Control Of Cardiac Contractility Research Articles

Phosphoinositide 3-Kinase C2alpha controls cardiac contractility through regulation of beta2-adrenergic receptor recycling

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Doctoral fellowship Introduction Phosphoinositide 3-kinase C2α (PI3KC2α) is a ubiquitously expressed class II PI3K isoform, which has been previously shown to be involved in the control of vesicular trafficking. However, its role in the myocardium has not been investigated. The primary aim of this study is to elucidate the mechanism by which mechanism PI3KC2α controls cardiac pathophysiology. Methods Cardiomyocyte-specific PI3KC2α knockout (PI3KC2α KO) animals were generated by crossing mice expressing a tamoxifen-inducible Cre recombinase under the control of the αMHC promoter with PI3KC2αflox/flox mice. Zebrafish embryos were injected with a PI3KC2α (PI3KC2α morphants) or a control morpholino (control morphants) at 1-cell stage. HEK293 with stable overexpression of GFP-tagged β2-AR were generated and transfected with either scramble or PI3KC2α siRNAs. Results Our findings indicate that PI3KC2α KO mice display a reduced cardiac contractility compared to wildtype animals. Similarly, PI3KC2α morphants zebrafish exhibit lower heart rate and fractional shortening compared to controls, both at baseline and after isoproterenol (ISO) stimulation. A similar unresponsiveness to ISO was found in vivo in PI3KC2α KO mice where chronic treatment with the β-adrenergic receptor (β-AR) agonist failed to induce the classical β-AR-mediated remodeling, characterized by an increase of the left ventricular mass and of cardiomyocyte area. These findings suggest that PI3KC2α plays a critical role in the regulation of β-AR signaling. In agreement, cAMP levels failed to increase in response to ISO treatment in PI3KC2α morphants. Furthermore, silencing of PI3KC2α in HEK293-GFP-β2-AR cells resulted in an increased GFP-β2-AR internalization compared to control cells, both at baseline and after ISO stimulation. Interestingly, preliminary findings indicate that overexpression of constitutively active Rab11Q70L induced a redistribution of GFP-β2-AR at the plasma membrane in PI3KC2α-silenced cells. Conclusion Overall, we identify a key role for PI3KC2α in the control of cardiac contractility through the regulation of β2-AR trafficking to the plasma membrane through a Rab11-dependent mechanism.

Mixed-solvent molecular dynamics simulation-based discovery of a putative allosteric site on regulator of G protein signaling 4.

Regulator of G protein signaling 4 (RGS4) is an intracellular protein that binds to the Gα subunit ofheterotrimeric G proteins and aids in terminating G protein coupled receptor signaling. RGS4 has been implicated in pain, schizophrenia, and the control of cardiac contractility. Inhibitors of RGS4 have been developed but bind covalently to cysteine residues on the protein. Therefore, we sought to identify alternative druggable sites on RGS4 using mixed-solvent molecular dynamics simulations, which employ low concentrations of organic probes to identify druggable hotspots on the protein. Pseudo-ligands were placed in consensus hotspots, and perturbation with normal mode analysis led to the identification and characterization of a putative allosteric site, which would be invaluable for structure-based drug design of non-covalent, small molecule inhibitors. Future studies on the mechanism of this allostery will aid in the development of novel therapeutics targeting RGS4.

Abstract P184: Coordinated Control of Cardiac Contractility By Multiple RGS Proteins

Signaling via heterotrimeric G proteins is critical to maintaining cardiovascular homeostasis. Abnormal G protein signaling due to the loss of G protein regulation by proteins such as regulators of G protein signaling (RGS) proteins is implicated in several cardiovascular disorders such as hypertension, cardiac hypertrophy, and heart failure. RGS act as GTPase activating proteins (GAPs) to control the kinetics and amplitude of G protein signaling. Multiple RGS proteins are prominently expressed in the cardiovascular system; however, it is unknown whether their activities/functions are coordinated to control G protein signaling and cardiovascular function. This study used mice concurrently lacking RGS2 and 5 ( Rgs2/5 dbKO) to determine how the dual loss of potent GAPs for Gq/11 and Gi/o class G proteins affects cardiovascular function. Blood pressure and heart rate in conscious, freely moving mice were monitored via radiotelemetry. Surgical implantation of the radiotelemetry device induced marked systolic blood pressure increase in Rgs2/5 dbKO mice (WT: 140 ± 6 vs. dbKO: 170 ± 2 mmHg; p <0.01) at baseline, which gradually declined but remained elevated above wild type (WT) control level several days later. Whereas all WT mice survived the surgery, ~70-80% of male Rgs2/5 dbKO mice died 72-96 hr post-surgery. When subjected to cardiac stress test using acute dobutamine infusion and echocardiography, male Rgs2/5 dbko mice showed hypocontractile response relative to WT mice. Freshly isolated ventricular cardiomyocytes from male Rgs2/5 dbKO mice showed decreased fractional shortening (WT: 16.1 ± 4.3 vs. dbKO: 7.4 ± 1.1 %; p <0.01) but high calcium transients (WT: 117 ± 20 vs. dbKO: 198 ± 50 au; p =0.07) at baseline, and application of electrical field stimulation or the non-selective β-adrenergic receptor agonist, isoproterenol (ISO), triggered premature calcium transients, tachyarrhythmia and death of cells from Rgs2/5 dbKO mice. Interestingly, cells from mice harboring just one copy of Rgs2 ( Rgs2+/-,Rgs5-/- ) but not Rgs5 ( Rgs2-/-,Rgs5+/- ) were resistant to low-dose ISO-induced arrhythmia. These results together suggest that RGS2 and 5 coordinate their activity to control cardiomyocyte excitation-contraction coupling and normal cardiac rhythm.

Oxidative Stress and Sarcomeric Proteins

Oxidative stress accompanies a wide spectrum of clinically important cardiac disorders, including ischemia/reperfusion, diabetes mellitus, and hypertensive heart disease. Although reactive oxygen species (ROS) can activate signaling pathways that contribute to ischemic preconditioning and cardioprotection, high levels of ROS induce structural modifications of the sarcomere that impact on pump function and the pathogenesis of heart failure. However, the precise nature of the redox-dependent change in contractility is determined by the source/identity of the oxidant species, the level of oxidative stress, and the chemistry/position of oxidant-induced posttranslational modifications on individual proteins within the sarcomere. This review focuses on various ROS-induced posttranslational modifications of myofilament proteins (including direct oxidative modifications of myofilament proteins, myofilament protein phosphorylation by ROS-activated signaling enzymes, and myofilament protein cleavage by ROS-activated proteases) that have been implicated in the control of cardiac contractility.

Imaging of the Ryanodine Receptor Distribution in Rat Cardiac Myocytes with Optical Single Channel Resolution

We have applied a new optical super-resolution technique based on single molecule localisation to examine the peripheral distribution of a cardiac signalling protein, the ryanodine receptor (RyR), in rat ventricular myocytes. Using high-resolution antibody labeling data we show that the new imaging approach, termed localization microscopy, can give novel insight into the distribution of large proteins, with optical single channel resolution. We present, to our knowledge, the first direct data showing evidence for a two-dimensional array-like arrangement of RyRs in cardiac muscle. Morphological analysis of peripheral RyR clusters in the surface membrane revealed a mean size of ∼14 RyRs per cluster, almost an order of magnitude smaller than previously estimated. Clusters were typically not circular (as previously assumed) but elongated with an average aspect ratio of 1.9. Edge-to-edge distances between adjacent RyR clusters were often less than 50 nm suggesting that peripheral RyR clusters may exhibit strong inter-cluster signalling. The cluster size varied widely and followed a near-exponential distribution. We show that this distribution is compatible with a stochastic cluster assembly process and construct simple cluster growth models that generate size distributions very similar to our experimental observations. Based on the placement and morphology of RyR clusters we suggest that calcium sparks may be the result of the concerted activation of several clusters forming a functional ‘supercluster’ whose gating is controlled by both cytosolic and sarcoplasmic reticulum luminal calcium levels. The new imaging approach can be extended to other cardiac proteins and should yield novel insight into excitation-contraction coupling and the control of cardiac contractility.

Functionally Opposing Roles of Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase in the Regulation of Cardiac Contractility

Extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38-MAPK) have been shown to regulate various cellular processes, including cell growth, proliferation, and apoptosis in the heart. However, the function of these signaling pathways in the control of cardiac contractility is unclear. Here, we characterized the contribution of ERK1/2 and p38-MAPK to the inotropic effect of endothelin-1 (ET-1). In isolated perfused rat hearts, infusion of ET-1 (1 nmol/L) for 10 minutes increased contractility and phosphorylation of ERK1/2 and their downstream target p90 ribosomal S6 kinase (p90RSK). Suppression of ERK1/2 activation prevented p90RSK phosphorylation and attenuated the inotropic effect of ET-1. Pharmacological inhibition of epidermal growth factor receptor kinase activity abolished ET-1-induced epidermal growth factor receptor transactivation and ERK1/2 and p90RSK phosphorylation and reduced ET-1-mediated inotropic response. Moreover, inhibition of the p90RSK target Na(+)-H(+) exchanger 1 attenuated the inotropic effect of ET-1. In contrast to ERK1/2 signaling, suppression of p38-MAPK activity further augmented ET-1-enhanced contractility, which was accompanied by increased phosphorylation of phospholamban at Ser-16. MAPKs play opposing roles in the regulation of cardiac contractility in that the ERK1/2-mediated positive inotropic response to ET-1 is counterbalanced by simultaneous activation of p38-MAPK. Hence, selective activation of ERK1/2 signaling and inhibition of p38-MAPK signaling may represent novel means to support cardiac function in disease.

Nitroxyl effects on myocardium provide new insights into the significance of altered myofilament response to calcium in the regulation of contractility

The idea that cardiac sarcomeres are not passive slaves to membrane related mechanisms modifying Ca2+ fluxes is generally accepted. There is substantial evidence that fundamental regulatory mechanisms including the Frank–Starling mechanism and neuro-humoral control of cardiac dynamics involve alterations in Ca2+ binding to troponin C (TnC) or transduction of the Ca–TnC signal to the promotion of ATP hydrolysis, force and shortening (Solaro, 2001; Kobayashi & Solaro 2005). In testimony to the recognition of the relative significance of these potential mechanisms in control of cardiac contractility, there has been a long-standing effort to develop inotropic agents acting directly on the myofilaments to modify their response to Ca2+ (Kass & Solaro, 2006). A paper by Dai et al. (2007) in this issue of The Journal of Physiology marks a new milestone in this search for ways to modify cardiac myofilament response to Ca2+. They demonstrated that a donor of nitroxyl (HNO) increases maximum tension and Ca2+ sensitivity of trabeculae sarcomeres functioning in situ. Although HNO also increased the peak amplitude of the Ca2+ transient, when compared to controls, the increase in tension was greater than expected from the rise in Ca2+. Except for the implication of thiols for functionally significant Cys residues in the sarcomeric proteins, the molecular mechanism of the effects of HNO remains obscure. An appealing property of HNO is that the enhancement of myofilament Ca2+ response occurs with a concomitant increase in Ca2+ flux into and out of the sarcoplasmic reticulum by a mechanism independent of β-adrenergic receptors, cAMP and cGMP. Other agents that increase myofilament response to Ca2+ have an associated inhibition of cAMP phospho-diesterases. An increase in cAMP increases the threat of arrythmias. The effects of HNO indicate a novel mechanism of enhancement of contractility without an effect on diastolic function and with a relative increase in the efficiency transduction of the Ca2+ binding signal. One aspect of this increase in efficiency is that the increase in myofilament Ca2+ response permits an increase in force generation while sparing the energy required in transporting Ca2+ to storage depots. There is also the possibility of increased economy in the unit change in ATP hydrolysis required for a unit change in tension (tension cost). Dai et al. (2007) reported no effect of HNO on ATPase rate of isolated myofibrils, but these preparations do not generate force. More rigorous tests are required in preparations bearing a load. The identification of the mechanism of the effect of HNO is necessarily couched in our understanding of the molecular mechanisms of the transitions of sarcomeres from diastolic to systolic states and how this transition is geared into the overt function of the heart to develop pressure, eject against the arterial impedance so as to satisfy metabolic needs of the body, and relax with a time course so as to not compromise filling for the next cycle. The critical parameter with regard to the relative role of the sarcomeres is not the kinetics of free Ca2+, detected by cytoplasmic probes, but the kinetics of Ca bound to TnC during the heart beat. Computations based on best estimates of the kinetics of Ca binding to the myofilaments during a beat of the ejecting ventricle indicate that Ca bound to TnC falls faster than systolic pressure and is near zero during isovolumic relaxation (Peterson et al. 1991; Hinken & Solaro, 2007). The mechanism that keeps the myofilaments activated with relatively little Ca bound resides in the ability of strongly bound crossbridges to promote Ca-binding affinity of TnC within a regulatory unit (actin: Tn complex: tropomyosin: myosin in a 7: 1: 1: 1 ratio) and to spread activation longitudinally along the thin filament by near neighbour cooperative mechanisms (Landesberg, 1996; Moss et al. 2004). Future investigation of HNO should include a detailed dissection of potential effects on thin and thick filament proteins as well as determination of effects on TnC–Ca and tension cost. Tropomyosin seems a likely target in view of its functionally significant and reactive Cys as well its key role as a determinant of Ca sensitivity of the number of cross-bridges reacting with the thin filament. It will also be of interest to know the effects of HNO in fast and slow skeletal muscle.

New milrinone analogues: in vitro study of structure-activity relationships for positive inotropic effect, antagonism towards endogenous adenosine, and inhibition of cardiac type III phosphodiesterase

Two mechanisms are responsible for the positive inotropic effect of the cardiotonic drug milrinone, i.e., inhibition of type III cAMP phosphodiesterase (PDE III), and displacement of endogenous adenosine from A(1) inhibitory receptor. Since PDE III inhibition may increase the likelihood of cardiac arrhythmias by increasing cAMP content, our attention focused on the synthesis of new compounds with more pronounced characteristics as adenosine antagonists. In this work, four new milrinone analogues were studied, in comparison with the parent drug, for their effects on the contractility of guinea pig isolated atrial preparations, their ability to antagonize endogenous adenosine at the level of A(1) receptor, and to inhibit the activity of PDE III partially purified from guinea pig heart. The new compounds present various chemical substitutions with respect to the parent drug: in compounds SF397 (methyl 5-cyano-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate) and SF399 (benzyl 5-cyano-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate), the 4-pyridil moiety of milrinone was replaced with a methoxycarbonyl and a benzyloxycarbonyl group, respectively; the same structural modifications were also associated with the replacement of the cyano-group in 5-position with an acetyl group in compounds SF416 (methyl 5-acetyl-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate) and SF419 (benzyl 5-acetyl-2-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate). All the new compounds had a marked positive inotropic effect, most of them also being more active and more potent than milrinone. When their affinity for A(1) receptor was assessed as the displacement of [(3)H] 8-cyclopentyl-1,3-dipropylxanthine ([(3)H]DPCPX) from cardiac membranes, SF397 and SF399 showed affinity (K(i) of about 600 nM) similar to that of milrinone (K(i) 550 nM). By contrast, SF416 and SF419 had very low (K(i) of about 10000 nM) or scarce (K(i) of about 2000 nM) anti-adenosine component, respectively. All the new compounds inhibited PDE III activity, their K(i) values proceeding in the following order: milrinone (3.80 microM) <SF397 (7.00 microM) <SF399 (8.80 microM) <SF416 (35.00 microM) SF419< (155.00 microM). To better characterize the mechanisms responsible for the positive inotropic response of the new compounds, we also investigated the effects of new analogues on some systems (ATP-dependent Ca(2+) uptake, Ca(2+)ATPase, Na(+)/K(+)ATPase, Na(+)/Ca(2+) exchange carrier) or a receptor (beta-adrenoceptor) mainly involved in the control of cardiac contractility. None of the tested compounds inhibited enzyme or transport systems; however, SF397, SF399 and SF416, although to different extents, had a direct beta-adrenergic action. Indications about structure-activity relationships are tentatively discussed, in order to obtain useful information for the design of new analogues with better pharmacological profiles.

Action potential control of cardiac contractility.

The transient and/or sustained trans-sarcolemmal voltage induced zero to peak tension changes in cardiac contractility at any given level of diastolic dimensions and associated physical-chemical conditions establish the action potential as the predominant controller of cardiac contractility. Demonstration that the inotropic increases in peak tension effects of extra cellular [Ca++] occur and are determined by the duration and degree of positivity of the plateau phase of the action potential document the predominant role of this phase of the action potential relative to control of cardiac contractility. The large increases in peak tension induced by prolongation of the plateau phase of the action potential by interpolated stimuli (i.e., extrasystolic potentiation) and the immediate maximal peak tension following sustained contracture induced by sarcolemmal depolarization highlight this fact. The demonstration that this prolonged action potential induced maximal tension capability (peak inotropic state) of ventricular fibers can be achieved within a single cycle suggests an analogous technic would provide objective noninvasive measurements of the cardiac reserve of humans in health and disease. A potentially valuable measurement, the temporal resolution requirements of which, although beyond the scope of current three-dimensional imaging systems, is achievable with current state of the art technology. The unique experimental and clinical diagnostic value of such a system for noninvasive analogous measurements in humans with congenital and acquired cardiac abnormalities is as yet, however, not widely recognized. Hence, objective confirmation of the unique potential value of such a system awaits development of the required high fidelity [X,Y,Z (1 mm3 voxel) spatial and (10 ms)] temporal resolution capability system.

Ca channels in cardiac myocytes: structure and function in Ca influx and intracellular Ca release.

There are Ca channels in the plasma membrane and also the sarcoplasmic reticulum (SR) membrane in cardiac myocytes. The relationship between channel structure, associated proteins and function of these Ca channels is discussed. The sarcolemmal Ca channels are crucial both to the basic cellular electrophysiological properties and control of cardiac contractility (via excitation-contraction coupling). The intracellular Ca release channels (or ryanodine receptors) respond to triggering events mediated by sarcolemmal ion currents and are largely responsible for releasing Ca which activates the myofilaments to produce contraction. Several possible mechanisms of excitation-contraction coupling are discussed. The Ca released from the SR can also feedback on several sarcolemmal ion currents and alter action potential configuration as well as contribute to arrhythmogenesis.

Modulation of cyclic nucleotide phosphodiesterase by dietary fats in rat heart.

Feeding oils of different fatty acid composition modifies the fatty acid composition of cardiac membrane phospholipids, thereby inducing changes in cardiac contractility and altering response of adenylate cyclase to catecholamines. In the present study, the effect of such dietary manipulations on cyclic nucleotide phosphodiesterase, which is involved in the control of cyclic nucleotide intracellular levels and in the control of cardiac contractility, was investigated. Rats were fed either a saturated fatty acid-enriched diet (8 weight percent [%] coconut oil + 2% sunflower oil), an n-6 fatty acid-enriched diet (10% sunflower oil) or an n-3 fatty acid-enriched diet (8% fish oil + 2% sunflower oil). The fatty acid composition of cardiac phospholipids, as well as the nonesterified fatty acid content of heart were markedly altered by the diets. The 18:2n-6 and 20:4n-6 content of cardiac phospholipids was markedly (-49%) depressed by fish oil as compared with sunflower oil feeding, but the nonesterified fatty acid level of heart membrane was lowest in coconut oil-fed rats. In addition, fish oil feeding more drastically depressed the n-6/n-3 fatty acid ratio in the nonesterified fatty acid pool than in cardiac phospholipids. Cyclic AMP phosphodiesterase activity was the lowest in both the particulate and soluble fractions of heart from rats fed sunflower oil, whereas cyclic GMP phosphodiesterase activity was not altered by the diets. Cyclic AMP phosphodiesterase activity was decreased by 18 and 12% in heart membranes of the sunflower oil group as compared to that of the coconut oil and fish oil groups, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 2-methyl-1,4-naphthoquinone (menadione) on myocardial contractility and cardiac sarcoplasmic reticulum Ca-ATPase

(1) In electrically driven guinea-pig left atria, menadione (2-methyl-1,4-naphthoquinone) (1 to 20 mumol/l) and menadione sodium bisulfite (30 to 200 mumol/l) produced marked positive inotropic effects. Endogenously released catecholamines and histamine contributed to 80-85% of the effect, the residual 15-20% appearing as a direct effect. (2) In electrically driven guinea-pig ventricular strips, low micromolar concentrations of menadione (0.05 to 0.3 mumol/l) exerted a catecholamine-mediated small positive inotropic effect. (3) In both myocardial preparations, the increase in force of contraction was followed by a non-reversible rise of resting force. In its effects on cardiac contractility menadione resembled the thiol group blocking agent p-chloromercuribenzoate and H2O2. Pretreatment of atria with glutathione prevented the increase in resting force, while dithiothreitol only slightly delayed it. By contrast, the pretreatment with the NAD(P)H-quinone reductase (DT-diaphorase) inhibitor, dicumarol, markedly increased the rate of appearance of the toxic effect of menadione. (4) Among enzymatic and transport systems involved in the onset and control of cardiac contractility, sarcoplasmic reticulum Ca-ATPase was significantly inhibited by menadione after a long contact time. The inhibition was concentration-dependent and persistent, and was antagonized by addition of glutathione. (5) On the basis of these results, the increase in resting force caused by menadione appears to be related to an impairment of the thiol groups of proteins (Ca-ATPase), presumably caused by the drug per se.

Comparative Pharmacology of Cardiac and Vascular Tissues in Heart Failure

The purpose of this paper is to review various factors of the control of cardiac contractility and of vascular tone that may be altered in heart failure and that are targets for pharmacotherapeutic interventions. This paper is divided into three main sections: pathophysiological aspects of heart failure, differences in the regulation of cardiac contraction and of vascular tone, and pharmacological principles in the management of heart failure.

Effects of N-chlorobenzyl analogues of amiloride on myocardial contractility, Na-Ca-exchange carrier and other cardiac enzymatic activities.

1. In electrically driven guinea pig left atria, micromolar concentrations (2 mumol/l to 80 mumol/l) of N-chlorobenzyl derivatives of amiloride (o-chlorobenzamil and 3',4'-dichlorobenzamil) produced quantitatively similar positive inotropic effects. Contracture developed with 3',4'-dichlorobenzamil. Endogenously released catecholamines contributed 30% to the positive inotropic effect of o-chlorobenzamil but did not contribute at all to the effect of 3',4'-dichlorobenzamil. When tested in the presence of the inhibitor of phosphodiesterase isobutylmethylxanthine, o-chlorobenzamil antagonized its positive inotropic effect, whereas 3',4'-dichlorobenzamil potentiated it. o-Chlorobenzamil also antagonized the positive inotropic effect of ouabain in that it shifted its concentration-effect curve to the right. Moreover, o-chlorobenzamil prevented the appearance of ouabain toxicity in terms of a rise in the resting force. 2. Also, in electrically driven guinea pig papillary muscle, micromolar concentrations (5 mumol/l to 30 mumol/l) of both N-chlorobenzyl derivatives of amiloride produced a positive inotropic effect. This effect was more marked with 3',4'-dichlorobenzamil than with o-chlorobenzamil and was associated for both compounds with lengthening of relaxation time. 3. o-Chlorobenzamil and 3',4'-dichlorobenzamil influenced, though not to the same extent, several systems involved in the onset and in the control of cardiac contractility. 3',4'-Dichlorobenzamil inhibited with the same potency Na-K-ATPase, sarcotubular Ca-ATPase, Na-Ca-exchange carrier, cAMP-dependent phosphodiesterase isolated from bovine heart and oxidative phosphorylation of mitochondria isolated from rat liver. Low micromolar concentrations of o-chlorobenzamil mainly inhibited Na-Ca-exchange carrier and cAMP-dependent phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of cardiac contractility at the cellular level.

Recent experiments involving the measurement of intracellular activities in Na+ and Ca2+; functional studies; descriptions of the Na+ and Ca2+ fluxes from intact cardiac tissue; studies of isolated cardiac sarcolemmal vesicles, isolated sarcoplasmic reticulum (SR) vesicles, and isolated mitochondria; and investigations of the behavior of cardiac contractile proteins have revealed a plurality of processes associated with the activation and control of the contraction and relaxation of cardiac muscle. Which of these processes predominates, even in intact cardiac muscle, would seem to depend critically on the experimental conditions. As the cycle of contraction and relaxation essentially involves the reversible binding of Ca2+ to the contractile proteins as a final step, if a number of processes influence this binding they must interact. Quantitative consideration of available information suggests that in the beating mammalian heart the bulk of the activating Ca2+ cycles between the SR and the contractile proteins with the sarcolemmal Ca2+ pump and Na+-Ca2+ exchange operating to maintain the Ca2+ economy of the heart and affecting contraction only by influencing the filling of the SR with Ca2+ and possibly its release. In amphibian heart the sarcolemmal Na+-Ca2+ exchange would seem to be more directly involved, adding or subtracting from the Ca2+ liberated from the SR. The Ca2+ uptake by mitochondria may become particularly important when the intracellular sodium concentration alters.

Characterization of the phosphoprotein profile in spontaneously beating cultured rat heart cells.

AbstractCulture of neonatal rat hearts yields a fairly homogeneous population of synchronous and spontaneously beating myocardial cells. Incubation of the culture with [32P]-orthophosphate followed by polyacrylamide gel electrophoresis and autoradiography yields a reproducible profile of both high- and low-molecular-weight phosphoproteins. This phosphoprotein profile is clearly distinct from that observed with mesenchymal cells, the major contaminant of the heart cell cultures. By comparison with purified standard proteins, and by copurification with neonatal rat myosin, we have identified one of these phosphoproteins as the 20,000-dalton myosin light chain. The demonstration of phosphoproteins in intact contracting myocardial cells provides a system to study the complex interaction of the phosphoproteins involved in the control of cardiac contractility.

Mechanism of reflex control of cardiac contractility by carotid sinus baroreceptors.

Effects of the carotid sinus baroreflex (CSR) on left ventricular dP/dt, mean arterial pressure (MAP), and heart rate (HR) were examined by changing the pressure in the vascularly isolated carotid sinus in anesthetized cats. When the carotid sinus pressure (CSP) was lowered, a significant increase was observed in dP/dt and MAP. These reflex changes were significantly increased after vagotomy. In vagotomized cats, cardiac sympathectomy attenuated but did not abolish the dP/dt and HR responses to CSR. The MAP response was little affected. The remaining response of dP/dt and HR were virtually abolished by subsequent adrenalectomy or beta-adrenergic blockade. Adrenalectomy reduced the MAP response by 39%. The effect of cardiac sympathectomy or adrenalectomy on the dP/dt response was greater when either procedure was performed in combination with the other. These results indicate that 1) the vagi interact with CSR and 2) both cardiac sympathetic nerves and adrenal secretion of catecholamines contribute to the reflex control of cardiac contractility by CSR in anesthetized cats. However, the relative contribution of either reflex system appears to vary depending on whether the other system in intact.

Role of carotid sinus and cardiopulmonary reflexes on left ventricular dP/dt in cats

Interaction between vagally mediated cardiopulmonary receptors and carotid sinus baroreceptors was studied in regard to cardiac contractility in anesthetized cats using vagal cold block and carotid occlusion. Cold block of the vagi produced a small but significant increase in left ventricular dP/dt and in arterial pressure. Carotid occlusion caused a greater increase in both parameters. The effects of each of these individual procedures were significantly greater when either intervention was tested in combination with the other. Cardiac sympathectomy reduced but did not abolish the reflex increase in cardiac contractility due to either procedure. Subsequent beta-adrenergic blockade eliminated the reflex change in the contractility. The study demonstrates that 1) both cardiopulmonary and carotid sinus baroreceptors participate in the neural control of cardiac contractility with the latter's role being dominant; 2) there is an apparent interaction between the two reflex mechanisms in which one reflex system acts to compensate the other's effects.

Membrane control of cardiac contractility

ALL myocardial cells contract in every cardiac cycle and the contractility of the heart cannot be modulated as in skeletal muscle by varying the number of active cells, so each cardiac cell must be capable of a broad range of contractile states. Such versatility could result from changes in the action potential, in the amount of Ca2+ released for activation of the contraction, or in the contractile proteins themselves. Phosphorylation of the inhibitory subunit of troponin seems to occur in parallel with the increase in contractility of isolated hearts perfused with catecholamines1,2, and studies using isolated proteins indicate that the phosphorylation may be associated with a change in ATPase activity3,4, but a direct demonstration that the functional properties of contractile proteins themselves can be altered in a manner compatible with a physiologically important regulatory mechanism has not yet been produced. We report here studies of cardiac cells with membranes that have been made hyperpermeable, in which the response of the contractile system to Ca ions, the presumed trigger for activation of contraction, may be altered markedly with respect to both the concentration of calcium required for activation and the maximum generated force by the microenvironment of the contractile proteins and by reactions involving the cell's membranes. Cyclic nucleotides may be involved in some of these regulatory reactions.

Excitation-contraction coupling and control of cardiac contractility.

Excitation-contraction coupling and control of cardiac contractility.