Abstract MP135: Cardiac Sarcoplasmic Reticulum Calcium Cycling is Regulated by Kinase Independent Function of Pi3kgamma

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Maradumane L Mohan ◽  
Conner P Witherow ◽  
Robert S Papay ◽  
Sathyamangla V Naga Prasad
Keyword(s):  
Intracellular Calcium ◽  
Calcium Release ◽  
Cardiac Contractility ◽  
Underlying Mechanism ◽  
Calcium Handling ◽  
Ventricular Contractility ◽  
Independent Function ◽  
Calcium Cycling ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Phosphoinositide 3 Kinase

Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ -/- ) results in increased cAMP levels and enhanced ventricular contractility. We investigated whether the lack of PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling. Isolated cardiomyocytes from PI3Kγ -/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) following caffeine induced calcium release indicating that PI3Kγ locally regulates the function of SR. The intracellular calcium remained at elevated levels in the cardiomyocytes of PI3Kγ -/- for a prolonged period after caffeine treatment. This could be due to changes in phosphorylation of SERCA2, Ryanodine receptor (RyR 2 ) or phospholamban (PLN). In fact, when we looked at phosphorylation of PLN in cardiac lysates, a major regulator of cardiac contractility and relaxation, PI3Kγ -/- mice showed significantly reduced PLN phosphorylation compared to littermate controls. Previous studies from our laboratory suggested that absence of PI3Kγ leads to increase in protein phosphatase (PP) activity which could be possible reason for rapid dephosphorylation of PLN, resulting in inhibition of SERCA2 pump. We observed increased SR associated PP activity and PLN associated PP activity in PI3Kγ -/- mice. We also observed increased association of PP-1 and PP2A with PLN in the absence of PI3Kγ. The altered calcium handling in the cardiomyocytes of PI3Kγ -/- mice could be restored to the level of WT controls by okadaic acid mediated inhibition of PP, suggesting that PI3Kγ plays a role in regulating PP activity associated with SR. To test whether PI3Kγ activity is required for PLN dephosphorylation and SR calcium cycling, we used mice with cardiac specific overexpression of kinase dead PI3Kγ (PI3Kγ inact ) in global PI3Kγ -/- mice (PI3Kγ inact /PI3Kγ -/- ). PI3Kγ inact /PI3Kγ -/- mice showed restored PLN phosphorylation, improved caffeine induced calcium reuptake, decreased SR and PLN associated PP activity. These studies show a novel regulation of PP and SR calcium regulation by kinase independent function of PI3Kγ. The underlying mechanism of PP regulation by PI3Kγ will be presented.

scholarly journals Kinase independent function of PI3Kγ modulates calcium re-uptake by regulating phospholamban function

2021 ◽  
Author(s):  
Maradumane L Mohan ◽  
Conner P Witherow ◽  
Robert S Papay ◽  
Yu Sun ◽  
Kate Stenson ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Protein Phosphatase ◽  
Cardiac Contractility ◽  
Ventricular Rate ◽  
Independent Function ◽  
Cardiac Stress ◽  
Adult Cardiomyocytes ◽  
Genetic Deletion ◽  
Phosphoinositide 3 Kinase ◽  
Camp Levels

Rationale Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ-/-) results in increased cAMP levels and enhanced ventricular rate/contractility. Whether PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling is not known. Objective To understand the mechanism of PI3Kγ mediated regulation of cardiac contractility. Methods and Results Caffeine treatment of adult cardiomyocytes from PI3Kγ-/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) indicating that PI3Kγ locally regulates SR function. This resulted in elevated levels of intracellular calcium for prolonged period following caffeine. Our findings show that delayed re-uptake of calcium was caused by changes in phosphorylation of phospholamban (PLN), a major regulator of SR calcium reuptake. PI3Kγ-/- cardiomyocytes show significantly reduced PLN phosphorylation due to increase in PLN-associated protein phosphatase (PP) activity as reflected by decreased demethylated-PP2A. Consistently, the altered calcium regulation in the cardiomyocytes of PI3Kγ-/- can be restored by inhibition of PP by okadaic acid. Unexpectedly, cardiomyocyate-specific overexpression of kinase-dead PI3Kγ (PI3Kγinact) in the global PI3Kγ-/- cardiomyocytes normalized caffeine induced calcium reuptake, restored PLN phosphorylation, and decreased PLN-associated PP activity reflected by increased demethylated-PP2A. Conclusions These studies bring-to-fore an unrecognized regulation of PLN by PI3Kγ through PP2A with implications in deleterious cardiac remodeling as PI3Kγ is significantly upregulated following cardiac stress.

Cardiac excitation–contraction coupling: new developments

1988 ◽  
Vol 66 (9) ◽  
pp. 1217-1217
Author(s):  
D. Bose
Keyword(s):  
Intracellular Calcium ◽  
Calcium Release ◽  
Myocardial Cell ◽  
Cardiac Contraction ◽  
Calcium Handling ◽  
Patch Clamp Technique ◽  
Regulatory Processes ◽  
Technological Advances ◽  
Calcium Indicator ◽  
Cardiac Excitation

Over 100 years have elapsed since Sidney Ringer made the serendipitous discovery that calcium played a crucial role in amphibian cardiac contraction. Since then we have learned that this ion is an obligatory requirement for cardiac muscle of all species, and that the regulation of intracellular calcium levels is considerably more complex in the mammalian heart than previously thought. Part of this complexity is due to the involved design requirements of mammalian physiological processes. Another element of complexity is introduced by the quantitative differences in the involvement of various regulatory processes in different species. Finally, many significant technological advances in methods for investigating cardiac cellular functions have provided exciting experimental data. However, these data must be integrated into a unifying framework of knowledge of cardiac functions. Among the exciting recent developments are the use of a patch clamp technique to discover different kinds of calcium channels, a highly refined skinned fiber technique to study calcium-induced calcium release, and calcium indicator dyes and laser diffraction and scattering techniques to study the dynamics of calcium handling by the cell. These studies have not only provided clues about the normal functioning of the myocardial cell but have also reinforced the notion that altered function of the sarcoplasmic reticulum during intracellular calcium overload can influence sarcolemmal electrical function.This symposium, organized by the Pharmacological Society of Canada, examined some of the more recent technological advances in the field to provide a glimpse not only of the "state of the art" but also of future directions.This symposium was made possible by generous financial assistance from Boehringer Ingelheim (Canada) Ltd., Bristol-Myers Pharmaceutical Group, Canadian and Manitoba Heart Foundations, Ciba-Geigy Canada Ltd., Du Pont Canada Inc., Hoffmann LaRoche Ltd., Merck-Frosst Canada Inc., Miles Laboratories Inc., Nordic Laboratories Inc., Pfizer (Canada) Inc., Rhône-Poulenc Pharmaceuticals Inc., G. D. Searle of Canada, Ltd., Squibb Canada Inc., Sterling Drugs Ltd./Winthrop Laboratories, the Upjohn Company of Canada, and the University of Manitoba Pharmacology Department.

Abstract MP236: PI3Kgamma-PP2A Axis In Regulation Of SERCA Function

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Maradumane L Mohan ◽  
Conner P Witherow ◽  
Robert Papay ◽  
Yu Sun ◽  
Kate Stenson ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Protein Phosphatase ◽  
Significant Interaction ◽  
Cardiac Contractility ◽  
Ventricular Rate ◽  
Cardiac Stress ◽  
Adult Cardiomyocytes ◽  
Genetic Deletion ◽  
Phosphoinositide 3 Kinase ◽  
Camp Levels

Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ -/- ) results in increased cAMP levels and enhanced ventricular rate/contractility. We investigated whether PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling. Caffeine treatment of adult cardiomyocytes from PI3Kγ -/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) indicating that PI3Kγ locally regulates SR function. This resulted in elevated levels of intracellular calcium for prolonged period following caffeine. Our findings show that delayed re-uptake of calcium was caused by changes in phosphorylation of phospholamban (PLN), a major regulator of SR calcium reuptake. PI3Kγ -/- cardiomyocytes showed significantly reduced PLN phosphorylation due to increase in PLN-associated protein phosphatase (PP) activity as reflected by decreased demethylated-PP2A. Abrogation of PLN phosphorylation in the PI3Kγ -/- cardiomyocytes shows that the loss in the steady-state phosphorylation of PLN leads to increased inhibition of SERCA. This inhibition is reflected by the slow reuptake of calcium by the SR in the PI3Kγ -/- cardiomyocytes. Concomitantly, significant interaction was observed between SERCA and PLN in the PI3Kγ -/- hearts compared to the controls. Consistently, the altered calcium regulation in the cardiomyocytes of PI3Kγ -/- can be restored by inhibition of PP by okadaic acid. Unexpectedly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ inact ) in the global PI3Kγ -/- cardiomyocytes normalized caffeine induced calcium reuptake, restored PLN phosphorylation, and decreased PLN-associated PP activity reflected by increased demethylated-PP2A. These studies bring-to-fore an unrecognized kinase-independent regulation of PLN by PI3Kγ through PP2A with implications in deleterious cardiac remodeling as PI3Kγ is significantly upregulated following cardiac stress.

Carnosine is a novel peptide modulator of intracellular calcium and contractility in cardiac cells

1997 ◽  
Vol 272 (1) ◽  
pp. H462-H468 ◽  
Author(s):  
G. P. Zaloga ◽  
P. R. Roberts ◽  
K. W. Black ◽  
M. Lin ◽  
G. Zapata-Sudo ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Cardiac Contractility ◽  
Cardiac Cells ◽  
Dependent Manner ◽  
Calcium Release Channel ◽  
Release Channel ◽  
Contractile Failure

Myocardial contractile failure is a common cause of morbidity and mortality in patients with ischemic heart disease and systemic inflammatory states such as sepsis. Accumulating evidence indicates that contractile failure is associated with dysregulation of myoplasmic calcium levels. In a search for biochemical causes for contractile dysfunction, we found that the dipeptide carnosine improves cardiac contractility and tested the possibility that carnosine plays a role in the regulation of intracellular calcium. Carnosine increased contractility in a dose-dependent manner (1-10 mM) in isolated perfused rat hearts. and it also increased free intracellular calcium levels in isolated myocytes. Carnosine increased myocyte tension via calcium release from the ryanodine receptor calcium release channel in skinned myocardial fibers and increased open-state probability and dwell time of the isolated ryanodine receptor calcium release channel in lipid bilayers. In addition. we report that carnosine sensitizes the contractile proteins so calcium. These results suggest a novel role for carnosine as a modulator of intracellular calcium and contractility in cardiac tissue.

Intracellular calcium handling heterogeneities in intact guinea pig hearts

2004 ◽  
Vol 286 (2) ◽  
pp. H648-H656 ◽  
Author(s):  
Rodolphe P. Katra ◽  
Etienne Pruvot ◽  
Kenneth R. Laurita
Keyword(s):  
Guinea Pig ◽  
Intracellular Calcium ◽  
Calcium Release ◽  
Optical Mapping ◽  
Contractile Function ◽  
Calcium Transient ◽  
Calcium Transients ◽  
Calcium Handling ◽  
Excitation Light ◽  
Intact Heart

Regional heterogeneities of ventricular repolarizing currents and their role in arrhythmogenesis have received much attention; however, relatively little is known regarding heterogeneities of intracellular calcium handling. Because repolarization properties and contractile function are heterogeneous from base to apex of the intact heart, we hypothesize that calcium handling is also heterogeneous from base to apex. To test this hypothesis, we developed a novel ratiometric optical mapping system capable of measuring calcium fluorescence of indo-1 at two separate wavelengths from 256 sites simultaneously. With the use of intact Langendorff-perfused guinea pig hearts, ratiometric calcium transients were recorded under normal conditions and during administration of known inotropic agents. Ratiometric calcium transients were insensitive to changes in excitation light intensity and fluorescence over time. Under control conditions, calcium transient amplitude near the apex was significantly larger (60%, P < 0.01) compared with the base. In contrast, calcium transient duration was significantly longer (7.5%, P < 0.03) near the base compared with the apex. During isoproterenol (0.05 μM) and verapamil (2.5 μM) administration, ratiometric calcium transients accurately reflected changes in contractile function, and, the direction of base-to-apex heterogeneities remained unchanged compared with control. Ratiometric optical mapping techniques can be used to accurately quantify heterogeneities of calcium handling in the intact heart. Significant heterogeneities of calcium release and sequestration exist from base to apex of the intact heart. These heterogeneities are consistent with base-to-apex heterogeneities of contraction observed in the intact heart and may play a role in arrhythmogenesis under abnormal conditions.

Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin αIIbβ3

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 151-158 ◽  
Author(s):  
Cindy L. Yap ◽  
Karen E. Anderson ◽  
Sascha C. Hughan ◽  
Sacha M. Dopheide ◽  
Hatem H. Salem ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Platelet Adhesion ◽  
Calcium Release ◽  
Platelet Glycoprotein ◽  
Integrin Αiibβ3 ◽  
Real Time Analysis ◽  
Lipid Product ◽  
Cell Arrest ◽  
Phosphoinositide 3 Kinase ◽  
Static Conditions

Platelet adhesion and aggregation at sites of vascular injury are critically dependent on the interaction between von Willebrand factor (VWF) and 2 major platelet adhesion receptors, glycoprotein (GP) Ib/V/IX and integrin αIIbβ3. GP Ib/V/IX binding to VWF mediates platelet tethering and translocation, whereas activation of integrin αIIbβ3 promotes cell arrest. To date, the signaling pathways used by the VWF-GP Ib/V/IX interaction to promote activation of integrin αIIbβ3, particularly under shear, have remained poorly defined. In this study, the potential involvement of type 1 phosphoinositide (PI) 3–kinases in this process was investigated. Results show that platelet adhesion and spreading on immobilized VWF results in a specific increase in the PI 3–kinase lipid product, PtdIns(3,4)P2. Under static conditions, inhibiting PI 3–kinase with LY294002 or wortmannin did not prevent platelet adhesion, integrin αIIbβ3activation, or platelet spreading although it significantly delayed the onset of these events. In contrast, PI 3–kinase inhibition under shear dramatically reduced both platelet adhesion and spreading. Real-time analysis of intracellular calcium demonstrated that under static conditions inhibiting PI 3–kinase delayed the onset of intracellular fluxes in adherent platelets, but did not affect the final magnitude of the calcium response. However, under shear, inhibiting PI 3–kinase dramatically reduced intracellular calcium mobilization and integrin αIIbβ3 activation, resulting in impaired thrombus growth. The studies demonstrate a shear-dependent role for PI 3–kinase in promoting platelet adhesion on immobilized VWF. Under static conditions, platelets appear to mobilize intracellular calcium through both PI 3–kinase–dependent and –independent mechanisms, whereas under shear PI 3–kinase is indispensable for VWF-induced calcium release.

Rapid Endotoxin-induced Alterations in Myocardial Calcium Handling

2001 ◽  
Vol 95 (6) ◽  
pp. 1396-1405 ◽  
Author(s):  
Christof Stamm ◽  
Douglas B. Cowan ◽  
Ingeborg Friehs ◽  
Sabrena Noria ◽  
Pedro J. del Nido ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Intracellular Calcium ◽  
Left Ventricular ◽  
Calcium Handling ◽  
Tnf Alpha ◽  
Free Calcium ◽  
Myocardial Uptake ◽  
Calcium Cycling ◽  
Perfused Heart ◽  
Inotropic Effects

Background Bacterial endotoxin (lipopolysaccharide [LPS]) induces septic shock and depressed myocardial contractility. The mechanism of LPS-mediated cardiac dysfunction remains controversial. We hypothesized that LPS exerts significant effects on myocardial excitation-contraction coupling by rapid stimulation of tumor necrosis factor alpha (TNF-alpha) expression in the heart. Methods Isolated rat hearts were studied with and without recirculation of cell-free perfusate. The effects of LPS, exogenous TNF-alpha, anti-TNF-alpha antibody, and ceramidase inhibition were examined. Measurements included myocardial uptake of LPS, left ventricular contractility, myocardial oxygen consumption, intracellular calcium [Ca2+] cycling, and TNF-alpha concentrations in coronary perfusate and myocardium. Results Lipopolysaccharide was rapidly taken up by the perfused heart. With non-recirculating perfusion, LPS had no effect on contractility, oxygen consumption, coronary vascular resistance, or intracellular free calcium concentration ([Ca2+]i). However, with recirculating perfusion contractility was significantly impaired after 30 min of LPS, associated with lower [Ca2+]i levels and attenuated systolic rise in [Ca2+]i. Significant amounts of TNF-alpha accumulated in recirculating perfusate and myocardial tissue from LPS-perfused hearts. Ceramidase inhibition or neutralizing anti-TNF-alpha antibody inhibited the effects of LPS on contractility and [Ca2+]i. Recombinant rat TNF-alpha mimicked the LPS effects with faster onset. Conclusions Lipopolysaccharide exerts rapid, negative inotropic effects on the isolated whole rat heart. The reduction in contractility is associated with depressed intracellular calcium cycling. In response to LPS, TNF-alpha is rapidly released from the heart and mediates the effects of LPS via the sphingomyelinase pathway. The present study for the first time directly links LPS-stimulated TNF-alpha production, abnormal calcium cycling, and decreased contractility in intact hearts.

Sign in / Sign up

Export Citation Format

Share Document