scholarly journals Local hyperactivation of L-type Ca2+ channels increases spontaneous Ca2+ release activity and cellular hypertrophy in right ventricular myocytes from heart failure rats

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Roman Y. Medvedev ◽  
Jose L. Sanchez-Alonso ◽  
Catherine A. Mansfield ◽  
Aleksandra Judina ◽  
Alice J. Francis ◽  
...  
Keyword(s):  
Heart Failure ◽  
Structural Changes ◽  
Ventricular Myocytes ◽  
Cellular Mechanisms ◽  
Open Probability ◽  
Surface Flattening ◽  
Cellular Hypertrophy ◽  
T Tubules ◽  
Optimized Treatment ◽  
Ca2 Channels

AbstractRight ventricle (RV) dysfunction is an independent predictor of patient survival in heart failure (HF). However, the mechanisms of RV progression towards failing are not well understood. We studied cellular mechanisms of RV remodelling in a rat model of left ventricle myocardial infarction (MI)-caused HF. RV myocytes from HF rats show significant cellular hypertrophy accompanied with a disruption of transverse-axial tubular network and surface flattening. Functionally these cells exhibit higher contractility with lower Ca2+ transients. The structural changes in HF RV myocytes correlate with more frequent spontaneous Ca2+ release activity than in control RV myocytes. This is accompanied by hyperactivated L-type Ca2+ channels (LTCCs) located specifically in the T-tubules of HF RV myocytes. The increased open probability of tubular LTCCs and Ca2+ sparks activation is linked to protein kinase A-mediated channel phosphorylation that occurs locally in T-tubules. Thus, our approach revealed that alterations in RV myocytes in heart failure are specifically localized in microdomains. Our findings may indicate the development of compensatory, though potentially arrhythmogenic, RV remodelling in the setting of LV failure. These data will foster better understanding of mechanisms of heart failure and it could promote an optimized treatment of patients.

scholarly journals Altered Na/Ca exchange distribution in ventricular myocytes from failing hearts

2016 ◽  
Vol 310 (2) ◽  
pp. H262-H268 ◽  
Author(s):  
Hanne C. Gadeberg ◽  
Simon M. Bryant ◽  
Andrew F. James ◽  
Clive H. Orchard
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Coronary Artery Ligation ◽  
Sham Operation ◽  
Ca Current ◽  
Artery Ligation ◽  
Whole Cell ◽  
Rat Hearts ◽  
T Tubules

In mammalian cardiac ventricular myocytes, Ca efflux via Na/Ca exchange (NCX) occurs predominantly at T tubules. Heart failure is associated with disrupted t-tubular structure, but its effect on t-tubular function is less clear. We therefore investigated t-tubular NCX activity in ventricular myocytes isolated from rat hearts ∼18 wk after coronary artery ligation (CAL) or corresponding sham operation (Sham). NCX current ( INCX) and l-type Ca current ( ICa) were recorded using the whole cell, voltage-clamp technique in intact and detubulated (DT) myocytes; intracellular free Ca concentration ([Ca]i) was monitored simultaneously using fluo-4. INCX was activated and measured during application of caffeine to release Ca from sarcoplasmic reticulum (SR). Whole cell INCX was not significantly different in Sham and CAL myocytes and occurred predominantly in the T tubules in Sham myocytes. CAL was associated with redistribution of INCX and ICa away from the T tubules to the cell surface and an increase in t-tubular INCX/ ICa density from 0.12 in Sham to 0.30 in CAL myocytes. The decrease in t-tubular INCX in CAL myocytes was accompanied by an increase in the fraction of Ca sequestered by SR. However, SR Ca content was not significantly different in Sham, Sham DT, and CAL myocytes but was significantly increased by DT of CAL myocytes. In Sham myocytes, there was hysteresis between INCX and [Ca]i, which was absent in DT Sham but present in CAL and DT CAL myocytes. These data suggest altered distribution of NCX in CAL myocytes.

Chronic receptor-mediated activation of Gi/o proteins alters basal t-tubular and sarcolemmal L-type Ca2+ channel activity through phosphatases in heart failure

2012 ◽  
Vol 302 (8) ◽  
pp. H1645-H1654 ◽  
Author(s):  
Toshihide Kashihara ◽  
Tsutomu Nakada ◽  
Hisashi Shimojo ◽  
Miwa Horiuchi-Hirose ◽  
Simmon Gomi ◽  
...  
Keyword(s):  
Heart Failure ◽  
Current Density ◽  
Ventricular Myocytes ◽  
Heterotrimeric G Proteins ◽  
Channel Activity ◽  
Open Probability ◽  
Current Densities ◽  
Fractional Shortening ◽  
Pp2a Inhibitor

L-type Ca2+ channels (LTCCs) play an essential role in the excitation-contraction coupling of ventricular myocytes. We previously found that t-tubular (TT) LTCC current density was halved by the activation of protein phosphatase (PP)1 and/or PP2A, whereas surface sarcolemmal (SS) LTCC current density was increased by the inhibition of PP1 and/or PP2A activity in failing ventricular myocytes of mice chronically treated with isoproterenol (ISO mice). In the present study, we examined the possible involvement of inhibitory heterotrimeric G proteins (Gi/o) in these abnormalities by chronically administrating pertussis toxin (PTX) to ISO mice (ISO + PTX mice). Compared with ISO mice, ISO + PTX mice exhibited significantly higher fractional shortening of the left ventricle. The expression level of Gαi2 proteins was not altered by the treatment of mice with ISO and/or PTX. ISO + PTX myocytes had normal TT and SS LTCC current densities because they had higher and lower availability and/or open probability of TT and SS LTCCs than ISO myocytes, respectively. A selective PKA inhibitor, H-89, did not affect LTCC current densities in ISO + PTX myocytes. A selective PP2A inhibitor, fostriecin, did not affect SS or TT current density in control or ISO + PTX myocytes but significantly increased TT but not SS LTCC current density in ISO myocytes. These results indicate that chronic receptor-mediated activation of Gi/o in vivo decreases basal TT LTCC activity by activating PP2A and increases basal SS LTCC activity by inhibiting PP1 without modulating PKA in heart failure.

Abstract 14309: A Novel Fibroblast Activation Inhibitor, NM922, Attenuates Maladaptive Fibrotic Remodeling to Preserve Cardiac Function Following the Onset of Heart Failure

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Jessica M Bradley ◽  
Craig M Ziblich ◽  
Kazi N Islam ◽  
Amanda M Rushing ◽  
David J Polhemus ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Function ◽  
Structural Changes ◽  
Volume Fraction ◽  
Left Ventricular ◽  
Normal Fibroblast ◽  
Wall Thickening ◽  
Cellular Mechanisms ◽  
Collagen Formation ◽  
Failing Heart

Background: Cardiac fibroblasts are critical mediators of fibrotic remodeling in the failing heart. These maladaptive structural changes can worsen cardiac function accelerating the progression to decompensated heart failure (HF). We investigated the effects of a novel inhibitor of the conversion of normal fibroblast to the myofibroblast phenotype in the setting of pressure overload induced HF. Methods: Male C57BL/6J mice (10 wks) were subjected to transverse aortic constriction (TAC; 27 g needle) and NM922 (NovoMedix, LLC50 mg/kg/d i.p.) or VEH (DMSO + HS-15) was administered daily starting at 6 wks post TAC. Echocardiography was assessed at baseline and for 16 wks post TAC. At the 16 wk endpoint, mice were sacrificed and hearts were collected for biochemical and molecular assessment. Results: NM922 significantly attenuated TAC-induced left ventricular (LV) dilation at 16 wks post TAC (LVEDD: 3.5 ± 0.1 vs. 4.5 ± 0.2 mm, p < 0.01; LVESD: 2.5 ± 0.2 vs. 3.8 ± 0.3 mm, p < 0.01) compared to VEH. NM922 treated mice displayed reduced wall thickening (LVPWd: 1.0 ± 0.03 vs. 1.2 ± 0.05 mm; p < 0.05) at 10 wks post TAC compared to VEH. LV ejection fraction (LVEF) was preserved in NM922 treated mice at 8-16 wks post TAC compared to VEH (*p < 0.05; **p < 0.001) compared to VEH. Treatment with NM922 resulted in reductions in heart (8.5 ± 0.5 vs. 12.0 ± 0.9 mg/mm; p < 0.01) and lung (8.2 ± 0.3 vs. 11.5 ± 0.6 mg/mm; p < 0.0001) weights compared to VEH. Picrosirius Red staining revealed that NM922 reduced cardiac interstitial collagen volume fraction by 50% (p < 0.05 vs. VEH). Circulating BNP levels trended toward lower (p = 0.08) in the NM922 mice when compared to VEH. Conclusion: Chronic treatment with NM922 following the onset of cardiac hypertrophy and HF resulted in attenuated myocardial collagen formation and adverse remodeling with preservation of LVEF. Future studies are aimed at further elucidation of the molecular and cellular mechanisms by which this novel agent protects the failing heart.

scholarly journals Junctophilin-2 tethers T-tubules and recruits functional L-type calcium channels to lipid rafts in adult cardiomyocytes

2020 ◽  
Author(s):  
Claire Poulet ◽  
Jose Sanchez-Alonso ◽  
Pamela Swiatlowska ◽  
Florence Mouy ◽  
Carla Lucarelli ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Lipid Rafts ◽  
Ventricular Myocytes ◽  
Ryanodine Receptors ◽  
Calcium Signalling ◽  
Super Resolution ◽  
Scaffolding Protein ◽  
Open Probability ◽  
T Tubules ◽  
Membrane Treatment

Abstract Aim In cardiomyocytes, transverse tubules (T-tubules) associate with the sarcoplasmic reticulum (SR), forming junctional membrane complexes (JMCs) where L-type calcium channels (LTCCs) are juxtaposed to Ryanodine receptors (RyR). Junctophilin-2 (JPH2) supports the assembly of JMCs by tethering T-tubules to the SR membrane. T-tubule remodelling in cardiac diseases is associated with downregulation of JPH2 expression suggesting that JPH2 plays a crucial role in T-tubule stability. Furthermore, increasing evidence indicate that JPH2 might additionally act as a modulator of calcium signalling by directly regulating RyR and LTCCs. This study aimed at determining whether JPH2 overexpression restores normal T-tubule structure and LTCC function in cultured cardiomyocytes. Methods and results Rat ventricular myocytes kept in culture for 4 days showed extensive T-tubule remodelling with impaired JPH2 localization and relocation of the scaffolding protein Caveolin3 (Cav3) from the T-tubules to the outer membrane. Overexpression of JPH2 restored T-tubule structure and Cav3 relocation. Depletion of membrane cholesterol by chronic treatment with methyl-β-cyclodextrin (MβCD) countered the stabilizing effect of JPH2 overexpression on T-tubules and Cav3. Super-resolution scanning patch-clamp showed that JPH2 overexpression greatly increased the number of functional LTCCs at the plasma membrane. Treatment with MβCD reduced LTCC open probability and activity. Proximity ligation assays showed that MβCD did not affect JPH2 interaction with RyR and the pore-forming LTCC subunit Cav1.2, but strongly impaired JPH2 association with Cav3 and the accessory LTCC subunit Cavβ2. Conclusions JPH2 promotes T-tubule structural stability and recruits functional LTCCs to the membrane, most likely by directly binding to the channel. Cholesterol is involved in the binding of JPH2 to T-tubules as well as in the modulation of LTCC activity. We propose a model where cholesterol and Cav3 support the assembly of lipid rafts which provide an anchor for JPH2 to form JMCs and a platform for signalling complexes to regulate LTCC activity.

scholarly journals Caveolin-3 KO disrupts t-tubule structure and decreases t-tubular ICa density in mouse ventricular myocytes

2018 ◽  
Vol 315 (5) ◽  
pp. H1101-H1111 ◽  
Author(s):  
Simon M. Bryant ◽  
Cherrie H. T. Kong ◽  
Judy J. Watson ◽  
Hanne C. Gadeberg ◽  
David M. Roth ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Ventricular Myocytes ◽  
Wild Type ◽  
Tubule Formation ◽  
Membrane Area ◽  
Cellular Hypertrophy ◽  
Channel Expression ◽  
T Tubules ◽  
And Function

Caveolin-3 (Cav-3) is a protein that has been implicated in t-tubule formation and function in cardiac ventricular myocytes. In cardiac hypertrophy and failure, Cav-3 expression decreases, t-tubule structure is disrupted, and excitation-contraction coupling is impaired. However, the extent to which the decrease in Cav-3 expression underlies these changes is unclear. We therefore investigated the structure and function of myocytes isolated from the hearts of Cav-3 knockout (KO) mice. These mice showed cardiac dilatation and decreased ejection fraction in vivo compared with wild-type control mice. Isolated KO myocytes showed cellular hypertrophy, altered t-tubule structure, and decreased L-type Ca2+ channel current ( ICa) density. This decrease in density occurred predominantly in the t-tubules, with no change in total ICa, and was therefore a consequence of the increase in membrane area. Cav-3 KO had no effect on L-type Ca2+ channel expression, and C3SD peptide, which mimics the scaffolding domain of Cav-3, had no effect on ICa in KO myocytes. However, inhibition of PKA using H-89 decreased ICa at the surface and t-tubule membranes in both KO and wild-type myocytes. Cav-3 KO had no significant effect on Na+/Ca2+ exchanger current or Ca2+ release. These data suggest that Cav-3 KO causes cellular hypertrophy, thereby decreasing t-tubular ICa density. NEW & NOTEWORTHY Caveolin-3 (Cav-3) is a protein that inhibits hypertrophic pathways, has been implicated in the formation and function of cardiac t-tubules, and shows decreased expression in heart failure. This study demonstrates that Cav-3 knockout mice show cardiac dysfunction in vivo, while isolated ventricular myocytes show cellular hypertrophy, changes in t-tubule structure, and decreased t-tubular L-type Ca2+ current density, suggesting that decreased Cav-3 expression contributes to these changes in cardiac hypertrophy and failure.

scholarly journals Reduced junctional Na+/Ca2+-exchanger activity contributes to sarcoplasmic reticulum Ca2+ leak in junctophilin-2-deficient mice

2014 ◽  
Vol 307 (9) ◽  
pp. H1317-H1326 ◽  
Author(s):  
Wei Wang ◽  
Andrew P. Landstrom ◽  
Qiongling Wang ◽  
Michelle L. Munro ◽  
David Beavers ◽  
...  
Keyword(s):  
Heart Failure ◽  
Sarcoplasmic Reticulum ◽  
Single Channel ◽  
Rapid Development ◽  
Mouse Heart ◽  
Cellular Mechanisms ◽  
Open Probability ◽  
Deficient Mice

Expression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca2+ leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca2+ release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca2+ imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca2+ leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na+/Ca2+-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd2+ administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca2+ leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca2+. This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca2+ leak and heart failure development.

scholarly journals Phorbol ester and endothelin-1 alter functional expression of Na+/Ca2+ exchange, K+, and Ca2+ currents in cultured neonatal rat myocytes

2011 ◽  
Vol 300 (2) ◽  
pp. H617-H626 ◽  
Author(s):  
José L. Puglisi ◽  
Weilong Yuan ◽  
Valeriy Timofeyev ◽  
Richard E. Myers ◽  
Nipavan Chiamvimonvat ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Ionic Currents ◽  
Functional Expression ◽  
Neonatal Rat ◽  
Delayed Rectifier ◽  
Electrophysiological Properties ◽  
Endothelin 1 ◽  
Cellular Hypertrophy ◽  
Myocyte Function

Endothelin-1 (ET-1) and activation of protein kinase C (PKC) have been implicated in alterations of myocyte function in cardiac hypertrophy and heart failure. Changes in cellular Ca2+ handling and electrophysiological properties also occur in these states and may contribute to mechanical dysfunction and arrhythmias. While ET-1 or PKC stimulation induces cellular hypertrophy in cultured neonatal rat ventricular myocytes (NRVMs), a system widely used in studies of hypertrophic signaling, there is little data about electrophysiological changes. Here we studied the effects of ET-1 (100 nM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 μM) on ionic currents in NRVMs. The acute effects of PMA or ET-1 (≤30 min) were small or insignificant. However, PMA or ET-1 exposure for 48–72 h increased cell capacitance by 100 or 25%, respectively, indicating cellular hypertrophy. ET-1 also slightly increased Ca2+ current density (T and L type). Na+/Ca2+ exchange current was increased by chronic pretreatment with either PMA or ET-1. In contrast, transient outward and delayed rectifier K+ currents were strongly downregulated by PMA or ET-1 pretreatment. Inward rectifier K+ current tended toward a decrease at larger negative potential, but time-independent outward K+ current was unaltered by either treatment. The enhanced inward and reduced outward currents also result in action potential prolongation after PMA or ET-1 pretreatment. We conclude that chronic PMA or ET-1 exposure in cultured NRVMs causes altered functional expression of cardiac ion currents, which mimic electrophysiological changes seen in whole animal and human hypertrophy and heart failure.

Decrease in the density of t-tubular L-type Ca2+ channel currents in failing ventricular myocytes

2011 ◽  
Vol 300 (3) ◽  
pp. H978-H988 ◽  
Author(s):  
Miwa Horiuchi-Hirose ◽  
Toshihide Kashihara ◽  
Tsutomu Nakada ◽  
Nagomi Kurebayashi ◽  
Hisashi Shimojo ◽  
...  
Keyword(s):  
Heart Failure ◽  
Sarcoplasmic Reticulum ◽  
Current Density ◽  
Ventricular Myocytes ◽  
Osmotic Shock ◽  
Adrenergic Stimulation ◽  
Whole Cell ◽  
Subunit Expression ◽  
Dependent Protein ◽  
T Tubules

In some forms of cardiac hypertrophy and failure, the gain of Ca2+-induced Ca2+ release [CICR; i.e., the amount of Ca2+ released from the sarcoplasmic reticulum normalized to Ca2+ influx through L-type Ca2+ channels (LTCCs)] decreases despite the normal whole cell LTCC current density, ryanodine receptor number, and sarcoplasmic reticulum Ca2+ content. This decrease in CICR gain has been proposed to arise from a change in dyad architecture or derangement of the t-tubular (TT) structure. However, the activity of surface sarcolemmal LTCCs has been reported to increase despite the unaltered whole cell LTCC current density in failing human ventricular myocytes, indicating that the “decreased CICR gain” may reflect a decrease in the TT LTCC current density in heart failure. Thus, we analyzed LTCC currents of failing ventricular myocytes of mice chronically treated with isoproterenol (Iso). Although Iso-treated mice exhibited intact t-tubules and normal LTCC subunit expression, acute occlusion of t-tubules of isolated ventricular myocytes with osmotic shock (detubulation) revealed that the TT LTCC current density was halved in Iso-treated versus control myocytes. Pharmacological analysis indicated that kinases other than PKA or Ca2+/calmodulin-dependent protein kinase II insufficiently activated, whereas protein phosphatase 1/2A excessively suppressed, TT LTCCs in Iso-treated versus control myocytes. These results indicate that excessive β-adrenergic stimulation causes the decrease in TT LTCC current density by altering the regulation of TT LTCCs by protein kinases and phosphatases in heart failure. This phenomenon might underlie the decreased CICR gain in heart failure.

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