Search

Background Patients with repair of tetralogy of Fallot (rToF) who are approaching adulthood often exhibit pulmonary valve regurgitation, leading to right ventricle (RV) dilatation and dysfunction. The regurgitation can be corrected by pulmonary valve replacement (PVR), but the optimal surgical timing remains under debate, mainly because of the poorly understood nature of RV remodeling in patients with rToF. The goal of this study was to probe for pathologic molecular, cellular, and tissue changes in the myocardium of patients with rToF at the time of PVR. Methods and Results We measured contractile function of permeabilized myocytes, collagen content of tissue samples, and the expression of mRNA and selected proteins in RV tissue samples from patients with rToF undergoing PVR for severe pulmonary valve regurgitation. The data were compared with nondiseased RV tissue from unused donor hearts. Contractile performance and passive stiffness of the myofilaments in permeabilized myocytes were similar in rToF-PVR and RV donor samples, as was collagen content and cross-linking. The patients with rToF undergoing PVR had enhanced mRNA expression of genes associated with connective tissue diseases and tissue remodeling, including the small leucine-rich proteoglycans ASPN (asporin), LUM (lumican), and OGN (osteoglycin), although their protein levels were not significantly increased. Conclusions RV myofilaments from patients with rToF undergoing PVR showed no functional impairment, but the changes in extracellular matrix gene expression may indicate the early stages of remodeling. Our study found no evidence of major damage at the cellular and tissue levels in the RV of patients with rToF who underwent PVR according to current clinical criteria.

The acceleration of myocardial relaxation produced by β-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca 2+ sensitivity. Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca 2+ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca 2+ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated (using sequential incubations in λ-phosphatase, PKD, and PKA, respectively). Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca 2+ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca 2+ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics ( k tr ). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca 2+ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca 2+ sensitivity., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Experimental data from human cardiac myocytes at body temperature is crucial for a quantitative understanding of clinically relevant cardiac function and development of whole-organ computational models. However, such experimental data is currently very limited. Specifically, important measurements to characterize changes in tension development in human cardiomyocytes that occur with perturbations in cell length are not available. To address this deficiency, in this study we present an experimental data set collected from skinned human cardiac myocytes, including the passive and viscoelastic properties of isolated myocytes, the steady-state force calcium relationship at different sarcomere lengths, and changes in tension following a rapid increase or decrease in length, and after constant velocity shortening. This data set is, to our knowledge, the first characterization of length and velocity-dependence of tension generation in human skinned cardiac myocytes at body temperature. We use this data to develop a computational model of contraction and passive viscoelasticity in human myocytes. Our model includes troponin C kinetics, tropomyosin kinetics, a three-state crossbridge model that accounts for the distortion of crossbridges, and the cellular viscoelastic response. Each component is parametrized using our experimental data collected in human cardiomyocytes at body temperature. Furthermore we are able to confirm that properties of length-dependent activation at 37°C are similar to other species, with a shift in calcium sensitivity and increase in maximum tension. We revise our model of tension generation in the skinned isolated myocyte to replicate reported tension traces generated in intact muscle during isometric tension, to provide a model of human tension generation for multi-scale simulations. This process requires changes to calcium sensitivity, cooperativity, and crossbridge transition rates. We apply this model within multi-scale simulations of biventricular cardiac function and further refine the parametrization within the whole organ context, based on obtaining a healthy ejection fraction. This process reveals that crossbridge cycling rates differ between skinned myocytes and intact myocytes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Impaired shortening deactivation of cardiac myocytes could sustain myocardial contraction, preserving ejection fraction at the expense of diastolic dysfunction. We examined the relationship between first-phase ejection fraction (EF1), the fraction of left ventricular volume ejected from the start of systole to the time of the first peak in left ventricular pressure (corresponding to the time of maximal ventricular shortening) to the duration of myocardial contraction and diastolic function in patients with hypertension (n=163), and varying degrees of diastolic dysfunction. Left ventricular systolic pressure was estimated by carotid tonometry; time-resolved left ventricular cavity and wall volume were obtained by echocardiography with speckle wall tracking. Measurements were repeated after nitroglycerin, a drug known to influence ventricular dynamics, in a subsample (n=18) of patients. EF1 and time of onset of ventricular relaxation (as determined from the temporal pattern of myocardial wall stress) were independently correlated with diastolic relaxation as measured by tissue Doppler early diastolic mitral annular velocity (E', standardized regression coefficients 0.48 and -0.34 for EF1 and time of onset of ventricular relaxation, respectively, each P <0.001, irrespective of adjustment for age, sex, antihypertensive treatment, measures of afterload, and ventricular geometry) and with diastolic function measured by the ratio of transmitral Doppler early filling velocity (E) to E' (E/E', regression coefficients -0.34 and 0.34, respectively, each P <0.001). Nitroglycerin increased EF1, decreased time of onset of ventricular relaxation, and improved diastolic function (each P <0.05). Hypertensive patients with diastolic dysfunction exhibit reduced EF1 which may sustain myocardial contraction, preserving systolic ejection fraction at the expense of impaired diastolic function., (© 2017 The Authors.)

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca(2+) sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca(2+) sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca(2+) transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E'/A', and higher E/E' ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca(2+) sensitivity, faster Ca(2+) transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca(2+) sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca(2+) transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

It is now generally accepted that phosphorylation of cMyBP-C is critically important in maintaining normal cardiac function. Although much of the work to date on phospho-regulation of cMyBP-C has focused on the role of protein kinase A (PKA, also known as cAMP-dependent protein kinase), recent evidence suggests that a number of non-PKA serine/threonine kinases, such as Ca(2+)/calmodulin-dependent protein kinase II, protein kinase C, protein kinase D and the 90-kDa ribosomal S6 kinase are also capable of targeting this key regulatory sarcomeric protein. This article reviews such evidence and proposes a hypothetical role for some of the pertinent signalling pathways in phospho-regulation of cMyBP-C in the setting of heart failure.

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.

Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, increased ventricular stiffness and impaired diastolic filling. We investigated to what extent myocardial functional defects can be explained by alterations in the passive and active properties of human cardiac myofibrils. Skinned ventricular myocytes were prepared from patients with obstructive HCM (two patients with MYBPC3 mutations, one with a MYH7 mutation, and three with no mutation in either gene) and from four donors. Passive stiffness, viscous properties, and titin isoform expression were similar in HCM myocytes and donor myocytes. Maximal Ca(2+)-activated force was much lower in HCM myocytes (14 ± 1 kN/m(2)) than in donor myocytes (23 ± 3 kN/m(2); P<0.01), though cross-bridge kinetics (k(tr)) during maximal Ca(2)(+) activation were 10% faster in HCM myocytes. Myofibrillar Ca(2)(+) sensitivity in HCM myocytes (pCa(50)=6.40 ± 0.05) was higher than for donor myocytes (pCa(50)=6.09 ± 0.02; P<0.001) and was associated with reduced phosphorylation of troponin-I (ser-23/24) and MyBP-C (ser-282) in HCM myocytes. These characteristics were common to all six HCM patients and may therefore represent a secondary consequence of the known and unknown underlying genetic variants. Some HCM patients did however exhibit an altered relationship between force and cross-bridge kinetics at submaximal Ca(2+) concentrations, which may reflect the primary mutation. We conclude that the passive viscoelastic properties of the myocytes are unlikely to account for the increased stiffness of the HCM ventricle. However, the low maximum Ca(2+)-activated force and high Ca(2+) sensitivity of the myofilaments are likely to contribute substantially to any systolic and diastolic dysfunction, respectively, in hearts of HCM patients., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Aims: Esmolol, an ultra-short-acting beta-blocker, acts as a cardioplegic agent at millimolar concentrations. We investigated the mechanism by which esmolol induces diastolic ventricular arrest., Methods and Results: In unpaced Langendorff-perfused rat hearts, esmolol (0.03-3 mmol/L) had a profound negative inotropic effect resulting in diastolic arrest at 1 mmol/L and above. This inhibition of contraction was maintained during ventricular pacing. At 3 mmol/L, esmolol also abolished action potential conduction. To determine the cellular mechanism for the negative inotropism, we measured contraction (sarcomere shortening) and the calcium transient (fura-2 fluorescence ratio; Ca(tr)) in electrically-stimulated rat ventricular myocytes at 23 and 34 degrees C. The decrease in contraction (by 72% at 23 degrees C, from 0.16 +/- 0.01 to 0.04 +/- 0.01 microm, P < 0.001) was similar to that of isolated hearts and was caused by a large decrease in Ca(tr) (from 0.13 +/- 0.02 to 0.07 +/- 0.02, P < 0.001). There was no additional effect on myofilament Ca(2+) sensitivity. Esmolol's effects on contraction and Ca(tr) were not shared or altered by the beta-blocker, atenolol (1 mmol/L). Sarcoplasmic reticulum inhibition with thapsigargin did not alter the inhibitory effects of esmolol. Whole-cell voltage-clamp experiments revealed that esmolol inhibited the L-type calcium current (I(Ca,L)) and the fast sodium current (I(Na)), with IC(50) values of 0.45 +/- 0.05 and 0.17 +/- 0.025 mmol/L, respectively., Conclusion: Esmolol at millimolar concentrations causes diastolic ventricular arrest by two mechanisms: at 1 mmol/L (and below), the pronounced negative inotropic effect is due largely to inhibition of L-type Ca(2+) channels; additionally, higher concentrations prevent action potential conduction, probably due to the inhibition of fast Na(+) channels.

Fish myocytes continue to develop active tension when stretched to sarcomere lengths (SLs) on the descending limb of the mammalian length-tension relationship. A greater length-dependent activation in fish than mammals could account for this because the increase in Ca(2+) sensitivity may overcome the tendency for force to fall due to reduced cross-bridge availability at SLs above optimal myofilament overlap. We stretched skinned fish and rat ventricular myocytes over a wide range of SLs, including those on the descending limb of the mammalian length-tension relationship. We found that fish myocytes developed greater active tension than rat myocytes at physiological Ca(2+) concentrations at long SLs as a result of a higher Ca(2+) sensitivity and a steeper relationship between Ca(2+) sensitivity and SL. We also investigated the diastolic properties of fish and rat myocytes at long SLs by measuring titin-based passive tension, titin isoform expression and titin phosphorylation. Fish myocytes produced higher titin-based passive tension despite expressing a higher proportion of a long N2BA-like isoform (38.0+/-2% of total vs 0% in rat). However, titin phosphorylation in fish myocytes was lower than in rat, which may explain some of the difference in passive tension between species. The high level of titin-based passive tension and the differential phosphorylation of sarcomeric proteins in fish myocytes may contribute to the enhanced length-dependent activation and underlie the extended range of in vivo stroke volumes found in fish compared with mammals., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser(22)/Ser(23), reduces myofilament Ca(2+) sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser(22)/Ser(23) remains to be established. To determine the role of cTnI phosphorylation at Ser(22)/Ser(23) in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser(22)/Ser(23) are substituted by nonphosphorylatable Ala (cTnI-Ala(2)). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser(22)/Ser(23) and decreased the Ca(2+) sensitivity of force. In contrast, PKD had no effect on the Ca(2+) sensitivity of force in myocardium from cTnI-Ala(2) mice, in which Ser(22)/Ser(23) were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala(2) mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser(273), Ser(282), and Ser(302), and revealed that PKD phosphorylates only Ser(302). Furthermore, PKD phosphorylated Ser(302) selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala(2) mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca(2+) sensitivity through cTnI phosphorylation at Ser(22)/Ser(23) but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser(302), which may mediate the latter effect.

Background: Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca(2+) sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca(2+)-regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation., Methods and Results: Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca(2+)-activated force was similar in cTnC G159D and donor myocytes, but the Ca(2+) sensitivity of cTnC G159D myocytes was higher (EC(50) G159D/donor=0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca(2+) sensitivity (EC(50) G159D/donor=0.55 + or - 0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca(2+) sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca(2+) sensitivity was increased (EC(50) P/unP=4.7 + or - 1.9) but not with cTnC G159D troponin (EC(50) P/unP=1.2 + or - 0.1)., Conclusions: We propose that uncoupling of the relationship between phosphorylation and Ca(2+) sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation.

Elective temporary cardiac arrest (cardioplegia) is often required during cardiac surgery. In the 1970 s, the development of hyperkalaemic cardioplegic solutions revolutionised cardiac surgery by offering effective chemically-induced cardiac arrest and myocardial protection during global ischaemia. Despite remaining the most widely-used cardioplegic technique, hyperkalaemia can have detrimental effects due to the Na and Ca loading of the cardiac cell induced by depolarisation of the cell membrane. Efforts over the last two decades to establish better cardioplegic agents have mainly remained limited to animal experiments. The failure of these approaches to progress to clinical trials may be due to a lack of clear criteria that a cardioplegic agent should meet at a cellular level and, more importantly, at a system level. In this review we attempt to define the criteria for the optimal cardioplegic agent. We also assess the suitability and clinical potential of previously-studied cardioplegic agents and suggest cellular targets, particularly those involved in cardiac excitation-contraction coupling, that may prove to be attractive options for the development of new cardioplegic drugs. Finally, we propose a multicellular target approach using a combination of pharmacological agents in order to offer better cardioplegic solutions.

Aims: Sepsis-associated cardiac dysfunction represents an intrinsic impairment of cardiomyocyte function due in part to a decrease in myofilament Ca(2+) sensitivity associated with a sustained increase in cardiac troponin I (cTnI) phosphorylation at Ser23/24. Dephosphorylation of cTnI is under regulatory control. Thus, muscarinic and adenosine A(1)-receptor agonists antagonize beta-adrenergic stimulation via activation of protein phosphatase 2A (PP2A). The aim of this study was to determine whether modulation of PP2A and thus cTnI phosphorylation could improve sepsis-induced contractile dysfunction., Methods and Results: Cardiomyocytes were isolated from control or septic mice 16-18 h after an injection of vehicle or lipopolysaccharide (LPS; 9 mg/kg ip) respectively. Protein expression and phosphatase activity were determined in homogenates of control and septic hearts. Our data showed that LPS significantly increased cTnI phosphorylation at Ser23/24 in cardiomyocytes and reduced contraction amplitude without affecting Ca(2+)-transients. Treatment of cardiomyocytes with the A(1) agonist cyclopentyladenosine (CPA) or the protein kinase A inhibitor H89 significantly attenuated the LPS-induced contractile dysfunction without effect on Ca(2+)-transients. Co-treatment with CPA and H89 completely reversed the contractile dysfunction. Increased cTnI phosphorylation in septic hearts was associated with a significant reduction in the protein expression of both the catalytic and regulatory subunits (B56 alpha) of PP2A and a decrease in PP2A activity. CPA treatment of septic hearts increased PP2A activity. An increase in the protein expression of demethylated PP2A and a decrease in the PP2A-methyltransferase (PPMT; the methyltransferase that catalyses this reaction) were also observed., Conclusion: These data support the hypothesis that sustained cTnI phosphorylation underlies the contractile dysfunction seen in sepsis.

Many of the links between the genotype and phenotype in hypertrophic cardiomyopathy remain unexplained. In this unique longitudinal study we have investigated a patient with classical clinical phenotypic features of hypertrophic obstructive cardiomyopathy, with a known mutation in MYBPC3, the most commonly affected gene in this disease. By collecting cardiac tissue from the patient at the time of surgical myectomy for relief of left ventricular outflow tract obstruction, we have been able to examine the structure of the myocytes and the functional differences that occur in MyBP-C mutated HCM cardiac tissue from single protein level, onto single cardiomyocyte contractility, through to whole organ function as assessed clinically by echocardiography.

Protein kinase D (PKD) is a serine/threonine kinase with emerging myocardial functions; in skinned adult rat ventricular myocytes (ARVMs), recombinant PKD catalytic domain phosphorylates cardiac troponin I at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity. We used adenoviral gene transfer to determine the effects of full-length PKD on protein phosphorylation, sarcomere shortening and [Ca(2+)](i) transients in intact ARVMs. In myocytes transduced to express wild-type PKD, the heterologously expressed enzyme was activated by endothelin 1 (ET1) (5 nmol/L), as reflected by PKD phosphorylation at Ser744/Ser748 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). The ET1-induced increase in cellular PKD activity was accompanied by increased cardiac troponin I phosphorylation at Ser22/Ser23; this measured approximately 60% of that induced by isoproterenol (10 nmol/L), which activates cAMP-dependent protein kinase (PKA) but not PKD. Phosphorylation of other PKA targets, such as phospholamban at Ser16, phospholemman at Ser68 and cardiac myosin-binding protein C at Ser282, was unaltered. Furthermore, heterologous PKD expression had no effect on isoproterenol-induced phosphorylation of these proteins, or on isoproterenol-induced increases in sarcomere shortening and relaxation rate and [Ca(2+)](i) transient amplitude. In contrast, heterologous PKD expression suppressed the positive inotropic effect of ET1 seen in control cells, without altering ET1-induced increases in relaxation rate and [Ca(2+)](i) transient amplitude. Complementary experiments in "skinned" myocytes confirmed reduced myofilament Ca(2+) sensitivity by ET1-induced activation of heterologously expressed PKD. We conclude that increased myocardial PKD activity induces cardiac troponin I phosphorylation at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity, suggesting that altered PKD activity in disease may impact on contractile function.

Here we demonstrate that type I protein kinase A is redoxactive, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. This oxidative disulfide formation causes a subcellular translocation and activation of the kinase, resulting in phosphorylation of established substrate proteins. The translocation is mediated at least in part by the oxidized form of the kinase having an enhanced affinity for alpha-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of beta-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase.

Myosin binding protein-C (MyBP-C) is a poorly understood component of the thick filament in striated muscle sarcomeres. Its C terminus binds tightly to myosin, whereas the N terminus contains binding sites for myosin S2 and possibly for the thin filament. To study the role of the N-terminal domains of cardiac MyBP-C (cMyBP-C), we added human N-terminal peptide fragments to human and rodent skinned ventricular myocytes. At concentrations >10 micromol/L, the N-terminal C0C2 peptide activated force production in the absence of calcium (pCa 9). Force at the optimal concentration (80 micromol/L) of C0C2 was approximately 60% of that in maximal Ca2+ (pCa 4.5), but the rate constant of tension redevelopment (ktr) matched or exceeded (by up to 80%) that produced by Ca2+ alone. Experiments using different N-terminal peptides suggested that this activating effect of C0C2 resulted from binding by the pro/ala-rich C0-C1 linker region, rather than the terminal C0 domain. At a lower concentration (1 micromol/L), exogenous C0C2 strongly sensitized cardiac myofibrils to Ca2+ at a sarcomere length (SL) of 1.9 microm but had no significant effect at SL 2.3 microm. This differential effect caused the normal SL dependence of myofibrillar Ca2+ sensitivity to be reduced by 80% (mouse myocytes) or abolished (human myocytes) in 1 micromol/L C0C2. These results suggest that cMyBP-C provides a regulatory pathway by which the thick filament can influence the activation of the thin filament, separately from its regulation by Ca2+. Furthermore, the N-terminal region of cMyBP-C can influence the SL-tension (Frank-Starling) relationship in cardiac muscle.

Gram negative endotoxemia is associated with an intrinsic impairment of cardiomyocyte contraction, in part due to a reduction in myofilament Ca2+ responsiveness. Endotoxemic rat hearts show increased cardiac troponin I (cTnI) phosphorylation at serines 23 and 24, residues required for the protein kinase A (PKA)-dependent reduction of myofilament Ca2+ sensitivity after beta-adrenoceptor stimulation. To investigate the functional significance of increased TnI phosphorylation in endotoxemia, we studied the contractile effects of systemic bacterial lipopolysaccharide (LPS) treatment in transgenic mice (TG) with cardiac-specific replacement of cTnI by slow skeletal TnI (ssTnI, which lacks the PKA phosphorylation sites) and matched nontransgenic littermates (NTG) on a CD1 background. In wild-type CD1 mice treated with LPS (6 mg/kg ip), after 16-18 h there was a significant reduction in the maximum rates of left ventricular pressure development and pressure decline in isolated Langendorff-perfused hearts compared with saline-treated controls and a decrease in isolated myocyte unloaded sarcomere shortening from 6.1 +/- 0.2 to 3.9 +/- 0.2% (1 Hz, 32 degrees C, P<0.05). Similarly, in NTG myocytes, endotoxemia reduced myocyte shortening by 42% from 6.7 +/- 0.2 to 3.9 +/- 0.1% (P<0.05) with no change in intracellular Ca2+ transients. However, in the TG group, LPS reduced myocyte shortening by only 13% from 7.5 +/- 0.2 to 6.5 +/- 0.2% (P<0.05). LPS treatment significantly reduced the positive inotropic effect of isoproterenol in NTG myocytes but not in TG myocytes, even though isoproterenol-induced increases in Ca2+ transient amplitude were similar in both groups. Only LPS-treated NTG hearts showed a significant increase in cTnI phosphorylation. Investigation of the sarcomere shortening-Ca2+ relationship in Triton-skinned cardiomyocytes revealed a significant reduction in myofilament Ca2+ sensitivity after LPS treatment in NTG myocytes, an effect that was substantially attenuated in TG myocytes. In conclusion, the replacement of cTnI with ssTnI in the heart provides significant protection against endotoxemia-induced cardiac contractile dysfunction, most probably by preserving myofilament Ca2+ responsiveness due to prevention of phosphorylation of TnI at PKA-sensitive sites.

Protein kinase D (PKD) is a serine kinase whose myocardial substrates are unknown. Yeast 2-hybrid screening of a human cardiac library, using the PKD catalytic domain as bait, identified cardiac troponin I (cTnI), myosin-binding protein C (cMyBP-C), and telethonin as PKD-interacting proteins. In vitro phosphorylation assays revealed PKD-mediated phosphorylation of cTnI, cMyBP-C, and telethonin, as well as myomesin. Peptide mass fingerprint analysis of cTnI by liquid chromatography-coupled mass spectrometry indicated PKD-mediated phosphorylation of a peptide containing Ser22 and Ser23, the protein kinase A (PKA) targets. Ser22 and Ser23 were replaced by Ala, either singly (Ser22Ala or Ser23Ala) or jointly (Ser22/23Ala), and the troponin complex reconstituted in vitro, using wild-type or mutated cTnI together with wild-type cardiac troponin C and troponin T. PKD-mediated cTnI phosphorylation was reduced in complexes containing Ser22Ala or Ser23Ala cTnI and completely abolished in the complex containing Ser22/23Ala cTnI, indicating that Ser22 and Ser23 are both targeted by PKD. Furthermore, troponin complex containing wild-type cTnI was phosphorylated with similar kinetics and stoichiometry (approximately 2 mol phosphate/mol cTnI) by both PKD and PKA. To determine the functional impact of PKD-mediated phosphorylation, Ca2+ sensitivity of tension development was studied in a rat skinned ventricular myocyte preparation. PKD-mediated phosphorylation did not affect maximal tension but produced a significant rightward shift of the tension-pCa relationship, indicating reduced myofilament Ca2+ sensitivity. At submaximal Ca2+ activation, PKD-mediated phosphorylation also accelerated isometric crossbridge cycling kinetics. Our data suggest that PKD is a novel mediator of cTnI phosphorylation at the PKA sites and may contribute to the regulation of myofilament function.

In cardiac muscle, beta-adrenergic stimulation increases contractile force and accelerates relaxation. The relaxant effect is thought to be due primarily to stimulation of Ca(2+) uptake into the sarcoplasmic reticulum (SR), although changes in myofilament properties may also contribute. The present study investigated the contribution of the myofilaments to the beta-adrenergic response in isolated rat cardiac trabeculae undergoing either isometric or work-loop contractions (involving simultaneous force generation and shortening) at different stimulation frequencies (range 0.25-4.5 Hz). SR-dependent effects were eliminated by treatment with ryanodine (1 microM) and cyclopiazonic acid (30 microM). In isometric contractions during SR inhibition, isoprenaline increased the force but did not alter the time course of the twitch. In contrast, in work-loop contractions, the positive inotropic effect was accompanied by a reduced diastolic force between beats, most apparent at higher frequencies (e.g. diastolic stress fell from 58.6 +/- 5.5 to 28.8 +/- 5.8 mN mm(-2) at 1.5 Hz). This relaxant effect contributed to a beta-adrenoceptor-mediated increase in net work and power output at higher frequencies, by reducing the amount of work required to re-lengthen the muscle. Consequently, the frequency for maximum power output increased from 1.1 +/- 0.1 to 1.6 +/- 0.1 Hz. We conclude that the contribution of myofilament properties to the relaxant effect of beta-stimulation may be of greater significance when force and length are changing simultaneously (as occurs in the heart) than during force development under isometric conditions.

Phosphorylation of cardiac myofibrils by cAMP-dependent protein kinase (PKA) can increase the intrinsic rate of myofibrillar relaxation, which may contribute to the shortening of the cardiac twitch during beta-adrenoceptor stimulation. However, it is not known whether the acceleration of myofibrillar relaxation is due to phosphorylation of troponin I (TnI) or of myosin binding protein-C (MyBP-C). To distinguish between these possibilities, we used transgenic mice that overexpress the nonphosphorylatable, slow skeletal isoform of TnI in the myocardium and do not express the normal, phosphorylatable cardiac TNI: The intrinsic rate of relaxation of myofibrils from wild-type and transgenic mice was measured using flash photolysis of diazo-2 to rapidly decrease the [Ca(2+)] within skinned muscles from the mouse ventricles. Incubation with PKA nearly doubled the intrinsic rate of myofibrillar relaxation in muscles from wild-type mice (relaxation half-time fell from approximately 150 to approximately 90 ms at 22 degrees C) but had no effect on the relaxation rate of muscles from the transgenic mice. In parallel studies with intact muscles, we assessed crossbridge kinetics indirectly by determining f(min) (the frequency for minimum dynamic stiffness) during tetanic contractions. Stimulation of beta-adrenoceptors with isoproterenol increased f(min) from 1.9 to 3.1 Hz in muscles from wild-type mice but had no effect on f(min) in muscles from transgenic mice. We conclude that the acceleration of myofibrillar relaxation rate by PKA is due to phosphorylation of TnI, rather than MyBP-C, and that this may be due, at least in part, to faster crossbridge cycle kinetics.

1. We studied the effects of alpha1- or beta-adrenoceptor stimulation on the contractility of isolated rat ventricular trabeculae at 24 degrees C using the work-loop technique, which simulates the cyclical changes in length and force that occur during the cardiac cycle. Some muscles were injected with fura-2 to monitor the intracellular Ca2+ transient. 2. Comparison of twitch records revealed that peak force was greater and was reached earlier in work-loop contractions than in corresponding isometric contractions. This was attributed to the changes in muscle length and velocity during work-loop contractions, since the Ca2+ transients were largely unaffected by the length changes. 3. Stimulation of alpha1-adrenoceptors (with 100 microM phenylephrine) increased net work, power production, the frequency for maximum work, and the frequency for maximum power production (fopt). The increase in net work was due to the positive inotropic effect of phenylephrine, which was similar at all frequencies investigated (0. 33-4.5 Hz). The increase in fopt was attributed to an abbreviation of twitch duration induced by alpha1-stimulation at higher frequencies (> 1 Hz), even though the twitch became longer at 0.33 Hz. 4. beta-Adrenoceptor stimulation (with 5 microM isoprenaline) produced marked increases in net work, power output, the frequency for net work, and fopt. These effects were attributed both to the positive inotropic effect of beta-stimulation, which was greater at higher frequencies, and to the reduction in twitch duration. beta-stimulation also abolished the frequency-dependent acceleration of twitch duration. 5. The increase in power output and fopt with alpha1- as well as beta-adrenoceptor stimulation suggested that both receptor types may contribute to the effects of catecholamines, released during stress or exercise, although the greater effects of beta-stimulation are likely to predominate.

The isometric force-frequency relationship of isolated rat ventricular trabeculae (diameter <250 micrometer) was examined at 24, 30, and 37 degreesC at stimulation frequencies (0.1-12 Hz) encompassing the physiological range. Some muscles were microinjected with fura PE3 to monitor the diastolic and systolic intracellular concentration of Ca2+ ([Ca2+]i). At a near-physiological external Ca2+ concentration ([Ca2+]o) of 1 mM, a positive force-frequency relationship was demonstrated at all temperatures. The force-frequency relationship became negative at high frequencies (e. g., >6 Hz at 30 degreesC) at 1 mM [Ca2+]o or at low frequencies at 8 mM [Ca2+]o. The twitch and Ca2+ transient became shorter as stimulation frequency increased; these changes were related to changes in systolic, rather than diastolic, [Ca2+]i and were not blocked by inhibitors of Ca2+/calmodulin-dependent protein kinase II. The positive force-frequency relationship of rat trabeculae was caused by a frequency-dependent loading of the sarcoplasmic reticulum (SR) with Ca2+. We suggest that at high frequencies, or under conditions of Ca2+ overload, this loading saturates. Processes that tend to decrease SR Ca2+ release will then predominate, resulting in a negative force-frequency relationship.

We examined the influences of Ca2+ and crossbridge kinetics on the maximum rate of force development during Ca2+ activation of cardiac myofibrils and on the maximum rate of relaxation. Flash photolysis of diazo-2 or nitrophenyl-EGTA was used to produce a sudden decrease or increase, respectively, in [Ca2+] within Triton-skinned trabeculae from rat and guinea pig hearts (22 degrees C). Trabeculae from both species had similar Ca2+ sensitivities, suggesting that the rate of dissociation of Ca2+ from troponin C (k(off)) is similar in the 2 species. However, the rate of relaxation after diazo-2 photolysis was 5 times faster in the rat (16.1 +/- 0.9 s(-1), mean +/- SEM, n = 11) than in the guinea pig (2.99 +/- 0.26 s(-1), n = 7). This indicates that the maximum relaxation rate is limited by crossbridge kinetics rather than by k(off). The maximum rates of rapid activation by Ca2+ after nitrophenyl-EGTA photolysis (k(act)) and of force redevelopment after forcible crossbridge dissociation (k(act)) were similar and were approximately 5-fold faster in rat (k(act)= 14.4 +/- 0.9 s(-1), k(tr)= 13.0 +/- 0.6 s(-1)) than in guinea pig (k(act)= 2.57 +/- 0.14 s(-1), k(tr)= 2.69 +/- 0.30 s(-1)) trabeculae. This too may be mainly due to species differences in crossbridge kinetics. Both k(act) and k(tr) increased as [Ca2+] increased. This Ca2+ dependence of the rates of force development is consistent with current models for the Ca2+ activation of the crossbridge cycle, but these models do not explain the similarity in the maximal rates of activation and relaxation within a given species.

1. Changes in cytosolic [Ca2+] ([Ca2+]i) were measured in isolated rat trabeculae that had been micro-injected with fura-2 salt, in order to investigate the mechanism by which twitch force changes following an alteration of muscle length. 2. A step increase in length of the muscle produced a rapid potentiation of twitch force but not of the Ca2+ transient. The rapid rise of force was unaffected by inhibiting the sarcoplasmic reticulum (SR) with ryanodine and cyclopiazonic acid. 3. The force-[Ca2+]i relationship of the myofibrils in situ, determined from twitches and tetanic contractions in SR-inhibited muscles, showed that the rapid rise of force was due primarily to an increase in myofibrillar Ca2+ sensitivity, with a contribution from an increase in the maximum force production of the myofibrils. 4. After stretch of the muscle there was a further, slow increase of twitch force which was due entirely to a slow increase of the Ca2+ transient, since there was no change in the myofibrillar force-[Ca2+]i relationship. SR inhibition slowed down, but did not alter the magnitude of, the slow force response. 5. During the slow rise of force there was no slow increase of diastolic [Ca2+]i, whether or not the SR was inhibited. The same was true in unstimulated muscles. 6. We conclude that the rapid increase in twitch force after muscle stretch is due to the length-dependent properties of the myofibrils. The slow force increase is not explained by length dependence of the myofibrils or the SR, or by a rise in diastolic [Ca2+]i. Evidence from tetani suggests the slow force responses result from increased Ca2+ loading of the cell during the action potential.

During heart failure, force production by the heart decreases. This may be overcome by Ca2+-sensitizing drugs, which increase myofibril Ca2+ sensitivity without necessarily altering intracellular Ca2+ concentration. However, Ca2+ sensitizers slow the relaxation of intact cardiac muscle. We used diazo-2, a caged chelator of Ca2+, to study the effects of the Ca2+ sensitizers caffeine and CGP 48506 on the intrinsic relaxation rate of cardiac myofibrils. Trabeculae from rat right ventricles were skinned by 1% Triton X-100 and were activated in a 10-microL bath. In steady state experiments, CGP 48506 (10 micromol/L) shifted the force-pCa curve leftward by 0.41+/-0.03 pCa units (mean+/-SEM, n=6). An identical shift was induced by caffeine (20 mmol/L). Photolysis of diazo-2 by a flash of light (160 mJ, 310 to 400 nm) caused an immediate decrease in Ca2+-activated force produced by the trabeculae. Relaxation was fitted by a double-exponential decay, and the rate constants were found to be independent of force and preflash Ca2+ concentration. The initial fast rate, corresponding to myofibrillar relaxation, was increased from 17.3+/-2.0 to 30.9+/-3.7 s(-1) (n=4) by caffeine but was unaffected by CGP 48506 (16.6+/-1.7 and 14.4+/-2.3 s(-1) in the absence and presence of drug, respectively; n=5). Thus, myofibril relaxation need not be slowed by Ca2+-sensitizing agents but can even be accelerated. Despite similarities in their effects on myofibril Ca2+ sensitivity, caffeine and CGP 48506 affect the myofibrils at least partly via different mechanisms.

Objective: Our aims were: (1) to examine the effect of pH (7.4-6.5) on Ca2+ release from the sarcoplasmic reticulum (SR) of cardiac muscle, and (2) to see if these effects were altered by phosphate (Pi)., Methods: Rat ventricular trabeculae were permeabilised with saponin. Ca(2+)-induced Ca2+ release (CICR) from the SR was triggered by flash photolysis of nitr-5. Under similar loading conditions, SR Ca2+ loading was assessed using caffeine to release the Ca2+ in the SR. Force and fluo-3 fluorescence (a measure of the cytosolic [Ca2+]) were monitored., Results: SR Ca2+ loading was optimal at pH 7.1 and was significantly reduced at pH 7.4, 6.8 and 6.5. CICR was the same at pH 7.4 as at pH 7.1, but was reduced, by more than Ca2+ loading, in acidic solutions. These differential effects on loading and CICR suggested that Ca2+ activation of the Ca2+ release channel was decreased (by > 50%) as pH was lowered from 7.4 to 6.5. A direct effect on the Ca2+ release channel was confirmed by the finding that Ca2+ release was slower in acidic solutions. Acidosis also slowed the re-uptake of Ca2+ into the SR after CICR, which may account for the reduced Ca2+ loading at low pH. As observed previously, Pi (20 mM) by itself decreased SR Ca2+ loading. However, the inhibitory effects of acidosis and Pi on SR Ca2+ loading were independent., Conclusions: A fall of pH over the range 7.4-6.5 directly inhibits the SR Ca2+ release channel. In addition, acidosis inhibits SR Ca2+ accumulation by a mechanism independent of that of Pi. Both effects of acidosis would act to decrease SR Ca2+ release and so would contribute to the negative inotropic actions of intracellular acidosis in intact cardiac muscle.

1. We studied the effects of flash photolysis on the novel enantiomeric cardiac inotropes EMD 57033 (a calcium sensitizer) and EMD 57439 (a phosphodiesterase III inhibitor) in rat isolated ventricular trabeculae. 2. In skinned trabeculae, EMD 57439 had no effect on force production, consistent with lack of an active cyclic AMP system in this preparation. In contrast, EMD 57033 potentiated force at partial and maximal activation. A single flash of near u.v. light given at partial activation (30-70%) reduced force potentiation by 52.4 +/- 5.2%. No effect was produced by flashes in the presence of EMD 57439 or in the absence of either drug. 3. The time course of relaxation induced by EMD 57033 photolysis was indistinguishable from that obtained on deactivating the muscle by rapidly lowering Ca2+ using photolysis of the caged chelator of calcium, diazo-2. 4. In intact, twitching trabeculae, EMD 57033 increased diastolic and peak force and slowed relaxation. These effects were simultaneously reduced by a light flash. In these muscles EMD 57439 reduced force, without affecting the twitch time course. These effects were also reduced by a light flash. 5. The u.v. absorbance spectra of EMD 57033 and EMD 57439 were identical. After photolysis optical density decreased substantially and the peak shifted from 320 nm to 280 nm. 6. The proton n.m.r. spectra of these compounds were identical. The main change post-photolysis was a decrease in the proton signal associated with the enantiomeric carbon atom. 7. This novel manipulation of the molecular structure of EMD 57033 and EMD 57439 within an experiment thus provides direct evidence linking calcium sensitization to a particular molecular structure. The three main effects of the sensitizer on the twitch were simultaneously abolished and may be mechanistically linked. Flash photolysis may be a useful tool for further investigations of the actions of these compounds. In particular, flash photolysis of the sensitizer represents a novel method of rapidly deactivating cardiac muscle.

1. Some rabbit masseter fibres express the alpha-cardiac myosin heavy chain (MHC). To compare the biochemical and physiological properties of these fibres with other skeletal fibre types, we examined the histochemical and immunohistochemical staining characteristics, maximum velocity of shortening (V(zero)) and MHC isoform content of fibres from rabbit masseter and soleus muscles. 2. The fibre-type composition of muscle sections was determined with MHC antibodies and myofibrillar ATPase histochemistry. Fibres we designated 'type alpha-cardiac' were different from type I and type II fibres in that they stained positively with the alpha-cardiac MHC antibody and they maintained. ATPase reactivity after acid and alkali pre-incubations. Samples of superficial masseter contained a few type I fibres, with the majority of fibres classified as either type IIA or type alpha-cardiac. Soleus samples contained type I, IIA and IIC fibres. 3. The V(zero) of chemically skinned fibres was determined by the slack-test method. Each fibre was subsequently characterized as type I, IIA, IIC or alpha-cardiac from MHC identification using gel electrophoresis (SDS-PAGE). In masseter fibres the V(zero) values were (in muscle lengths s-1): type I, 0.54 +/- 0.05 (mean +/- S.D., n = 3); type IIA, 1.23 +/- 0.34 (n = 27); type alpha-cardiac, 0.78 +/- 0.08 (n = 9). In soleus fibres V(zero) values were: type I, 0.55 +/- 0.06 (n = 14); type IIA, 0.89 +/- 0.04 (n = 8); type IIC, 0.73 (n = 2). 4. We conclude that the rabbit masseter muscle contains an 'alpha-cardiac' fibre type that is distinct from other skeletal fibres. This fibre type expresses only the alpha-cardiac MHC, has unusual myofibrillar ATPase reactivity and has a V(zero) intermediate between type I and type II fibres.

The Ca2+ sensitivity of cardiac myofibrillar force production can be decreased by acidosis or inorganic phosphate (P(i)) and increased by caffeine. To investigate whether the source of tissue influences the potency of these agents, we compared the actions of acidosis (change of pH from 7.0 to 6.2), P(i) and caffeine (both 20 mM) on force production of skinned cardiac muscles from adult ventricle, adult atrium and neonate ventricle of the rat. Maximum Ca(2+)-activated force was reduced by all three interventions and the responses of the different muscle types to a given intervention were similar. Acidosis reduced myofibrillar Ca2+ sensitivity by 1.09 and 1.04 pCa units in adult ventricle and atrium, respectively, and P(i) reduced it by 0.19 and 0.22 pCa units. However, each effect was only one-third as great in the neonate ventricle, which showed falls of 0.33 pCa units for acidosis and 0.06 for P(i). In contrast, caffeine raised the Ca2+ sensitivity by the same amount (approximately 0.4 pCa units) in all three muscle types. The differential effect between adult and neonate seen with both acidosis and P(i) suggests some similarity in the mechanisms by which these factors decrease Ca2+ sensitivity. In contrast, the equal effects of caffeine on neonate and adult suggests that caffeine acts by a completely different mechanism. The lower pH- and P(i)-sensitivity of the neonatal ventricle can help to explain why neonatal and adult myocardium exhibit differential force responses to ischaemia (or hypoxia alone).

Purpose: To ascertain if the inotropic effects of altered intracellular pH on detrusor smooth muscle could be explained by a change in Ca(2+)-sensitivity of the contractile proteins., Materials and Methods: Guinea pig detrusor smooth muscle was permeabilized with alpha-toxin and exposed to solutions mimicking the composition of the intracellular compartment and of varying [Ca2+]. Isometric tension was measured during exposure to solutions of varying [Ca2+] at pH 6.3, 7.1 and 7.5., Results: At pH 7.1 the pCa (-log10[Ca2+]) required for half-maximal activation (pCa50) was 6.00 +/- 0.07 at 22C. A Hill coefficient of unity suggested lack of cooperativity in myofilament Ca2+ activation. A decrease of pH from 7.1 to 6.3 had no significant effect on the pCa50 value or the maximum Ca2+ activated force. An increase to pH 7.5 decreased the pCa50 value by 0.65 +/- 0.20 units but left maximum force unaffected., Conclusions: The reduced Ca2+ sensitivity of detrusor myofilaments at alkaline pH could partly explain the negative inotropic effect of intracellular alkalosis in intact muscle. The positive inotropic effect of intracellular acidosis cannot, however, be explained by alteration to myofilament Ca2+ sensitivity.

Objective: The aim was to investigate whether, and how, increases in inorganic phosphate (Pi) and ADP, similar to those occurring intracellularly during early myocardial ischaemia, affect the calcium handling of the sarcoplasmic reticulum., Methods: Rat ventricular trabeculae were permeabilised with saponin. The physiological process of calcium induced calcium release (CICR) from the muscle sarcoplasmic reticulum was triggered via flash photolysis of the "caged Ca2+", nitr-5. Alternatively, calcium release was induced by rapid application of caffeine to give an estimate of sarcoplasmic reticular calcium loading. The initial rate of sarcoplasmic reticular calcium pumping was also assessed by photolysis of caged ATP at saturating [Ca2+]. Myoplasmic [Ca2+] (using fluo-3) and isometric force were measured., Results: Pi (2-20 mM) significantly depressed the magnitude of CICR and the associated force transient. Sarcoplasmic reticular calcium loading was inhibited even more than CICR by Pi, suggesting that reduced calcium loading could account for all of the inhibitory effect of Pi on CICR and that Pi may slightly activate the calcium release mechanism. The reduced sarcoplasmic reticular calcium loading seemed to be due to a fall in the free energy of ATP hydrolysis (delta GATP) available for the calcium pump, since equal decreases in delta GATP produced by adding both Pi and ADP in various ratios caused similar falls in the calcium loading of the sarcoplasmic reticulum. The caged ATP experiments indicated that Pi (20 mM) did not affect the rate constant of sarcoplasmic reticular calcium uptake. ADP (10 mM) alone, or with 1 mM Pi, inhibited calcium loading. In spite of this, ADP (10 mM) did not alter CICR and, when 1 mM Pi was added, ADP increased CICR above control., Conclusions: An increase in intracellular Pi reduces sarcoplasmic reticular calcium loading and thus depresses the CICR. This could be an important contributing factor in the hypoxic or ischaemic contractile failure of the myocardium. However the detrimental effect of Pi may be offset to some extent by a stimulatory action of ADP on the calcium release mechanism of CICR.

1. We investigated the effects of acidosis, inorganic phosphate (Pi) and caffeine on the Ca2+ affinity of isolated fast-twitch skeletal and cardiac troponin C (TnC), labelled with fluorescent probes to report Ca2+ binding to the regulatory sites. We also measured the effects of these interventions on the maximum force development and the Ca2+ sensitivity of skinned fibres from fast-twitch skeletal muscle and cardiac muscle, as has been done previously. The two types of experiment were carried out under similar solution conditions, so that we could assess the contribution of any direct actions on TnC to the modulation of Ca2+ sensitivity in the skinned muscle fibres. 2. In skinned fibres, acidosis (decreasing pH from 7.0 to 6.2) and Pi (20 mM) suppressed maximum force to the same extent within a given muscle type, but had greater effects on cardiac fibres compared with skeletal fibres. Caffeine (20 mM) depressed maximum force equally in cardiac and skeletal muscle. Thus, the fall of force induced by acidosis or Pi may involve a different mechanism from that induced by caffeine. 3. Skinned skeletal fibres were more Ca2+ sensitive than cardiac fibres by 0.29 pCa units (pCa = -log10[Ca2+]). Isolated skeletal TnC also had a greater Ca2+ affinity than cardiac TnC, by 0.20 pCa units. These results suggest that the Ca2+ sensitivity of skinned fibres is at least partly determined by the type of TnC present. 4. Acidosis reduced the Ca2+ sensitivity of force in skinned fibres profoundly and had a 2-fold greater effect in cardiac muscle than skeletal muscle (falls in pCa for 50% activation, pCa50, were 1.09 and 0.55, respectively). Acidosis also reduced the Ca2+ affinity of TnC, again having double the effect on the pCa50 for cardiac TnC (0.58) as on that for skeletal TnC (0.28). The greater effect of acidosis on cardiac skinned fibres, compared with skeletal, may be partly explained, therefore, by the type of TnC present, and one-half of the effect on fibres may be attributed to the direct effect of H+ on TnC. 5. Pi reduced the Ca2+ sensitivity of force in skeletal and cardiac skinned fibres by 0.30 and 0.19 pCa units, respectively. However, the Ca2+ affinity of isolated cardiac and skeletal TnC was unaffected by Pi, indicating that the decrease in muscle Ca2+ sensitivity is not mediated by a direct action of Pi on TnC. 6. Caffeine increased the Ca2+ sensitivity of cardiac skinned fibres by 0.31 pCa units, which was 3 times greater than for the skeletal fibres (0.09 pCa units).(ABSTRACT TRUNCATED AT 400 WORDS)

1. The fall of maximum Ca(2+)-activated force of cardiac myofibrils at short muscle lengths could be due to a reduction of cross-bridge cycling or to development of an opposing (restoring) force. To try to distinguish between these possibilities, we measured simultaneously myofibrillar force development and MgATPase activity (a measure of cross-bridge cycling) in rat skinned trabeculae at different muscle lengths. ATPase activity was measured photometrically from the utilization of NADH in a coupled enzyme assay. Muscle length was varied to give estimated 0.2 micron changes in sarcomere length (SL) over the range 1.4-2.4 microns. 2. Both Ca(2+)-activated force development and ATPase activity were optimal at a muscle length (Lo) where the resting SL was 2.2 microns. At Lo the maximum ATPase activity at 21 degrees C was 0.56 +/- 0.05 mM s-1 (mean +/- S.E.M., n = 6), which was equivalent to an ATP turnover per myosin S1 head of 3.3 s-1. 3. The relationship between ATPase activity and SL was curved, with rather little change in ATPase activity over the SL range 2.0-2.4 microns, but significant falls at 1.8 microns and below. At 65% of Lo (corresponding to a mean active SL of approximately 1.4 microns), the ATPase activity was only 50% of its value at 2.2 microns SL. 4. Force development decreased linearly as SL was reduced below 2.2 microns. Force fell by more than ATPase activity, particularly at SL 1.6 and 1.8 microns. 5. The fall of ATPase activity indicates that some of the decline of force production at short SL results from a fall in the net rate of cross-bridge cycling. This is probably the result of double overlap of thin filaments. However, the differential effect on force and ATPase reveals that, in the intermediate range of SL, decreased cross-bridge cycling can account for only part of the fall of force; the remainder is probably due to an increase in a restoring force, which may arise from deformation of the connective tissue in the muscle preparations used.

An alteration in the length of isolated cardiac muscle produces an immediate change in twitch force, then a slow further change in the same direction. We have found that the slow changes in force in rabbit papillary muscles are blocked or reversed by the beta-agonist, isoprenaline (1 microM). The abolition of the slow responses by isoprenaline was not due to saturation of the myofibrils with Ca2+, as the blockade continued if the extracellular [Ca2+] was reduced in the presence of isoprenaline so that twitch force was < 50% maximal. Ryanodine (1 microM) did not block the slow responses, suggesting that the sarcoplasmic reticulum does not mediate the responses. These results suggest that changes of intracellular [cAMP] may mediate, or at least modulate, the slow force responses to a length change in cardiac muscle.

Possible interactions between the effects of pH and phosphate (Pi) on the maximum force development of cardiac myofibrils were investigated in rat skinned trabeculae in solutions of different pH (7.4-6.2) and [Pi] (where [] denote concentration). At pH 7.0 there was an inverse linear relationship between force and log [Pi] over the [Pi] range 0.2-20 mM; its slope (-0.46/decade) was twice that found previously for skeletal muscle [21]. Acidosis depressed force substantially, but the relative change of force was unaffected by Pi addition (0, 5, 20 mM); there was no evidence for the synergism between acidosis and Pi that would be expected if some of the inhibition by acidosis was due to protonation of Pi to the putative inhibitory form, H2PO4-. It was taken into account that even without Pi addition, there was enough Pi inside the muscle from various sources to produce significant changes in [H2PO4-] as the pH was varied. The results suggest that H+ and Pi inhibit maximum force development of cardiac myofibrils independently, by different mechanisms. From this it is argued that H+ and Pi may be released at different steps in the crossbridges cycle. In the myocardium Pi and H+ probably exert tonic inhibitory influences on cardiac myofibrils under all conditions.

It has been known for over 100 years that acidosis decreases the contractility of cardiac muscle. However, the mechanisms underlying this decrease are complicated because acidosis affects every step in the excitation-contraction coupling pathway, including both the delivery of Ca2+ to the myofilaments and the response of the myofilaments to Ca2+. Acidosis has diverse effects on Ca2+ delivery. Actions that may diminish Ca2+ delivery include 1) inhibition of the Ca2+ current, 2) reduction of Ca2+ release from the sarcoplasmic reticulum, and 3) shortening of the action potential, when such shortening occurs. Conversely, Ca2+ delivery may be increased by the prolongation of the action potential that is sometimes observed and by the rise of diastolic Ca2+ that occurs during acidosis. This rise, which will increase the uptake and subsequent release of Ca2+ by the sarcoplasmic reticulum, may be due to 1) stimulation of Na+ entry via Na(+)-Ca2+ exchange; 2) direct inhibition of Na(+)-Ca2+ exchange; 3) mitochondrial release of Ca2+; and 4) displacement of Ca2+ from cytoplasmic buffer sites by H+. Acidosis inhibits myofibrillar responsiveness to Ca2+ by decreasing the sensitivity of the contractile proteins to Ca2+, probably by decreasing the binding of Ca2+ to troponin C, and by decreasing maximum force, possibly by a direct action on the cross bridges. Thus the final amount of force developed by heart muscle during acidosis is the complex sum of these changes.

The ability of Ca2+ or inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to release Ca2+ from cardiac sarcoplasmic reticulum (SR) was investigated using saponin-skinned ventricular trabeculae from rats. To overcome diffusion delays, rapid increases in the concentrations of Ca2+ and Ins(1,4,5)P3 were produced by laser photolysis of "caged Ca2+" (Nitr-5) and "caged Ins(1,4,5)P3". Photolysis of Nitr-5 to produce a small jump in [Ca2+] from pCa 6.8 to 6.4 induced a large and rapid force response (t1/2 = 0.89 s at 12 degrees C); the source of the Ca2+ that activated the myofibrils was judged to be the SR, since it was blocked by 0.1 mM ryanodine or 5 mM caffeine. A smaller, slower, and less consistent release of SR Ca2+ was produced by photorelease of Ins(1,4,5)P3. The results demonstrate that these caged compounds can be used to study excitation-contraction coupling in skinned multicellular preparations of cardiac muscle. The data are consistent with a major role for Ca2(+)-induced Ca2+ release in cardiac activation, whereas the role for Ins(1,4,5)P3 may be to modulate, rather than directly stimulate, SR Ca2+ release.

Thin ventricular trabeculae from rat hearts were skinned with the non-ionic detergent Triton X-100. The isometric force development of these muscles was investigated over a range of Ca2+ concentrations (0.2-200 microM) in the presence of various concentrations of creatine phosphate (CP), creatine and inorganic phosphate (Pi). The addition of Pi (1-30 microM) at constant ionic strength reduced the maximum Ca2+-regulated force (obtained at 200 microM-Ca2+). At 20 mM-Pi force was 31 +/- 5% (mean +/- S.E. of mean, n = 7) of that in Pi-free solution. This inhibitory action of Pi was not due to an inhibition of creatine kinase activity or to a reduction in the amount of free energy available for work from ATP hydrolysis. Increases in Pi concentration over the range 0-20 mM shifted the sigmoid relationship between force and [Ca2+] to higher Ca2+ concentrations. The [Ca2+] required for 50% activation rose from 8.3 +/- 1.5 microM (mean +/- S.E. of mean, n = 7) in Pi-free solutions to 19.8 +/- 4.2 microM in 20 mM-Pi. An increase in CP concentration in the range 10-30 mM had a small inhibitory effect on maximum Ca2+-regulated force but did not alter the force--[Ca2+] relationship. Creatine (0-30 mM) was without effect on the skinned muscles. The inhibitory effects of Pi suggest that a net hydrolysis of CP to Pi and creatine in the myoplasm of intact cardiac cells could reduce force development at a given myoplasmic [Ca2+], especially if the latter was below the level needed to fully activate the myofibrils. This suggestion is discussed in relation to the CP hydrolysis and decrease in force development that are observed in cardiac muscle during hypoxia or ischaemia.

We believe that the weight of present evidence suggests that acute contractile failure in hypoxic or ischaemic cardiac muscle is accompanied by a fall in A but this fall is not the direct cause of the reduced force production. The main established causes of acute contractile failure are the effects of intracellular pH and of Pi accumulation on the contractile proteins and, at a later stage, failure of calcium release. It is worth stressing the role of Pi accumulation both because the effects of Pi have only been recognised relatively recently and because it has a large depressant effect. Pi accumulation is the best candidate to explain the rapid fall of force which occurs during hypoxia and ischaemia when ATP levels are unchanged and before pH changes are substantial.

When the pH surrounding myofilaments of striated muscle is reduced there is an inhibition of both the actin-myosin reaction as well as the Ca2+-sensitivity of the myofilaments. Although the mechanism for the effect of acidic pH on Ca2+-sensitivity has been controversial, we have evidence for the hypothesis that acidic pH reduces the affinity of troponin C (TNC) for Ca2+. This effect of acidic pH depends not only on a direct effect of protons on Ca2+-binding to TNC, but also upon neighboring thin filament proteins, especially TNI, the inhibitory component of the TN complex. Using fluorescent probes that report Ca2+-binding to the regulatory sites of skeletal and cardiac TNC, we have shown, for example, that acidic pH directly decreases the Ca2+-affinity of TNC, but only by a relatively small amount. However, with TNC in whole TN or in the TNI-TNC complex, there is about a 2-fold enhancement of the effects of acidic pH on Ca2+-binding to TNC. Acidic pH decreases the affinity of skeletal TNI for skeletal TNC, and also influences the micro-environment of a probe positioned at Cys-133 of TNI, a region of interaction with TNC. Other evidence that the effects of acidic pH on Ca2+-TNC activation of myofilaments are influenced by TNI comes from studies with developing hearts. In contrast, to the case with the adult preparations, Ca2+-activation of detergent extracted fibers prepared from dog or rat hearts in the peri-natal period are weakly affected by a drop in pH from 7.0 to 6.5. This difference in the effect of acidic pH appears to be due to a difference in the isoform population of TNI, and not to differences in isotype population or amount of TNC.

McClellan and Winegrad (1980, J. Gen. Physiol., 75:283-295) have reported that in rat ventricular muscles that have reportedly been made "hyperpermeable" to small ions such as Ca2+, CaEGTA2-, and MgATP2- by a soak in EGTA, the maximum Ca2+-regulated force can be permanently increased by a short exposure to positively inotropic drugs, such as epinephrine or cAMP plus theophylline, in the presence of the detergent Triton X-100. The experiments reported here were begun as an attempt to repeat and extend this important observation. However, no evidence could be found for a potentiation of force that was not merely produced by Triton alone. In addition, the thickest muscles used (250-440 microns diameter) exhibited very low values for force per unit cross-sectional area, which suggested that either Ca2+ reached only a fraction of the myofibrils or the myofibrils were in a state of low contractility. The results of further experiments that were designed to test the permeability characteristics of these EGTA-treated muscles indicated that the movement of certain ions into these preparations was restricted, even in thin muscles (80-200 microns diameter). The rate of development of Ca2+-regulated force was slow (t1/2 approximately equal to 1-3 min), but was greatly accelerated after the muscles had been superfused with Triton X-100 (t1/2 approximately equal to 10-20 s). Removal of creatine phosphate (CP) in the presence of MgATP produced a partial rigor contracture in the EGTA-treated muscles. The results were consistent with the suggestion that the EGTA-treated muscles were permeable to some extent to Ca2+ and HCP2- ions but not to CaEGTA2- and MgATP2-. Thus, it would seem unlikely that the [Ca2+], [MgATP2-], and [Mg2+] in the immediate vicinity of the myofibrils in these preparations can be adequately controlled by the solution bathing the muscles.

1. Ferret ventricular muscles were skinned by prolonged application of Triton X-100. Aequorin was allowed to diffuse into the myoplasmic space and the resulting light emission was used to monitor the myoplasmic [Ca2+]. The muscle was then activated with a lightly buffered Ca2+ solution and the changes in myoplasmic [Ca2+] and tension in response to length changes were investigated. 2. A sudden reduction in muscle length led to a rapid increase in myoplasmic [Ca2+] to a new level which was maintained as long as muscle length was reduced and which was reversed when the muscle was stretched back to the control length. The rate of increase of [Ca2+] when the muscle length was reduced was greater than the rate of decrease in [Ca2+] when the muscle was stretched. 3. Increasing the concentration of EGTA in the activating solution, so as to increase its Ca2+-buffering capacity, eliminated the changes in myoplasmic [Ca2+] in response to a length change but had little effect on developed tension. 4. On stretching the muscle there was a slow component of recovery of tension with a time course broadly similar to the rate of decrease of myoplasmic [Ca2+]. The time course of tension redevelopment and of the accompanying reduction in myoplasmic [Ca2+] both decreased to a similar extent when the [Ca2+] used to activate the muscle was increased. 5. Step reductions of length of increasing amplitude caused increases in myoplasmic [Ca2+] which were larger in proportion to the size of the step. 6. Step reductions of length of equal size but from different starting lengths caused changes in myoplasmic [Ca2+] the amplitude of which correlated with the change in tension rather than the change in length. 7. The increase in myoplasmic [Ca2+] when muscle length is reduced suggests that Ca2+ is released from a site in the muscle, probably troponin C. The time course and magnitude of the changes in myoplasmic [Ca2+] correlate more closely with the changes in developed tension than muscle length.

In preparations of isolated myofibrils from rabbit white skeletal muscle, strands could be seen attached to the myofibrils at the level of the Z-band. The membranous nature of these strands was suggested by their appearance and was confirmed by their removal with the detergent, Triton X-100. The membrane appeared to be connected directly with the Z-band, with no intervening filaments or cables. It is suggested that these direct connections serve to anchor the sarcoplasmic reticulum to the myofibrils.

A simple electronic circuit is described that produces a ramp voltage, the amplitude of which is proportional to the duration of the action potential. The circuit is ideal for monitoring changes in the duration of the action potential on slowly moving chart paper.

To investigate the extent to which the properties of the cardiac myofibrils contribute to the length-force relation of cardiac muscle, we determined the sarcomere length-force relations for rat ventricular trabeculae both before and after the muscles were skinned with the detergent Triton X-100. Sarcomere length was measured continuously by laser diffraction. In the unskinned trabeculae stimulated at 0.2 Hz, the relation between active force and sarcomere length at an extracellular calcium concentration of 1.5 mM was curved away from the sarcomere length axis, with zero force at sarcomere length of 1.5-1.6 micron. At 0.3 mM calcium, the sarcomere length-force relation was curved toward the sarcomere length axis. Chemical skinning of the muscle with 1% Triton X-100 in a "relaxing solution" caused an increase in intensity and decrease in dispersion of the first order diffraction beam, indicating an increased uniformity of sarcomere length in the relaxed muscle. During calcium-regulated contractures in the skinned muscles, the central sarcomeres shortened by up to 20%. As the calcium concentration was increased over the range 1-50 microM, the relation between steady calcium-regulated force and sarcomere length shifted to higher force values and changed in shape in a manner similar to that observed for changes in extracellular calcium concentration before skinning. The sarcomere length-force relations for the intact muscles at an extracellular calcium concentration of 1.5 mM were similar to the curves at calcium concentration of 8.9 microM in the skinned preparations, whereas the curves at an extracellular calcium concentration of 0.3 mM in intact muscles fell between the relations at calcium concentrations of 2.7 and 4.3 microM in the skinned preparations. A factor contributing to the shape of the curves in the skinned muscle at submaximal calcium concentrations was that the calcium sensitivity of force production increased with increasing sarcomere length. The calcium concentration required for 50% activation decreased from 7.71 +/- 0.52 microM to 3.77 +/- 0.33 microM for an increase of sarcomere length from 1.75 to 2.15 micron. The slope of the force-calcium concentration relation increased from 2.82 to 4.54 with sarcomere length between 1.75 and 2.15 micron. This change in calcium sensitivity was seen over the entire range of sarcomere lengths corresponding to the ascending limb of the cardiac length-force relation. It is concluded that the properties of the cardiac contractile machinery (including the length-dependence of calcium sensitivity) can account for much of the shape of the ascending limb in intact cardiac muscle.