thapsigargin and Heart-Failure

Mouse models of heart disease are extensively employed. The echocardiographic characterization of contractile function is usually focused on systolic function with fewer studies assessing diastolic function. Furthermore, the applicability of diverse echocardiographic parameters of diastolic function that are commonly used in humans has not been extensively evaluated in different pathophysiological models in mice.. We used high resolution echocardiography to evaluate parameters of diastolic function in mouse models of chronic pressure overload (aortic constriction), volume overload (aorto-caval shunt), heart failure with preserved ejection fraction (HFpEF; DOCA-salt hypertension), and acute sarcoplasmic reticulum dysfunction induced by thapsigargin - all known to exhibit diastolic dysfunction. Left atrial area increased in all three chronic models while mitral E/A was difficult to quantify at high heart rates. Isovolumic relaxation time (IVRT) and Doppler E/E' increased significantly and the peak longitudinal strain rate during early filling (peak reverse longitudinal strain rate) decreased significantly after aortic constriction, with the changes being proportional to the magnitude of hypertrophy. In the HFpEF model, reverse longitudinal strain rate decreased significantly but changes in IVRT and E/E' were non-significant, consistent with less severe dysfunction. With volume overload, there was a significant increase in reverse longitudinal strain rate and decrease in IVRT, indicating a restrictive physiology. Acute thapsigargin treatment caused significant prolongation of IVRT and decrease in reverse longitudinal strain rate.. These results indicate that the combined measurement of left atrial area plus reverse longitudinal strain rate and/or IVRT provide an excellent overall assessment of diastolic function in the diseased mouse heart, allowing distinction between different types of pathophysiology.

Topics: Animals; Cardiomegaly; Diastole; Disease Models, Animal; Echocardiography; Heart Diseases; Heart Failure; Mice, Inbred C57BL; Observer Variation; Pressure; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Stroke Volume; Systole; Thapsigargin

Ca(2+) release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. We found that the Golgi apparatus was the source of prolonged Ca(2+) release events that originated near the nuclei of primary cardiomyocytes. Golgi Ca(2+) release was unaffected by depletion of sarcoplasmic reticulum Ca(2+), and disruption of the Golgi apparatus abolished Golgi Ca(2+) release without affecting sarcoplasmic reticulum function, suggesting functional and spatial independence of Golgi and sarcoplasmic reticulum Ca(2+) stores. β1-Adrenoceptor stimulation triggers the production of the second messenger cAMP, which activates the Epac family of Rap guanine nucleotide exchange factors and the kinase PKA (protein kinase A). Phosphodiesterases (PDEs), including those in the PDE3 and PDE4 families, degrade cAMP. Activation of β1-adrenoceptors stimulated Golgi Ca(2+) release, an effect that required activation of Epac, PKA, and the kinase CaMKII. Inhibition of PDE3s or PDE4s potentiated β1-adrenergic-induced Golgi Ca(2+) release, which is consistent with compartmentalization of cAMP signaling near the Golgi apparatus. Interventions that stimulated Golgi Ca(2+) release appeared to increase the trafficking of vascular endothelial growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane of cardiomyocytes. In cardiomyocytes from rats with heart failure, decreases in the abundance of PDE3s and PDE4s were associated with increased Golgi Ca(2+) release events. These data suggest that the Golgi apparatus is a focal point for β1-adrenergic-stimulated Ca(2+) signaling and that the Golgi Ca(2+) store functions independently from the sarcoplasmic reticulum and the global Ca(2+) transients that trigger contraction in cardiomyocytes.

Topics: Adrenergic beta-Agonists; Animals; Calcium; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Heart Failure; Immunoblotting; Isoproterenol; Male; Microscopy, Confocal; Microscopy, Electron; Monocrotaline; Myocytes, Cardiac; Phosphoric Diester Hydrolases; Rats, Wistar; Receptors, Adrenergic, beta-1; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Thapsigargin

Myocardial C-type natriuretic peptide (CNP) levels are increased in heart failure. CNP can induce negative inotropic (NIR) and positive lusitropic responses (LR) in normal hearts, but its effects in failing hearts are not known. We studied the mechanism of CNP-induced NIR and LR in failing hearts and determined whether sarcoplasmatic reticulum Ca(2+) ATPase2 (SERCA2) activity is essential for these responses.. Contractility, cGMP levels, Ca(2+) transient amplitudes and protein phosphorylation were measured in left ventricular muscle strips or ventricular cardiomyocytes from failing hearts of Wistar rats 6 weeks after myocardial infarction.. CNP increased cGMP levels, evoked a NIR and LR in muscle strips, and caused phospholamban (PLB) Ser(16) and troponin I (TnI) Ser(23/24) phosphorylation in cardiomyocytes. Both the NIR and LR induced by CNP were reduced in the presence of a PKG blocker/cGMP analogue (Rp-8-Br-Pet-cGMPS) and the SERCA inhibitor thapsigargin. CNP increased the amplitude of the Ca(2+) transient and increased SERCA2 activity in cardiomyocytes. The CNP-elicited NIR and LR were not affected by the L-type Ca(2+) channel activator BAY-K8644, but were abolished in the presence of isoprenaline (induces maximal activation of cAMP pathway). This suggests that phosphorylation of PLB and TnI by CNP causes both a NIR and LR. The NIR to CNP in mouse heart was abolished 8 weeks after cardiomyocyte-specific inactivation of the SERCA2 gene.. We conclude that CNP-induced PLB and TnI phosphorylation by PKG in concert mediate both a predictable LR as well as the less expected NIR in failing hearts.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium-Binding Proteins; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Heart Failure; Isoproterenol; Male; Mice; Mice, Knockout; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, C-Type; Phosphorylation; Rats; Rats, Wistar; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Thionucleotides; Troponin I

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys(27) of PLB was cross-linked to the Ca(2+) pump at the cytoplasmic extension of M4 (at or near Lys(328)) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å). Cross-linking was augmented by ATP but abolished by Ca(2+) or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca(2+) affinity conformation of the Ca(2+)-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca(2+)-ATPase activity and Ca(2+) transport. The results suggest that the inhibitory effect of PLB on Ca(2+)-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca(2+) to the Ca(2+) pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca(2+) pump are largely unchanged in failing myocardium.

Topics: Animals; Antibodies, Monoclonal, Murine-Derived; Calcium; Calcium-Binding Proteins; Cell Line; Cross-Linking Reagents; Heart Failure; Humans; Ion Transport; Microsomes; Muscle Proteins; Myocardium; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spodoptera; Thapsigargin

Heart failure increases autonomic nerve activities and changes intracellular calcium (Ca(i)) dynamics.. The purpose of this study was to investigate the hypothesis that abnormal Ca(i) dynamics are responsible for triggered activity in the pulmonary veins (PVs) during acetylcholine infusion in a canine model of heart failure.. Simultaneous optical mapping of Ca(i) and membrane potential was performed in isolated Langendorff-perfused PV-left atrial (LA) preparations from nine dogs with ventricular pacing-induced heart failure. Mapping was performed at baseline, during acetylcholine (1 micromol/L) infusion (N = 9), and during thapsigargin and ryanodine infusion (N = 6).. Acetylcholine abbreviated the action potential. In four tissues, long pauses were followed by elevated diastolic Ca(i), late phase 3 early afterdepolarizations, and atrial fibrillation (AF). The incidence of PV focal discharges during AF was increased by acetylcholine from 2.4 +/- 0.6 beats/s (N = 4) to 6.5 +/- 2.2 beats/s (N = 8; P = .003). PV focal discharge and PV-LA microreentry coexisted in 6 of 9 preparations. The spatial distribution of dominant frequency demonstrated a focal source pattern, with the highest dominant frequency areas colocalized with PV focal discharge sites in 35 (95%) of 37 cholinergic AF episodes (N = 8). Thapsigargin and ryanodine infusion eliminated focal discharges in 6 of 6 preparations and suppressed the inducibility of AF in 4 of 6 preparations. PVs with focal discharge have higher densities of parasympathetic nerves than do PVs without focal discharges (P = .01), and periodic acid-Schiff (PAS)-positive cells were present at the focal discharge sites.. Ca(i) dynamics are important in promoting triggered activity during acetylcholine infusion in PVs from pacing-induced heart failure. PV focal discharge sites have PAS-positive cells and high densities of parasympathetic nerves.

Topics: Acetylcholine; Animals; Calcium; Calcium-Transporting ATPases; Cardiac Pacing, Artificial; Dogs; Enzyme Inhibitors; Heart Failure; Heart Ventricles; Models, Animal; Pulmonary Veins; Ryanodine; Stroke Volume; Thapsigargin; Vasodilator Agents; Ventricular Function, Left

The flat or negative force frequency relationship (FFR) is a hallmark of the failing heart. Either decreases in SERCA2a expression, increases in Na(+)/Ca(2+) exchanger (NCX) expression or elevated Na(+)(i) have been independently proposed as mediators of the negative FFR.. To determine whether each one of these mechanisms is sufficient to account for the negative FFR of the failing heart or on the contrary, various mechanisms, acting in concert are required. SERCA2a was pharmacologically inhibited with thapsigargin (TG) or cyclopiazonic acid (CPA) or by using siRNA technology; Na(+)(i) was increased with either ouabain (Oua) or monensin and NCX protein was overexpressed by gene transfer (Ad.NCX), to mimic in nonfailing cat myocytes the phenotype of the failing heart and examine their effect on the FFR. The positive FFR of healthy myocytes remained unaffected after either SERCA2a inhibition, Na(+)(i) elevation, or NCX overexpression. However, the combination of TG + Oua, Oua + Ad.NCX, or TG + Ad.NCX, converted the positive FFR to negative. Moreover, the FFR became negative at lower frequencies, when the 3 interventions were combined.. Ca(2+) handling has to be altered at several levels to explain the negative FFR of the failing heart. These anomalies in Ca(2+) homeostasis acting in synergy have additive effects.

Topics: Animals; Blotting, Western; Calcium; Calcium-Transporting ATPases; Cats; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Fluorescent Dyes; Gene Expression; Heart Failure; Indoles; Intracellular Fluid; Myocardium; Myocytes, Cardiac; RNA, Small Interfering; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium; Thapsigargin

In chronic heart failure, the lung endothelial permeability response to angiotensin II or thapsigargin-induced store depletion is ablated, although the mechanisms are not understood.. To determine whether the ablated permeability response to store depletion during heart failure was due to impaired expression of store operated Ca2+ channels in lung endothelium.. Heart failure was induced by aortocaval fistula in rats. Permeability was measured in isolated lungs using the filtration coefficient and a low Ca2+/Ca2+ add-back strategy to identify the component of the permeability response dependent on Ca2+ entry.. In fistulas, right ventricular mass and left ventricular end diastolic pressure were increased and left ventricular shortening fraction decreased compared with shams. Thapsigargin-induced store depletion increased lung endothelial permeability in shams, but not in fistulas. Permeability increased in both groups after the Ca2+ ionophore A23187 or 14,15-epoxyeicosatrienoic acid, independent of store depletion. A diacylglycerol analog had no impact on permeability. Increased distance between the endoplasmic reticulum and the plasmalemmal membrane was ruled out as a mechanism for the loss of the permeability response to store depletion. Endothelial expression of the endoplasmic reticulum Ca2+ ATPase was not altered in fistulas compared with shams, whereas the store-operated canonical transient receptor potential channels 1, 3, and 4 were downregulated in extraalveolar vessel endothelium.. We conclude that the adaptive mechanism limiting store depletion-induced endothelial lung injury in the aortocaval model of heart failure involves downregulation of store-operated Ca2+ channels.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Calcimycin; Calcium Channels; Calcium-Transporting ATPases; Disease Models, Animal; Endothelium; Enzyme Inhibitors; Heart Failure; Ionophores; Lung; Permeability; Rats; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Tissue Culture Techniques; Vasodilator Agents

The release of intracellular stores of Ca(2+) occurs virtually in all types of cells by a means of amplifying external signals that modulate intracellular signaling events. In cardiac myocytes, type 2 ryanodine receptor (RyR(2)) is activated during excitation-contraction (E-C) coupling by Ca(2+)-induced Ca(2+) release (CICR) triggered by Ca(2+) influx across the sarcolemma. The hyperadrenergic state of heart failure results in leaky RyR(2) channels attributable to PKA hyperphosphorylation and depletion of the stabilizing FK506 binding protein, FKBP12.6. Dysregulation of sarcoplasmic reticulum (SR) Ca(2+) release via RyR(2) could contribute to defects in Ca(2+) signaling in failing hearts. Researchers tested the hypothesis that improved cardiac muscle function attributable to beta-AR blockade is associated with restoration of normal RyR(2) channel function in patients with heart failure. The authors aimed to observe change of RyR in junior mouse with HF and the effect of beta-adrenoreceptor blocker on RyR in HF in this experiment.. The animal model of congestive heart failure was established by constriction of abdominal aorta. Five weeks old mice were randomly divided into 3 groups: (1) HF group without treatment (n = 20); (2) HF group treated with carvedilol (n = 20); (3) Sham-operated group (n = 20). Carvedilol was administered through direct gastric gavage. After 4 weeks of treatment the high frequency ultrasound was performed. Myocardial SR was fractionated with velocity centrifugation. The time courses of Ca(2+) uptake and leak were determined by fluorescent spectrophotometr.. Compared with the sham-operated group, left ventricular diastolic dimension (LVEDD) (P < 0.05), left ventricular systolic dimension (LVESD), interventricular septal thickness at end-diastole (IVSTd), interventricular septal thickness at end-systole (IVSTs), left ventricular posterior wall thickness at end-diastole (LVPWTd), and left ventricular posterior wall thickness at end-systole (LVPWTs) were all significantly increased (P < 0.01). Ejection fraction (EF) and fractional shortening (FS) were decreased (P < 0.01) in HF group without treatment. LVEDD (P < 0.05), LVESD, IVSTd, IVSTs, LVPWTd and LVPWTs were all prominently decresed (P < 0.01). EF and FS were increased (P < 0.01) in cases of HF treated with carvedilol when compared with HF group without treatment. After adding thapsigargin to the buffer including SR of three groups, there were fewer Ca(2+) leak in sham-operated group and HF group treated with carvedilol than that of HF group without treatment (P < 0.01), while after adding FK506 and thapsigargin together to the buffer including SR of three groups, there were marked Ca(2+) leak in sham-operated group and HF group treated with carvedilol (P < 0.01). However, there was no additional increase in Ca(2+) leak in HF group compared with that of the group where only thapsigargin was added (P > 0.05).. There is more cardiac Ca(2+) leak in HF. Carvedilol can inhibite Ca(2+) leak by restoring the contactation of FKBP12.6 back to RyR in HF to improve cardiac function and prevent left ventricle from remodeling.

Topics: Adrenergic beta-Antagonists; Animals; Animals, Newborn; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Carbazoles; Carvedilol; Disease Models, Animal; Heart Failure; Myocardial Contraction; Myocytes, Cardiac; Propanolamines; Rats; Rats, Wistar; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Spectrometry, Fluorescence; Thapsigargin; Ultrasonography

The endoplasmic reticulum (ER) is recognized as an organelle that participates in folding secretory and membrane proteins. The ER responds to stress by upregulating ER chaperones, but prolonged and/or excess ER stress leads to apoptosis. However, the potential role of ER stress in pathophysiological hearts remains unclear.. Mice were subjected to transverse aortic constriction (TAC) or sham operation. Echocardiographic analysis demonstrated that mice 1 and 4 weeks after TAC had cardiac hypertrophy and failure, respectively. Cardiac expression of ER chaperones was significantly increased 1 and 4 weeks after TAC, indicating that pressure overload by TAC induced prolonged ER stress. In addition, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells increased, and caspase-3 was cleaved in failing hearts. The antagonism of angiotensin II type 1 receptor prevented upregulation of ER chaperones and apoptosis in failing hearts. On the other hand, angiotensin II upregulated ER chaperones and induced apoptosis in cultured adult rat cardiac myocytes. We also investigated possible signaling pathways for ER-initiated apoptosis. The CHOP- (a transcription factor induced by ER stress), but not JNK- or caspase-12-, dependent pathway was activated in failing hearts by TAC. Pharmacological ER stress inducers upregulated ER chaperones and induced apoptosis in cultured cardiac myocytes. Finally, mRNA levels of ER chaperones were markedly increased in failing hearts of patients with elevated brain natriuretic peptide levels.. These findings suggest that pressure overload by TAC induces prolonged ER stress, which may contribute to cardiac myocyte apoptosis during progression from cardiac hypertrophy to failure.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Aortic Valve Stenosis; Apoptosis; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Disease Progression; Endoplasmic Reticulum; Gene Expression Regulation; Heart Failure; Humans; Imidazoles; Ligation; Male; Mice; Mice, Inbred C57BL; Molecular Chaperones; Myocytes, Cardiac; Natriuretic Peptide, Brain; Olmesartan Medoxomil; Rats; Rats, Inbred WKY; RNA, Messenger; Signal Transduction; Stress, Physiological; Tetrazoles; Thapsigargin; Tunicamycin

We examined mechanical alternans and electromechanical restitution in normal and failing rat hearts. Alternans occurred at 5 Hz in failing versus 9 Hz in control hearts and was reversed by 300 nM isoproterenol, 6 mM extracellular Ca(2+), 300 nM -BAY K 8644, or 50 nM ryanodine. Restitution curves comprised phase I, which was completed before relaxation of the steady-state beat, and phase II, which occurred later. Phase I action potential area and developed pressure ratios were significantly reduced in the failing versus control hearts. Phase II was a monoexponential increase in relative developed pressure as the extrasystolic interval was increased. The plateau of phase II was significantly elevated in failing hearts. Thapsigargin (3 microM) plus ryanodine (200 nM) potentiated phase I to a significantly greater extent in control versus failing hearts and abolished phase II in both groups. The results suggest that both regulation of Ca(2+) influx across the sarcolemma and Ca(2+) release by the sarcoplasmic reticulum may contribute to altered excitation-contraction coupling in the failing spontaneously hypertensive heart failure prone rat heart.

Topics: Action Potentials; Animals; Calcium; Heart Failure; Heart Rate; Hypertension; Isoproterenol; Male; Rats; Rats, Inbred BN; Rats, Inbred Strains; Rats, Inbred WF; Rats, Wistar; Ryanodine; Thapsigargin; Ventricular Dysfunction, Left; Ventricular Function, Left

Rest- and stimulation frequency-dependent potentiation of contractile force is blunted in failing human myocardium. These alterations have been related to reduced sarcoplasmic reticulum (SR) Ca(2+)-reuptake and enhanced transsarcolemmal Ca(2+)-elimination by Na+/Ca(2+)-exchange. We investigated whether inotropic interventions that enhance SR Ca(2+)-uptake, or reduce Ca(2+)-elimination by Na+/Ca(2+)-exchange, normalize impaired post-rest and force-frequency behavior in left ventricular muscle strips from failing human hearts.. We tested the influence of [10]-gingerol which activates SR Ca(2+)-ATPase (10 mumol/l; n = 13), and isoproterenol which activates cAMP-dependent pathways (0.01, 0.1, 1 mumol/l; n = 40) on post-rest and force-frequency behavior. Ouabain which blocks Na+/K(+)-ATPase (0.03 mumol/l; n = 16) was used to test the effects of inhibiting Ca(2+)-elimination by Na+/Ca(2+)-exchange. For comparison, the effects of blocking SR Ca(2+)-uptake by thapsigargin (10 mumol/l; n = 14) were tested. In addition, Ca(2+)-uptake in myocardial homogenates was measured for gingerol (10 mumol/l; n = 6).. Gingerol, isoproterenol (0.1, 1 mumol/l) and ouabain exerted significant positive inotropic effects under basal experimental conditions and normalized post-rest behavior. In contrast, force-frequency relation was only slightly improved by gingerol and isoproterenol (0.01 mumol/l). Ouabain and isoproterenol (1 mumol/l) further deteriorated force-frequency relation due to frequency-dependent significant increases in diastolic tension. Thapsigargin exerted negative inotropic effects and significantly deteriorated post-rest and force-frequency behavior. In addition, gingerol increased SR Ca(2+)-uptake significantly in myocardial homogenates.. Inotropic interventions that stimulate SR Ca(2+)-ATPase or inhibit Na+/Ca(2+)-exchange normalize impaired post-rest behavior. Force-frequency behavior is only slightly improved by stimulation of SR Ca(2+)-ATPase but not by inhibition of Na+/Ca(2+)-exchange. This dissociation between post-rest and force-frequency behavior results from diastolic dysfunction at high stimulation rates.

Topics: Adult; Analysis of Variance; Calcium; Calcium-Transporting ATPases; Cardiotonic Agents; Catechols; Cyclic AMP; Enzyme Inhibitors; Fatty Alcohols; Heart Failure; Humans; Isoproterenol; Middle Aged; Myocardial Contraction; Myocardium; Ouabain; Sarcoplasmic Reticulum; Sodium-Potassium-Exchanging ATPase; Stimulation, Chemical; Thapsigargin

Defective excitation-contraction coupling in heart failure is generally associated with both a reduction in sarcoplasmic reticulum (SR) Ca(2+) uptake and a greater dependence on transsarcolemmal Na(+)-Ca(2+) exchange (NCX) for Ca(2+) removal. Although a relative increase in NCX is expected when SR function is impaired, few and contradictory studies have addressed whether there is an absolute increase in NCX activity. The present study examines in detail NCX density and function in left ventricular midmyocardial myocytes isolated from normal or tachycardic pacing-induced failing canine hearts. No change of NCX current density was evident in myocytes from failing hearts when intracellular Ca(2+) ([Ca(2+)](i)) was buffered to 200 nmol/L. However, when [Ca(2+)](i) was minimally buffered with 50 micromol/L indo-1, Ca(2+) extrusion via NCX during caffeine application was doubled in failing versus normal cells. In other voltage-clamp experiments in which SR uptake was blocked with thapsigargin, both reverse-mode and forward-mode NCX currents and Ca(2+) transport were increased >2-fold in failing cells. These results suggest that, in addition to a relative increase in NCX function as a consequence of defective SR Ca(2+) uptake, there is an absolute increase in NCX function that depends on [Ca(2+)](i) in the failing heart.

Topics: Animals; Buffers; Caffeine; Calcium; Cardiac Pacing, Artificial; Cells, Cultured; Disease Models, Animal; Dogs; Electric Stimulation; Enzyme Inhibitors; Female; Heart Failure; Ion Transport; Male; Membrane Potentials; Myocardium; Nickel; Phosphodiesterase Inhibitors; Sarcoplasmic Reticulum; Sodium-Calcium Exchanger; Thapsigargin

To investigate how the morphological and physiological properties of single myocytes isolated from the hypertrophied, failing left ventricles (LV) differ from those of normal or hypertrophied not failing ventricles.. Single myocytes were isolated separately from right (RV) and left ventricles (LV) of male spontaneously hypertensive rats (SHR) or Wistar-Kyoto (WKY) rats at the age of 6 and 12 months and of SHRs which developed or not developed heart failure at the age of 20-24 months. We measured cells dimensions, range and kinetics of electrically stimulated or initiated by caffeine contractions and Ca2+ transients, and investigated the response of cells to thapsigargin.. The transversal dimensions of the LV myocytes of 6 months old SHRs showed approximately 20% increase with respect to transversal dimensions of their RV myocytes and LV and RV myocytes of WKY rats. The difference did not change with progressing age and in the heart failure. The LV myocytes of 6 or 12 months old SHRs showed slowed kinetics of the Ca2+ transients and of contraction and relaxation and decreased contractile response to 2 s superfusion with 15 mM caffeine preceded by 5 mM Ni2+ used as an index of the sarcoplasmic reticulum (SR) Ca2+ content. Despite of this the range of shortening and relative contribution of the SR to contraction (assessed by measuring of the residual contractile response to electrical stimulation in cells poisoned with thapsigargin) or relaxation (assessed by calculation of the ratio of rate constants of the electrically stimulated and stimulated by 30 s superfusion with caffeine Ca2+ transients) was not altered in the hypertrophied myocytes. Properties of the LV myocytes of the 20-24 old SHRs with or without heart failure did not differ from those of LV myocytes of younger SHRs. The contractile response to caffeine of their RV myocytes dropped to the level of that in the LV myocytes.. Our results suggest that transition from the compensated hypertrophy to the heart failure in 20-24 months old SHRs did not result from the further changes in properties of the surviving myocytes. Data from literature suggest that myocyte apoptosis and remodeling of the extramyocyte space is the more likely reason.

Topics: Animals; Apoptosis; Caffeine; Calcium; Cell Count; Cell Size; Central Nervous System Stimulants; Electric Stimulation; Enzyme Inhibitors; Heart Failure; Hypertrophy, Left Ventricular; Male; Muscle Fibers, Skeletal; Muscle Proteins; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sarcoplasmic Reticulum; Thapsigargin; Ventricular Dysfunction, Left; Ventricular Function, Right

To examine the contribution of reverse mode sodium-calcium (Na-Ca) exchange to contractions in isolated left-ventricular myocytes from failing human heart.. Low resistance patch pipettes were used to dialyze cells with Na-free or high-Na pipette solution ([Na]pipette = 0 and 20 mmol/L, respectively) to reduce or enhance Na-Ca exchange. Whole-cell membrane-potential, membrane-current and cell-shortening data were simultaneously acquired during whole-cell voltage clamp protocols. Thapsigargin (100 nmol/L) and nifedipine (1 mumol/L) were also used to inhibit sarcoplasmic reticulum (SR) Ca-ATPase and L-type Ca channels, respectively.. Two types of contractions were observed. Rapid phasic contractions were seen in both Na-free and high-Na cells. Slow tonic contractions were seen only in high-Na cells. Phasic contractions demonstrated bell-shaped voltage dependence over the voltage range that corresponds to the activity of the L-type Ca channel. Although the voltage dependence of phasic contractions were similar Na-free and high-Na cells, phasic contractions in high-Na cells were larger than phasic contractions in Na-free cells. Phasic contractions were sensitive to inhibition of SR Ca-ATPase and L-type Ca channels. Tonic contractions were not inhibited by either thapsigargin or nifedipine. In thapsigargin-treated high-Na cells, tonic contraction magnitude increased exponentially with test-potential.. The increases in phasic contraction magnitude observed in high-Na cells compared to Na-free cells were most likely due to increased SR Ca loading resulting from increased reverse-mode Na-Ca exchange. Our results also suggest that tonic contractions in high-Na cells were mediated by Ca entry via reverse-mode Na-Ca exchange and were not the result of either SR Ca release or L-type Ca channel activity.

Topics: Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Cells, Cultured; Cytosol; Enzyme Inhibitors; Heart Failure; Humans; Myocardial Contraction; Myocardium; Nifedipine; Sarcoplasmic Reticulum; Sodium; Sodium-Calcium Exchanger; Thapsigargin

beta-Receptor desensitisation, low basal cAMP, and a negative force-frequency relationship are characteristic changes in human heart failure. Isolated cardiomyocytes from noradrenaline-treated guinea pigs also show these features. We tested the hypothesis that low basal cAMP underlies the loss of contractile response to increasing stimulation frequency in this model.. Isolated cardiomyocytes were obtained from noradrenaline-treated (NA) and sham-operated (SHAM) guinea pigs. They were stimulated from 0.1-2 Hz and contraction amplitude was monitored with a video edge-detection system.. NA cells had less positive amplitude-frequency responses (AFR) compared to SHAMs at 2 mM (P = 0.002, n = 17), or midrange Ca2+ concentrations (EC40-EC60) (P < 0.001, n = 13). When the cAMP agonist, 8-CPT-cAMP (CPT, 10 microM) or high Ca2+ (above EC75) was added to NA cells the AFR was normalised to that of SHAM myocytes (NA vs. SHAM P = ns). In control experiments the cAMP antagonists, Rp-cAMPS (Rpc) and Rp-8-CPT-cAMPS (Rp8, 100 microM), blocked the positive inotropic effects of CPT at 0.5 Hz (control pD2 = 4.36 +/- 0.06, Rp8 pD2 = 3.68 +/- 0.08, P < 0.0001), n = 6 paired). Rpc (100 microM) completely but reversibly blocked the effect of maximal isoprenaline in control experiments (P < 0.0001). Neither antagonist reduced the AFR compared to time-matched controls (P = ns, n = 6). Blockade of SERCA2a with thapsigargin resulted in a significant reduction in the AFR (ANOVA P < 0.0001).. The results are consistent with sarcoplasmic reticulum (SR) function being a more important determinant of the amplitude-frequency relationship than tonic levels of cAMP under basal conditions. Reversal of AFR depression by CPT may result from stimulation of SR Ca2+ uptake.

Topics: Adrenergic beta-Agonists; Animals; Calcium; Calcium-Transporting ATPases; Cell Size; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Guinea Pigs; Heart Failure; Isoproterenol; Male; Myocardial Contraction; Myocardium; Norepinephrine; Thapsigargin; Thionucleotides

Sarcoplasmic reticulum (SR) Ca2+-ATPase gene expression is reduced in the failing myocardium. However, the functional relevance of these changes to myocardial contractility is not yet established. We assessed myocardial contractile function by analyzing sarcomere motion of isolated myocytes and also quantified SR Ca2+ regulatory protein gene expression by Northern blot analysis in the same hearts obtained from 10 dogs with pacing-induced heart failure (HF; 240 beats/min, 4 wk) and 7 control dogs. Sarcomere-shortening velocity was depressed in HF myocytes, accompanied by the prolongation of intracellular Ca2+ concentration ([Ca2+]i) transient measured by indo 1 fluorescence ratio. SR Ca2+-ATPase mRNA levels (normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA) were significantly depressed in HF, and calsequestrin mRNA was increased. For control and HF dogs, sarcomere-shortening velocity correlated positively with Ca2+-ATPase mRNA levels (r = 0.73, n = 17, P < 0.01) but not with calsequestrin mRNA. Ca2+-ATPase mRNA levels were correlated with 45Ca2+ uptake rate by SR, which was also reduced in HF. Moreover, the inhibition of SR Ca2+-ATPase with thapsigargin or cyclopiazonic acid reproduced in normal myocytes the abnormalities observed in HF myocytes, such as depressed contractility and the prolonged [Ca2+]i transient duration. A downregulation of Ca2+-ATPase gene expression and a resultant decrease in Ca2+ uptake by SR may be responsible for the contractile dysfunction and the alterations of [Ca2+]i transient in HF.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Calsequestrin; Cells, Cultured; Disease Models, Animal; Dogs; Echocardiography; Heart; Heart Failure; Myocardial Contraction; Myocardium; RNA, Messenger; Sarcomeres; Sarcoplasmic Reticulum; Tachycardia; Thapsigargin; Time Factors; Transcription, Genetic; Ventricular Function, Left

Available information regarding the cellular and molecular mechanisms for reduced myocardial function after myocardial infarction (MI) is scarce. In rats with congestive heart failure (CHF), we examined cardiomyocytes isolated from the non-infarcted region of the left ventricle 6 weeks after ligation of the left coronary artery. Systolic left-ventricular pressure was reduced and diastolic pressure was markedly increased in the CHF-rats. The cardiomyocytes isolated from the CHF-hearts had increased resting length, reduced fractional shortening by 31% and a 34% increase in time to 90% relaxation compared to sham cells (P<0.01 for all). Peak L-type calcium currents were not significantly changed, but peak calcium transients measured with fura-2 were reduced by 19% (P<0.01). Moreover, the decline of the calcium transients as measured by the time constant of a monoexponential function was significantly increased by 26% (P<0.01). We also examined the contribution of the Ca2+-ATPase of the sarcoplasmic reticulum (SR) in the removal of cytosolic Ca2+ during relaxation by superfusing cells with 1 microM thapsigargin that effectively inhibits the Ca2+-ATPase. Relaxation time in CHF-cells was significantly less prolonged when this drug was used (P<0.01). This suggests that other mechanisms, probably the Na+-Ca2+ exchanger, contribute significantly to the relaxation rate in CHF. Simultaneous measurements of fura-2 transients and mechanical shortening did not reveal any alteration in the calcium-myofilament sensitivity in CHF. Our study clearly shows reduced shortening and prolonged relaxation in cardiomyocytes isolated from non-infarcted region of the left ventricle in heart failure. Moreover, we were able to relate the observed cardiomyocyte dysfunction to changes in specific steps in the excitation-contraction coupling.

Topics: Actin Cytoskeleton; Animals; Calcium; Calcium Channels; Calcium-Transporting ATPases; Colforsin; Fura-2; Heart Failure; Heart Ventricles; Hemodynamics; Male; Muscle Contraction; Myocardial Infarction; Myocardium; Patch-Clamp Techniques; Rats; Rats, Wistar; Sarcoplasmic Reticulum; Sodium; Thapsigargin

There has been debate regarding the level of sarcoplasmic reticulum (SR) Ca2+ ATPase protein in heart failure. We have used the SR Ca2+ ATPase inhibitor thapsigargin to investigate the functional contribution of this uptake system to contraction and relaxation in myocytes from failing and non-failing human ventricle.. Myocytes were isolated from 28 failing and 18 non-failing ventricles and stimulated at 0.2 Hz, 32 degrees C in Krebs-Henseleit solution. Contraction amplitude and speed were compared before and after treatment with 1 mumol/l thapsigargin for 20 min to deplete SR Ca2+ stores.. Initial beat duration was longer in myocytes from failing hearts. Addition of thapsigargin significantly prolonged the beat in myocytes from both groups, but the increase was greater in non-failing hearts (beat duration increased by 0.79 +/- 0.12 s in myocytes from non-failing hearts compared with 0.37 +/- 0.12 s in those from failing, P < 0.02). The contraction amplitude increased at high stimulation frequencies in myocytes from non-failing hearts (from 2.6% shortening at 0.1 Hz to 4.6% at 1 Hz, P < 0.001, n = 9), but not in those from failing hearts (1.8% at 0.1 Hz compared with 1.7% at 1 Hz, n = 5). Thapsigargin abolished the positive staircase in the non-failing, but had no effect in the failing group. Contraction amplitude following a rest interval was significantly depressed relative to steady-state levels in myocytes from the non-failing hearts (44.8 +/- 10.3% at 3 min), but not in failing (102 +/- 18%, P < 0.01 compared to non-failing at 3 min). Following thapsigargin treatment, there were no longer significant differences between failing and non-failing myocytes in the time course of the beat, frequency response or post-rest behaviour.. The differences between myocytes from failing and non-failing hearts were reduced by inhibition of SR function. These results are consistent with the hypothesis that the initial differences had been due to decreased SR Ca2+ uptake.

Topics: Aged; Calcium; Calcium-Transporting ATPases; Cell Size; Cells, Cultured; Electric Stimulation; Female; Heart Failure; Humans; Male; Middle Aged; Myocardial Contraction; Myocardium; Sarcoplasmic Reticulum; Thapsigargin

The purpose of this study was to examine the activity and expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase in left ventricular (LV) myocardium of dogs with chronic heart failure (HF). LV and right ventricular (RV) tissue specimens were obtained from six normal (NL) control dogs and six dogs with chronic HF (LV ejection fraction, 23 +/- 2%) produced by multiple sequential intracoronary microembolizations. Thapsigargin-sensitive Ca(2+)-ATPase activity was measured in isolated SR membrane fractions prepared from LV and RV myocardium. Ca(2+)-ATPase expression, using a specific dog myocardium monoclonal antibody, was measured in sodium dodecyl sulfate (SDS) extract prepared from LV and RV myocardium. Ca(2+)-ATPase activity in both ventricles of NL or HF dogs increased with increasing Ca2+ concentration and reached a plateau at 3 microM Ca2+. The maximal velocity (Vmax, mumol Pi released.min-1.mg-1) of Ca(2+)-ATPase activity was significantly lower in LV of HF dogs compared with NL (0.15 +/- 0.01 vs. 0.23 +/- 0.01, P < 0.05), whereas the affinity of the Ca2+ pump for Ca2+ was unchanged. LV tissue levels of Ca(2+)-ATPase (densitometric units/5 micrograms noncollagen protein) were also significantly lower in LV myocardium of HF dogs compared with NL (3.52 +/- 0.43 vs. 5.53 +/- 0.47, P < 0.05). No significant differences in Ca(2+)-ATPase activity or expression were observed in RV myocardium of HF dogs compared with NL. We conclude that SR Ca(2+)-ATPase activity and protein levels are reduced in LV myocardium of dogs with chronic HF. This abnormality of the SR Ca2+ pump of the failed LV can result in impaired Ca2+ uptake and ultimately to Ca2+ overload and global LV dysfunction.

Topics: Animals; Blood Pressure; Blotting, Western; Calcium-Transporting ATPases; Calsequestrin; Coronary Angiography; Diastole; Dogs; Female; Heart Failure; Heart Ventricles; Hemodynamics; Intracellular Membranes; Kinetics; Male; Myocardium; Sarcoplasmic Reticulum; Systole; Thapsigargin; Ventricular Function, Left

Rapid cardiac pacing has been used as a model for experimentally-induced cardiomyopathy. However, its relevance to human heart failure is not clear at present because little is known about changes in size and function of ventricular myocytes. We have therefore studied the responses to graded increases in frequency and calcium in canine ventricular myocytes from failing hearts. The aim of our study was to evaluate the resemblance between canine pacing-induced and human end-stage heart failure.. Myocytes were isolated from the left ventricular wall of dogs that were in heart failure after 6 weeks of pacing at 250 beats/min. Cell shortening was measured by edge detection.. Clinical signs of failure included dyspnea, ascites, and heart dilatation; the hemodynamic parameters were: LVdP/dtmax 1613 +/- 149 vs. 4713 +/- 304 mmHg/s in 6 control dogs; LVEDP 17.2 +/- 4.4 vs 5.6 +/- 1.1 mmHg; LV volume 60.5 +/- 6.2 vs. 30-35 ml. Myocytes from failing hearts were longer and thinner than those from controls (from factor: 0.40 +/- 0.01 vs. 0.47 +/- 0.01, P < 0.001, > 30 cells/heart). With 6 mM Ca2+ and at 0.5 Hz, contraction amplitude was significantly attenuated in myocytes from failing hearts: 6.6 +/- 0.9% cell shortening vs. 10.0 +/- 0.8% in controls (P < 0.05). This deficit was exacerbated at higher stimulation rates. Time-to-peak contraction and time-to-50% relaxation were not altered. There was no difference in sensitivity to thapsigargin.. As with cells from human failing hearts, contraction amplitude showed rate-dependent depression in this animal model, whereas features like slowing of contraction and relaxation and reduced sensitivity to thapsigargin, were not reproduced.

Topics: Animals; Anti-Arrhythmia Agents; Calcium; Calcium-Transporting ATPases; Cell Size; Cells, Cultured; Dogs; Electric Stimulation; Heart Failure; Myocardial Contraction; Myocardium; Thapsigargin