Sarcoplasmic Reticulum Content Research Articles

The effects of doxorubicin on calcium handling and contractility in sheep ventricular myocytes; role of oxidative stress

Anthracyclines such as doxorubicin (DOX) are effective chemotherapeutics and contribute to improved cancer survival rates in children and adults. However, anthracyclines exhibit acute and chronic cardiotoxicity which can produce heart failure in cancer survivors. While the cellular basis remains unclear, previous studies show anthracyclines perturb aspects of excitation-contraction coupling (ECC), though to our knowledge this has yet to be demonstrated in a large animal model. Furthermore, the extent to which altered ECC is dependent on anthracycline-induced reactive oxygen species (ROS) production / oxidative stress remains ambiguous. This is important given the key role of oxidative stress in other cardiotoxic processes. Therefore, we aimed to (1) characterise the effects of DOX on Ca handling and contractility in sheep ventricular myocytes and (2) quantify the dependence of any effect on oxidative stress.Sheep myocytes were enzymatically isolated in accordance with the Animals (Scientific Procedures) Act, UK, 1986. Cells were loaded with fura-2 (0.1 μM) then field stimulated at 0.5 Hz. Fura-2 was excited sequentially at 340 nm and 380 nm (200 Hz) and changes of cytoplasmic Ca inferred from the ratio of emitted light. Sarcomere length was measured simultaneously using a Myocam-S CCD camera and SarcLen acquisition module (Ion Optix, USA). Changes to sarcoplasmic reticulum (SR) Ca content were estimated by rapid application of caffeine (10 mM). Two-way ANOVA was used for statistical analysis.1 nM DOX reduced the amplitude of systolic Ca and shortening by 39 ± 3 % (n=49, p<0.001) and 42 ± 4 % (n=45, p<0.001) respectively. The amplitude of the caffeine-evoked Ca transient decreased by 33 ± 5 % (n=24, p<0.001). The rate of decay of the systolic Ca transient ( k sys ) represents the combined activity of SERCA and NCX and was reduced by 17 ± 3 % (n=46, p<0.001). The rate of decay of the caffeine-evoked Ca transient ( k caff ) represents NCX alone and was increased by 64 ± 26 % (n=23, p=0.002).Subtraction of k caff from k sys indicates SERCA activity ( k SERCA ), which was decreased by 34 ± 8 % (n=20, p<0.001). When the experiment was repeated in the presence of the antioxidant N-acetylcysteine (NAC), the effect of DOX on systolic Ca, systolic shortening and the caffeine-evoked Ca transient was attenuated by 52 % (n=29, p<0.001), 58 % (n=29, p<0.001) and 45 % (n=15, p=0.003) respectively. Furthermore, NAC prevented the effect of DOX on k sys , k caff and k SERCA .These data suggest DOX increases NCX activity and reduces SERCA activity which decreases SR Ca content thence systolic Ca and contractility. The effect of DOX on NCX and SERCA appears to be entirely ROS / oxidative stress dependent. However, other non-oxidative mechanisms likely contribute to net DOX-mediated negative cellular inotropy. Our next experiments will investigate this and the sources of anthracycline-induced oxidative stress. This work was funded by Kidscan childrens cancer research, UK. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Calcium signaling consequences of RyR2-S4938F mutation expressed in hiPSC-CMs.

Ryanodine receptor 2 (RyR2) is the major Ca2+ release channel of cardiac sarcoplasmic reticulum (SR) that regulates the rhythm and strength of the heartbeat, but its malfunction generates arrhythmia leading to heart failure. Here we report on a catecholaminergic polymorphic ventricular tachycardia (CPVT1)-associated RyR2 mutation (S4938F) located in RyR2 carboxyl-terminal that was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing. Ca2+ signaling and electrophysiological properties of beating cardiomyocytes carrying S4938F mutation were studied using total internal fluorescence microscopy (TIRF) and patch clamp technique. ICa densities measured either by depolarizations to zero mV or repolarizations from +100 mV to −50 mV in mutant hiPSC-CMs were comparable to WT cells, but their accompanying Ca2+-transients detected by Fura-2 (cytosolic Ca2+) or ER-GCaMP6 (SR Ca2+ release) were significantly suppressed, suggesting ICa induced Ca2+ release (CICR) was compromised. Even though 5mM caffeine triggered Fura-2 or ER-GCaMP6 signals and the frequency of spontaneously generated calcium transients of mutant cells were comparable to those of WT cells, the 4-CmC triggered cytosolic or SR Ca2+ signals and magnitude of spontaneously occurring Ca2+-transients were significantly suppressed. Spontaneously occurring Ca2+ sparks in S4938F hiPSC-CMs had longer durations but their frequency was comparable to those of WT cells. Isoprenaline increased the beating frequency that led to arrhythmic beating and cessation of excitability. Our data suggests that S4938F-RyR2 mutation does not significantly affect Ca2+content of SR, but decreases its Ca2+ sensitivity resulting in suppressed ICa-gated and spontaneous Ca2+ release from SR. Supported by NIH grant R01HL153504.

Modulating mitochondrial phosphate transport shapes intracellular Ca signaling to impact arrhythmogenesis.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Our recent study suggests that mitochondria function as a Ca buffer to absorb RyR2-mediated aberrant Ca release and thus mitigate its detrimental consequences in CPVT. Mitochondrial phosphate (Pi) transport has been shown to play a role in both mitochondrial Ca uptake and buffering. We hypothesize that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT by altering mitochondria Ca uptake and buffering. Ventricular myocytes were isolated from a genetic CPVT model of CASQ2 knock out (Cnull) mouse. Fluorescent imaging experiments were performed with live cells to examine intracellular Ca dynamics and cellular arrhythmic burden. Mitochondrial Pi transport was modulated by a pharmacological inhibitor or manipulating cytosolic Pi concentration. An inhibitor of mitochondrial Pi carrier N-ethylmaleimide (NEM) significantly exacerbated Ca handling of intact Cnull cells, resulting in more than 60% of cells displaying pacing-independent Ca oscillations. The NEM treatment also significantly increased the amplitude of cytosolic Ca transient and SR Ca content. Consistent with results obtained in intact cells, NEM treatment exacerbated arrhythmogenic Ca waves and reduced mitochondrial Ca uptake in permeabilized Cnull cells. Moreover, inhibiting mitochondrial Pi transport by lowering cytosolic Pi concentration also increased the frequency of Ca waves and reduced mitochondria Ca uptake in permeabilized Cnull cells. On the contrary, increasing cytosolic Pi concentration reduced the frequency of Ca waves and increased mitochondrial Ca uptake in permeabilized Cnull cells. Our results suggest that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT cells through altering mitochondrial Ca uptake and buffering. Enhancing mitochondrial Pi transport may represent a promising therapeutic strategy to reduce Ca waves and cardiac arrhythmias in CPVT.

Abstract 13377: Modulating Mitochondrial Phosphate Transport Shapes Intracellular Ca Signaling to Impact Arrhythmogenesis

Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Our recent study suggests that mitochondria function as a Ca buffer to absorb RyR2-mediated aberrant Ca release and thus mitigate its detrimental consequences in CPVT. Mitochondrial phosphate (Pi) transport has been shown to play a role in both mitochondrial Ca uptake and buffering. Hypothesis: We hypothesize that modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT by altering mitochondrial Ca uptake and buffering. Methods: Ventricular myocytes were isolated from a genetic CPVT model of CASQ2 knockout (Cnull) mouse. Fluorescent imaging experiments were performed with live cells to examine intracellular Ca dynamics and cellular arrhythmic burden. Mitochondrial Pi transport was modulated by a pharmacological inhibitor or manipulating cytosolic Pi concentration. Results: In the presence of β agonist isoproterenol, an inhibitor of mitochondrial Pi carrier N-ethylmaleimide (NEM) significantly exacerbated Ca handling of intact Cnull cells, resulting in more than 60% of cells displaying pacing-independent Ca oscillations. The NEM treatment also significantly increased the amplitude of cytosolic Ca transient and SR Ca content. Consistent with results obtained in intact cells, NEM treatment exacerbated arrhythmogenic Ca waves and reduced mitochondrial Ca uptake in permeabilized Cnull cells. Moreover, inhibiting mitochondrial Pi transport by lowering cytosolic Pi concentration also increased the frequency of Ca waves and reduced mitochondria Ca uptake in permeabilized Cnull cells. On the contrary, increasing cytosolic Pi concentration reduced the frequency of Ca waves and increased mitochondrial Ca uptake in permeabilized Cnull cells. Conclusions: Modulating mitochondrial Pi transport impacts arrhythmogenesis in CPVT cells through altering mitochondrial Ca uptake and buffering. Enhancing mitochondrial Pi transport may represent a promising therapeutic strategy to reduce arrhythmogenic Ca waves and cardiac arrhythmias in CPVT.

PDE1 inhibition and adenosine A2A receptor activation synergistically potentiates spontaneous calcium waves and transients in mouse ventricular myocytes

Abstract Background The phosphodiesterase-1C (PDE1C) and the adenosine A2A receptor (A2AR) are located in a macromolecular cluster and expected to modulate cAMP-dependent regulation of the intracellular calcium homeostasis in cardiac myocytes. In this contect, atrial fibrillation has been associated with a high incidence of spontanoeus arrhythmogenic calcium waves caused by excessive A2AR activation. On the other hand, inhibition of PDE1C and A2AR activation has also been reported to produce a synergistic and protective effect on cardiomyocyte survival, but little is known about the corresponding effects on the intracellular calcium homeostasis. Purpose This study, therfore, aimed to investigate how PDE1 inhibition and/or A2AR activation modulate sarcoplasmic reticulum (SR) calcium homeostasis in mouse ventricular myocytes. Methods Mouse ventricular myocytes (47 cells from 20 mice) were loaded with the calcium indicator Rhod-2 and spontaneous calcium release events at rest or caffeine induced calcium transients were visualized using resonance-scanning confocal microscopy at a frame rate of 150 Hz. The SR calcium content was estimated as the ratio of the peak fluorescence and the fluoresence the end of a 10 mM caffeine pulse (20s). Calcium leak was estimated as ratio of the flourescence emmision at baseline before and at the end of the caffeine pulse. Results The incidence of spontaneous calcium waves or transients increased from 0.7±0.3 events/min in control to 1.1±0.5 events/min and 3.4±1.0 events/min (p<0.01) after inhibition of PDE1 with the selective antagonist ITI-214 and the A2AR agonist CGS-21680 respectively. Concomitant PDE1 inhibition and A2AR activation synergistically increased the event frequency to 8.4±2.3 events/min (p<0.05). There were no significant differences in the corresponding SR calcium contents (4.4±0.3, 4.5±0.4, 4.7±0.4 and 4.1±0.2 for control, ITI-214, CGS-21680 and ITI-214+CGS-21680 respectively). However, there was a parallel increase in calcium leak from 8.7±4.2% in control to 21.8±9.8% with ITI-214, 17.6±7.2% with CGS-21680 and 37.5±11.1% when myocytes were exposed to both compounds (p<0.05), suggesting that the higher incidence of spontaneous calcium waves and transients observed upon PDE1 inhibition and/or A2AR activation is likely caused by a lowering of the threshold for spontaneous SR calcium release rather than by SR calcium overloading. Conclusion PDE1 inhibition exacerbates the incidence of spontaneous arrhythmogenic SR calcium release events in myocytes with high levels of A2AR activation, suggesting that PDE1 inhibition under these conditions increases the risk of arrhythmia. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Science and Innovation

NR1D1 controls skeletal muscle calcium homeostasis through myoregulin repression.

The sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions, such as myopathies. Here, we investigated whether the nuclear receptor subfamily 1 group D member (NR1D1, also called REV-ERBα) regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that NR1D1 deficiency in mice impaired sarco/endoplasmic reticulum calcium ATPase–dependent (SERCA-dependent) SR calcium uptake. NR1D1 acts on calcium homeostasis by repressing the SERCA inhibitor myoregulin through direct binding to its promoter. Restoration of myoregulin counteracted the effects of NR1D1 overexpression on SR calcium content. Interestingly, myoblasts from patients with Duchenne muscular dystrophy displayed lower NR1D1 expression, whereas pharmacological NR1D1 activation ameliorated SR calcium homeostasis and improved muscle structure and function in dystrophic mdx/Utr+/– mice. Our findings demonstrate that NR1D1 regulates muscle SR calcium homeostasis, pointing to its therapeutic potential for mitigating myopathy.

Activation of GPER limits adverse changes to Ca2+ signalling and arrhythmogenic activity in cardiomyocytes of ovariectomised guinea pigs

Abstract Background Post-menopausal women have an enhanced risk of developing cardiovascular disease and disturbances of cardiac rhythm, generally attributed to declining oestrogen levels during menopause. In an animal model that mimics menopause, the long-term withdrawal of oestrogen dysregulated Ca2+ signalling and increased the formation of a pro-arrhythmic substrate. Selective targeting of the membrane bound G-protein coupled oestrogen receptor 1 (GPER) eliminated such arrhythmogenic activity associated with the loss of oestrogen. Purpose We aim to assess the “cardioprotective” role of GPER in response to oestrogen withdrawal. Methods Ovariectomy (OVx) or sham surgeries were conducted on female guinea pigs. Left ventricular cardiomyocytes were isolated 150-days post-operatively for experimental use. GPER expression was quantified by western blot. Myocytes were incubated in solutions containing GPER agonist G-1 for >2h before recording some electrophysiological and Ca2+ regulatory parameters. Results GPER expression was 32% higher in OVx. OVx cardiomyocytes had prolonged action potential duration (APD) compared with sham and in the presence of G-1, APD90 shortened by 12% and 22% in sham and OVx, respectively. G-1 reduced early after depolarisation (EAD) formation by >99% in OVx. OVx cells had larger sarcoplasmic reticulum (SR) Ca2+ content (by 13%), compared with sham. While G-1 had little effect on SR content, it reduced Ca2+ transient amplitude (by 40%), SR fractional release (by 11%) and sarcomere shortening (by 29%) in OVx cells. The frequency of occurrence of spontaneous Ca2+ waves evoked by periods of rapid stimulation reduced by 40% and wave-free survival time prolonged in OVx cells incubated with G-1. Conclusions In the hearts of an animal species whose electrophysiology and intracellular Ca2+ regulation is akin to humans, we show that following oestrogen deficiency, GPER expression increased and its activation induces negative inotropic responses in cardiomyocytes. It limits the adverse changes to Ca2+ signalling and induces anti-arrhythmogenic behaviours in OVx. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): British Heart Foundation

The effect of oestrogen withdrawal on cardiac Ca2+ regulation and the influence of GPER1

Abstract Background Post-menopausal women have an enhanced risk of developing heart failure, attributed to declining oestrogen levels during menopause. However, the signalling mechanisms remain undetermined. Purpose We aim to determine the role of G-protein coupled oestrogenic receptor 1 (GPER1) in intracellular Ca2+ regulation and the consequences of hormonal changes that may exacerbate the pathophysiology of heart failure. Methods Ovariectomy (OVx) (mimics menopausal hormone changes) or sham surgeries were conducted on female guinea pigs. Left ventricular cardiomyocytes were isolated 150-days post-operatively for experimental use. Cellular t-tubule network and structural integrity was measured using fluorescent di-8-ANEPPs staining and scanning ion conductance microscopy. GPER1 expression and localisation was measured by Western blot and immunostaining. The role of GPER1 activation was measured using selective agonist G-1 in electrophysiological and Ca2+-sensitive dye fluorescence experiments. Results Following oestrogen withdrawal, the t-tubule network density decreased by 13% and z-groove index reduced by 15%. GPER1 predominantly localised to the peri-nuclear endoplasmic reticulum and its expression increased by 32% in OVx. Action potential duration (APD) prolonged in OVx and following GPER1 activation, APD90 shortened by 11% and 25% in sham and OVx respectively. OVx cells had larger peak inward Ca2+ current (ICaL) (by 22%) and sarcoplasmic reticulum (SR) Ca2+ content (by 13%), compared with sham. While GPER1 activation had little effect on peak ICaL or SR content, it reduced Ca2+ transient amplitude (by 20%), SR fractional release (by 11%) in OVx cells. The frequency of occurrence of spontaneous Ca2+ waves evoked by periods of rapid stimulation reduced by 40% and wave-free survival time prolonged in OVx cells following GPER1 activation. Conclusions In the hearts of an animal species whose electrophysiology and intracellular Ca2+ regulation is akin to humans, we show that following oestrogen deficiency, the t-tubule network is down-regulated and becomes disorganised, GPER1 expression is increased and its activation induces negative inotropic responses in cardiomyocytes. This may limit the adverse changes to Ca2+ signalling reported in OVx that could be pro-arrhythmic and exacerbate the progression to heart failure. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation

Redox-Resistant SERCA [Sarco(endo)plasmic Reticulum Calcium ATPase] Attenuates Oxidant-Stimulated Mitochondrial Calcium and Apoptosis in Cardiac Myocytes and Pressure Overload-Induced Myocardial Failure in Mice.

SERCA [sarco(endo)plasmic reticulum calcium ATPase] is regulated by oxidative posttranslational modifications at cysteine 674 (C674). Because sarcoplasmic reticulum (SR) calcium has been shown to play a critical role in mediating mitochondrial dysfunction in response to reactive oxygen species, we hypothesized that SERCA oxidation at C674 would modulate the effects of reactive oxygen species on mitochondrial calcium and mitochondria-dependent apoptosis in cardiac myocytes. Adult rat ventricular myocytes expressing wild-type SERCA2b or a redox-insensitive mutant in which C674 is replaced by serine (C674S) were exposed to H2O2 (100 µmol/Lμ). Free mitochondrial calcium concentration was measured in adult rat ventricular myocytes with a genetically targeted fluorescent probe, and SR calcium content was assessed by measuring caffeine-stimulated release. Mice with heterozygous knock-in of the SERCA C674S mutation were subjected to chronic ascending aortic constriction. In adult rat ventricular myocytes expressing wild-type SERCA, H2O2 caused a 25% increase in mitochondrial calcium concentration that was associated with a 50% decrease in SR calcium content, both of which were prevented by the ryanodine receptor inhibitor tetracaine. In cells expressing the C674S mutant, basal SR calcium content was decreased by 31% and the H2O2-stimulated rise in mitochondrial calcium concentration was attenuated by 40%. In wild-type cells, H2O2 caused cytochrome c release and apoptosis, both of which were prevented in C674S-expressing cells. In myocytes from SERCA knock-in mice, basal SERCA activity and SR calcium content were decreased. To test the effect of C674 oxidation on apoptosis in vivo, SERCA knock-in mice were subjected to chronic ascending aortic constriction. In wild-type mice, ascending aortic constriction caused myocyte apoptosis, LV dilation, and systolic failure, all of which were inhibited in SERCA knock-in mice. Redox activation of SERCA C674 regulates basal SR calcium content, thereby mediating the pathologic reactive oxygen species-stimulated rise in mitochondrial calcium required for myocyte apoptosis and myocardial failure.

Calcium entry units (CEUs): perspectives in skeletal muscle function and disease

In the last decades the term Store-operated Ca2+ entry (SOCE) has been used in the scientific literature to describe an ubiquitous cellular mechanism that allows recovery of calcium (Ca2+) from the extracellular space. SOCE is triggered by a reduction of Ca2+ content (i.e. depletion) in intracellular stores, i.e. endoplasmic or sarcoplasmic reticulum (ER and SR). In skeletal muscle the mechanism is primarily mediated by a physical interaction between stromal interaction molecule-1 (STIM1), a Ca2+ sensor located in the SR membrane, and ORAI1, a Ca2+-permeable channel of external membranes, located in transverse tubules (TTs), the invaginations of the plasma membrane (PM) deputed to propagation of action potentials. It is generally accepted that in skeletal muscle SOCE is important to limit muscle fatigue during repetitive stimulation. We recently discovered that exercise promotes the assembly of new intracellular junctions that contains colocalized STIM1 and ORAI1, and that the presence of these new junctions increases Ca2+ entry via ORAI1, while improving fatigue resistance during repetitive stimulation. Based on these findings we named these new junctions Ca2+ Entry Units (CEUs). CEUs are dynamic organelles that assemble during muscle activity and disassemble during recovery thanks to the plasticity of the SR (containing STIM1) and the elongation/retraction of TTs (bearing ORAI1). Interestingly, similar structures described as SR stacks were previously reported in different mouse models carrying mutations in proteins involved in Ca2+ handling (calsequestrin-null mice; triadin and junctin null mice, etc.) or associated to microtubules (MAP6 knockout mice). Mutations in Stim1 and Orai1 (and calsequestrin-1) genes have been associated to tubular aggregate myopathy (TAM), a muscular disease characterized by: (a) muscle pain, cramping, or weakness that begins in childhood and worsens over time, and (b) the presence of large accumulations of ordered SR tubes (tubular aggregates, TAs) that do not contain myofibrils, mitochondria, nor TTs. Interestingly, TAs are also present in fast twitch muscle fibers of ageing mice. Several important issues remain un-answered: (a) the molecular mechanisms and signals that trigger the remodeling of membranes and the functional activation of SOCE during exercise are unclear; and (b) how dysfunctional SOCE and/or mutations in Stim1, Orai1 and calsequestrin (Casq1) genes lead to the formation of tubular aggregates (TAs) in aging and disease deserve investigation.

Nogo-C causes post-MI arrhythmia through increasing calcium leakage from sarcoplasmic reticulum

Disturbance of calcium homeostasis in cardiomyocytes plays a pivotal role in the pathogenesis of ventricular arrhythmias after myocardial infarction(MI). Nogo-C is a sarcoplasmic reticulum (SR) protein ubiquitously expressed in tissues including heart. We previously reported that Nogo-C played a critical role in the pathogenesis of MI through inducing cardiomyocyte apoptosis. However, the role and mechanisms of Nogo-C in the regulation of calcium homeostasis and in the pathogenesis of arrhythmia post-MI remain unclear. In the present study, wildtype and Nogo-C knockout mice were subjected with MI operation followed by intraperitoneal injection of caffeine and isoproterenol to induce arrhythmia. We found that Nogo-C knockout mice displayed lower incidence of arrhythmia compared with the high incidence of arrhythmia of wildtype mice. Moreover, Nogo-C overexpression by adenovirus in cardiomyocytes caused decreased SR calcium content, reduced contractile ability, and increased diastolic cytoplasma calcium concentration, as compared with control cells. Oxalate-facilitated 45Ca uptake assay showed no difference of SR calcium reuptake ability between control and Nogo-C overexpressing cardiomyocytes, indicating that the decreased SR calcium content was not through disturbing SR calcium reuptake in Nogo-C overexpressing cells. In contrast, the rate of SR calcium leak, spontaneous calcium wave, and calcium spark markedly increased in Nogo-C overexpressing cardiomyocytes, suggesting that the increased SR calcium leak causes the decreased SR calcium content. Furthermore, We found that Nogo-C overexpression markedly increased, while Nogo-C knockdown decreased Sec61 protein, a newly found calcium leak channel, in cardiomyocytes. Nogo-C bound with Sec61 through its transmembrane domains loop-6 and loop-8, thus inhibited the ubiquitination of Sec61, leading to the increased calcium leak from SR. Together, our data show that Nogo-C is increased in MI hearts, and the increased Nogo-C inhibits the degradation of Sec61 by ubiquitination, leading to increased SR calcium leak, thus promoting arrhythmia after MI.

Role of Intracellular Ca2+-overload in Cardiac Dysfunction in Heart Disease

Various heart diseases such as genetically-determined heart failure, acute myocardial infarction, ischemia-reperfusion injury and catecholamine-induced cardiomyopathies are associated with cardiac dysfunction, cellular damage, subcellular derangements and metabolic alterations. Since increase in myocardial Ca2+ is accompanied by these abnormalities, it is generally held that intracellular Ca2+-overload plays an important role in the pathogenesis of cardiac dysfunction as well as cellular and metabolic defects in different cardiovascular diseases. This view is supported by observations in hearts subjected to Ca2+-paradox, where reperfusion of Ca2+-free perfused hearts with Ca2+-containing medium was found to produce a marked increase in myocardial Ca2+-content, cellular damage and cardiac contracture. The intracellular Ca2+-overload in the heart has also been shown to produce mitochondrial Ca2+-overload, depress ATP production, release different toxic substances and induce cardiomyocyte apoptosis. By virtue of its ability to depress cardiac gene expression and increase proteolysis of sarcolemma (SL) sarcoplasmic reticulum (SR) and myofibrils (MF), the intracellular Ca2+-overload has been reported to reduce SL, SR and MF protein content and activities. Such remodeling of subcellular organelles is associated with dramatic alterations in Ca2+ -handling by SL and SR membranes as well as interaction of Ca2+ with MF for the impairment of cardiac function. Thus, it is evident that mitochondrial Ca2+-overload, and subcellular remodeling for Ca2+-handling defects are responsible for the occurrence of cardiac dysfunction, metabolic derangements and cellular damage during the development of heart disease.

Total Calcium Content of Sarcoplasmic Reticulum and Mitochondria in Ryanodine Receptor Variant Muscle

Ryanodine receptor (RyR) mutations can induce susceptibility to malignant hyperthermia or myopathies. The mutation of the RyR can cause an increased leak of Ca2+ from the sarcoplasmic reticulum (SR). The leak of SR Ca2+ is associated with an increase in cytoplasmic free Ca2+, which may activate cytoplasmic proteases or potentially enter the mitochondria to increase the load of calcium inside this organelle. A major gap in our understanding the issues associated with RyR variants is the effect on Ca2+-handling by the cellular organelles, which we could better address if we had information on the total calcium content of the SR and mitochondria under conditions of known RyR mutations and associated Ca2+ leak. We determined the RyR Ca2+ leak in extensor digitorum longus muscle fibres of wild-type (WT), heterozygous (HET) and homozygous (HOM) mice for the RyR1 mutant p.G2435R (Lopez et al 2018, BJA) using recently developed techniques (Cully et al 2018, PNAS). We observed an increasing leak with increasing number of mutated RyR alleles, from WT to HET to HOM mice. Next, we used a membrane-lysis method to release all the total compartmentalized calcium onto the contractile apparatus to use the ensuing force response to determine SR calcium content (Fryer & Stephenson, 1996. J.Physiol; Lamboley et al 2013, J.Physiol). We modified this technique to isolate the depletion of mitochondrial calcium to allow determination of total calcium within this compartment. Our results show a progressive decrease in the SR total calcium content with increasing RyR Ca2+ leak. The mitochondrial total calcium content increased significantly from WT, to that in the HET, to that in the HOM mice.

Spatial Arrangement of TRPC1, 3 and 6 Channels in Rabbit Ventricular Cardiomyocytes

Transient receptor potential canonical (TRPC) 1, 3 and 6 channels are mechanosensitive cation channels abundantly expressed in the mammalian heart. A physiological role of the channels in regulation of intracellular calcium and contractility in cardiomyocytes was proposed. Increased TRPC expression and activity was linked to cardiac hypertrophy and heart failure. Understanding the precise location of TRPCs is essential to investigations of their functional role in normal and diseased hearts. We studied the spatial arrangement of TRPC channels in left ventricular myocardium from sham and transverse aortic constriction (TAC) adult rabbit using single-molecule localization microscopy. We acquired 3-dimensional images from tissue labelled for TRPC1, 3 and 6, along with sarco/endoplasmic reticulum ATPase (SERCA) as a marker of sarcoplasmic reticulum (SR) or wheat germ agglutinin (WGA) as a marker of sarcolemma including transverse tubular system. Proximity of TRPCs with SERCA and WGA was quantified with nearest neighbor analyses. The images revealed that TRPC1, 3 and 6 localize in intracellular regularly distributed striations. We found high proximity of TRPC1, 3 and 6 with SERCA and lower proximity with WGA in sham animals. In TAC animals we found decreased proximity of TRPCs with SERCA. Our studies suggest that TRPC1, 3 and 6 reside in the SR membrane in adult rabbit cardiomyocytes. This finding agrees with our functional studies testing the hypothesis that TRPC channels act as mechanical modulators of SR calcium content. We also revealed spatial remodeling associated with cardiac hypertrophy. Our study sheds light on the physiological function of TRPCs in cardiomyocytes and their role in hypertrophic remodeling in cardiac disease.

The Role of NAADP-Mediated Endo-Lysosomal Calcium Release in the Cardiac Atria

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a highly potent calcium-mobilising second messenger which stimulates calcium release from endo-lysosomes via the opening of TPC2 channels. In cardiac ventricular myocytes, lysosomes form nano-junctions with the sarcoplasmic reticulum (SR) and mitochondria and exposure to NAADP (synthesised by CD38 upon beta-adrenergic stimulation), leads to an augmented calcium transient and SR calcium content, and contributes to the hypertrophy and arrhythmogenesis associated with chronic beta-adrenergic stress. We utilised transmission electron microscopy (TEM) and optical mapping in lapine and murine models to investigate endo-lysosomal calcium signalling in the cardiac atria. TEM of fixed, imbedded rabbit atrial myocytes or tissue (n=3 animals) confirmed that lysosomes form nano-junctions with both the SR (median distance 17nm, IQR 13-21nm, n=15 lysosomes) and mitochondria (median distance 13nm, IQR 10-57nm, n=16 lysosomes). These are close enough to have the potential for localised calcium signalling. In intact murine left atria, 10nM isoprenaline caused a 59.3±4.8% increase in calcium transient amplitude (n=6, fluorophore rhod-2). Abolishing endo-lysosomal calcium storage using 5µM Bafilomycin A1 (Baf) significantly reduced the calcium transient response to isoprenaline (to 35.3±2.7%, n=6, P<0.01). In CD38KO mice, left atrial responses were significantly reduced (35.9±2.6%, n=6) in comparison to wild-type controls (59.3±4.9%, n=6, P<0.01) and there was no longer an effect of Baf on isoprenaline response (35.9±2.6% and 33.6±4.0% in CD38KO control and Baf respectively, P>0.05, both n=6). Our data suggest that, endo-lysosomes can form nanojunctions for calcium signalling within cardiac atrial myocytes and that the endo-lysosomal NAADP pathway is functionally relevant during beta-adrenergic stimulation. The changes to and effects of this stereotyped architecture and pathway in atrial disease states are unknown and will be a vital avenue for future research.

Ultrastructure of primary pacemaking cells in rabbit sino-atrial node cells indicates limited sarcoplasmic reticulum content.

The main mammalian heart pacemakers are spindle‐shaped cells compressed into tangles within protective layers of collagen in the sino‐atrial node (SAN). Two cell types, “dark” and “light,” differ on their high or low content of intermediate filaments, but share scarcity of myofibrils and a high content of glycogen. Sarcoplasmic reticulum (SR) is scarce. The free SR (fSR) occupies 0.04% of the cell volume within ~0.4 µm wide peripheral band. The junctional SR (jSR), constituting peripheral couplings (PCs), occupies 0.03% of the cell volume. Total fSR + jSR volume is 0.07% of cell volume, lower than the SR content of ventricular myocytes. The average distance between PCs is 7.6 µm along the periphery. On the average, 30% of the SAN cells surfaces is in close proximity to others. Identifiable gap junctions are extremely rare, but small sites of close membrane‐to‐membrane contacts are observed. Possibly communication occurs via these very small sites of contact if conducting channels (connexons) are located within them. There is no obvious anatomical detail that might support ephaptic coupling. These observations have implications for understanding of SAN cell physiology, and require incorporation into biophysically detailed models of SAN cell behavior that currently do not include such features.

Increase in phospholamban content in mouse skeletal muscle after denervation.

It is well-known that denervation of motor nerves induces atrophy and decreases contractile force of the skeletal muscle. However, it is not completely understood how denervation alters calcium handling in the skeletal muscle. We investigated the effect of denervation on the expression and function of proteins involved in calcium handling. Two weeks after denervation of the right sciatic nerve in mice, we observed a significant decrease in mass and cross-sectional area of the ipsilateral tibialis anterior (TA) and flexor digitorum brevis (FDB) muscles. Also, we observed a significant decrease in the specific tetanus contractile force in the ipsilateral TA muscle. Calcium imaging of the ipsilateral FDB showed that the peak twitch and tetanus calcium concentrations were significantly decreased due to a decrease in calcium content of the sarcoplasmic reticulum (SR). Denervation reduced the maximum rate of sarcoplasmic/endoplasmic calcium ATPase (SERCA) activity. Interestingly, the amount of phospholamban (PLN), but not its transcripts, was increased in the ipsilateral vs. contralateral side after denervation, suggesting that denervation impairs constitutive regulation of PLN. Immunohistochemical analysis revealed increased PLN in all major fiber types in TA with IIx fibers showing a threefold higher expression than the contralateral side. These results suggest that the abnormal increase in PLN in the ipsilateral fast-twitch fibers may be involved in decreased SERCA activity, SR calcium content, peak calcium transients, and contractile forces of denervated muscles.

Phosphodiesterase-5 inhibition with sildenafil suppresses triggered ventricular arrhythmias and calcium waves via reduced sarcoplasmic reticulum content in the large mammal

Phosphodiesterase-5 inhibition with sildenafil suppresses triggered ventricular arrhythmias and calcium waves via reduced sarcoplasmic reticulum content in the large mammal

The Anti-Cancer Multikinase Inhibitor Sorafenib Impairs Cardiac Contractility by Reducing Phospholamban Phosphorylation and Sarcoplasmic Calcium Transients

Tyrosine-kinase inhibitors (TKIs) have revolutionized cancer therapy in recent years. Although more targeted than conventional chemotherapy, TKIs exhibit substantial cardiotoxicity, often manifesting as hypertension or heart failure. Here, we assessed myocyte intrinsic cardiotoxic effects of the TKI sorafenib and investigated underlying alterations of myocyte calcium homeostasis. We found that sorafenib reversibly decreased developed force in auxotonically contracting human myocardia (3 µM: −25 ± 4%, 10 µM: −29 ± 7%, 30 µM: −43 ± 12%, p < 0.01), reduced peak cytosolic calcium concentrations in isolated cardiomyocytes (10 µM: 52 ± 8.1% of baseline, p < 0.001), and slowed cytosolic calcium removal kinetics (RT50, RT10, Tau, p < 0.05). Beta-adrenergic stimulation induced augmentation of calcium transient (CaT) amplitude was attenuated in sorafenib-treated cells (2.7 ± 0.3-fold vs. 3.6 ± 0.2-fold in controls, p < 0.001). Sarcoplasmic reticulum (SR) calcium content was reduced to 67 ± 4% (p < 0.01), and SR calcium re-uptake slowed (p < 0.05). Sorafenib significantly reduced serine 16 phosphorylation of phospholamban (PLN, p < 0.05), while PLN threonine 17 and CaMKII (T286) phosphorylation were not altered. Our data demonstrate that sorafenib acutely impairs cardiac contractility by reducing S16 PLN phosphorylation, leading to reduced SR calcium content, CaT amplitude, and slowed cytosolic calcium removal. These results indicate myocyte intrinsic cardiotoxicity irrespective of effects on the vasculature and chronic cardiac remodeling.

SOCE Is Important for Maintaining Sarcoplasmic Calcium Content and Release in Skeletal Muscle Fibers

Store-operated Ca2+ entry (SOCE) is a Ca2+-entry process activated by the depletion of intracellular stores and has an important role in many cell types. In skeletal muscle, however, its role during physiological muscle activation has been controversial. To address this question, sarcoplasmic reticulum (SR) calcium release in a mouse strain with a naturally occurring mutation in the myostatin gene (Compact (Cmpt)) leading to a hypermuscular yet reduced muscle-force phenotype was compared to that in wild-type mice. To elicit Ca2+ release from the SR of flexor digitorum brevis (FDB) fibers, either a ryanodine receptor agonist (4-chloro-meta-cresol) or depolarizing pulses were used. In muscles from Cmpt mice, endogenous protein levels of STIM1 and Orai1 were reduced, and consequently, SOCE after 4-chloro-meta-cresol-induced store depletion was suppressed. Although the voltage dependence of SR calcium release was not statistically different between wild-type and Cmpt fibers, the amount of releasable calcium was significantly reduced in the latter, indicating a smaller SR content. To assess the immediate role of SOCE in replenishing the SR calcium store, the evolution of intracellular calcium concentration during a train of long-lasting depolarizations to a maximally activating voltage was monitored. Cmpt mice exhibited a faster decline in calcium release, suggesting a compromised ability to refill the SR. A simple model that incorporates a reduced SOCE as an important partner in regulating immediate calcium influx through the surface membrane readily accounts for the steady-state reduction in SR calcium content and its more pronounced decline after calcium release.