Sarcoplasmic reticulum Ca2+-ATPase modulates cardiac contraction and relaxation

The cardiac SR Ca(2+)-ATPase (SERCA2a) regulates intracellular Ca(2+)-handling and thus, plays a crucial role in initiating cardiac contraction and relaxation. SERCA2a may be modulated through its accessory phosphoprotein phospholamban or by direct phosphorylation through Ca(2+)/calmodulin dependent protein kinase II (CaMK II). As an inhibitory component phospholamban, in its dephosphorylated form, inhibits the Ca(2+)-dependent SERCA2a function, while protein kinase A dependent phosphorylation of the phospho-residues serine-16 or Ca(2+)/calmodulin-dependent phosphorylation of threonine-17 relieves this inhibition. Recent evidence suggests that direct phosphorylation at residue serine-38 in SERCA2a activates enzyme function and enhances Ca(2+)-reuptake into the sarcoplasmic reticulum (SR). These effects that are mediated through phosphorylation result in an overall increased SR Ca(2+)-load and enhanced contractility. In human heart failure patients, as well as animal models with induced heart failure, these modulations are altered and may result in an attenuated SR Ca(2+)-storage and modulated contractility. It is also believed that abnormalities in Ca(2+)-cycling are responsible for blunting the frequency potentiation of contractile force in the failing human heart. Advanced gene expression and modulatory approaches have focused on enhancing SERCA2a function via overexpressing SERCA2a under physiological and pathophysiological conditions to restore cardiac function, cardiac energetics and survival rate.     

Abnormal Ca2+ release from cardiac sarcoplasmic reticulum in tachycardia-induced heart failure

OBJECTIVE: In heart failure, little information is available as to the Ca2+ release function of sarcoplasmic reticulum (SR), which plays a major role in cardiac contractile function. Here, we assessed the rapid kinetics of drug-induced Ca2+ release from cardiac SR in combination with a measurement of ryanodine binding in heart failure. METHODS: The SR vesicles were isolated from dog left ventricular (LV) muscles (normal (N), n = 10; pacing induced heart failure (HF), n = 10). The time course of SR Ca2+ release was continuously monitored by a stopped-flow apparatus using arsenazoIII as a Ca2+ indicator, and Ca2+ uptake and [3H]ryanodine binding assays were done using a filtration method. RESULTS: The amount of Ca2+ uptake was reduced in HF to 55% of N (P < 0.05). Even the more marked and earlier appeared decrease was seen in the rate constant and the initial rate of polylysine (PL; a specific release trigger)-induced Ca2+ release (P < 0.05). However, the PL concentration dependency of the initial rate shifted towards lower concentrations of PL in HF than in N ([PL] at half maximum stimulation = 0.13 vs. 0.35 microM). The [3H]ryanodine binding assay revealed a lower Bmax (pmol/mg) in HF than in N (0.91 +/- 0.19 vs. 2.64 +/- 0.59, P < 0.05), but no difference in Kd (nM) (0.95 +/- 0.29 vs. 0.90 +/- 0.11, P = n.s.). The [PL] dependency on the enhancement of [3H]ryanodine binding again showed a shift towards lower [PL] in HF than in N. CONCLUSIONS: In pacing-induced heart failure, the Ca2+ releasing function of SR is disturbed, which may result in an intra-cellular Ca2+ transient that was slowed down.     

Altered hepatocellular Ca2+ regulation during hemorrhagic shock and resuscitation

The present study evaluated the effect of the benzothiazepine Ca²⁺ channel blocker diltiazem (DZ) on altered hepatocellular Ca²⁺ regulation and oxidant injury during hemorrhagic shock/resuscitation. In anesthetized, male Sprague-Dawley rats, hemorrhagic shock was induced by rapid blood withdrawal and maintaining the mean arterial blood pressure at 40 mm Hg over 60 minutes. Rats were then resuscitated with 60% of shed blood and threefold the shed blood volume of Ringer's lactate. At the end of ischemia, and 60 or 300 minutes after resuscitation, hepatocytes were isolated by liver collagenase perfusion. Hepatocellular Ca²⁺ exchange (Ca²⁺_ex), rate of cellular Ca²⁺ influx (Ca²⁺_in), and Ca²⁺ membrane flux (Ca²⁺_flux) were determined using 45Ca incubation techniques. Hepatocyte oxidant injury was evaluated by fluorometrically measuring thiobarbituric acid reactive substances and oxidized/reduced glutathione. Both hemorrhage and hemorrhage/resuscitation increased hepatocellular Ca²⁺_in, Ca²⁺_ex, and Ca²⁺_flux. In contrast to control and sham-operated rats, in vitro stimulation by the Ca²⁺ agonist epinephrine (100 nmol/L) of hepatocytes from either hemorrhaged or resuscitated rats did not further increase Ca²⁺_in. Administration of DZ (.8 mg/kg) with resuscitation significantly decreased cellular Ca²⁺_ex and Ca²⁺_flux, but did not restore impaired epinephrine-induced Ca²⁺_in. DZ prevented hepatocyte lipid peroxidation and glutathione oxidation. These findings suggest hepatocellular Ca²⁺ overload and impaired Ca²⁺ signaling during hemorrhage/resuscitation. Increased Ca²⁺ uptake could be because of a receptor-gated Ca²⁺ influx and/or oxygen-free radical induced membrane Ca²⁺ leaks.(Hepatology 1997 Feb;25(2):379-84)     

Altered phosphatase activity in heart failure, influence on Ca 2+ movement

Many cardiac proteins undergo reversible phosphorylation. While the protein kinases which bring about phosphorylations are well studied, less effort has been put into the dephosphorylating phosphatases (for an earlier review compare 14). An important event in the heart, which is controlled by phosphorylation, is the uptake of Ca ²⁺ by the sarcoplasmic reticulum (SR). This process is brought about by a SR Ca ²⁺ ATPase (SERCA) and accounts for relaxation. The amount of Ca ²⁺ pumped by SERCA is enhanced when phospholamban (PLB), an intrinsic protein of the SR, is phosphorylated and is diminished when PLB is dephosphorylated. PLB is dephosphorylated by protein phosphatases (PPs) like PP1. As the activity of PP1 is enhanced in heart failure, subsequent dephosphorylation by of, e.g., PLB may explain the impaired relaxation of the human heart. Thus, PPs may play an important role in the etiology and/or symptoms of heart failure.     

Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure

Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin-dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.     

Ca2+-handling in heart failure – a review focusing on Ca2+ sparks

[Ca ²⁺] _i-transients have been shown to be altered in isolated ventricular myocytes from terminally failing human myocardium. It has been demonstrated that one reason for this alteration is a reduction in the Ca ²⁺ content of the sarcoplasmic reticulum (SR). Further investigations were done to investigate, whether there may be an additional defect of the Ca ²⁺-release mechanisms from the SR. These release mechanisms were investigated through the recording of Ca ²⁺ sparks in single human myocytes. In cardiac myocytes, Ca ²⁺ sparks are elementary units of Ca ²⁺ release, which occur spontaneously, or which are triggered by Ca ²⁺ influx through L-type Ca ²⁺-channels (Ca ²⁺-induced Ca ²⁺ release). Ca ²⁺ sparks have been investigated in various animal models of cardiac hypertrophy and cardiac failure and results were conflicting. Discrepancies may be explained by different species and also by the mechanisms underlying hypertrophy and heart failure. This review summarizes our current knowledge on Ca ²⁺ sparks in heart failure.     

Correlation between myofilament response to Ca2+ and altered dynamics of contraction and relaxation in transgenic cardiac cells that express beta-tropomyosin

We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.     

Altered Na+/Ca2+-exchanger activity due to downregulation of Na+/K+-ATPase alpha2-isoform in heart failure

AIMS: The Na+/K+-ATPase (NKA) alpha2-isoform is preferentially located in the t-tubules of cardiomyocytes and is functionally coupled to the Na+/Ca(+-exchanger (NCX) and Ca2+ regulation through intracellular Na+ concentration ([Na+]i). We hypothesized that downregulation of the NKA alpha2-isoform during congestive heart failure (CHF) disturbs the link between Na+ and Ca2+, and thus the control of cardiomyocyte contraction. METHODS AND RESULTS: NKA isoform and t-tubule distributions were studied using immunocytochemistry, confocal and electron microscopy in a post-infarction rat model of CHF. Sham-operated rats served as controls. NKA and NCX currents (I NKA and I NCX) were measured and alpha2-isoform current (I NKA,alpha2) was separated from total I NKA using 0.3 microM ouabain. Detubulation of cardiomyocytes was performed to assess the presence of alpha2-isoforms in the t-tubules. In CHF, the t-tubule network had a disorganized appearance in both isolated cardiomyocytes and fixed tissue. This was associated with altered expression patterns of NKA alpha1- and alpha2-isoforms. I NKA,alpha2 density was reduced by 78% in CHF, in agreement with decreased protein expression (74%). When I NKA,alpha2 was blocked in Sham cardiomyocytes, contractile parameters converged with those observed in CHF. In Sham, abrupt activation of I NKA led to a decrease in I NCX, presumably due to local depletion of [Na+]i in the vicinity of NCX. This decrease was smaller when the alpha2-isoform was downregulated (CHF) or inhibited (ouabain), indicating that the alpha2-isoform is necessary to modulate local [Na+]i close to NCX. CONCLUSION: Downregulation of the alpha2-isoform causes attenuated control of NCX activity in CHF, reducing its capability to extrude Ca2+ from cardiomyocytes.     

Bidirectional regulation of Ca2+ sparks by mitochondria-derived reactive oxygen species in cardiac myocytes

AIMS: The cardiac ryanodine receptor (RyR) Ca(2+) release channel homotetramer harbours approximately 21 potentially redox-sensitive cysteine residues on each subunit and may act as a sensor for reactive oxygen species (ROS), linking ROS homeostasis to the regulation of Ca(2+) signalling. In cardiac myocytes, arrayed RyRs or Ca(2+) release units are packed in the close proximity of mitochondria, the primary source of intracellular ROS production. The present study investigated whether and how mitochondria-derived ROS regulate Ca(2+) spark activity in intact cardiac myocytes. METHODS AND RESULTS: Bidirectional manipulation of mitochondrial ROS production in intact rat cardiac myocytes was achieved by photostimulation and pharmacological means. Simultaneous measurement of intracellular ROS and Ca(2+) signals was performed using confocal microscopy in conjunction with the indicators 5-(-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (for ROS) and rhod-2 (for Ca(2+)). Photoactivated or antimycin A (AA, 5 microg/mL)-induced mitochondrial ROS production elicited a transient increase in Ca(2+) spark activity, followed by gradual spark suppression. Intriguingly, photoactivated mitochondrial ROS oscillations subsequent to the initial peaks mirrored phasic depressions of the spark activity, suggesting a switch of ROS modulation from spark-activating to spark-suppressing. Partial deletion of Ca(2+) stores in the sarcoplasmic reticulum contributed in part to the gradual, but not the phasic, spark depression. H(2)O(2) at 200 microM elicited a bidirectional effect on sparks and produced sustained spark activation at 50 microM. Lowering basal mitochondrial ROS production, scavenging baseline ROS, and applying the sulphydryl-reducing agent dithiothreitol diminished the incidence of spontaneous Ca(2+) sparks and abolished the Ca(2+) spark responses to mitochondrial ROS. CONCLUSION: Mitochondrial ROS exert bidirectional regulation of Ca(2+) sparks in a dose- and time (history)-dependent manner, and basal ROS constitute a hitherto unappreciated determinant for the production of spontaneous Ca(2+) sparks. As such, ROS signalling may play an important role in Ca(2+) homeostasis as well as Ca(2+) dysregulation in oxidative stress-related diseases.     

Ca2+-activated K+ channels mediate relaxation of forearm veins in chronic renal failure

BACKGROUND: In arteries, agonists such as acetylcholine release an endothelium-derived hyperpolarizing factor (EDHF) that is neither nitric oxide nor prostacyclin. OBJECTIVES: To examine the responses to acetylcholine in segments of forearm veins from patients with chronic renal failure who either had never received dialysis or had undergone long-term dialysis, and to determine the contribution of nitric oxide and EDHF to endothelium-dependent relaxation in veins from patients with chronic renal failure. METHODS: Isometric tension was recorded in rings of forearm vein from 34 non-dialysed patients, 27 dialysed patients and 14 multiorgan donors (controls). RESULTS: Relaxation in response to acetylcholine was reduced in veins of non-dialysed and dialysed patients. The inhibitors of nitric oxide synthase NG-monomethyl-l-arginine (l-NMMA) and NG,NG-dimethyl-l-arginine (ADMA) reduced by 50% the maximum relaxation in response to acetylcholine in veins from controls and non-dialysed patients; the remaining relaxation was inhibited by 20 mmol/l KCl or by the K+ channel blockers tetraethylammonium chloride, iberiotoxin, charybdotoxin and the combination of barium plus ouabain, but not by apamin or glibenclamide. Relaxation in veins from dialysed patients was inhibited by K+ channel blockade but not by l-NMMA or ADMA. CONCLUSIONS: The results demonstrate that the endothelium-dependent relaxation in forearm veins from controls and non-dialysed patients is mediated by release of nitric oxide and EDHF. In contrast, the relaxation in veins from dialysed patients is mediated mainly by EDHF. EDHF-induced relaxation involves activation of large-conductance Ca2+-activated K+ channels.     

Altered beta-adrenergic receptor gene regulation and signaling in chronic heart failure

J. D. Port and M. R. Bristow. Altered Beta-adrenergic Receptor Gene Regulation and Signaling in Chronic Heart Failure. Journal of Molecular and Cellular Cardiology (2001) 33, 887-905. Beta adrenergic receptors (beta -ARs) are critical regulators of cardiac function in both normal and pathophysiological states. Under normal conditions, beta -ARs and their signaling pathways modulate both the rate and force of myocardial contraction and relaxation, allowing individuals to respond appropriately to physiological stress or exercise. However, in chronic heart failure, sustained activation of the beta -AR signaling pathways can have overtly negative biological consequences. This notion is reinforced by the positive outcomes of a number of clinical trials demonstrating the usefulness of beta-blocker therapy in chronic congestive heart failure. During the last few years, significant progress has been made in understanding the molecular biological basis of beta -AR function, both at the biochemical and genetic levels. In this review, the biological basis of adrenergic signaling and how this changes in heart failure is discussed. Aspects of adrenergic receptor pharmacology relevant to heart failure are reviewed, including the recently emerging differences described for beta(1)- v beta(2)-AR signaling pathways. Highlighting these differences is recent evidence that over-stimulation of the beta(1)-AR pathway in cardiac myocytes appears to be pro-apoptotic, whereas stimulation of the beta(2)-AR pathway may be anti-apoptotic. Overview of beta -AR gene regulation, transgenic models of beta -AR overexpression, and beta -AR polymorphisms as they relate to heart failure progression are also discussed.     

Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.     

Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy

Mutations in cardiac Troponin T (TnT) are responsible for approximately 15% of all cases of familial hypertrophic cardiomyopathy (FHC). This review summarizes recent data from in vitro assays, transgenic models and clinical studies on how TnT mutations alter the regulation of cardiac muscle contraction. Each TnT mutation has somewhat different effects on myofilament properties (increased myofilament Ca(2)+ sensitivity, decreased maximal force, decreased binding affinity to the thin filament, impaired pH-regulation). But when the in vitro data are correlated with the results from the transgenic models, essentially all mutations can be predicted to result in: (1) impaired relaxation, (2) reduced diastolic compliance, (3) reduced contractile reserve, (4) preserved systolic function under baseline conditions, and (5) cardiac dysfunction under inotropic stimulation. Thus, the alterations of myofilament function caused by TnT mutations likely play an important role in the pathogenesis of FHC.     

Role of Ca2+ availability to myofilaments and their sensitivity to Ca2+ in myocyte contractile dysfunction in heart failure

OBJECTIVE: Contractile function is depressed at the isolated myocyte level in heart failure (HF), which could result from the decreased availability of intracellular calcium ([Ca2+]i) to the myofibrils and/or the depressed sensitivity of myofilaments to [Ca2+]i. However, the cellular basis of contractile dysfunction remains unestablished. METHODS: We isolated left ventricular myocytes from dogs with rapid pacing-induced HF. Cell shortening and [Ca2+]i transients were measured by indo-1 fluorescence and the myofilament Ca2+ sensitivity was analyzed by the shortening-[Ca2+]i relation in intact myocytes as well as by the pCa tension relation in skinned cells. RESULTS: Peak cell shortening magnitude was depressed in HF, associated with a parallel decrease of [Ca2+]i transient amplitude. There was a significant positive correlation between these two variables (r = 0.71, P < 0.01). In contrast, myofibrillar sensitivity to Ca2+, determined by both intact and skinned myocytes, was comparable between control and HF. Further, there was no significant difference in Ca2+ sensitivity between control and HF even at shorter (1.8 microns) or longer (2.2 microns) sarcomere length. CONCLUSIONS: Using both intact and skinned cellular preparations, a potential defect in myocyte contractile function in HF was a reduction in Ca2+ availability to the myofilaments, rather than the inherent defects in myofilament sensitivity to Ca2+.