SR Ca2 Research Articles

Ca2+ waves and the arrhythmogenicity paradigm: role of SERCA stimulation in a CPVT mouse model

Channelopathies of the cardiac ryanodine receptor (RyR2) can predispose to life threatening catecholaminergic polymorphic ventricular tachycardias (CPVTs) during exercise or stress, however the underlaying mechanism involving complex signaling pathways is not yet fully understood. Furthermore, the resulting arrhythmogenic events on the cellular level are most likely quite diverse, depending on the mutated residue on the RyR2. Obvious possibilities are the phosphorylation of the mutated RyR2s or the stimulation of the SR Ca2+ pump (SERCA), which could increase SR Ca2+ loading.

FRET assays for RyR-targeted drug discovery platforms

Using fluorescence lifetime FRET-based high-throughput screening (HTS) assays, we have identified compounds that modulate the sarcoplasmic reticulum's (SR) Ca2+ gatekeeper, the ryanodine receptor (RyR) channel. Intracellular Ca2+ regulation is critical for striated muscle function, and SR Ca2+ release via opening of RyR is essential for triggering muscle contraction. Under cellular rest, increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, and neurodegenerative diseases.

Nanoscale Organization, Regulation, and Dynamic Reorganization of Cardiac Calcium Channels.

The architectural specializations and targeted delivery pathways of cardiomyocytes ensure that L-type Ca2+ channels (CaV1.2) are concentrated on the t-tubule sarcolemma within nanometers of their intracellular partners the type 2 ryanodine receptors (RyR2) which cluster on the junctional sarcoplasmic reticulum (jSR). The organization and distribution of these two groups of cardiac calcium channel clusters critically underlies the uniform contraction of the myocardium. Ca2+ signaling between these two sets of adjacent clusters produces Ca2+ sparks that in health, cannot escalate into Ca2+ waves because there is sufficient separation of adjacent clusters so that the release of Ca2+ from one RyR2 cluster or supercluster, cannot activate and sustain the release of Ca2+ from neighboring clusters. Instead, thousands of these Ca2+ release units (CRUs) generate near simultaneous Ca2+ sparks across every cardiomyocyte during the action potential when calcium induced calcium release from RyR2 is stimulated by depolarization induced Ca2+ influx through voltage dependent CaV1.2 channel clusters. These sparks summate to generate a global Ca2+ transient that activates the myofilaments and thus the electrical signal of the action potential is transduced into a functional output, myocardial contraction. To generate more, or less contractile force to match the hemodynamic and metabolic demands of the body, the heart responds to β-adrenergic signaling by altering activity of calcium channels to tune excitation-contraction coupling accordingly. Recent accumulating evidence suggests that this tuning process also involves altered expression, and dynamic reorganization of CaV1.2 and RyR2 channels on their respective membranes to control the amplitude of Ca2+ entry, SR Ca2+ release and myocardial function. In heart failure and aging, altered distribution and reorganization of these key Ca2+ signaling proteins occurs alongside architectural remodeling and is thought to contribute to impaired contractile function. In the present review we discuss these latest developments, their implications, and future questions to be addressed.

Abstract 9052: Upregulation of Cardiac SERCA 2a is a Key Molecular Mechanism of the Protective Effect on Myocardial Aging in Beta3-Adrenergic Receptor Deficiency Mice

Background: We have shown previously that aging-induced cardiomyocyte dysfunction and impaired β-adrenergic regulation were prevented in β 3 -adrenergic receptor (AR) knockout (β 3 KO) aged mice. However, the molecular mechanism is unclear. We hypothesize that reversal of aging-induced down-regulation of SR Ca 2+ -ATPase (SERCA 2a) and β 1 -ARs by β 3 -AR deficiency may play a key role for the protective effect. Methods: We compared cardiac SERCA 2a, phospholamban (PLB), β 1 - and β 3 -AR protein expression and myocyte contractile and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to isoproterenol (ISO, 10 -8 M) in left ventriclar (LV) myocytes obtained from 2 young (Y)(~4-6 mo) and 2 aged (A) (~18-24 mo) groups (7/group) of wild-type (WT) and β 3 KO mice, respectively. Results: Compared with YWT, AWT myocytes had significantly decreased SERCA 2a (66%, AWT: 0.21 vs YWT: 0.61), SERCA 2a/PLB ratio (0.76 vs 1.77) and β 1 -AR (33%, 0.39 vs 0.58), but increased β 3 -AR (93%, 0.27 vs 0.14). These changes were associated with reduced basal cell contraction (dL/dt max ) (AWT: 84.3 vs YWT: 124.8 μm/s), relaxation (dR/dt max ) (60.1 vs 89.8 μm/s) and [Ca 2+ ] iT (0.17 vs 0.22). This was accompanied by diminished ISO-stimulated positive inotropic effect; in AWT myocytes, ISO (10 -8 M) caused significantly less increases in dL/dt max (AWT: 33% vs YWT: 81%), dR/dt max (24% vs 58%), and [Ca 2+ ] iT (14% vs 34%). Compared with YWT, Yβ 3 KO did not alter basal contraction and ISO response of myocytes, but SERCA 2a (Yβ 3 KO: 1.2 vs YWT: 0.61) and SERCA 2a/PLB ratio were doubled with relatively unchanged β 1 -AR (0.62 vs 0.58); Aβ 3 KO mice had similar alterations. Importantly, in contrast to AWT, in Aβ 3 KO myocytes, the increased SERCA 2a (1.1) and β 1 -AR (0.61) correlated with normal basal cell contraction and relaxation with preserved ISO-stimulated positive inotropic response. ISO caused similar increases in dL/dt max (84% vs 82%) and [Ca 2+ ] iT (32% vs 34%) compared to Yβ 3 KO mice. Conclusions: Chronic β 3 -AR deficiency upregulates cardiac SERCA 2a, prevents aging-induced downregulation of β 1 -ARs, and leads to the preservation of myocyte function, [Ca 2+ ] iT , and β-adrenergic responsiveness in aged hearts. Thus, antagonizing β 3 -AR could be a new therapeutic strategy for age-related decline in myocardial function.

Abstract 9024: Molecular Mechanism of Chronic Beta3-Adrenergic Receptor Antagonist Caused Regression of Diabetic Cardiomyopathy in a Murine Model With Type 2 Diabetes

Background: Diabetic cardiomyopathy (DCM) is a serious complication of diabetes (DM). As yet, no effective therapeutic strategies exist. Recently, we found that DM-induced LV dysfunction was prevented by chronic treatment with a β 3 -adrenergic receptor (AR) antagonist (β 3 ANT). However, the molecular mechanism is unclear. We hypothesized that reversal of DM-induced detrimental alterations on the expressions of cardiac SR Ca 2+ -ATPase (SERCA2a), β-ARs, nitric oxide synthase (NOS) by β 3 ANT may play a key role for its salutary role in DCM. Methods: The protein levels of myocyte β 1 - and β 3 - AR, SERCA2a, NOS and myocyte functional responses were determined in 3 groups (7/group) wild-type female mice over 14 weeks (W): Type 2 DM ( T2 ), 14 W fed a high-fat diet (HFD), but after HFD for 4 W receiving streptozotocin (STZ, 40 mg/kg/day, i.p. 5 days); T2β 3 ANT , T2 mice at 10 W received L-748,337, a selective β 3 ANT (10 -7 M/kg/day, mini-pump) for 4 W; and Vehicle controls (C). Results: Versus C, T2DM was induced in mice received HFD and low dose STZ with significantly elevated blood glucose (~340 to 380 mg/dl). Only T2 mice had significantly decreased protein levels of LV myocyte β 1 -AR (T2: 0.35 vs C: 0.49) and SERCA2a (0.19 vs 0.29), but increased β 3 -AR (0.25 vs 0.14) and iNOS (0.25 vs 0.15) without significant changes in eNOS (0.14 vs 0.13) and nNOS. These changes were followed by significantly reduced basal cell contraction (dL/dt max , 75.4 vs 133.7 μm/s), relaxation (dR/dt max , 59.6 vs 113.8 μ m/s) and [Ca 2+ ] iT (0.16 vs 0.21). The isoproterenol (ISO, 10 -8 M)-stimulated increases in dL/dt max (38% vs 58%), dR/dt max (34% vs 55%) and [Ca 2+ ] iT (19% vs 30%) were also significantly reduced. By contrary, versus C, T2β 3 ANT myocytes had similar protein levels of β 1 -AR (0.52), SERCA2a (0.75) and iNOS (0.36) with preserved normal basal cell contractility (127.8 μm/s), relaxation (109.4 μm/s) and [Ca 2+ ] iT (0.21). ISO caused increases in dL/dt max (57%), dL/dt max (52%), and [Ca 2+ ] iT (30%) were also closed to control values. Conclusions: Chronic β 3 ANT prevents DCM-caused downregulations of cardiac β 1 -ARs and SERCA2a, and normalized upregulations of iNOS and β 3 -AR, thus leading to the preservation of normal myocyte contractile function, [Ca 2+ ] iT , and β-adrenergic reserve in a mouse model of T2DM.

Fragmentation and roles of junctophilin1 in muscle of patients with cytosolic leak of stored calcium

The mechanisms that link the primary increase in SR Ca2+ leak of MH susceptibility and related conditions to their disease phenotypes are not well understood. We found that abnormal Ca2+ homeostasis in MHS individuals induces proteolysis of junctophilin1 (JPh1), an essential structural protein of EC coupling (Perni, in 2017). Guo (in 2018) and Lahiri (in 2020) reported similar fragmentation of JPh2 in stressed hearts. Western blot of patients' muscle with domain-specific antibodies showed a deficit of full-length JPh1 and excess of a 44-kD C-terminal fragment (JPh44) in MHS subjects. While JPh1 was located in T-SR junctions, JPh44 was found anywhere within the I band, and at high densities within nuclei-a location forbidden for JPh1. Expression and cleavage in mice of a JPh1 plasmid tagged at both ends showed that its N-terminal fragment remained in triads, and the C-terminal fragment, orthologue to JPh44, entered nuclei, which indicates that JPh44 is the C-terminal cleavage product. Endogenous calpain1 appeared in T-SR junctions, colocalized with JPh1. On muscle extracts and primary cultures, Ca2+-activated calpain1 cleaved a 44-kD JPh1 piece, consistent with the C-terminal fragment that starts at Ser241, the highest probability cleavage site found by calpain1 algorithms. Completing the identification of Ser241 as the likely start of JPh44, the tagged deletion plasmid GFP-JPh1_Δ1-240, expressed in mice, copied the location and migration of JPh44. Expression of GFP-JPh1_Δ1-240 in C2C12 myoblasts reduced by more than twofold the transcription of PI3K-Akt genes that inhibit muscle uptake and storage of glucose, including GSK3β, an inhibitor of glycogen synthase that is activated in MHS patients. In agreement with the genetic profile, GSK3β protein content decreased upon expression of GFP-JPh1_Δ1-240. In sum, the identified gene control roles of JPh44 oppose the deleterious effects of chronically elevated cytosolic [Ca2+], including late-onset hyperglycemia and type-2 diabetes (Tammineni, in 2020).

Mitochondrial calpain inhibition restores defective SR-mitochondrial crosstalk in CPVT rat myocytes

Cardiac RYR2-mediated sarcoplasmic Ca2+ (SR) release is essential for matching increased energy demand during fight-or-flight response with mitochondrial metabolic output by delivering Ca2+ into the mitochondrial matrix to activate Ca2+-dependent Krebs cycle dehydrogenases. RYR2 complex gain-of-function mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) have been linked to mitochondrial structural damage and enhanced production of reactive oxygen species (ROS). Despite being critical for arrhythmogenesis in CPVT, the exact causes of these phenomena remain undetermined. Taking advantage of a new rat model of CPVT induced by heterozygous RYR2 gain-of-function mutation S2222L, we tested how RYR2 overactivity alters mitochondrial Ca2+ and ROS handling, and how these changes cause mitochondrial structural defects. Injection of epinephrine (1 mg/kg) and caffeine (120 mg/kg) induced bigamy and bidirectional VT in vivo in 100% of CPVT rats. Simultaneous whole-cell patch clamp and confocal Ca2+-imaging demonstrated that under β-adrenergic stimulation with isoproterenol (50 nM), CPVT ventricular myocytes (VMs) exhibited severe Ca2+ mishandling and high propensity for generation of spontaneous Ca2+ waves (SCWs) that cause arrhythmogenic afterdepolarizations. Diminished Ca2+ transient amplitude in CPVT VMs resulted in a significant reduction in mitochondrial matrix-[Ca2+], and thereby a mito-ROS surge, visualized using matrix-targeted biosensors mtRCaMP1h and MLS-HyPer, respectively. Importantly, using novel Ca2+-biosensors targeted to intermembrane space (IMS-GECO), we uncovered that [Ca2+] in this compartment reaches 1 µM, sufficient for activation of Ca2+-dependent protease μ-calpain. Adenoviral overexpression of IMS-targeted calpastatin, an endogenous calpain inhibitor, reduced mito-ROS, restored cytosolic Ca2+ transient amplitude and SR Ca2+ content, and reduced RYR2-mediated SCWs in CPVT VMs. These changes were paralleled by restored expression levels of OPA1, a mitochondrial structural protein responsible for tight cristae organization. Our data suggest that enhanced mito-ROS due to matrix-[Ca2+] reduction in CPVT VMs and unexpectedly high IMS-[Ca2+] promotes IMS-calpain-mediated degradation of OPA1, resulting in mitochondrial structural damage that contributes to proarrhythmic remodeling.

Loss of α-actinin-3 confers protection from eccentric contraction damage in fast-twitch EDL muscles from aged mdx dystrophic mice by reducing pathological fibre branching.

The common null polymorphism (R577X) in the ACTN3 gene is present in over 1.5 billion people worldwide and results in the absence of the protein α-actinin-3 from the Z-discs of fast-twitch skeletal muscle fibres. We have previously reported that this polymorphism is a modifier of dystrophin-deficient Duchenne Muscular Dystrophy. To investigate the mechanism underlying this, we use a double knockout (dk)Actn3KO/mdx (dKO) mouse model, which lacks both dystrophin and sarcomere α-actinin-3. We used dKO mice and mdx dystrophic mice at 12 months (aged) to investigate the correlation between morphological changes to the fast-twitch dKO EDL and the reduction in force deficit produced by an in vitro eccentric contraction protocol. In the aged dKO mouse, we found a marked reduction in fibre branching complexity that correlated with protection from eccentric contraction induced force deficit. Complex branches in the aged dKO EDL fibres (28%) were substantially reduced compared to aged mdx EDL fibres (68%), and this correlates with a graded force loss over three eccentric contractions for dKO muscles (~36% after first contraction, ~66% overall) compared to an abrupt drop in mdx upon the first eccentric contraction (~75% after first contraction, ~89% after three contractions). In dKO, protection from eccentric contraction damage was linked with a doubling of SERCA1 pump density the EDL. We propose that the increased oxidative metabolism of fast-twitch glycolytic fibres characteristic of the null polymorphism (R577X) and increase in SR Ca2+ pump proteins reduces muscle fibre branching and decreases susceptibility to eccentric injury in the dystrophinopathies.

Mutation in RyR2-FKBP Binding site alters Ca2+ signaling modestly but increases “arrhythmogenesis” in human stem cells derived cardiomyocytes

Aims: To gain insights into FKBP regulation of cardiac ryanodine receptor (RyR2) and Ca2+ signaling, we introduced the point mutation (N771D-RyR2) corresponding to skeletal muscle mutation (N760D-RyR1) associated with central core disease (CCD) via CRISPR/Cas9 gene-editing in the RyR2 FKBP binding site expressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). Patients inflicted with CCD and other hereditary skeletal muscle diseases often show higher incidence of atrial or ventricular arrhythmias. Methods and Results: Ca2+ imaging of voltage-clamped N771D-RyR2 mutant compared to WT hiPSCCMs showed: (1) ∼30% suppressed ICa with no significant changes in the gating kinetics of ICa; (2) 29% lower SR Ca2+ content and 33% lower RyR2 Ca2+ leak; (3) higher CICR gain and 30–35% increased efficiency of ICa-triggered Ca2±release; (4) higher incidence of aberrant SR Ca2+ releases, DADs, and Ca2+ sparks; (5) no change in fractional Ca2+-release, action potential morphology, sensitivity to isoproterenol, and sarcomeric FKBP-binding pattern. Conclusions: The more frequent spontaneous Ca2+ releases and longer Ca2+ sparks underlie the increased incidence of DADs and cellular arrhythmogenesis of N771D-RyR2 mutant. The smaller RyR2 Ca2±leak and SR content result from suppressed ICathat is compensated by higher CICR gain.

Sarcoplasmic Reticulum Calcium Release Is Required for Arrhythmogenesis in the Mouse.

Dysfunctional sarcoplasmic reticulum Ca2+ handling is commonly observed in heart failure, and thought to contribute to arrhythmogenesis through several mechanisms. Some time ago we developed a cardiomyocyte-specific inducible SERCA2 knockout mouse, which is remarkable in the degree to which major adaptations to sarcolemmal Ca2+ entry and efflux overcome the deficit in SR reuptake to permit relatively normal contractile function. Conventionally, those adaptations would also be expected to dramatically increase arrhythmia susceptibility. However, that susceptibility has never been tested, and it is possible that the very rapid repolarization of the murine action potential (AP) allows for large changes in sarcolemmal Ca2+ transport without substantially disrupting electrophysiologic stability. We investigated this hypothesis through telemetric ECG recording in the SERCA2-KO mouse, and patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling of isolated SERCA2-KO myocytes. While the SERCA2-KO animals exhibit major (and unique) electrophysiologic adaptations at both the organ and cell levels, they remain resistant to arrhythmia. A marked increase in peak L-type calcium (ICaL) current and slowed ICaL decay elicited pronounced prolongation of initial repolarization, but faster late repolarization normalizes overall AP duration. Early afterdepolarizations were seldom observed in KO animals, and those that were observed exhibited a mechanism intermediate between murine and large mammal dynamical properties. As expected, spontaneous SR Ca2+ sparks and waves were virtually absent. Together these findings suggest that intact SR Ca2+ handling is an absolute requirement for triggered arrhythmia in the mouse, and that in its absence, dramatic changes to the major inward currents can be resisted by the substantial K+ current reserve, even at end-stage disease.

Abstract P348: Pathophysiological Basis Of A Compound Variant In Calcium And Sarcomere Regulation Causing Cardiac Arrhythmias And Hypertrophic Cardiomyopathy

Rationale: Hypertrophic cardiomyopathy (HCM) is common inheritable heart disease. HCM is highly associated with arrhythmias and/or sudden death. Studies show that molecular defects in calcium handling impairing the cardiomyocyte contractility is a predominant cause. However, the pathophysiology underlying HCM with arrhythmias is not well understood, hindering the identification of novel therapies. Objective: To investigate the pathophysiological consequences of compound variants, consisting of Histidine Rich Calcium Binding Protein gene ( HRC S96A ) and an intronic 25bp deletion in cardiac myosin binding protein-C ( MYBPC3 Δ25bp ). Methods and Results: Clinical data revealed that co-segregation of HRC S96A and MYBPC3 ΔInt32 results in cardiac arrhythmia, heart failure, and sudden cardiac death in South Asians. To determine the cellular/molecular trigger underlying the pathophysiology of this dual variant, we used humanized, knock-in, heterozygous mouse models, including HRC S81A (equivalent to HRC S96A ) MYBPC3 Δ25bp , HRC S81A / MYBPC3 Δ25bp (double variant, DV), and wild-type controls. Echocardiography revealed a significant decrease in the percentage of ejection fraction and fractional shortening in DV mice, as well as the presence of diastolic dysfunction, at 12 weeks of age, compared to single-variant and wild-type mice. Electrocardiogram tracing of DV mice showed the presence of stress-induced arrhythmias, such as ventricular tachycardia after caffeine and epinephrine administration. Using isolated cardiomyocytes in vitro , Calcium transient experiments indicated a significant decrease in fractional shortening, Ca 2+ transient amplitude, and a higher number of after-contractions in cardiomyocytes from DV mice. DV mouse hearts showed increased phosphorylation of CaMKII and SR Ca 2+ leak by cardiomyocytes. Inclusion of the CaMKII inhibitor KN-93 rescued the increases in SR Ca 2+ leak and in aftercontractions. Conclusion: Impaired Ca 2+ -handling, owing to the HRC S96A variant, aggravates SR Ca 2+ leak and aftercontractions in MYBPC3 Δ25bp cardiomyocytes, subsequently triggering cardiac arrhythmias and sudden death in vivo .

Acute Genetic Ablation of Cardiac Sodium/Calcium Exchange in Adult Mice: Implications for Cardiomyocyte Calcium Regulation, Cardioprotection, and Arrhythmia.

BackgroundSodium‐calcium (Ca2+) exchanger isoform 1 (NCX1) is the dominant Ca2+ efflux mechanism in cardiomyocytes and is critical to maintaining Ca2+ homeostasis during excitation‐contraction coupling. NCX1 activity has been implicated in the pathogenesis of cardiovascular diseases, but a lack of specific NCX1 blockers complicates experimental interpretation. Our aim was to develop a tamoxifen‐inducible NCX1 knockout (KO) mouse to investigate compensatory adaptations of acute ablation of NCX1 on excitation‐contraction coupling and intracellular Ca2+ regulation, and to examine whether acute KO of NCX1 confers resistance to triggered arrhythmia and ischemia/reperfusion injury.Methods and ResultsWe used the α‐myosin heavy chain promoter (Myh6)‐MerCreMer promoter to create a tamoxifen‐inducible cardiac‐specific NCX1 KO mouse. Within 1 week of tamoxifen injection, NCX1 protein expression and current were dramatically reduced. Diastolic Ca2+ increased despite adaptive reductions in Ca2+ current and action potential duration and compensatory increases in excitation‐contraction coupling gain, sarcoplasmic reticulum Ca2+ ATPase 2 and plasma membrane Ca2+ ATPase. As these adaptations progressed over 4 weeks, diastolic Ca2+ normalized and SR Ca2+ load increased. Left ventricular function remained normal, but mild fibrosis and hypertrophy developed. Transcriptomics revealed modification of cardiovascular‐related gene networks including cell growth and fibrosis. NCX1 KO reduced spontaneous action potentials triggered by delayed afterdepolarizations and reduced scar size in response to ischemia/reperfusion.ConclusionsTamoxifen‐inducible NCX1 KO mice adapt to acute genetic ablation of NCX1 by reducing Ca2+ influx, increasing alternative Ca2+ efflux pathways, and increasing excitation‐contraction coupling gain to maintain contractility at the cost of mild Ca2+‐activated hypertrophy and fibrosis and decreased survival. Nevertheless, KO myocytes are protected against spontaneous action potentials and ischemia/reperfusion injury.

Wenxin Keli for the Treatment of Arrhythmia-Systems Pharmacology and In Vivo Pharmacological Assessment.

This study employed a systems pharmacology approach to identify the active compounds and action mechanisms of Wenxin Keli for arrhythmia treatment. Sixty-eight components identified in vivo and in vitro by UPLC/Q-TOF-MS were considered the potential active components of Wenxin Keli. Network pharmacology further revealed 33 key targets and 75 KEGG pathways as possible pathways and targets involved in WK-mediated treatment, with the CaMKII/CNCA1C/Ca2+ pathway being the most significantly affected. This finding was validated using an AC-induced rat arrhythmias model. Pretreatment with Wenxin Keli reduced the malignant arrhythmias and shortened RR, PR, and the QT interval. Wenxin Keli exerted some antiarrhythmic effects by inhibiting p-CaMKII and intracellular Ca2+ transients and overexpressing CNCA1C. Thus, suppressing SR Ca2+ release and maintaining intracellular Ca2+ balance may be the primary mechanism of Wenxin Keli against arrhythmia. In view of the significance of CaMKII and NCX identified in this experiment, we suggest that CaMKII and NCX are essential targets for treating arrhythmias.

Uptake-leak balance of SR Ca2+ determines arrhythmogenic potential of RyR2R420Q+/- cardiomyocytes

Uptake-leak balance of SR Ca2+ determines arrhythmogenic potential of RyR2R420Q+/- cardiomyocytes

Description of the Mechanism of Positive Inotropic Action of the Isoquinoline Alkaloid F-18

This study evaluated the mechanism of inotropic effect of an isoquinoline alkaloid derivative, 1-(2´-bromine-4´,5´-dimethoxyphenyl)-6,7-dimethoxy-1,2,3,4- tetrahydroisoquinoline (F-18) using electrically stimulated rat left ventricular papillary muscle of rat. The F-18 alkaloid have been shown to have positive inotropic effect on papillary muscle contraction activity, IC50 value -14,6 µM. Са2+L-channel blocker - nifedipine was used in experiments. Inotropic effects of F-18 isoquinoline alkaloid on cardiomyocytes were suggested, based on results obtained in experiments carried in cardiomyocytes SR Ca2+- transport systems modulation.

Finite element analysis predicts Ca2+ microdomains within tubular-sarcoplasmic reticular junctions of amphibian skeletal muscle

A finite element analysis modelled diffusional generation of steady-state Ca2+ microdomains within skeletal muscle transverse (T)-tubular-sarcoplasmic reticular (SR) junctions, sites of ryanodine receptor (RyR)-mediated SR Ca2+ release. It used established quantifications of sarcomere and T-SR anatomy (radial diameter d=220 , mathrm{n}mathrm{m}; axial distance w=12 , mathrm{n}mathrm{m}). Its boundary SR Ca2+ influx densities,{J}_{mathrm{influx}}, reflected step impositions of influxes, it {Phi }_{mathrm{influx}}={J}_{mathrm{influx}}left(frac{pi {d}^{2}}{4}right), deduced from previously measured Ca2+ signals following muscle fibre depolarization. Predicted steady-state T-SR junctional edge [Ca2+], [Ca2+]edge, matched reported corresponding experimental cytosolic [Ca2+] elevations given diffusional boundary effluxit it {Phi }_{mathrm{efflux}}=frac{D [ {{{mathrm{Ca}}^{2+}}}]_{mathrm{edge}}}{lambda } (pi dw), established cytosolic Ca2+ diffusion coefficients (D = 4 times {10}^{7} mathrm{nm}^{2}/mathrm{s}) and exit length lambda = 9.2 , mathrm{n}mathrm{m}. Dependences of predicted [Ca2+]edge upon {J}_{mathrm{influx}} then matched those of experimental [Ca2+] upon Ca2+ release through their entire test voltage range. The resulting model consistently predicted elevated steady-state T-SR junctional ~ µM-[Ca2+] elevations radially declining from maxima at the T-SR junction centre along the entire axial T-SR distance. These [Ca2+] heterogeneities persisted through 104- and fivefold, variations in D and w around, and fivefold reductions in d below, control values, and through reported resting muscle cytosolic [Ca2+] values, whilst preserving the flux conservation (it it {Phi }_{mathrm{influx}}={Phi }_{mathrm{efflux}}) condition, {left[mathrm{C}{mathrm{a}}^{2+}right]}_{mathrm{edge}}=frac{lambda {dJ}_{mathrm{influx}}}{4Dw}. Skeletal muscle thus potentially forms physiologically significant ~ µM-[Ca2+] T-SR microdomains that could regulate cytosolic and membrane signalling molecules including calmodulin and RyR, These findings directly fulfil recent experimental predictions invoking such Ca2+ microdomains in observed regulatory effects upon Na+ channel function, in a mechanism potentially occurring in similar restricted intracellular spaces in other cell types.

Description of the Mechanism of Positive Inotropic Action of the Isoquinoline Alkaloid F-18

This study evaluated the mechanism of inotropic effect of an isoquinoline alkaloid derivative, 1-(2´-bromine-4´,5´-dimethoxyphenyl)-6,7- dimethoxy-1,2,3,4- tetrahydroisoquinoline (F-18) using electrically stimulated rat left ventricular papillary muscle of rat. The F-18 alkaloid have been shown to have positive inotropic effect on papillary muscle contraction activity, IC50 value -14,6 μM. Са2+L-channel blocker - nifedipine was used in experiments. Inotropic effects of F-18 isoquinoline alkaloid on cardiomyocytes were suggested, based on results obtained in experiments carried in cardiomyocytes SR Ca2+- transport systems modulation.

Ticagrelor alleviates high-carbohydrate intake induced altered electrical activity of ventricular cardiomyocytes by regulating sarcoplasmic reticulum-mitochondria miscommunication.

Metabolic syndrome (MetS) is associated with additional cardiovascular risk in mammalians while there are relationships between hyperglycemia-associated cardiovascular dysfunction and increased platelet P2Y12 receptor activation. Although P2Y12 receptor antagonist ticagrelor (Tica) plays roles in reduction of cardiovascular events, its beneficial mechanism remains poorly understood. Therefore, we aimed to clarify whether Tica can exert a direct protective effect in ventricular cardiomyocytes from high-carbohydrate diet-induced MetS rats, at least, through affectingsarcoplasmic reticulum (SR)-mitochondria (Mit) miscommunication. Tica treatment of MetS rats (150mg/kg/day for 15 days) significantly reversed the altered parameters of action potentials by reversing sarcolemmal ionic currents carried by voltage-dependent Na+ and K+ channels, and Na+/Ca2+-exchanger in the cells, expressed P2Y12 receptors. The increased basal-cytosolic Ca2+ level and depressed SR Ca2+ load were also reversed in Tica-treated cells, at most, though recoveries in the phosphorylation levels of ryanodine receptors and phospholamban. Moreover, there were marked recoveries in Mit structure and function (including increases in both autophagosomes and fragmentations) together with recoveries in Mit proteins and the factors associated with Ca2+ transfer between SR-Mit. There were further significant recoveries in markers of both ER stress and oxidative stress. Taken into consideration the Tica-induced prevention of ER stress and mitochondrial dysfunction, our data provided an important document on the pleiotropic effects of Tica in the electrical activity of the cardiomyocytes from MetS rats. This protective effect seems through recoveries in SR-Mit miscommunication besides modulation of different sarcolemmal ion-channel activities, independent of P2Y12 receptor antagonism.

Ca2+ handling in induced-pluripotent cardiomyocytes from female CPVT patient, and comparison to male

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agence Nationale de la Recherche Introduction Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an inherited disease manifested as syncope or sudden death in apparently healthy children or young adults, in the absence of structural cardiac disease. The ryanodine receptor (RyR2) R420Q mutation was identified in a Spanish 14-year-old boy who died suddenly due to emotional stress. We generated human induced-pluripotent (h-iPS) derived cardiomyocytes from two brothers (see Yin’s poster in this meeting). As most of sudden death occurs in males, we had the hypothesis that there might be some sex-dependent differences. Purpose and methods We aim to elucidate the key role of [Ca2+]i in the genesis of cardiac arrhythmia in female RyR2R420Q CPVT patients . We generated here hiPSC-CM from two sisters of the same family, one exhibiting the mutation RyR2R420Q and the other without the mutation used as control. The experiments were conducted at 30-35 days after differentiation into cardiomyocytes. Using confocal microscopy, we assessed intracellular [Ca2+]i handling both spontaneous and during electrical pacing. To record [Ca2+]i transients, h-iPS-CMs were electrically field stimulated by two Pt electrodes at 1Hz. Spontaneous Ca2+ sparks and Ca2+ waves were recorded in quiescent cells, after [Ca2+]i transients recordings. For SR Ca2+ load quantification, h-iPS-CMs were rapidly perfused with 10 mmol/L caffeine. To induce β-adrenergic activation, 100nM isoproterenol (ISO) was added in some experiments. All confocal Ca2+ images analyses were performed by home-made routines using IDL software. Results First, we verify by immunofluorescence the expression of alpha-actinin in the differentiated h-iPS-CMs. Then, by confocal microscopy we found that woman CPVT h-iPS-CMs presented longer cycle length than wt h-iPS-CMs, correlating with the bradycardia observed in CPVT patients. The amplitude of the Ca2+ transients were significantly higher for CPVT h-iPS-CMs compared to wt; however, after ISO perfusion Ca2+ transients did not show a significant increase in CPVT h-iPS-CMs compared to CPVT h-iPS-CMs before ISO. SR Ca2+ load presented no differences between groups but the time decay constant of the caffeine-evoked Ca2+ transients were significantly faster in CPVT h-iPS-CMs whereas it became slower after ISO perfusion. In addition, analysis of Ca2+ pro-arrhythmogenic events were significantly augmented in CPVT h-iPS-CMs compared to wt h-iPS-CMs. After ISO perfusion, pro-arrhythmogenic events in CPVT cells presented no significant difference compared to CPVT cell before ISO perfusion. Although proarrhythmogenic events were similarly higher in both male and female hiPSC-CM, the Ca2+ handling characteristics were slightly different. Conclusion The RyR2R420Q mutation in woman CPVT h-iPS-CMs provides a reliable model to study CPVT in human context.

Bay 60-7550, a PDE2 inhibitor, exerts positive inotropic effect of rat heart by increasing PKA-mediated phosphorylation of phospholamban

This study investigated the hemodynamic effect of Bay 60-7550, a phosphodiesterase type 2 (PDE2) inhibitor, in healthy rat hearts both in vivo and ex vivo and its underlying mechanisms. In vivo rat left ventricular pressure-volume loop, Langendorff isolated rat heart, Ca2+ transient of left ventricular myocyte and Western blot experiments were used in this study. The results demonstrated that Bay 60-7550 (1.5 mg/kg, i. p.) increased the in vivo rat heart contractility by enhancing stroke work, cardiac output, stroke volume, end-diastolic volume, heart rate, and ejection fraction. The simultaneous aortic pressure recording indicated that the systolic blood pressure was increased and diastolic blood pressure was decreased by Bay 60-7550. Also, the arterial elastance which is proportional to the peripheral vessel resistance was significantly decreased. Bay 60-7550 (0.001, 0.01, 0.1, 1 μmol/l) also enhanced the left ventricular development pressure in non-paced and paced modes with a decrease of heart rate in non-paced model. Bay 60-7550 (1 μmol/l) increased SERCA2a activity and SR Ca2+ content and reduced SR Ca2+ leak rate. Furthermore, Bay 60-7550 (0.1 μmol/l) increased the phosphorylation of phospholamban at 16-serine without significantly changing the phosphorylation levels of phospholamban at 17-threonine and RyR2. Bay 60-7550 increased the rat heart contractility and reduced peripheral arterial resistance may be mediated by increasing the phosphorylation of phospholamban and dilating peripheral vessels. PDE2 inhibitors which result in a positive inotropic effect and a decrease in peripheral resistance might serve as a target for developing agents for the treatment of heart failure in clinical settings.