Inhibition of PAF-acether effects on isolated guinea pig hearts by zinc ions (Zn2+)

PAF-acether is a phospholipid synthesized by most animal tissues and exerting a strong decrease on the heart’s contractile force and coronary flow. PAF-acether (10⁻⁹ and 10⁻¹⁰ M) was administered to isolated guinea pig hearts perfused via the Langendorff apparatus with Chenoweth solution. Zinc (1.5 μM) is known to benefit heart function thus, Zn²⁺ (1.5, 7.5, and 30 μM) was added in the perfusing solution before or after PAF-acether administration. Contractile force, coronary flow, and heart rate were recorded by means of a Narco MK-IV Physiograph throughout all modes of perfusion. Calcium inhibitor (Verapamil 10⁻¹⁰ M) and Pb²⁺ Co²⁺ (1.5×10⁻⁶ M) were used subsequently in the perfusing solutions in order to elucidate some of the Zn and PAF interactions observed. All hearts were analyzed for their Zn and Ca content by means of an Atomic Absorption Spectrophotometry (AAS). Our data suggest that low concentrations of zinc (1.5 μM) can strongly inhibit PAF-induced decrease of contractile force and coronary flow. Zinc-inhibiting effects on PAF's negative inotropic action (myocytic level) is not exerted through Zn−Ca antagonism. Nevertheless, a Zn−Ca antagonism in the arteriolar level cannot be excluded. Zinc inhibits PAF selectively only if it is administered before PAF injection and this strongly suggests a receptor interaction between the metal and the phospholipid at the heart level.     

Subcellular calcium transport during contractile failure and recovery in heart perfused with Na-free medium

Perfusion of isolated rat hearts with Na-free medium resulted in an immediate increase in contractile force followed by a decline and complete loss of contractile force within 25 s. The recovery of the contractile force upon reperfusion was only partial if the duration of Na-free perfusions was 10 min or longer. Ca binding and uptake activities of mitochondria obtained from hearts perfused with Na-free medium did not change significantly. However, Ca binding and uptake activities of microsomes were depressed after 5 min of perfusion. The critical concentration of Na in the perfusion medium for inducing these changes was found to be less than 35 mM. The microsomal Ca-ATPase activity was decreased after 10 min of Na-free perfusion. Only partial recovery of microsomal Ca uptake was observed upon reperfusion of hearts preperfused with Na-free medium for 20 min or longer whereas Ca-ATPase activity in these hearts did not recover at all. These results suggest that the defect in the microsomal Ca transport may be secondary to the development of contractile failure and may partially be associated with the inability of Na-depleted hearts to recover fully their contractile force.     

The new HNO donor, 1-nitrosocyclohexyl acetate, increases contractile force in normal and β-adrenergically desensitized ventricular myocytes

Contractile dysfunction and diminished response to β-adrenergic agonists are characteristics for failing hearts. Chemically donated nitroxyl (HNO) improves contractility in failing hearts and thus may have therapeutic potential. Yet, there is a need for pharmacologically suitable donors. In this study we tested whether the pure and long acting HNO donor, 1-nitrosocyclohexyl acetate (NCA), affects contractile force in normal and pathological ventricular myocytes (VMs) as well as in isolated hearts. VMs were isolated from mice either subjected to isoprenaline-infusion (ISO; 30 μg/g per day) or to vehicle (0.9% NaCl) for 5 days. Sarcomere shortening and Ca²⁺ transients were simultaneously measured using the IonOptix system. Force of contraction of isolated hearts was measured by a Langendorff-perfusion system. NCA increased peak sarcomere shortening by + 40-200% in a concentration-dependent manner (EC₅₀ ∼55 μM). Efficacy and potency did not differ between normal and chronic ISO VMs, despite the fact that the latter displayed a markedly diminished inotropic response to acute β-adrenergic stimulation with ISO (1 μM). NCA (60 μM) increased peak sarcomere shortening and Ca²⁺ transient amplitude by ∼200% and ∼120%, respectively, suggesting effects on both myofilament Ca²⁺ sensitivity and sarcoplasmic reticulum (SR) Ca²⁺ cycling. Importantly, NCA did not affect diastolic Ca²⁺ or SR Ca²⁺ content, as assessed by rapid caffeine application. NCA (45 μM) increased force of contraction by 30% in isolated hearts. In conclusion, NCA increased contractile force in normal and β-adrenergically desensitized VMs as well as in isolated mouse hearts. This profile warrants further investigations of this HNO donor in the context of heart failure.     

Studies on the phosphorylation of the inhibitory subunit of troponin during modification of contraction in perfused rat heart.

1. Rat hearts were perfused with 32Pi, and contractile force was increased by positive inotropic agents (agents that increase contractility). The inhibitory subunit of troponin (troponin I) was then isolated by affinity chromatography in 8M-urea, and its 32P content measured. Incorporation of phosphate into the subunit was calculated on the basis of the [gamma-32P]ATP specific radioactivity in the hearts. 2. When hearts were perfused with 30 nM-DL-isoprenaline (N-isopropylnoradrenaline), there was an increase in contractile force over 30s which was paralleled by an increase in troponin I phosphorylation. When hearts were perfused for 25s with increasing concentrations of isoprenaline from 1 NM to 0.6 muM, there was again a parallel increase in contractile force and troponin I phosphorylation. The maximum phosphorylation observed was 1.5 mol of phosphate/mol of troponin I, which was reached after 25s with 0.1 muM-isoprenaline. 3. Hearts were stimulated with a 15s pulse perfusion of 30nM-DL-isoprenaline. There was an increase in contractile force which was followed by a return to the control value within 50s. Troponin I phosphorylation increased to a plateau value which was reached within 30s, and remained constant for 60s after the isoprenaline pulse. Phosphorylase a and 3':5'-cyclic AMP concentration showed changes similar to that of the contractile force. There was no change in 3':5'-cyclic GMP concentration. 4. When hearts stimulated with a 15S pulse of isoprenaline were subsequently perfused with 0.6 muM-acetylcholine, the changes in contractile force, phosphorylase a and 3':5'-cyclic AMP were very similar to those seen with the 15s pulse of isoprenaline alone. Troponin I phosphorylation increased to a maximum 30s after the end of the isoprenaline pulse, but then rapidly decreased during the subsequent 30s. This decrease was preceded by a 60% increase in the concentration of 3':5'-cyclic GMP. 5. Hearts were perfused with 0.2 muM-glucagon for periods up to 60s. Contractile force showed little change for the first 30s, but then increased rapidly. This was paralleled by changes in 3':5'-cyclic AMP concentration. Troponin I phosphorylation increased slowly, but the increase in contractile force had reached a maximum before significant phosphorylation had occurred. 6. It is concluded that under certain conditions, e.g. immediately after beta-adrenergic stimulation, there is a good correlation between contractile force and troponin I phosphorylation. However, under other conditions, e.g. when contractile force is decreasing after removal of beta-adrenergic stimulation or in the presence of glucagon, contractile force and troponin I phosphorylation are not well correlated. These results suggest that mechanisms for modifying cardiac contractility, other than troponin I phosphorylation, must be present in rat heart.     

Cellular mechanisms of burn-related changes in contractility and its prevention by mesenteric lymph ligation

Major burn injury results in impairment of left ventricular (LV) contractile function. There is strong evidence to support the involvement of gut-derived factor(s) transported in mesenteric lymph in the development of burn-related contractile dysfunction; i.e., mesenteric lymph duct ligation (LDL) prevents burn-related contractile depression. However, the cellular mechanisms for altered myocardial contractility of postburn hearts are largely unknown, and the cellular basis for the salutary effects of LDL on cardiac function have not been investigated. We examined contractility, Ca(2+) transients, and L-type Ca(2+) currents (I(Ca)) in LV myocytes isolated from four groups of rats: 1) sham burn, 2) sham burn with LDL (sham + LDL), 3) burn ( approximately 40% of total body surface area burn), and 4) burn with LDL (burn + LDL). Myocytes isolated from hearts at 24 h postburn had a depressed contractility ( approximately 20%) at baseline and blunted responsiveness to elevation of bath Ca(2+). Myocyte contractility was comparable in sham + LDL and sham burn hearts. LDL completely prevented burn-related changes in myocyte contractility. Mechanistically, the decrease in contractility in myocytes from postburn hearts occurred with a decrease in the amplitude of Ca(2+) transients ( approximately 20%) without changes in resting Ca(2+) or Ca(2+) content of the sarcoplasmic reticulum. On the other hand, I(Ca) density was decreased ( approximately 30%) in myocytes from postburn hearts, with unaltered voltage-dependent properties. Thus burn-related myocardial contractile dysfunction is linked with depressed myocyte contractility associated with a decrease in I(Ca) density. These findings also provide strong evidence that mesenteric lymph is involved in the onset of burn-related cardiomyocyte dysfunction.     

Dual role of endothelin-1 via ETA and ETB receptors in regulation of cardiac contractile function in mice

An increase in coronary perfusion pressure leads to increased cardiac contractility, a phenomenon known as the Gregg effect. Exogenous endothelin (ET)-1 exerts a positive inotropic effect; however, the role of endogenous ET-1 in the contractile response to elevated load is unknown. We characterized here the role of ETA and ETB receptors in regulation of contractility in isolated, perfused mouse hearts subjected to increased coronary flow. Elevation of coronary flow from 2 to 5 ml/min resulted in 80 +/- 10% increase in contractile force (P < 0.001). BQ-788 (ETB receptor antagonist) augmented the load-induced contractile response by 35% (P < 0.05), whereas bosentan (ETA/B receptor antagonist) and BQ-123 (ETA receptor antagonist) attenuated it by 34% and 56%, respectively (P < 0.05). CV-11974 (ANG II type 1 receptor antagonist) did not modify the increase in contractility. These results show that endogenous ET-1 is a key mediator of the Gregg effect in mouse hearts. Moreover, ET-1 has a dual role in the regulation of cardiac contractility: ETA receptor-mediated increase in contractile force is suppressed by ETB receptors.     

The effect of lanthanum on the mechanical function of the isolated perfused rat heart at different extracellular calcium concentrations.

The effects of 50 microM lanthanum (La3+) on the contractile force, rate and coronary flow of rat hearts perfused with solutions containing 2.5, 5, 7.5 mM calcium (Ca2+) have been investigated. La3+ produced a rapid and marked decrease in contractile force within 1-3 min ("early La(3+)-effect"). The inhibition of contractility by La3+ was reduced progressively when the Ca2+ ion concentration in the perfusion fluid was raised from 2.5 to 7.5 mM. However, after 10-80 min of La3+ perfusion the contractile force was increased significantly ("late La(3+)-effect"). Elevation of Ca2+ during exposure to La3+ increased its effect. During the late La(3+)-effect, a marked decrease in heart rate and a significant increase in time to reach peak tension, time for half relaxation and twitch duration was observed. High concentrations of perfusate Ca2+ decreased the chronotropic response to La3+, in contrast, elevated Ca2+ potentiated La(3+)-induced increase in time to reach peak tension, time for half relaxation and twitch duration. La3+ produced a significant decrease in coronary flow. High Ca2+ augmented the decrease coronary flow. The findings indicate that La3+ may produce marked effects on myocardial function. High extracellular Ca2+ reduces the La(3+)-induced initial decrease in force of contraction, but potentiates the late increase in contractile force by La3+. Elevated external Ca2+ also increases the effects of La3+ on twitch parameters, heart rate and coronary flow.     

Reversibility of ultrastructural, contractile function and Ca2+ transport changes in guinea pig hearts after global ischemia

To understand the subcellular basis of contractile failure due to ischemia-reperfusion injury, effects of 20, 60, and 90 min of global ischemia followed by 30 min of reperfusion were examined in isolated guinea pig hearts. Cardiac ultrastructure and function as well as Ca2+ transport abilities of both mitochondrial and microsomal fractions were determined in control, ischemic, and reperfused hearts. Hearts were unable to generate any contractile force after 20 min of ischemia and showed a 75% recovery upon reperfusion. However, there were no significant changes in the subcellular Ca2+ transport in the 20-min ischemic or reperfused hearts. When hearts were made ischemic for 60 and 90 min, the recovery of contractile force on reperfusion was 50 and 7%, respectively. There was a progressive decrease in mitochondrial and microsomal Ca2+ binding and uptake activities after 60 and 90 min of ischemia; these changes were evident at various times of incubation period and at different concentrations of Ca2+. Mitochondrial Ca2+ transport changes were only partially reversible upon reperfusion after 60 and 90 min of ischemia, whereas the microsomal Ca2+ binding, uptake and Ca2+ ATPase activities deteriorated further upon reperfusion of the 90-min ischemic hearts. Ultrastructural changes increased with the duration of the ischemic insult and reperfusion injury was extensive in the 90-min ischemic hearts. These data show that the lack of recovery of contractile function upon reperfusion after a prolonged ischemic insult was accompanied by defects in sarcoplasmic reticulum Ca2+ transporting properties and structural damage.     

Effect of Australian Propolis from Stingless Bees (Tetragonula carbonaria) on Pre-Contracted Human and Porcine Isolated Arteries

Bee propolis is a mixture of plant resins and bee secretions. While bioactivity of honeybee propolis has been reported previously, information is limited on propolis from Australian stingless bees (Tetragonula carbonaria). The aim of this study was to investigate possible vasomodulatory effects of propolis in KCl-precontracted porcine coronary arteries using an ex vivo tissue bath assay. Polar extracts of propolis produced a dose-dependent relaxant response (EC₅₀=44.7±7.0 μg/ml), which was unaffected by endothelial denudation, suggesting a direct effect on smooth muscle. Propolis markedly attenuated a contractile response to Ca²⁺ in vessels that were depolarised with 60 mM KCl, in Ca²⁺-free Krebs solution. Propolis (160 µg/ml) reduced vascular tone in KCl pre-contracted vessels to near-baseline levels over 90 min, and this effect was partially reversible with 6h washout. Some loss in membrane integrity, but no loss in mitochondrial function was detected after 90 min exposure of human cultured umbilical vein endothelial cells to 160 µg/ml propolis. We conclude that Australian stingless bee (T. carbonaria) propolis relaxes porcine coronary artery in an endothelial-independent manner that involves inhibition of voltage-gated Ca²⁺ channels. This effect is partially and slowly reversible upon washout. Further studies are required to determine the therapeutic potential of Australian stingless bee propolis for conditions in which vascular supply is compromised.     

The effects of Zn2+ on guinea pig isolated heart preparations

Isolated guinea pig hearts were perfused, by the Langendorff technique, with 30, 15, 7.5, and 1.5 μM Zn²⁺ in Chenoweth solution. Contractile force, coronary flow, and heart rate were recorded by means of Narco IV Physiograph. Calcium inhibitor (Verapamil 1 μM) and anion inhibitor (DIDS: 0.1, 1, and 5 μM) were used subsequently in the perfusing solutions in order to distinguish some of the possible mechanisms that Zn²⁺ uses to exert its action on cardiac myocytes. Isomolar to zinc concentration of Pb (II) and Co (II) were used to elucidate whether zinc effects on heart are specific for this metal. All hearts were used to estimate their zinc and calcium content by means of AAS (Atomic Absorption Spectrometry). Our findings suggest that the higher the Zn²⁺ concentration, the more toxic effects on heart are expressed by rapid reversible contractile force reduction and reversible specific changes of heart rate and flow. Zinc 1.5 μM in the perfusing solution benefits heart performance, but not significantly. Furthermore, the metal exerts specific effects on guinea pig heart, and it is rather possible that these effects on cardiac myocytes are held through cell membrane receptors.     

Specific uncoupling of excitation and contraction in mammalian cardiac tissue by lanthanum

Arterially cannulated rabbit interventricular septal tissue was exposed to 5–40 µM La in 2.5 mM Ca perfusate. Immediately following perfusion with La two concurrent events were consistently observed: (a) a rapid decline of active tension to a lesser steady-state value, and (b) an abrupt, release of short duration of tissue-bound Ca. The magnitude of both events was directly related to the [La]_o. If the duration of exposure to La was brief, contractility returned toward normal upon return to the La-free perfusate. Elevation of [Ca]_o during exposure to La counteracted its effect and induced a concurrent displacement of tissue-bound La. Cellular action potentials recorded during brief perfusion with La demonstrated that essentially normal regenerative depolarization was maintained. Analysis of the quantities of ⁴⁵Ca released following exposure to 10 µM La indicated that this La-susceptible Ca was being displaced from a homogeneous pool—the one previously shown by Langer to represent contractile dependent Ca. These data led to the following conclusions: During perfusion with 2.5 mM Ca contractile dependent Ca was derived primarily from "superficially" located sites. La effected the release of contractile dependent Ca by modifying the normal permselectivity of this "superficial" membrane for activator Ca. These and other data infer that contractile dependent Ca is derived primarily from superficially located sites.     

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Effects of sustained length-dependent activation on in situ cross-bridge dynamics in rat hearts

The cellular basis of the length-dependent increases in contractile force in the beating heart has remained unclear. Our aim was to investigate whether length-dependent mediated increases in contractile force are correlated with myosin head proximity to actin filaments, and presumably the number of cross-bridges activated during a contraction. We therefore employed x-ray diffraction analyses of beat-to-beat contractions in spontaneously beating rat hearts under open-chest conditions simultaneous with recordings of left ventricle (LV) pressure-volume. Regional x-ray diffraction patterns were recorded from the anterior LV free wall under steady-state contractions and during acute volume loading (intravenous lactate Ringers infusion at 60 ml/h, <5 min duration) to determine the change in intensity ratio (I_1,0/I_1,1) and myosin interfilament spacing (d_1,0). We found no significant change in end-diastolic (ED) intensity ratio, indicating that the proportion of myosin heads in proximity to actin was unchanged by fiber stretching. Intensity ratio decreased significantly more during the isovolumetric contraction phase during volume loading than under baseline contractions. A significant systolic increase in myosin head proximity to actin filaments correlated with the maximum rate of pressure increase. Hence, a reduction in interfilament spacing at end-diastole (∼0.5 nm) during stretch increased the proportion of cross-bridges activated. Furthermore, our recordings suggest that d_1,0 expansion was inversely related to LV volume but was restricted during contraction and sarcomere shortening to values smaller than the maximum during isovolumetric relaxation. Since ventricular volume, and presumably sarcomere length, was found to be directly related to interfilament spacing, these findings support a role for interfilament spacing in modulating cross-bridge formation and force developed before shortening.     

Cardiac Function Is Regulated by B56α-mediated Targeting of Protein Phosphatase 2A (PP2A) to Contractile Relevant Substrates

Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]_i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of β-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of −5 mV, the I_Ca,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser¹⁶ was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca²⁺ sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation.     

Hypoxia-induced skeletal muscle fiber dysfunction: role for reactive nitrogen species

Hypoxia impairs skeletal muscle function, but the precise mechanisms are incompletely understood. In hypoxic rat diaphragm muscle, generation of peroxynitrite is elevated. Peroxynitrite and other reactive nitrogen species have been shown to impair contractility of skinned muscle fibers, reflecting contractile protein dysfunction. We hypothesized that hypoxia induces contractile protein dysfunction and that reactive nitrogen species are involved. In addition, we hypothesized that muscle reoxygenation reverses contractile protein dysfunction. In vitro contractility of rat soleus muscle bundles was studied after 30 min of hyperoxia (Po2 approximately 90 kPa), hypoxia (Po2 approximately 5 kPa), hypoxia + 30 microM N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), hyperoxia + 30 microM L-NMMA, and hypoxia (30 min) + reoxygenation (15 min). One part of the muscle bundle was used for single fiber contractile measurements and the other part for nitrotyrosine detection. In skinned single fibers, maximal Ca2+-activated specific force (Fmax), fraction of strongly attached cross bridges (alphafs), rate constant of force redevelopment (ktr), and myofibrillar Ca2+ sensitivity were determined. Thirty minutes of hypoxia reduced muscle bundle contractility. In the hypoxic group, single fiber Fmax, alphafs, and ktr were significantly reduced compared with hyperoxic, L-NMMA, and reoxygenation groups. Myofibrillar Ca2+ sensitivity was not different between groups. Nitrotyrosine levels were increased in hypoxia compared with all other groups. We concluded that acute hypoxia induces dysfunction of skinned muscle fibers, reflecting contractile protein dysfunction. In addition, our data indicate that reactive nitrogen species play a role in hypoxia-induced contractile protein dysfunction. Reoxygenation of the muscle bundle partially restores bundle contractility but completely reverses contractile protein dysfunction.     

Dexamethasone treatment improves sarcoplasmic reticulum function and contractile performance in aged myocardium

Corticosteroids are thought to be involved in the maintenance of normal myocardial function by mechanisms incompletely understood. This study investigated the potential therapeutic benefit of the synthetic glucocorticoid, dexamethasone, in reversing age-associated deterioration in cardiac contractile performance and Ca2+ sequestration function of the sarcoplasmic reticulum. Dexamethasone was administered to senescent (26-28-month old), male Fischer 344 rats at a rate of 4 microg/h for 5 days via subcutaneously implanted osmotic mini pumps. Control rats received vehicle solution in similar manner. Contractile performance was assessed in Langendorff-perfused, electrically paced hearts from control and dexamethasone-treated rats. The results obtained showed that dexamethasone-treatment of aged rats resulted in significant improvement in myocardial contractile performance as evidenced by (i) increase (approximately 30-60%) in developed peak tension at a wide range of beating frequencies (2-6 Hz), (ii) unaltered time to peak tension, and (iii) decrease (approximately 8-15%) in time to half-relaxation. Also, SR isolated from dexamethasone-treated rats displayed approximately 2-fold higher rates of ATP-energized Ca2+ uptake compared to SR from control rats. The deficits in contractile performance of the senescent heart (prolonged contraction duration and diminished contractile force) are reversible through a glucocorticoid-mediated improvement in SR Ca2+ pump function.     

Changes in cardiac contractility related to calcium-mediated changes in phosphorylation of myosin-binding protein C.

Ca ions can influence the contraction of cardiac muscle by activating kinases that specifically phosphorylate the myofibrillar proteins myosin-binding protein C (MyBP-C) and the regulatory light chain of myosin (RLC). To investigate the possible role of Ca-regulated phosphorylation of MyBP-C on contraction, isolated quiescent and rhythmically contracting cardiac trabeculae were exposed to different concentrations of extracellular Ca and then chemically skinned to clamp the contractile system. Maximum Ca-activated force (F(max)) was measured in quiescent cells soaking in 1) 2.5 mM Ca for 120 min, 2) 1.25 mM for 120 min, or 3) 1.25 mM for 120 min followed by 10 min in 7.5 mM, and 4) cells rhythmically contracting in 2.5 mM for 20 min. F(max) was, respectively, 21.5, 10.5, 24.7, and 32.6 mN/mm(2). Changes in F(max) were closely associated with changes in the degree of phosphorylation of MyBP-C and occurred at intracellular concentrations of Ca below levels associated with phosphorylation of RLC. Monophosphorylation of MyBP-C by a Ca-regulated kinase is necessary before beta-adrenergic stimulation can produce additional phosphorylation. These results suggest that Ca-dependent phosphorylation of MyBP-C modulates contractility by changing thick filament structure.     

Increased tolerance to hypoxic metabolic inhibition and reoxygenation of cardiomyocytes from apolipoprotein E-deficient mice

Although hypercholesterolemia is a strong risk factor for cardiovascular disease, it has in some instances paradoxically been associated with reduced infarct size and preserved contractile function in isolated hearts after ischemia and reperfusion. To elucidate potential cellular protective mechanisms, myocytes of hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) and wild-type mice were subjected to hypoxic metabolic inhibition (I) with subsequent reoxygenation (R). Intracellular Ca2+ concentration ([Ca2+]i) and pH (pHi) were monitored as well as cell length and arrhythmic events. Force measurements in papillary muscles were also recorded, and myocardial expression of Na+/H+ exchanger 1 (NHE1) and three Ca2+ handling proteins [sarco(endo)plasmic reticulum Ca2+-ATPase, Na+/Ca2+ exchanger, and plasma membrane Ca2+-ATPase] was quantified. After 30 min of I and 35 min of R, Ca2+ overload was more pronounced in wild-type cells (P < 0.05). In these myocytes, pHi also dropped faster and remained below those values determined in ApoE-/- cells (P < 0.05). Furthermore, more wild-type myocytes remained in a contracted state (P < 0.05). This group also showed a higher incidence of arrhythmic events during R (P < 0.05). No group difference was found in the expression of the Ca2+ handling proteins. However, NHE1 protein was downregulated in hearts of ApoE-/- mice (P < 0.05). Histological results depict hyperplasia in ApoE-/- hearts without atherosclerosis of the coronaries. Contractile dysfunction was not observed in papillary muscles from ApoE-/- hearts. Our results suggest that downregulated myocardial NHE1 expression in hypercholesterolemic ApoE-/- mice could have contributed to increased tolerance to I/R. It remains to be elucidated whether NHE1 downregulation is a unique feature of these genetically altered animals.     

THE CONTRACTILE PROCESS IN THE CILIATE, STENTOR COERULEUS : I. The Role of Microtubules and Filaments

The structural basis for the function of microtubules and filaments in cell body contractility in the ciliate Stentor coeruleus was investigated. Cells in the extended state were obtained for ultrastructural analysis by treatment before fixation with a solution containing 10 mM EGTA, 50–80 mM Tris, 3 mM MgSO₄, 7.5 mM NH₄Cl, 10 mM phosphate buffer (pH 7.1). The response of Stentor to changes in the divalent cation concentrations in this solution suggests that Ca⁺² and Mg⁺² are physiologically important in the regulation of ciliate contractility. The generation of motive force for changes in cell length in Stentor resides in two distinct longitudinal cortical fiber systems, the km fibers and myonemes. Cyclic changes in cell length are associated with (a) the relative sliding of parallel, overlapping microtubule ribbons in the km fibers, and (b) a distinct alteration in the structure of the contractile filaments constituting the myonemes. The microtubule and filament systems are distinguished functionally as antagonistic contractile elements. The development of motive force for cell extension is accomplished by active microtubule-to-microtubule sliding generated by specific intertubule bridges. Evidence is presented which suggests that active shortening of contractile filaments, reflected in a reversible structural transformation of dense 4-nm filaments to tubular 10–12-nm filaments, provides the basis for rapid cell contraction.