Ca Waves Research Articles

Coexisting fast–slow dendritic traveling waves in a 3D-array electric field coupled neuronal network

Coexistence of fast and slow traveling waves without synaptic transmission has been found in hhhippocampal tissues, which is closely related to both normal brain activity and abnormal neural activity such as epileptic discharge. However, the propagation mechanism behind this coexistence phenomenon remains unclear. In this paper, a three-dimensional electric field coupled hippocampal neural network is established to investigate generation of coexisting spontaneous fast and slow traveling waves. This model captures two types of dendritic traveling waves propagating in both transverse and longitude directions: the N-methyl-D-aspartate (NMDA)-dependent wave with a speed of about 0.1 m/s and the Ca-dependent wave with a speed of about 0.009 m/s. These traveling waves are synaptic-independent and could be conducted only by the electric fields generated by neighboring neurons, which are basically consistent with the in vitro data measured experiments. It is also found that the slow Ca wave could trigger generation of fast NMDA waves in the propagation path of slow waves whereas fast NMDA waves cannot affect the propagation of slow Ca waves. These results suggest that dendritic Ca waves could acted as the source of the coexistence fast and slow waves. Furthermore, we also confirm the impact of cellular spacing heterogeneity on the onset of coexisting fast and slow waves. The local region with decreasing distances among neighbor neurons is more liable to promote the onset of spontaneous slow waves which, as sources, excite propagation of fast waves. These modeling studies provide possible biophysical mechanisms underlying the neural dynamics of spontaneous traveling waves in brain tissues.

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.

Alzheimer's disease-related presenilin 1 M146L mutation disrupts SERCA2a regulation and increases propensity of Ca waves in ventricular cardiomyocytes.

Presenilin 1 (PS1) is a transmembrane protein expressed in the endoplasmic reticulum (ER) of different cell types. It has been shown that Ca mishandling due to PS1 mutations contributes to the Alzheimer's disease (AD)-related neurodegeneration. PS1 is also expressed in the heart. Several clinical studies revealed that PS1 mutations are associated with cardiomyopathies. We previously showed that PS1 interacts with the cardiac ER Ca ATPase (SERCA2a) and regulates its function. Here, we studied the role of AD-related PS1M146L mutation in regulation of intracellular Ca homeostasis. Fluorescent resonance energy transfer (FRET) experiments in HEK293 cells transfected with fluorescently labeled SERCA2a and PS1M146L revealed that the mutation does not disrupt the interaction between these two proteins. Measurements of SERCA2a-mediated Ca transport showed that at low ER Ca loads ([Ca]ER≤0.15 mM), both PS1WT and PS1M146L enhance ER Ca uptake by a similar level (∼40%) compared to control (no PS1 expression). At high ER Ca loads ([Ca]ER≥0.35 mM), however, PS1WT reduced ER Ca uptake and ER Ca load, whereas PS1M146L was less effective in regulating SERCA2a function. We used in vivo gene delivery in mouse cardiac wall to study the effect of PS1WT and PS1M146L overexpression on Ca regulation in ventricular myocytes. We found that both PS1WT and PS1M146L localized predominantly at the sarcoplasmic reticulum. Analysis of cytosolic Ca dynamics revealed that PS1WT overexpression in cardiomyocytes increases diastolic Ca and decreases Ca transient amplitudes. In contrast, PS1M146L expression increased propensity of spontaneous Ca waves. These results illustrate that the lack of SERCA2a regulation by PS1M146L at high ER Ca loads can lead to ER Ca overload and Ca waves. These defects in Ca regulation might contribute to the onset of cardiomyopathies in patients with PS1 mutations.

The role of mitochondrial-associated-membranes in Ca-dependent arrhythmias.

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). Recent studies suggest that mitochondria function as a protective Ca buffer to absorb RyR2-mediated aberrant Ca release in CPVT. However, the molecular mechanism underlying the protective Ca buffering function of CPVT mitochondria remains unclear. We hypothesize that the tethering between SR and mitochondria, also known as mitochondria-associated-membranes (MAMs) are promoted in CPVT to facilitate SR-mitochondria Ca transfer. Moreover, manipulating SR-mitochondria tethering or MAMs impacts arrhythmogenesis in CPVT. Ventricular myocytes were isolated from a CPVT model of CASQ2 knockout (Cnull) mouse. Cellular immunofluorescence assays and western blots were employed to detect MAMs remodeling in CPVT cells. Pharmacological and genetic approaches were employed to disrupt or promote MAMs and examine its effect on cellular arrhythmogenesis using live-cell imaging. SR-mitochondria tethering was assessed by examining the interaction between RyR2 and Voltage-dependent anion channel (VDAC), a protein localized in the mitochondrial-outer-membrane. As compared with wild-type (WT), we detected a higher degree of colocalization of immunofluorescence between RyR2 and VDAC in Cnull cells, and increased RyR2-VDAC interactions. The expression of SR-mitochondria tethering protein Mitofusin2 was increased in MAMs isolated from CPVT hearts. When treated with colchicine to induce a partial disruption of MAMs, arrhythmogenic Ca waves were exacerbated in intact Cnull cells. In permeabilized cells, colchicine also increased the frequency of Ca waves and reduced mitochondrial Ca uptake. Manipulation of MAMs by genetically targeting Mitofusin 2 also affected the frequency of Ca waves. Our results suggest that SR-mitochondria tethering or MAMs shape intracellular Ca signaling to impact arrhythmogenesis. Targeting this signaling microdomain may represent a novel strategy to reduce Ca waves and Ca-dependent arrhythmias.

Dynamic calcium-calmodulin-dependent stabilization of leaky RyR2 in live cardiac myocytes.

Calmodulin (CaM) is a major regulator of cardiac ryanodine receptors (RyR2). CaM binding shifts RyR2 channel conformation to a state that suppresses diastolic SR Ca leak that can become pro arrhythmic as commonly observed in cardiac diseases such as heart failure (HF) or diabetic cardiomyopathy. In these diseases, CaM affinity for RyR2 is reduced, promoting a leaky pathological RyR2 structural state that is arrhythmogenic. Conversely, increased CaM binding can suppress RyR2-mediated Ca leak and promote a conformational shift to the stable physiological non-leaky state. Still, there are unknowns regarding the dynamic interplay between CaM-RyR2 during each heartbeat and in pathogenic RyR states in live cardiac myocytes. We hypothesize that high [Ca] elevations reached during Ca transients in a healthy beating myocyte would increase CaM-RyR binding throughout the cardiac cycle, promoting RyR stability. Further, the lower CaM-RyR2 affinity in disease, exacerbated by reduced Ca transient amplitude, resulting in lower CaM-RyR2 binding, destabilized RyR2s and enhance arrhythmogenic diastolic SR Ca leak. We used permeabilized cardiac myocytes from WT and HF mouse models as a controlled approach to measuring both the [Ca]i and [CaM] dependence of CaM-RyR2 binding via FRET between donor-labeled FKBP12.6 and acceptor-labeled CaM. Moreover, we simultaneously recorded [Ca]i transients within myocytes, enabling measurement of dynamic CaM-RyR2 saturation during periodic Ca waves that emulate paced Ca transients. We find that CaM-RyR2 FRET is reduced in the leaky pathological state and associated with alterations in Ca leak event frequencies. We infer that the healthy higher CaM-RyR2 affinity favors more complete saturation of RyR2 with CaM throughout the cardiac cycle, versus pathological states. These data suggest that a modest fraction of CaM-free RyR2s with pathological diastolic conformation might suffice to elicit proarrhythmic SR Ca leak in cardiac disease.

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.

Abstract 13523: The Role of Mitochondrial-Associated-Membranes in Ca-Dependent Arrhythmias

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). Recent studies suggest that mitochondria function as a protective Ca buffer to absorb RyR2-mediated aberrant Ca release in CPVT. However, the molecular mechanism underlying the protective Ca buffering function of CPVT mitochondria remains unclear. Hypothesis: We hypothesize that the tethering between SR and mitochondria, also known as mitochondria-associated-membranes (MAMs) are promoted in CPVT to facilitate SR-mitochondria Ca transfer. Moreover, manipulating SR-mitochondria tethering or MAMs impacts arrhythmogenesis in CPVT. Methods: Ventricular myocytes were isolated from a CPVT model of CASQ2 knockout (Cnull) mouse. Cellular immunofluorescence assays and western blots were employed to detect MAMs remodeling in CPVT cells. Pharmacological approach was employed to disrupt MAMs and examine its effect on cellular arrhythmogenesis using live-cell imaging. Results: SR-mitochondria tethering was assessed by examining the interaction between RyR2 and Voltage-dependent anion channel (VDAC), a protein localized in the mitochondrial-outer-membrane. As compared with wild-type (WT), we detected a higher degree of colocalization of immunofluorescence between RyR2 and VDAC in Cnull cells, as well as increased RyR2-VDAC interactions by proximity ligation assay. The expression of SR-mitochondria tethering protein Mitofusin2 was increased in MAMs isolated from CPVT hearts. Colchicine was employed to induce a partial disruption of MAMs in Cnull cells. In intact Cnull cells perfused with β agonist isoproterenol, colchicine exacerbated arrhythmogenic Ca waves. In permeabilized cells, colchicine increased the frequency of arrhythmogenic Ca waves and reduced mitochondrial Ca uptake. Conclusions: Our results support that MAMs are promoted in CPVT cells to facilitate SR-mitochondria Ca transfer so mitochondria can function as a protective Ca buffer. Pharmacological disruption of SR-mitochondria tethering limits SR-mitochondria Ca transfer, thus exacerbating cellular arrhythmic burden.

Commentary : Combating COVID-19-associated mucormycosis with limited resources.

Two long and critical years have passed since the onset of coronavirus disease 2019 (COVID-19). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused by COVID-19 has affected over 40 million of the Indian population, with over 500 thousand people losing their lives due to either by SARS-CoV-2 or other co-infections associated with it.[1] Invasive rhino-orbito-cerebral mucormycosis (ROCM) was identified as one of the fatal co-infections concurrent with or post COVID-19, with India alone contributing more than 81% of cases globally.[2] Diabetes mellitus, diabetic ketoacidosis, systemic corticosteroids, immunosuppression, malignancy, especially hematological, are all known risk factors of predisposing ROCM. The ROCM form of mucormycosis is more common in COVID-19 infection than other disseminated forms such as cutaneous or pulmonary (ROCM) associated with COVID19, that is, COVID-19-associated mucormycosis (CAM). The largest multicentric data of CAM was the Collaborative OPAI-IJO Study on Mucormycosis in COVID-19 (COSMIC), documenting 78% of patients to have diabetes mellitus. Fourteen percent of patients suffered death by CAM while 16% underwent orbital exenteration. twenty-one percent of patients lost their vision with stable disease post treatment.[3] The current series in this issue of IJO includes 49 cases of CAM treated over a six-month period in a single center. The authors noted that 67.7% of patients had diabetes mellitus and 28% of patients had hypertension as the commonest associations apart from 47% of the patients being COVID-19 positive. More than 38% received steroids, either intravenously (IV) or orally, to deal effectively with cytokine response driving COVID-19-related pneumonitis and acute respiratory distress syndrome. Supplemental oxygen or ventilatory support was required in 29% of the patients. Over 90% of the affected patients were either unvaccinated, and none were fully vaccinated. In ROCM care, a multidisciplinary approach as well as multiple medical and surgical options are often recruited for curbing the disease. Medical management demands specialized antifungals such as IV liposomal amphotericin B and other oral antifungal medications of the azole group on discharge from the hospital, such as posoconazole. Although authors have not faced any demand and supply mismatch in this particular study, medical practitioners from several parts of India faced severe shortages of amphotericin B (liposomal form) and were compelled to use the lyophilized form of the same. The latter form, being nephrotoxic, also demanded close monitoring of renal parameters, causing additional burden to an already strained health care system. The costs of medical and surgical treatment of ROCM, excluding hospital stay, may approach somewhere around 1000 USD.[4] CAM affected young males <60 years, who are the financial backbones of Indian families. The fact that this caused social strain during second wave of COVID-19 pandemic can’t be denied. This study has reciprocated the same increased incidence in males. A total of 57% received transcutaneous retrobulbar amphotericin B (TRAMB), though the exact number of patients in whom globe could be salvaged is not clearly attributed here. Effectivity of TRAMB versus exenteration has surely been debated ever since, due to lack of randomized control trial on the same.[56] In CAM, use of TRAMB has been studied widely across the country in multiple studies, but there is still a lack of uniform criteria of indications of TRAMB and its outcomes. In caring for around 49 patients with CAM, the authors had unique clinical expertise as well as a valuable database of patient outcomes. Like in any case series database, however, there were limitations to this study: lack of randomization of treatment, and detailing about imaging, as well as flaws in the lack of azole treatment in the stable period. Although the authors understandably sought to derive maximal insights from their data, mortality rate and exenteration rates are in co-ordination with other parallel studies. This study definitely adds to the literature in the form of long-term follow up and outcome of CAM patients, which has not been studied in detail until now.

Voltage-mediated mechanism for calcium wave synchronization and arrhythmogenesis in atrial tissue

A wide range of atrial arrythmias are caused by molecular defects in proteins that regulate calcium (Ca) cycling. In many cases, these defects promote the propagation of subcellular Ca waves in the cell, which can perturb the voltage time course and induce dangerous perturbations of the action potential (AP). However, subcellular Ca waves occur randomly in cells and, therefore, electrical coupling between cells substantially decreases their effect on the AP. In this study, we present evidence that Ca waves in atrial tissue can synchronize in-phase owing to an order-disorder phase transition. In particular, we show that, below a critical pacing rate, Ca waves are desynchronized and therefore do not induce substantial AP fluctuations in tissue. However, above this critical pacing rate, Ca waves gradually synchronize over millions of cells, which leads to a dramatic amplification of AP fluctuations. We exploit an underlying Ising symmetry of paced cardiac tissue to show that this transition exhibits universal properties common to a wide range of physical systems in nature. Finally, we show that in the heart, phase synchronization induces spatially out-of-phase AP duration alternans which drives wave break and reentry. These results suggest that cardiac tissue exhibits a phase transition that is required for subcellular Ca cycling defects to induce a life-threatening arrhythmia.

SR-Mitochondria Crosstalk Shapes Ca Signalling to Impact Pathophenotype in Disease Models Marked by Dysregulated Intracellular Ca Release.

Diastolic Ca release (DCR) from sarcoplasmic reticulum (SR) Ca release channel ryanodine receptor (RyR2) has been linked to multiple cardiac pathologies, but its exact role in shaping divergent cardiac pathologies remains unclear. We hypothesize that the SR-mitochondria interplay contributes to disease phenotypes by shaping Ca signalling. A genetic model of catecholaminergic polymorphic ventricular tachycardia (CPVT2 model of CASQ2 knockout) and a pre-diabetic cardiomyopathy model of fructose-fed mice (FFD), both marked by DCR, are employed in this study. Mitochondria Ca (mCa) is modulated by pharmacologically targeting mitochondria Ca uniporter (MCU) or permeability transition pore (mPTP), mCa uptake, and extrusion mechanisms, respectively. An MCU activator abolished Ca waves in CPVT2 but exacerbated waves in FFD cells. Mechanistically this is ascribed to mitochondria's function as a Ca buffer or source of reactive oxygen species (mtROS) to exacerbate RyR2 functionality, respectively. Enhancing mCa uptake reduced and elevated mtROS production in CPVT2 and FFD, respectively. In CPVT2, mitochondria took up more Ca in permeabilized cells, and had higher level of mCa content in intact cells vs. FFD. Conditional ablation of MCU in the CPVT2 model caused lethality and cardiac remodelling, but reduced arrhythmias in the FFD model. In parallel, CPVT2 mitochondria also employ up-regulated mPTP-mediated Ca efflux to avoid mCa overload, as seen by elevated incidence of MitoWinks (an indicator of mPTP-mediated Ca efflux) vs. FFD. Both pharmacological and genetic inhibition of mPTP promoted mtROS production and exacerbation of myocyte Ca handling in CPVT2. Further, genetic inhibition of mPTP exacerbated arrhythmias in CPVT2. In contrast to FFD, which is more susceptible to mtROS-dependent RyR2 leak, in CPVT2 mitochondria buffer SR-derived DCR to mitigate Ca-dependent pathological remodelling and rely on mPTP-mediated Ca efflux to avoid mCa overload. SR-mitochondria interplay contributes to the divergent pathologies by disparately shaping intracellular Ca signalling.

Increasing SERCA function promotes initiation of calcium sparks and breakup of calcium waves.

Increasing sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump activity enhances sarcoplasmic reticulum calcium (Ca) load, which increases both ryanodine receptor opening and driving force of Ca release flux. Both of these effects promote Ca spark formation and wave propagation. However, increasing SERCA activity also accelerates local cytosolic Ca decay as the wave front travels to the next cluster, which limits wave propagation. As a result, increasing SERCA pump activity has a biphasic effect on the propensity of arrhythmogenic Ca waves, but a monotonic effect to increase Ca spark frequency and amplitude. Waves of sarcoplasmic reticulum (SR) calcium (Ca) release can cause arrhythmogenic afterdepolarizations in cardiac myocytes. Ca waves propagate when Ca sparks at one Ca release unit (CRU) recruit new Ca sparks in neighbouring CRUs. Under normal conditions, Ca sparks are too small to recruit neighbouring Ca sparks where Ca sensitivity is also low. However, under pathological conditions such as a Ca overload or ryanodine receptor (RyR) sensitization, Ca sparks can be larger and propagate more readily as macro-sparks or full Ca waves. Increasing SERCA pump activity promotes SR Ca load, which promotes RyR opening and increases driving force of the Ca release flux from SR to cytosol, promoting Ca waves. However, high sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) activity can also decrease local cytosolic [Ca] as it approaches the next CRU, thereby reducing wave appearance and propagation. In this study, we use a physiologically detailed model of subcellular Ca cycling and experiments in phospholamban-knockout mice, to show how Ca waves are initiated and propagate and how different conditions contribute to the generation and propagation of Ca waves. We show that reducing diffusive coupling between Ca sparks by increasing SERCA activity prevents Ca waves by reducing [Ca] at the next CRU, as do Ca buffers, low intra-SR Ca diffusion and distance between CRUs. Increasing SR Ca uptake rate has a biphasic effect on Ca wave propagation; initially it enhances Ca spark probability and amplitude and CRU coupling, thereby promoting arrhythmogenic Ca wave propagation, but at higher levels SR Ca uptake can abort those arrhythmogenic Ca waves.

Mechanistic link between CaM-RyR2 interactions and the genesis of cardiac arrhythmia

In this study, we develop a computational model of the interaction between ryanodine receptor type 2 (RyR2) and calmodulin (CaM) to explore the mechanistic link between CaM-RyR2 interactions and cardiac arrhythmia. Our starting point is a biophysically based computational model of CaM binding to a single RyR2 subunit, which reproduces single-channel RyR2 measurements in lipid bilayers. We then integrate this CaM-RyR2 model into a spatially distributed whole-cell model of Ca cycling, which is used to investigate the relationship between CaM and Ca cycling homeostasis. We show that a reduction in CaM concentration leads to a substantial increase in the rate of spontaneous Ca sparks, and this induces a marked reduction in sarcoplasmic reticulum Ca load during steady-state pacing. Also, we show that a reduction in CaM modifies the RyR2 open probability, which makes the cell more prone to Ca wave propagation. These results indicate that aberrant Ca cycling activity during pacing is determined by the interplay between sarcoplasmic reticulum load reduction and the threshold for Ca wave propagation. Based on these results, we show that when CaM is reduced, Ca waves can occur in a cell and induce action potential perturbations that are arrhythmogenic. Thus, this study outlines a novel, to our knowledge, mechanistic link between CaM-RyR2 binding kinetics and the induction of arrhythmias in the heart.

Suppression of ventricular arrhythmia by mitochondrial calcium uptake via mitochondrial calcium uniporter in the ischemic heart failure mice

Abstract Background In heart failure (HF), diastolic calcium (Ca) leak from sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) causes delayed after depolarization (DAD), leading ventricular arrhythmias (VAs). Recent study reported that Ca uptake into mitochondria via mitochondrial calcium uniporter (MCU) suppress Ca waves (CaWs) and DAD in catecholaminergic polymorphic ventricular tachycardia, in which diastolic Ca leak is thought to be a major cause of VAs as in HF. However, such anti-arrhythmic effect of mitochondrial Ca uptake via MCU remains unclear in HF. Purpose We sought to investigate whether mitochondrial Ca uptake via MCU decreases CAWs and VAs incidence in ischemic HF mice. Methods Ten-week-old male C57BL/6J mice were divided into 2 groups; sham operation mice (Sham) or HF mice (HF) in which myocardial infarction was induced by left coronary artery ligation. After 4–6 weeks, cardiomyocyte or mitochondria was isolated respectively from the myocardium of Sham and the non-infarct myocardium of HF. Influence of MCU activation on Ca dynamics, VA inducibility and left ventricular hemodynamics were evaluated using Kaemenpferol, a MCU activator. Intracellular Ca dynamics and mitochondrial Ca uptake were measured in isolated cardiomyocytes loaded with Fluo-4 AM on an epifluorescence microscopy and by estimating the extra-mitochondrial Ca reduction with Fluo-5N on a spectrofluoro-photometer, respectively. VAs was induced by programmed stimulation in the Langendorff perfused hearts. Left ventricular (LV) pressure was measured using a microtip transducer catheter. Finally, the effect of intravenous administration of Kaempferol (5mg/kg) on hemodynamic parameters was examined 30 minutes after administration in Sham and HF. Results HF mice showed left ventricular dysfunction, as well as the increased heart and lung weights compared to Sham. MCU protein expression in cardiomyocytes did not differ between Sham and HF. Kaempferol increased mitochondrial Ca uptake in the isolated mitochondria both in Sham and HF. The number of the diastolic CaWs was higher in HF compared to Sham. Such increased number of CaWs in HF was attenuated by 10 μM Kaempferol, which was, however, abolished by a MCU blocker Ruthenium Red. The incidence of induced VA was significantly higher in HF than Sham, which was suppressed by Kaempferol. In vivo measurements, intravenous administration of Kaempferol did not show significant changes in hemodynamic parameters in Sham and HF mice. Conclusions Mitochondrial Ca uptake via MCU suppresses CaWs and VAs, but did not change LV hemodynamics in HF. Whereas traditional antiarrhythmic drugs have limited use in heart failure patients, a novel strategy that promotes Ca uptake into mitochondria might be a new and safer option for treating VAs in HF. Funding Acknowledgement Type of funding source: None

Iron Overload, Oxidative Stress and Calcium Mishandling in Cardiomyocytes: Role of the Mitochondrial Permeability Transition Pore.

Iron (Fe) plays an essential role in many physiological processes. Hereditary hemochromatosis or frequent blood transfusions often cause iron overload (IO), which can lead to cardiomyopathy and arrhythmias; however, the underlying mechanism is not well defined. In the present study, we assess the hypothesis that IO promotes arrhythmias via reactive oxygen species (ROS) production, mitochondrial membrane potential (∆Ψm) depolarization, and disruption of cytosolic Ca dynamics. In ventricular myocytes isolated from wild type (WT) mice, both cytosolic and mitochondrial Fe levels were elevated following perfusion with the Fe3+/8-hydroxyquinoline (8-HQ) complex. IO promoted mitochondrial superoxide generation (measured using MitoSOX Red) and induced the depolarization of the ΔΨm (measured using tetramethylrhodamine methyl ester, TMRM) in a dose-dependent manner. IO significantly increased the rate of Ca wave (CaW) formation measured in isolated ventricular myocytes using Fluo-4. Furthermore, in ex-vivo Langendorff-perfused hearts, IO increased arrhythmia scores as evaluated by ECG recordings under programmed S1-S2 stimulation protocols. We also carried out similar experiments in cyclophilin D knockout (CypD KO) mice in which the mitochondrial permeability transition pore (mPTP) opening is impaired. While comparable cytosolic and mitochondrial Fe load, mitochondrial ROS production, and depolarization of the ∆Ψm were observed in ventricular myocytes isolated from both WT and CypD KO mice, the rate of CaW formation in isolated cells and the arrhythmia scores in ex-vivo hearts were significantly lower in CypD KO mice compared to those observed in WT mice under conditions of IO. The mPTP inhibitor cyclosporine A (CsA, 1 µM) also exhibited a protective effect. In conclusion, our results suggest that IO induces mitochondrial ROS generation and ∆Ψm depolarization, thus opening the mPTP, thereby promoting CaWs and cardiac arrhythmias. Conversely, the inhibition of mPTP ameliorates the proarrhythmic effects of IO.

Tales of a Master’s Uncertainty

Tales of a Master’s Uncertainty Joshua Parker Philip Horne, editor. Tales from a Master’s Notebook: Stories Henry James Never Wrote. Vintage, 2018. xiv + 255 pp. $17.19 (hardcover). Mary F. Burns. At Chalk Farm. San Francisco: Word by Word, 2019. 187 pp. $8.95 (paperback). Mary F. Burns. The Grace of Uncertainty. San Francisco: Word by Word, 2019. 102 pp. $5.95 (paperback). Michael Woods’s foreword to this short story collection speaks of ghosts: “Tales from a Master’s Notebook is full of ghosts,” the “undead” (xii). Unlike novels, by definition offering new, original narratives, the tale, etymologically related to “talk, or telling,” is “an account of things in their due order,” or, perhaps more interestingly, “things divulged that were given secretly, gossip.” Its German root word is linked with reckoning and numbers. Here, eleven tales differing in tone, theme, and setting have a common source as unfinished story sketches in James’s nine surviving notebooks covering the years 1878 to 1911 (recently available online with all of James’s “original hieroglyphics”), which Philip Horne has been re-editing. “Why these particular volumes survive and the rest were chucked into the flames at Lamb House is something of a puzzle,” Horne writes. [End Page E-1] Horne lays out an overview of precursors who have taken up James’s unfinished plot sketches, from David Lodge to Joyce Carol Oates to John Banville (leaving aside facetious parodists like Max Beerbohm and H. G. Wells). Horne asked ten authors to choose a plot sketch from entries in James’s notebooks (James’s “germs”) and to make “whatever loose arrangements they wished to make, quite privately, with the spirit of James” (12). “Germs,” Horne calls them, quickly clarifying: as in the sense of seeds (3). Much as Roland Barthes suggested, the “soul” of any narrative function is “its seedlike quality, which enables the function to inseminate the narrative with an element that will later come to materiality, on the same level, or elsewhere on another level” (Barthes 244). You might best begin with the volume’s appendix. Read the plot sketches from James’s notebooks, then choose which tale you’d like to see fleshed out by the authors Horne has selected. James left open patches in the plots sketched in his notebooks. Would a husband take back a straying wife? How long will love wait? Will X marry Y, or will Y abscond with Z? At times James’s notes seem to indicate he felt his tales might incarnate better at a later date, even decades later. In the hands of these authors, James’s plots are, if not quite infected with James’s style, haunting. James haunts us, but these authors haunt him. I started with Colm Tóibín’s take on 34 D[e].V[ere].G[ardens]. Jan. 23, 1894: “Silence.” James’s 1894 journal records a story told to James by Lady Gregory: a wife’s passion leads her to run away from her husband and children. Her lover having deserted her, she returns home, on the condition that she leave the family forever once her children are grown—on a fixed date and hour. The hour strikes. Mayhem ensues as she leaves the family in “bewilderment and distress,” appalled and with “the most terrible effect on them” (CN 84–86). Horne’s footnotes are wonderful.1 Tóibín’s tale works in flashbacks, from the perspective of the wife, now an aging dowager. The Dowager describes in detail a miserable love life with her late husband, a man twice her age. Stylistically, James is in there. But there’s also Djuna Barnes, Ian McEwan, Lorrie Moore, and one senses how much the intervening waves of modernism, postmodernism, neorealism, and minimalism can’t help but color the style of contemporary authors, even those most eager to follow the lessons of the master. Spirits of the living mingle with those of the dead. Tóibín’s fictional husband, a retired MP, and his wife go to Cairo, where a young poet and his wife are by chance staying at the same hotel. The two couples dine together each night. The dinner scenes, as...

Remodeling Promotes Proarrhythmic Disruption of Calcium Homeostasis in Failing Atrial Myocytes

It is well known that heart failure (HF) typically coexists with atrial fibrillation (AF). However, until now, no clear mechanism has been established that relates HF to AF. In this study, we apply a multiscale computational framework to establish a mechanistic link between atrial myocyte structural remodeling in HF and AF. Using a spatially distributed model of calcium (Ca) signaling, we show that disruption of the spatial relationship between L-type Ca channels (LCCs) and ryanodine receptors results in markedly increased Ca content of the sarcoplasmic reticulum (SR). This increase in SR load is due to changes in the balance between Ca entry via LCCs and Ca extrusion due to the sodium-calcium exchanger after an altered spatial relationship between these signaling proteins. Next, we show that the increased SR load in atrial myocytes predisposes these cells to subcellular Ca waves that occur during the action potential (AP) and are triggered by LCC openings. These waves are common in atrial cells because of the absence of a well-developed t-tubule system in most of these cells. This distinct spatial architecture allows for the presence of a large pool of orphaned ryanodine receptors, which can fire and sustain Ca waves during the AP. Finally, we incorporate our atrial cell model in two-dimensional tissue simulations and demonstrate that triggered wave generation in cells leads to electrical waves in tissue that tend to fractionate to form wavelets of excitation. This fractionation is driven by the underlying stochasticity of subcellular Ca waves, which perturbs AP repolarization and consequently induces localized conduction block in tissue. We outline the mechanism for this effect and argue that it may explain the propensity for atrial arrhythmias in HF.

Exploring Impaired SERCA Pump-Caused Alternation Occurrence in Ischemia.

Impaired sarcoplasmic reticulum (SR) calcium transport ATPase (SERCA) gives rise to Ca2+ alternans and changes of the Ca2+release amount. These changes in Ca2+ release amount can reveal the mechanism underlying how the interaction between Ca2+ release and Ca2+ uptake induces Ca2+ alternans. This study of alternans by calculating the values of Ca2+ release properties with impaired SERCA has not been explored before. Here, we induced Ca2+ alternans by using an impaired SERCA pump under ischemic conditions. The results showed that the recruitment and refractoriness of the Ca2+ release increased as Ca2+ alternans occurred. This indicates triggering Ca waves. As the propagation of Ca waves is linked to the occurrence of Ca2+ alternans, the “threshold” for Ca waves reflects the key factor in Ca2+ alternans development, and it is still controversial nowadays. We proposed the ratio between the diastolic network SR (NSR) Ca content (Cansr) and the cytoplasmic Ca content (Cai) (Cansr/Cai) as the “threshold” of Ca waves and Ca2+ alternans. Diastolic Cansr, Cai, and their ratio were recorded at the onset of Ca2+ alternans. Compared with certain Cansr and Cai, the “threshold” of the ratio can better explain the comprehensive effects of the Ca2+ release and the Ca2+ uptake on Ca2+ alternans onset. In addition, these ratios are related with the function of SERCA pumps, which vary with different ischemic conditions. Thus, values of these ratios could be used to differentiate Ca2+ alternans from different ischemic cases. This agrees with some experimental results. Therefore, the certain value of diastolic Cansr/Cai can be the better “threshold” for Ca waves and Ca2+ alternans.

Coordinated calcium signalling in cochlear sensory and non‐sensory cells refines afferent innervation of outer hair cells

Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine‐tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca2+ signals originating in OHCs and non‐sensory cells. OHCs fire spontaneous Ca2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca2+ activity in the non‐sensory cells of the greater epithelial ridge cause, via ATP‐induced activation of P2X3 receptors, the increase and synchronization of the Ca2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience‐independent Ca2+ signals from sensory and non‐sensory cells.

The Role of Cysteine 3602 in RyR2 Regulation by Calmodulin and Oxidative Stress

In the skeletal ryanodine receptor (RyR1), cysteine 3635 (C3635) is considered to be an important residue responsible for the redox regulation of the channel. It has been shown that oxidation of C3635 causes intersubunit cross-linking and calmodulin (CaM) dissociation from RyR1. These structural modifications can lead to channel activation. While the role of RyR1 C3635 has been extensively studied, the functional significance of the corresponding cysteine 3602 (C3602) in the cardiac RyR (RyR2) remains unclear. To characterize the role of C3602 in RyR2 response to oxidative stress, we co-expressed either wild type (WT) GFP-RyR2 or the single mutant C3602A GFP-RyR2, together with the ER-targeted Ca sensor R-CEPIA1er and SERCA2a, in HEK293 cells. Cells transfected with either WT- or C3602A-RyR2 produced Ca waves that were sensitive to oxidative stress induced by diamide. Diamide equally increased the endoplasmic reticulum (ER) Ca leak in the cells expressing WT- and the C3602A-RyR2 mutant. Both WT-RyR2 and the mutant RyR2 expressed in HEK293 cells demonstrated the same level of disulfide cross-linking upon diamide treatment. Moreover, the nitric oxide donor NOC-12 had similar effect on ER Ca leak via WT and the mutant RyR2. Given the localization of C3602 within the CaM binding site in RyR2, we assessed the effect of C3602A mutation on the degree of CaM dissociation during oxidative stress. The C3602A mutation did not significantly affect CaM binding to RyR2 during oxidative stress, and the degree of this binding was comparable to the one observed in WT RyR2. Taken together, these results suggest that C3602 does not play a key role in RyR2 response to oxidative stress. Despite ∼70% homology between RyR1 and RyR2, these channels appear to exhibit important structural and functional differences, particularly with respect to redox regulation.

Size Matters: Ryanodine Receptor Cluster Size Heterogeneity Potentiates Calcium Waves

Ryanodine receptors (RyRs) mediate calcium (Ca)-induced Ca release and intracellular Ca homeostasis. In a cardiac myocyte, RyRs group into clusters of variable size from a few to several hundred RyRs, creating a spatially nonuniform intracellular distribution. It is unclear how heterogeneity of RyR cluster size alters spontaneous sarcoplasmic reticulum (SR) Ca releases (Ca sparks) and arrhythmogenic Ca waves. Here, we tested the impact of heterogeneous RyR cluster size on the initiation of Ca waves. Experimentally, we measured RyR cluster sizes at Ca spark sites in rat ventricular myocytes and further tested functional impacts using a physiologically detailed computational model with spatial and stochastic intracellular Ca dynamics. We found that the spark frequency and amplitude increase nonlinearly with the size of RyR clusters. Larger RyR clusters have lower SR Ca release threshold for local Ca spark initiation and exhibit steeper SR Ca release versus SR Ca load relationship. However, larger RyR clusters tend to lower SR Ca load because of the higher Ca leak rate. Conversely, smaller clusters have a higher threshold and a lower leak, which tends to increase SR Ca load. At the myocyte level, homogeneously large or small RyR clusters limit Ca waves (because of low load for large clusters but low excitability for small clusters). Mixtures of large and small RyR clusters potentiates Ca waves because the enhanced SR Ca load driven by smaller clusters enables Ca wave initiation and propagation from larger RyR clusters. Our study suggests that a spatially heterogeneous distribution of RyR cluster size under pathological conditions may potentiate Ca waves and thus afterdepolarizations and triggered arrhythmias.