Fredrik Swift

Altered Na+/Ca2+-exchanger activity due to downregulation of Na+/K+-ATPase α2-isoform in heart failure

AIMS The Na+/K+-ATPase (NKA) alpha2-isoform is preferentially located in the t-tubules of cardiomyocytes and is functionally coupled to the Na+/Ca(+-exchanger (NCX) and Ca2+ regulation through intracellular Na+ concentration ([Na+]i). We hypothesized that downregulation of the NKA alpha2-isoform during congestive heart failure (CHF) disturbs the link between Na+ and Ca2+, and thus the control of cardiomyocyte contraction. METHODS AND RESULTS NKA isoform and t-tubule distributions were studied using immunocytochemistry, confocal and electron microscopy in a post-infarction rat model of CHF. Sham-operated rats served as controls. NKA and NCX currents (I NKA and I NCX) were measured and alpha2-isoform current (I NKA,alpha2) was separated from total I NKA using 0.3 microM ouabain. Detubulation of cardiomyocytes was performed to assess the presence of alpha2-isoforms in the t-tubules. In CHF, the t-tubule network had a disorganized appearance in both isolated cardiomyocytes and fixed tissue. This was associated with altered expression patterns of NKA alpha1- and alpha2-isoforms. I NKA,alpha2 density was reduced by 78% in CHF, in agreement with decreased protein expression (74%). When I NKA,alpha2 was blocked in Sham cardiomyocytes, contractile parameters converged with those observed in CHF. In Sham, abrupt activation of I NKA led to a decrease in I NCX, presumably due to local depletion of [Na+]i in the vicinity of NCX. This decrease was smaller when the alpha2-isoform was downregulated (CHF) or inhibited (ouabain), indicating that the alpha2-isoform is necessary to modulate local [Na+]i close to NCX. CONCLUSION Downregulation of the alpha2-isoform causes attenuated control of NCX activity in CHF, reducing its capability to extrude Ca2+ from cardiomyocytes.

Sodium accumulation promotes diastolic dysfunction in end-stage heart failure following Serca2 knockout

Alterations in trans‐sarcolemmal and sarcoplasmic reticulum (SR) Ca2+ fluxes may contribute to impaired cardiomyocyte contraction and relaxation in heart failure. We investigated the mechanisms underlying heart failure progression in mice with conditional, cardiomyocyte‐specific excision of the SR Ca2+‐ATPase (SERCA) gene. At 4 weeks following gene deletion (4‐week KO) cardiac function remained near normal values. However, end‐stage heart failure developed by 7 weeks (7‐week KO) as systolic and diastolic performance declined. Contractions in isolated myocytes were reduced between 4‐ and 7‐week KO, and relaxation was slowed. Ca2+ transients were similarly altered. Reduction in Ca2+ transient magnitude resulted from complete loss of SR Ca2+ release between 4‐ and 7‐week KO, due to loss of a small remaining pool of SERCA2. Declining SR Ca2+ release was partly offset by increased L‐type Ca2+ current, which was facilitated by AP prolongation in 7‐week KO. Ca2+ entry via reverse‐mode Na+–Ca2+ exchange (NCX) was also enhanced. Up‐regulation of NCX and plasma membrane Ca2+‐ATPase increased Ca2+ extrusion rates in 4‐week KO. Diastolic dysfunction in 7‐week KO resulted from further SERCA2 loss, but also impaired NCX‐mediated Ca2+ extrusion following Na+ accumulation. Reduced Na+‐K+‐ATPase activity contributed to the Na+ gain. Normalizing [Na+] by dialysis increased the Ca2+ decline rate in 7‐week KO beyond 4‐week values. Thus, while SERCA2 loss promotes both systolic and diastolic dysfunction, Na+ accumulation additionally impairs relaxation in this model. Our observations indicate that if cytosolic Na+ gain is prevented, up‐regulated Ca2+ extrusion mechanisms can maintain near‐normal diastolic function in the absence of SERCA2.

There Goes the Neighborhood: Pathological Alterations in T-Tubule Morphology and Consequences for Cardiomyocyte Ca2+ Handling

T-tubules are invaginations of the cardiomyocyte membrane into the cell interior which form a tortuous network. T-tubules provide proximity between the electrically excitable cell membrane and the sarcoplasmic reticulum, the main intracellular Ca2+ store. Tight coupling between the rapidly spreading action potential and Ca2+ release units in the SR membrane ensures synchronous Ca2+ release throughout the cardiomyocyte. This is a requirement for rapid and powerful contraction. In recent years, it has become clear that T-tubule structure and composition are altered in several pathological states which may importantly contribute to contractile defects in these conditions. In this review, we describe the “neighborhood” of proteins in the dyadic cleft which locally controls cardiomyocyte Ca2+ homeostasis and how alterations in T-tubule structure and composition may alter this neighborhood during heart failure, atrial fibrillation, and diabetic cardiomyopathy. Based on this evidence, we propose that T-tubules have the potential to serve as novel therapeutic targets.

Mutual antagonism between IP3RII and miRNA-133a regulates calcium signals and cardiac hypertrophy

IP3RII-induced calcium release decreases miR-133a expression, which further increases IP3RII levels and calcium release and thereby promotes hypertrophic heart remodeling.

Dual Serotonergic Regulation of Ventricular Contractile Force Through 5-HT2A and 5-HT4 Receptors Induced in the Acute Failing Heart

Cardiac responsiveness to neurohumoral stimulation is altered in congestive heart failure (CHF). In chronic CHF, the left ventricle has become sensitive to serotonin because of appearance of Gs-coupled 5-HT4 receptors. Whether this also occurs in acute CHF is unknown. Serotonin responsiveness may develop gradually or represent an early response to the insult. Furthermore, serotonin receptor expression could vary with progression of the disease. Postinfarction CHF was induced in male Wistar rats by coronary artery ligation with nonligated sham-operated rats as control. Contractility was measured in left ventricular papillary muscles and mRNA quantified by real-time reverse-transcription PCR. Myosin light chain-2 phosphorylation was determined by charged gel electrophoresis and Western blotting. Ca2+ transients in CHF were measured in field stimulated fluo-4-loaded cardiomyocytes. A novel 5-HT2A receptor-mediated inotropic response was detected in acute failing ventricle, accompanied by increased 5-HT2A mRNA levels. Functionally, this receptor dominated over 5-HT4 receptors that were also induced. The 5-HT2A receptor-mediated inotropic response displayed a triphasic pattern, shaped by temporally different activation of Ca2+-calmodulin-dependent myosin light chain kinase, Rho-associated kinase and inhibitory protein kinase C, and was accompanied by increased myosin light chain-2 phosphorylation. Ca2+ transients were slightly decreased by 5-HT2A stimulation. The acute failing rat ventricle is, thus, dually regulated by serotonin through Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT4 receptors.

Extreme sarcoplasmic reticulum volume loss and compensatory T-tubule remodeling after Serca2 knockout

Cardiomyocyte contraction and relaxation are controlled by Ca2+ handling, which can be regulated to meet demand. Indeed, major reduction in sarcoplasmic reticulum (SR) function in mice with Serca2 knockout (KO) is compensated by enhanced plasmalemmal Ca2+ fluxes. Here we investigate whether altered Ca2+ fluxes are facilitated by reorganization of cardiomyocyte ultrastructure. Hearts were fixed for electron microscopy and enzymatically dissociated for confocal microscopy and electrophysiology. SR relative surface area and volume densities were reduced by 63% and 76%, indicating marked loss and collapse of the free SR in KO. Although overall cardiomyocyte dimensions were unaltered, total surface area was increased. This resulted from increased T-tubule density, as revealed by confocal images. Fourier analysis indicated a maintained organization of transverse T-tubules but an increased presence of longitudinal T-tubules. This demonstrates a remarkable plasticity of the tubular system in the adult myocardium. Immunocytochemical data showed that the newly grown longitudinal T-tubules contained Na+/Ca2+-exchanger proximal to ryanodine receptors in the SR but did not contain Ca2+-channels. Ca2+ measurements demonstrated a switch from SR-driven to Ca2+ influx-driven Ca2+ transients in KO. Still, SR Ca2+ release constituted 20% of the Ca2+ transient in KO. Mathematical modeling suggested that Ca2+ influx via Na+/Ca2+-exchange in longitudinal T-tubules triggers release from apposing ryanodine receptors in KO, partially compensating for reduced SERCA by allowing for local Ca2+ release near the myofilaments. T-tubule proliferation occurs without loss of the original ordered transverse orientation and thus constitutes the basis for compensation of the declining SR function without structural disarrangement.

Hypokalaemia induces Ca2+ overload and Ca2+ waves in ventricular myocytes by reducing Na+,K+-ATPase α2 activity.

Hypokalaemia is a risk factor for development of ventricular arrhythmias. In rat ventricular myocytes, low extracellular K+ (corresponding to clinical moderate hypokalaemia) increased Ca2+ wave probability, Ca2+ transient amplitude, sarcoplasmic reticulum (SR) Ca2+ load and induced SR Ca2+ leak. Low extracellular K+ reduced Na+,K+‐ATPase (NKA) activity and hyperpolarized the resting membrane potential in ventricular myocytes. Both experimental data and modelling indicate that reduced NKA activity and subsequent Na+ accumulation sensed by the Na+, Ca2+ exchanger (NCX) lead to increased Ca2+ transient amplitude despite concomitant hyperpolarization of the resting membrane potential. Low extracellular K+ induced Ca2+ overload by lowering NKA α2 activity. Triggered ventricular arrhythmias in patients with hypokalaemia may therefore be attributed to reduced NCX forward mode activity linked to an effect on the NKA α2 isoform.

ICaL inhibition prevents arrhythmogenic Ca2+ waves caused by abnormal Ca2+ sensitivity of RyR or SR Ca2+ accumulation

AIMS Arrhythmogenic Ca(2+) waves result from uncontrolled Ca(2+) release from the sarcoplasmic reticulum (SR) that occurs with increased Ca(2+) sensitivity of the ryanodine receptor (RyR) or excessive Ca(2+) accumulation during β-adrenergic stimulation. We hypothesized that inhibition of the L-type Ca(2+) current (I(CaL)) could prevent such Ca(2+) waves in both situations. METHODS AND RESULTS Ca(2+) waves were induced in mouse left ventricular cardiomyocytes by isoproterenol combined with caffeine to increase RyR Ca(2+) sensitivity. I(CaL) inhibition by verapamil (0.5 µM) reduced Ca(2+) wave probability in cardiomyocytes during electrostimulation, and during a 10 s rest period after ceasing stimulation. A separate type of Ca(2+) release events occurred during the decay phase of the Ca(2+) transient and was not prevented by verapamil. Verapamil decreased Ca(2+) spark frequency, but not in permeabilized cells, indicating that this was not due to direct effects on RyR. The antiarrhythmic effect of verapamil was due to reduced SR Ca(2+) content following I(CaL) inhibition. Computational modelling supported that the level of I(CaL) inhibition obtained experimentally was sufficient to reduce the SR Ca(2+) content. Ca(2+) wave prevention through reduced SR Ca(2+) content was also effective in heterozygous ankyrin B knockout mice with excessive SR Ca(2+) accumulation during β-adrenergic stimulation. CONCLUSION I(CaL) inhibition prevents diastolic Ca(2+) waves caused by increased Ca(2+) sensitivity of RyR or excessive SR Ca(2+) accumulation during β-adrenergic stimulation. In contrast, unstimulated early Ca(2+) release during the decay of the Ca(2+) transient is not prevented, and merits further study to understand the full antiarrhythmic potential of I(CaL) inhibition.

Slowed relaxation and preserved maximal force in soleus muscles of mice with targeted disruption of the Serca2 gene in skeletal muscle

Non‐technical summary  Muscle function depends on tightly regulated Ca2+ movement between the intracellular sarcoplasmic reticulum (SR) Ca2+ store and cytoplasm in muscle cells. Disturbances in these processes have been linked to impaired muscle function and muscle disease. We disrupted the gene for the SERCA2 SR Ca2+ pump in mouse skeletal muscle to study how decreased transport of Ca2+ into the SR would affect soleus muscle function. We found that the SERCA2 content was strongly reduced in the 40% fraction of soleus muscle fibres normally expressing SERCA2. Muscle relaxation was slowed, supporting the hypothesis that reduced SERCA2 would reduce Ca2+ transport into the SR and prolong muscle relaxation time. Surprisingly, the muscles maintained maximal force, despite the fact that less SERCA2 in these fibres would be expected to lower the amount of Ca2+ released during contraction, and thereby lower the maximal force. Our findings raise important questions regarding the roles of SERCA2 and SR in muscle function.

Calcium release units in heart failure: that's about the size of it.

This editorial refers to ‘Ultrastructural remodelling of Ca2+ signalling apparatus in failing heart cells’ by H.-D. Wu et al ., pp. 430–438, this issue. Cardiac remodelling, often defined as changes in the mass and shape of the whole heart,1 has been a focus of research over several decades. Despite considerable efforts, the cellular and molecular mechanisms that lead to contractile dysfunction of the remodelled heart are still not clear. Remodelling may also occur at the level of the cardiomyocyte and is then termed ultrastructural remodelling. Studies have revealed substantial ultrastructural alterations in several parts of the cardiomyocytes during cardiac disease, including the sarcoplasmic reticulum (SR)2 and transverse (T)-tubules,3,4 and in the positioning of ion channels and pumps that are crucial in regulating myocardial contraction and relaxation. Thus, it is likely that ultrastructural remodelling can explain several important aspects of systolic and diastolic heart failure of various aetiologies. The machinery that couples the electrical activation of the cardiomyocyte to mechanical contraction is set to control transient rises of [Ca2+] in the cytosol. Research in the past decades has revealed that the global rise of [Ca2+] in the cytosol that activates myofilaments is regulated by mechanisms that are confined within spatially constrained microdomains in the cardiomyocyte. These microdomains occur at sites where Ca2+ channels in the T-tubule membrane come very close to Ca …