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.

scholarly journals Loss of Rad-GTPase produces a novel adaptive cardiac phenotype resistant to systolic decline with aging

2015 ◽  
Vol 309 (8) ◽  
pp. H1336-H1345 ◽  
Author(s):  
Janet R. Manning ◽  
Catherine N. Withers ◽  
Bryana Levitan ◽  
Jeffrey D. Smith ◽  
Douglas A. Andres ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Output ◽  
Calcium Current ◽  
Ventricular Myocytes ◽  
Morphological Changes ◽  
Heart Function ◽  
Early Time ◽  
Cardiac Phenotype ◽  
Systolic And Diastolic Function

Rad-GTPase is a regulator of L-type calcium current (LTCC), with increased calcium current observed in Rad knockout models. While mouse models that result in elevated LTCC have been associated with heart failure, our laboratory and others observe a hypercontractile phenotype with enhanced calcium homeostasis in Rad−/−. It is currently unclear whether this observation represents an early time point in a decompensatory progression towards heart failure or whether Rad loss drives a novel phenotype with stable enhanced function. We test the hypothesis that Rad−/− drives a stable nonfailing hypercontractile phenotype in adult hearts, and we examine compensatory regulation of sarcoplasmic reticulum (SR) loading and protein changes. Heart function was measured in vivo with echocardiography. In vivo heart function was significantly improved in adult Rad−/− hearts compared with wild type. Heart wall dimensions were significantly increased, while heart size was decreased, and cardiac output was not changed. Cardiac function was maintained through 18 mo of age with no decompensation. SR releasable Ca2+ was increased in isolated Rad−/− ventricular myocytes. Higher Ca2+ load was accompanied by sarco/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) protein elevation as determined by immunoblotting and a rightward shift in the thapsigargan inhibitor-response curve. Rad−/− promotes morphological changes accompanied by a stable increase in contractility with aging and preserved cardiac output. The Rad−/− phenotype is marked by enhanced systolic and diastolic function with increased SR uptake, which is consistent with a model that does not progress into heart failure.

Short-term in vivo inhibition of nitric oxide synthase with L-NAME influences the contractile function of single left ventricular myocytes in rats

2011 ◽  
Vol 89 (4) ◽  
pp. 305-310 ◽  
Author(s):  
Wellington Lunz ◽  
Antônio José Natali ◽  
Miguel Araújo Carneiro ◽  
Luciano dos Santos Aggum Capettini ◽  
Marcelo Perim Baldo ◽  
...  
Keyword(s):  
Maximum Rate ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Left Ventricular ◽  
Regulatory Proteins ◽  
Hemodynamic Parameters ◽  
Short Term ◽  
Oxide Synthase ◽  
Fractional Shortening

The main purpose of this study was to investigate the effects of short-term L-NAME treatment on the contractile function of left ventricle (LV) myocytes and the expression of proteins related to Ca2+ homeostasis. Data from Wistar rats treated with L-NAME (L group, n = 20; 0.7 g/L in drinking water; 7 days) were compared with results from untreated controls (C group, n = 20). Cardiomyocytes from the L group showed increased (p < 0.05) fractional shortening (23%) and maximum rate of shortening (20%) compared with the C group. LV from the L group also showed increased (p < 0.05) expression of the ryanodine receptor 2 and Na+/Ca2+ exchanger proteins (76% and 83%, respectively; p < 0.05). However, the L and C groups showed similar in vivo hemodynamic parameters of cardiac function. In conclusion, short-term NOS inhibition determines an increased expression of Ca2+ regulatory proteins, which contributes to improving cardiomyocyte contractile function, preserving left ventricular function.

scholarly journals Effects of Bepridil on Stretch-Activated BKca Channels and Stretch-Induced Extrasystoles in Isolated Chick Hearts

2017 ◽  
pp. 459-465 ◽  
Author(s):  
H. JIN ◽  
G. IRIBE ◽  
K. NARUSE
Keyword(s):  
Membrane Potential ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Heart Rhythm ◽  
Channel Activity ◽  
Open Probability ◽  
Bkca Channels ◽  
Chick Heart ◽  
Lv Volume ◽  
Stretch Activated Channels

Various types of mechanosensitive ion channels, including cationic stretch-activated channels (SACNS) and stretch-activated BKca (SAKca) channels, modulate heart rhythm. Bepridil has been used as an antiarrhythmic drug with multiple pharmacological effects; however, whether it is effective for mechanically induced arrhythmia has not been well investigated. To test the effects of Bepridil on SAKca channels activity, cultured chick embryonic ventricular myocytes were used for single-channel recordings. Bepridil significantly reduced the open probability of the SAKca channel (PO). Next, to test the effects of bepridil on stretch-induced extrasystoles (SIE), we used an isolated 2-week-old Langendorff-perfused chick heart. The left ventricle (LV) volume was rapidly changed, and the probability of SIE was calculated in the presence and absence of bepridil, and the effect of the drug was compared with that of Gadolinium (Gd3+). Bepridil decreased the probability of SIE despite its suppressive effects on SAKca channel activity. The effects of Gd3+, which blocks both SAKca and SACNS, on the probability of SIE were the same as those of bepridil. Our results suggest that bepridil blocks not only SAKca channels but possibly also blocks SACNS, and thus decreases the stretch-induced cation influx (stabilizing membrane potential) to compensate and override the effects of the decrease in outward SAKca current (destabilizing membrane potential).

Abstract 17473: GRK2 in the Mitochondria: Uncovering Novel Mechanisms in Heart Failure

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Kimberly M Ferrero ◽  
Gizem Kayki Mutlu ◽  
Jessica M Pfleger ◽  
Douglas G Tilley ◽  
Walter J Koch
Keyword(s):  
Heart Failure ◽  
Mitochondrial Dysfunction ◽  
Ventricular Myocytes ◽  
Atp Synthesis ◽  
Neonatal Rat ◽  
Negative Effects ◽  
Protein Kinase C Inhibitor ◽  
Neonatal Rat Ventricular Myocytes

Introduction: During heart failure, levels and activity of G protein-coupled receptor kinase 2 (GRK2) increase. GRK2 is canonically studied in the phosphorylation of GPCRs and β-adrenergic desensitization. Noncanonical activities of GRK2 are being uncovered, however. Our lab has recently discovered that in cardiac myocytes, GRK2 translocates to the mitochondria ( mtGRK2 ) following injury and is associated with negative effects on metabolism and cell survival. Hypothesis: GRK2 plays a role in regulating mitochondrial function following cardiac stress and contributes to HF pathogenesis in a novel manner, by interacting with a novel group of mitochondrial proteins involved in pro-death signaling, bioenergetics and substrate utilization. Methods: Mitochondrial translocation of GRK2 was validated with either protein kinase C inhibitor (chelerythine) administration or hypoxia/reoxygenation stress in primary neonatal rat ventricular myocytes or a cardiac-like cell line. Immunoprecipitation of the GRK2 interactome basally and under stress conditions was conducted endogenously in vitro, in vivo , and with purified recombinant GRK2 peptides. Proteins were separated via SDS-PAGE and potential binding partners were identified by mass spectroscopy (LCMS) and proteomics analysis conducted with Ingenuity Pathway (IPA; Qiagen) software to determine which partners in the GRK2 interactome were potentially involved in mitochondrial dysfunction. Results: Subunits of Complexes I, II, IV and V of the electron transport chain were identified as potential mtGRK2 interacting partners. Several mtGRK2-ETC interactions were increased following oxidative stress-induced translocation of GRK2. Finally, mtGRK2 appears to phosphorylate some of the interactome partners identified in mitochondrial dysfunction. Conclusions: The phosphorylation of subunits of the ATP synthesis machinery by mtGRK2, or other mechanisms of interaction between these proteins, may be regulating some of the phenotypic effects of HF previously observed by our lab, such as increased ROS production and reduced fatty acid metabolism. Further research is essential to elucidate the novel role of GRK2 in regulating mitochondrial bioenergetics and cell death in failing hearts.

scholarly journals Regulation of Na channels of the rat cortical collecting tubule by aldosterone.

1993 ◽  
Vol 102 (1) ◽  
pp. 25-42 ◽  
Author(s):  
J Pácha ◽  
G Frindt ◽  
L Antonian ◽  
R B Silver ◽  
L G Palmer
Keyword(s):  
Membrane Surface ◽  
Plasma Aldosterone ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Open Probability ◽  
Collecting Tubule ◽  
Salt Depletion ◽  
Cortical Collecting Tubule

The activity of apical membrane Na channels in the rat cortical collecting tubule was studied during manipulation of the animals' mineralocorticoid status in vivo using a low-Na diet or the diuretic furosemide. Tubules were isolated and split open to expose the luminal membrane surface. Induction of Na channel activity was studied in cell-attached patches of the split tubules. No activity was observed with control animals on a normal diet. Channel activity could be induced by putting the animals on the low-Na diet for at least 48 h. The mean number of open channels per patch (NPo) was maximal after 1 wk on low Na. Channels were also induced within 3 h after injection of furosemide (20 mg/kg body wt per d). NPo was maximal 48 h after the first injection. In both cases, increases in NPo were primarily due to increases in the number of channels per patch (N) at a constant open probability (Po). With salt depletion or furosemide injection NPo is a saturable function of aldosterone concentration with half-maximal activity at approximately 8 nM. When animals were salt repleted after 1-2 wk of salt depletion, both plasma aldosterone and NPo fell markedly within 6 h. NPo continued to decrease over the next 14 h, while plasma aldosterone rebounded partially. Channel activity may be dissociated from aldosterone concentrations under conditions of salt repletion.

scholarly journals Loss of primary cilia increases polycystin-2 and TRPV4 and the appearance of a nonselective cation channel in the mouse cortical collecting duct

2019 ◽  
Vol 317 (3) ◽  
pp. F632-F637 ◽  
Author(s):  
Takamitsu Saigusa ◽  
Qiang Yue ◽  
Marlene A. Bunni ◽  
P. Darwin Bell ◽  
Douglas C. Eaton
Keyword(s):  
Collecting Duct ◽  
Cation Channel ◽  
Conditional Knockout ◽  
Nonselective Cation Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Duct Cells ◽  
Collecting Ducts ◽  
Collecting Duct Cells

Flow-related bending of cilia results in Ca2+ influx through a polycystin-1 (Pkd1) and polycystin-2 (Pkd2) complex, both of which are members of the transient receptor potential (TRP) family (TRPP1 and TRPP2, respectively). Deletion of this complex as well as cilia result in polycystic kidney disease. The Ca2+ influx pathway has been previously characterized in immortalized collecting duct cells without cilia and found to be a 23-pS channel that was a multimere of TRPP2 and TRPV4. The purpose of the present study was to determine if this TRPP2 and TRPV4 multimere exists in vivo. Apical channel activity was measured using the patch-clamp technique from isolated split-open cortical collecting ducts from adult conditional knockout mice with ( Ift88flox/flox) or without ( Ift88−/−) cilia. Single tubules were isolated for measurements of mRNA for Pkd1, Pkd2, Trpv4, and epithelial Na+ channel subunits. The predominant channel activity from Ift88flox/flox mice was from epithelial Na+ channel [5-pS Na+-selective channels with long mean open times (475.7 ± 83.26 ms) and open probability > 0.2]. With the loss of cilia, the predominant conductance was a 23-pS nonselective cation channel (reversal potential near 0) with a short mean open time (72 ± 17 ms), open probability < 0.08, and a characteristic flickery opening. Loss of cilia increased mRNA levels for Pkd2 and Trpv4 from single isolated cortical collecting ducts. In conclusion, 23-pS channels exist in vivo, and activity of this channel is elevated with loss of cilia, consistent with previous finding of an elevated-unregulated Ca2+-permeable pathway at the apical membrane of collecting duct cells that lack cilia.

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.

Normal contractions triggered by I Ca,L in ventricular myocytes from rats with postinfarction CHF

2002 ◽  
Vol 283 (3) ◽  
pp. H1225-H1236 ◽  
Author(s):  
Ivar Sjaastad ◽  
Janny Bøkenes ◽  
Fredrik Swift ◽  
J. Andrew Wasserstrom ◽  
Ole M. Sejersted
Keyword(s):  
Heart Failure ◽  
Congestive Heart Failure ◽  
Coronary Artery ◽  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Cardiac Contractility ◽  
Left Ventricular ◽  
Coronary Artery Ligation ◽  
Artery Ligation ◽  
Fractional Shortening

Attenuated L-type Ca2+ current ( I Ca,L), or current-contraction gain have been proposed to explain impaired cardiac contractility in congestive heart failure (CHF). Six weeks after coronary artery ligation, which induced CHF, left ventricular myocytes from isoflurane-anesthetized rats were current or voltage clamped from −70 mV. In both cases, contraction and contractility were attenuated in CHF cells compared with cells from sham-operated rats when cells were only minimally dialyzed using high-resistance microelectrodes. With patch pipettes, cell dialysis caused attenuation of contractions in sham cells, but not CHF cells. Stepping from −50 mV, the following variables were not different between sham and CHF, respectively: peak I Ca,L (4.5 ± 0.3 vs. 3.8 ± 0.3 pApF−1 at 23°C and 9.4 ± 0.5 vs. 8.4 ± 0.5 pApF−1 at 37°C), the bell-shaped voltage-contraction relationship in Cs+ solutions (fractional shortening, 15.2 ± 1.0% vs. 14.3 ± 0.7%, respectively, at 23°C and 7.5 ± 0.4% vs. 6.7 ± 0.5% at 37°C) and the sigmoidal voltage-contraction relationship in K+ solutions. Caffeine-induced Ca2+ release and sarcoplasmic reticulum Ca2+-ATPase-to-phospholamban ratio were not different. Thus CHF contractions triggered by I Ca,L were normal, and the contractile deficit was only seen in undialyzed cardiomyocytes stimulated from −70 mV.

Evaluation of T- and L-type Ca2+ currents in shark ventricular myocytes

1995 ◽  
Vol 269 (5) ◽  
pp. H1695-H1703 ◽  
Author(s):  
J. Maylie ◽  
M. Morad
Keyword(s):  
Action Potential ◽  
Time Constant ◽  
Current Density ◽  
Ventricular Myocytes ◽  
Squalus Acanthias ◽  
Na Channel ◽  
Na Current ◽  
Peak Current Density ◽  
Peak Current ◽  
Current Densities

Two types of Ca2+ currents with characteristics of T- and L-type Ca2+ currents were recorded in ventricular myocytes of dogfish (Squalus acanthias). The T-type Ca2+ current activated near -70 mV and had a peak current density of 9.8 pA/pF at -34 mV. The L-type Ca2+ current activated near -50 mV and had a peak current density of 10.6 pA/pF near 0 mV. The threshold for activation of the T-type Ca2+ current was 20 mV negative to that of the tetrodotoxin-sensitive Na+ current. Inactivation of the T-type Ca2+ current was rapid with a limiting time constant of 5 ms at positive potentials. The T-type Ca2+ current was not modulated by isoproterenol or acetylcholine. In dogfish the T-type Ca2+ channel has current densities equivalent to the L-type channel and is likely to activate before the Na+ channel, contributing significantly to generation of the foot of the action potential.

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