents under conditions of electroneutral Na+-Na+ exchange were studied. Transient pump currents elicited by voltage jumps displayed an initial peak and then decayed monoexponentially. Moved charge (Q) and the rate constant of current decay varied with membrane potential (V). The Q-V relationship followed a Boltzmann distribution characterized by the midpoint voltage (V0·5) and the maximum amount of movable charge (ΔQmax). Forskolin (2-10 μM) shifted V0·5 to more negative values while ΔQmax was not affected (n = 11). The effects of forskolin on transient pump currents were mimicked by 8-bromo-cAMP (500 μM; n = 2) and abolished by a peptide inhibitor of PKA (PKI, 10 μM; n = 5).We conclude that activation of the cAMP-PKA pathway in guinea-pig ventricular myocytes increases Na+-K+ pump current at least in part by modulating partial reactions in the Na+ limb of the pump cycle. Under physiological conditions, the observed stimulation of the cardiac Na+-K+ pump may serve to shorten the action potential duration and to counteract the increased passive sarcolemmal Na+ and K+ fluxes during sympathetic stimulation of the heart

Die Regulation des nucleären Kalziumtransienten in ventrikulären Kardiomyozyten

Zusammengefasst stellt die Arbeit das cytoplasmatische Ca als Hauptdeterminante des nucleären Ca-Anstiegs heraus und liefert den Nachweis, dass die SERCA-Funktion die passive Ca-Propagation vom Cytoplasma in den Kern direkt moduliert. Darüber hinaus spielt die perinucleäre Ca-Beladung unter physiologischen Bedingungen nur eine untergeordnete Rolle bei der Regulation des nucleären Ca. Schließlich konnten die wichtigsten Ergebnisse aus den Studien an WKY-Ratten in SHR reproduziert werden, was die Befunde bestätigt und auf ihre Übertragbarkeit und Validität hindeutet