Changes In Myofilament Ca2 Research Articles

Depressed myocardial cross-bridge cycling kinetics in a female guinea pig model of diastolic heart failure.

Cardiac hypertrophy is associated with diastolic heart failure (DHF), a syndrome in which systolic function is preserved but cardiac filling dynamics are depressed. The molecular mechanisms underlying DHF and the potential role of altered cross-bridge cycling are poorly understood. Accordingly, chronic pressure overload was induced by surgically banding the thoracic ascending aorta (AOB) in ∼400 g female Dunkin Hartley guinea pigs (AOB); Sham-operated age-matched animals served as controls. Guinea pigs were chosen to avoid the confounding impacts of altered myosin heavy chain (MHC) isoform expression seen in other small rodent models. In vivo cardiac function was assessed by echocardiography; cardiac hypertrophy was confirmed by morphometric analysis. AOB resulted in left ventricle (LV) hypertrophy and compromised diastolic function with normal systolic function. Biochemical analysis revealed exclusive expression of β-MHC isoform in both sham control and AOB LVs. Myofilament function was assessed in skinned multicellular preparations, skinned single myocyte fragments, and single myofibrils prepared from frozen (liquid N2) LVs. The rates of force-dependent ATP consumption (tension-cost) and force redevelopment (Ktr), as well as myofibril relaxation time (Timelin) were significantly blunted in AOB, indicating reduced cross-bridge cycling kinetics. Maximum Ca2+ activated force development was significantly reduced in AOB myocytes, while no change in myofilament Ca2+ sensitivity was observed. Our results indicate blunted cross-bridge cycle in a β-MHC small animal DHF model. Reduced cross-bridge cycling kinetics may contribute, at least in part, to the development of DHF in larger mammals, including humans.

Mutation location of HCM-causing troponin T mutations defines the degree of myofilament dysfunction in human cardiomyocytes

BackgroundThe clinical outcome of hypertrophic cardiomyopathy patients is not only determined by the disease-causing mutation but influenced by a variety of disease modifiers. Here, we defined the role of the mutation location and the mutant protein dose of the troponin T mutations I79N, R94C and R278C. Methods and resultsWe determined myofilament function after troponin exchange in permeabilized single human cardiomyocytes as well as in cardiac patient samples harboring the R278C mutation. Notably, we found that a small dose of mutant protein is sufficient for the maximal effect on myofilament Ca2+-sensitivity for the I79N and R94C mutation while the mutation location determines the magnitude of this effect. While incorporation of I79N and R94C increased myofilament Ca2+-sensitivity, incorporation of R278C increased Ca2+-sensitivity at low and intermediate dose, while it decreased Ca2+-sensitivity at high dose. All three cTnT mutants showed reduced thin filament binding affinity, which coincided with a relatively low maximal exchange (50.5 ± 5.2%) of mutant troponin complex in cardiomyocytes. In accordance, 32.2 ± 4.0% mutant R278C was found in two patient samples which showed 50.0 ± 3.7% mutant mRNA. In accordance with studies that showed clinical variability in patients with the exact same mutation, we observed variability on the functional single cell level in patients with the R278C mutation. These differences in myofilament properties could not be explained by differences in the amount of mutant protein. ConclusionsUsing troponin exchange in single human cardiomyocytes, we show that TNNT2 mutation-induced changes in myofilament Ca2+-sensitivity depend on mutation location, while all mutants show reduced thin filament binding affinity. The specific mutation-effect observed for R278C could not be translated to myofilament function of cardiomyocytes from patients, and is most likely explained by other (post)-translational troponin modifications. Overall, our studies illustrate that mutation location underlies variability in myofilament Ca2+-sensitivity, while only the R278C mutation shows a highly dose-dependent effect on myofilament function.

Effect of Truncated Mutations in the Titin Gene on Cardiac Function

Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and impaired systolic function, where up to 40% of cases are associated with mutations. Truncating mutations in the TTN gene are the most common cause of familial DCM. While it is known that titin is the major contributor to myocardial stiffness and is an essential component of the Frank-Starling mechanism of the heart, its function is not fully established. Titin mechanical properties are particularly important in assisting with myofibril viscoelasticity and consequently contribute to the work output characteristic of myocardium. In contrast, changes in myofilament Ca2+-sensitivity and loss of its modulation by phosphorylation are involved in the proposed mechanism for disease-causing mutations in contractile proteins but not necessary in the TTN gene. We studied mechanical properties and isometric contractility in single cardiac myofibrils of DCM patients with and without TTN mutations. We found that all studied DCM mutations in contractile protein genes (TNNC1, TNNI3, and MYH7) including truncations in the titin gene (TTN), result in lower passive stiffness compared to donor samples. Furthermore we showed that the myofilament Ca2+-sensitivity was higher than donor and without any difference in myofibril kinetics. In fact, heart function is highly coordinated and strongly dependent on spatiotemporal control during the cardiac cycle so data obtained in isometric conditions may be inadequate to understand the effect of mutations. To further characterize mutations and the impact of myofibril length changes on cardiac output, we now use cyclic length and Ca2+ concentration changes to imitate the changes in single myofibril similar to those that have place during the cardiac work-producing cycle.

There is a Limit to the Changes in Myofilament Ca2+-Sensitivity due to Myopathies

We have investigated Ca2+-sensitivity changes in cardiac and skeletal muscle due to mutations or phosphorylation of troponin I (TnI) using the in vitro motility assay.In normal heart, TnI phosphorylation alters myofibrillar Ca2+-sensitivity and increases the speed of Ca2+-dissociation (lusitropic effect). When end-stage heart failure samples with a reduced level of phosphorylation were looked at, the average Ca2+-sensitivity change was x2.6±0.24.Eight dilated cardiomyopathy (DCM) mutations have been looked at in all components of the thin filament (Actin E361G, TnT R141W and ΔK210, TnI K36Q, TnC G159D and Tm E40K, E54K and D230N). Although they all have differing effects on Ca2+-sensitivity (some increased it and some decreased it) the average Ca2+-sensitivity change was x2.0±0.16.The actin E99K hypertrophic cardiomyopathy (HCM) mutation causes an increase in Ca2+-sensitivity of x2.5 but a literature search looking at 21 other HCM mutations in every sarcomeric protein has found an average Ca2+-sensitivity change of x1.8±0.13.A wide range of skeletal myopathies were looked at which either caused a gain of function (TPM2 ΔK49, ΔE139, ACTA1 K326N and TPM3 ΔK7) or loss of function (TPM2 E117K, R91P and TPM3 A4V, L100M and R167C). The mean changes of Ca2+-sensitivity were x1.8±0.3 and x2.7±0.4 respectively.It is remarkable that mutations causing a range of different striated muscle myopathies have such a similar and limited effect on myofilament Ca2+-sensitivity and that phosphorylation of troponin I regulates Ca2+-sensitivity over the same range. We have found in DCM and HCM that Ca2+-sensitivity is uncoupled from the level of TnI phosphorylation and tends to the Ca2+-sensitivity characteristic of phosphorylated wild-type muscle at all phosphorylation levels. It is tempting to speculate that the 2-fold change represents a transition between two activation states of the thin filament.

Testosterone regulates cardiac contractile activation by modulating SERCA but not NCX activity

Alterations in intracellular Ca(2+) transients of cardiomyocytes in orchidectomized (ORX) rats could be a cause of cardiac dysfunction in the hypogonadal condition. To investigate the role of male sex hormones in intracellular Ca(2+) homeostasis during relaxation, Ca(2+)-handling activities by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and the Na(+)/Ca(2+) exchanger (NCX) were evaluated in the ventricular muscle of 10-wk-old ORX rats with and without testosterone supplementation (2.5 mg/kg testosterone propionate, 2 times/wk). ORX induced a 50% decrease in contraction force accompanied by a prolonged time to achieve 50% relaxation (T(50)) in isolated intact ventricular trabeculae, which was partially corrected by testosterone administration. Maximum active tension was also suppressed in ORX rats without changes in myofilament Ca(2+) sensitivity and passive stiffness of the heart. Using a sarcoplasmic reticulum-enriched membrane preparation, the maximum thapsigargin-sensitive SERCA activity of the ORX rat was 27% lower with an increased Ca(2+) sensitivity, which was prevented by testosterone treatment. However, neither changes in SERCA content nor its modulating components, sarcolipin and heat shock protein 20, were detected in the ORX rat, but there was a significant decrease in the phosphorylated Thr(17) form of phospholamban. Despite a lower level of NCX protein in the heart of ORX rats, prolonged T(50) disappeared after an incubation with thapsigargin (10 μM), implying a lack of effect of male sex hormone deficiency on NCX function. These findings indicate that male sex hormones can regulate cardiac relaxation by acting mainly through SERCA. However, a detailed mechanism of SERCA modulation under male sex hormone deficiency status remains to be explored.

Abstract 18131: Increased Cardiomyocyte Stiffness in Cardio-Renal Disease

Cardio-renal disease has been associated with diastolic rather than systolic cardiac dysfunction. As diastolic dysfunction cannot be attributed to adverse remodelling, the present study investigated if intrinsic cardiomyocyte stiffness is a determinant of diastolic dysfunction in cardio-renal disease. Thereto, cardiomyocyte function was studied in rats with cardio-renal disease, which was induced by myocardial infarction in rats with chronic renal dysfunction (Munich Wistar Fromter; RD+CD). This group was compared to rats with solely cardiac disease (CD; age-matched Wistar rats), solely renal disease (RD) and healthy rats (Ctrl). Twelve weeks after surgery force was measured in left ventricular Triton-permeabilized cardiomyocytes at different calcium concentrations to assess myofilament function. Maximal force did not differ among groups. In contrast, myocardial infarction significantly increased passive force (CD vs Ctrl; figure), which was even more pronounced in rats with renal dysfunction (RD+CD vs RD; figure). No change in myofilament Ca 2+ -sensitivity was observed in the CD group, whereas Ca 2+ -sensitivity of force was significantly reduced in RD+CD compared to Ctrl. The changes in passive force and Ca 2+ -sensitivity could not be corrected by protein kinase A, which mimics beta-adrenergic stimulation. In contrast, a 2-times increase in expression and activity of protein kinase C (PKC) alpha was found in RD+CD myocardium (Figure). In conclusion, diastolic dysfunction in cardio-renal disease may be partly attributed to increased intrinsic cardiomyocyte stiffness, rather than to adverse remodelling. The increase in cardiomyocyte stiffness may result from increase PKCalpha-mediated phosphorylation of titin, and could represent a target for treatment of diastolic dysfunction in cardio-renal disease.

Increased myocardial stiffness with maintenance of length-dependent calcium activation by female sex hormones in diabetic rats

A decrease in peak early diastolic filling velocity in postmenopausal women implies a sex hormone-related diastolic dysfunction. The regulatory effect of female sex hormones on cardiac distensibility therefore was evaluated in ovariectomized rats by determining the sarcomere length-passive tension relationship of ventricular skinned fiber preparations. Diabetes also was induced in the rat to assess the protective significance of female sex hormones on diastolic function. While ovariectomy had no effect on myocardial stiffness, collagen content, or titin ratio, a significant increase in myocardial stiffness was observed in diabetic rat only when female sex hormones were intact. The increased stiffness in diabetic-sham rats was accompanied by an elevated collagen content resulting from increases in the levels of procollagen and Smad2. Surprisingly, the increased myocardial stiffness in diabetic-sham rats was accompanied by a shift toward a more compliant N2BA of cardiac titin isoforms. The pCa-active tension relationship was analyzed at fixed sarcomere lengths of 2.0 and 2.3 μm to determine the magnitude of changes in myofilament Ca(2+) sensitivity between the two sarcomere lengths. Interestingly, high expression of N2BA titin was associated with a suppressed magnitude of changes in myofilament Ca(2+) sensitivity only in the diabetic-ovariectomized condition. Estrogen supplementation in diabetic-ovariectomized rats partially increased myocardial stiffness but completely reversed the change in myofilament Ca(2+) sensitivity. These results indicate a restrictive adaptation of myocardium governed by female sex hormones to maintain myofilament activity in compensation to the pathophysiological induction of cardiac dilatation by the diabetic condition.

Cardiac-Specific Helical Structure in Troponin I Imparts Unique, Cardiac-Specific Contractile Function

A coiled-coil between helical motifs of cardiac troponin I (cTnI) and cardiac troponin T (termed cH2(I) and cH2(T), respectively) forms a centrally positioned interface within the core domain of cardiac troponin. The centralized position and cardiac-specific structure of cH2(I) suggest an important, cardiac-specific regulatory role for this region of troponin I. However, whether the cardiac-specific structure of cH2(I) imparts unique, cardiac-specific function to cardiac myofilaments is not currently known. To better understand the functional significance of the structure of cH2(I), we studied the effects of replacing native troponin in detergent-skinned rat cardiac muscle fiber bundles with complexes containing chimeric cTnI in which cH2(I) was replaced with the corresponding helix from rat slow skeletal (ssH2(I)) or fast skeletal (fsH2(I)) troponin I. Ca2+- and length-mediated myofilament force, ATPase activity, and length-dependent contractile dynamics were estimated at different levels of activation in muscle fibers reconstituted with wild-type or chimeric cTnI. Our preliminary work demonstrates that fiber bundles containing chimeric cTnI produced the same maximal force and ATPase activity, but exhibited significant differences in myofilament responsiveness to Ca2+ and changes in muscle length, when compared to bundles reconstituted with wild-type cTnI. Fiber bundles containing chimeric cTnI exhibited altered cooperativity and sensitivity of force production to Ca2+. For example, myofilament cooperativity was lower and the sarcomere length-dependent change in myofilament Ca2+ sensitivity was less pronounced in fiber bundles reconstituted with ssH2(I) chimeric cTnI. Furthermore, length-dependent recruitment dynamics and the nonlinear effect by which crossbridge distortion influences crossbridge recruitment were both blunted in fiber bundles containing chimeric cTnI. These results indicate that replacing cH2(I) with either ssH2(I) or fsH2(I) results in aberrant cardiac contractile function, and suggest that the structure of cH2(I) is important to impart unique, cardiac-specific function in cardiac myofilaments.

Abstract 1478: AMP-Activated Protein Kinase Phosphorylates Cardiac Troponin I and Alters Contractility in Murine Ventricular Myocytes

In the heart, AMP-activated protein kinase (AMPK) is critical in the regulation of energy balance and in myocardial signaling, and is activated especially during ischaemia and exercise. It is a αβγ heterotrimer, with α being the catalytic subunit. Mutations affecting the regulatory γ2 subunit have been shown to cause a cardiac phenotype of hypertrophy and conduction disease, thus suggesting a specific role for this subunit in the heart. The γ isoforms are highly conserved at their C-termini but very different at their N-termini; hence we have investigated whether the unique N-terminus of γ2 could be involved in conferring substrate specificity or in determining intracellular localization. We have undertaken a GAL4 -based yeast two-hybrid assay to screen a human heart cDNA library using the N-terminal 273 residues of γ2 as bait. Five proteins were identified as true interactors in the yeast assay, one of these being cardiac troponin I (cTnI). In vitro studies showed that cTnI (isolated or reconstituted as troponin complex) is a good substrate for AMPK. Studies using site-specific cTnI mutants and mass spectrometry identified Ser-150 as the principal residue phosphorylated by AMPK. Actomyosin ATPase assays showed that phosphorylation at this residue resulted in increased Ca 2+ sensitivity of contractile regulation (ΔpCa 50 =+0.07). Treatment of cardiomyocytes with the AMPK activator AICAR for 15 min resulted in prolonged relaxation (time to 50% relaxation, TR50) in field stimulated left ventricular myocytes of C57B/6 mice (TR50 in ms: 29.8±1.4 in control and 38.0±2.4 with AICAR, n=11, 3 Hz and 35 o C). There was a tendency towards an increase in contraction with AICAR. Pretreatment of myocytes with AMPK inhibitor compound C (10 μM) abolished the effects of AICAR on myocytes (TR50 in ms: 30.9±1.7 before AICAR and 31.5±2.6 after AICAR, n=9). The effect of AICAR was mediated without altering the amplitude of the Ca 2+ transient, suggesting that it may be caused by a change in myofilament Ca 2+ -sensitivity and consistent with AMPK phosphorylation of cTnI. We hypothesize that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.

Characterization and mechanism of P2X receptor-mediated increase in cardiac myocyte contractility

Cardiac P2X purinergic receptors can mediate an increase in myocyte contractility and a potentially important role in the heart. The P2X(4) receptor (P2X(4)R) is an important subunit of native cardiac P2X receptors. With transgenic mice with cardiac-specific overexpression of P2X(4)R (Tg) used as a model, the objectives here were to characterize the P2X receptor-mediated cellular contractile and Ca(2+) transient effects and to determine the mechanism underlying the receptor-induced increase in myocyte contractility. In response to the agonist 2-methylthioATP (2-meSATP), Tg myocytes showed an increased intracellular Ca(2+) transient, as defined by fura 2 fluorescence ratio, and an enhanced contraction shortening that were unaccompanied by cAMP accumulation or L-type Ca(2+) channel activation. The increased Ca(2+) transient was not associated with any alteration in action potential duration, resting membrane potential, or diastolic fluorescence ratio or rates of rise and decline of the Ca(2+) transient. Simultaneous Ca(2+) transient and contraction measurements did not show any agonist-mediated change in myofilament Ca(2+) sensitivity. However, activation of the overexpressed P2X(4) receptor caused an enhanced SR Ca(2+) loading, as evidenced by a 2-meSATP-evoked increase in the caffeine-induced inward current and Ca(2+) transient. Similar data were obtained in wild-type mouse ventricular myocytes. Thus an increased SR Ca(2+) content, occurring in the absence of cAMP accumulation or L-type Ca(2+) channel activation, is the principal mechanism by which cardiac P2X receptor mediates a stimulatory effect on cardiac myocyte contractility.

Coupling a change in intraluminal pressure to vascular smooth muscle depolarization: still stretching for an explanation

pressure-induced (myogenic) constriction of arterioles is believed to be a fundamental contributor to the local regulation of microvascular blood flow and pressure. Myogenic constriction provides a level of tone upon which vasodilators can act to lower resistance and match blood flow to metabolic

Targeted Disruption of PKA Binding to AKAPs Increases Cardiac Contractility and Decreases PKA-Dependent Phosphorylation, In Vivo

Beta-adrenergic receptor (ß-AR) stimulation signals through PKA-dependent phosphorylation to enhance contractility and increase rate of relaxation of the heart. PKA signaling is conferred in part by PKA binding to A-kinase anchoring proteins (AKAPs). AKAPs are targeted to specific intracellular locations, resulting in localization of PKA with its target substrates. Substrate phosphorylation leads to changes in myofilament Ca2+ sensitivity and availability. Thus agents that modify PKA signaling would be expected to mediate an altered inotropic response.

Chronic hypoxia modulates relations among calcium, myosin light chain phosphorylation, and force differently in fetal and adult ovine basilar arteries

The present study tests the hypothesis that age-related differences in contractility of cerebral arteries from hypoxic animals involve changes in myofilament Ca2+ sensitivity. Basilar arteries from term fetal and nonpregnant adult sheep maintained 110 days at 3,820 m were used in measurements of cytosolic calcium concentration ([Ca2+]i), myosin light chain phosphorylation, and contractile tensions induced by graded concentrations of K+ or serotonin (5-HT). Slopes relating [Ca2+]i to tension were similar in fetal (0.83 +/- 0.07) and adult (1.02 +/- 0.08) arteries for K+ contractions but were significantly greater for fetal (3.77 +/- 0.64) than adult (2.00 +/- 0.13) arteries for 5-HT contractions. For both K+ and 5-HT contractions, these relations were left shifted in fetal compared with adult arteries, indicating greater Ca2+ sensitivity in fetal arteries. In contrast, slopes relating [Ca2+]i and %myosin phosphorylation for K+ contractions were less in fetal (0.37 +/- 0.08) than adult (0.81 +/- 0.07) arteries, and the fetal curves were right shifted. For 5-HT contractions, the slope of the Ca2+-phosphorylation relation was similar in fetal (0.33 +/- 0.09) and adult (0.33 +/- 0.23) arteries, indicating that 5-HT depressed Ca2+-induced myosin phosphorylation in adult arteries. For slopes relating %myosin phosphorylation and tension, fetal values (K+: 1.52 +/- 0.22, 5-HT: 7.66 +/- 1.70) were less than adult values (K+: 2.13 +/- 0.30, 5-HT: 8.29 +/- 2.40) for both K+- and 5-HT-induced contractions, although again fetal curves were left shifted relative to the adult. Thus, in hypoxia-acclimatized basilar arteries, a downregulated ability of Ca2+ to promote myosin phosphorylation is offset by an upregulated ability of phosphorylated myosin to produce force yielding an increased fetal myofilament Ca2+ sensitivity. Postnatal maturation reprioritizes the mechanisms regulating hypoxic contractility through changes in the source of activator Ca2+, the pathways governing myosin light chain phosphorylation, and its interaction with actin.

Ketamine Attenuates Acetylcholine-induced Contraction by Decreasing Myofilament Ca2+Sensitivity in Pulmonary Veins

The authors investigated the extent and cellular mechanisms by which the intravenous anesthetic ketamine alters acetylcholine-induced contraction in pulmonary veins (PVs). They tested the hypothesis that ketamine inhibits acetylcholine contraction in PVs. Canine PV rings with endothelium (E+) and without endothelium (E-) were isolated for measurement of isometric tension. The effects of ketamine (10(-5) m approximately 10(-3) m) on acetylcholine contraction were assessed in E+ and E- rings. The effects of inhibiting nitric oxide synthase on ketamine-induced changes in acetylcholine contraction were investigated in E+ rings, whereas the effects of Ca2+ influx and Ca2+ release were investigated in E- rings. In fura-2 loaded E- PV strips, the effects of ketamine (10(-4) m) on the intracellular Ca2+ concentration-tension relation (i.e., myofilament Ca sensitivity) were assessed in the presence or absence of acetylcholine. The roles of the protein kinase C and rho-kinase signaling pathways in ketamine-induced changes in myofilament Ca2+ sensitivity were also investigated. Ketamine caused dose-dependent (P < 0.001) inhibition of acetylcholine contraction in E+ and E- PV rings. The ketamine-induced attenuation of acetylcholine contraction was still observed after inhibition of nitric oxide synthase (P = 0.002), Ca2+ influx (P < 0.001), and Ca2+ release (P = 0.021). Ketamine alone had no effect on myofilament Ca2+ sensitivity (P = 0.892) but attenuated (P = 0.038) the acetylcholine-induced increase in myofilament Ca2+ sensitivity. This attenuation was still observed after rho-kinase inhibition (P = 0.039), whereas it was abolished by protein kinase C inhibition (P = 0.798). Ketamine attenuates acetylcholine contraction by inhibiting the acetylcholine-induced increase in myofilament Ca2+ sensitivity, which is mediated by the protein kinase C signaling pathway.

Chronic SR Ca2+-ATPase inhibition causes adaptive changes in cellular Ca2+ transport.

Phospholamban, the critical regulator of the cardiac SERCA2a Ca2+ affinity, is phosphorylated at Ser16 and Thr17 during beta-adrenergic stimulation (eg, isoproterenol). To assess the impact of nonphosphorylatable phospholamban, a S16A, T17A double-mutant (DM) was introduced into phospholamban knockout mouse hearts. Transgenic lines expressing DM phospholamban at levels similar to wild types (WT) were identified. In vitro phosphorylation confirmed that DM phospholamban could not be phosphorylated, but produced the same shift in EC50 of SERCA2a for Ca2+ as unphosphorylated WT phospholamban. Rates of basal twitch [Ca2+]i decline were not different in DM versus WT cardiomyocytes. Isoproterenol increased the rates of twitch [Ca2+]i decline in WT, but not DM myocytes, confirming the prominent role of phospholamban phosphorylation in this response. Increased L-type Ca2+ current (ICa) density, with unaltered characteristics, was the major compensation in DM myocytes. Consequently, the normal beta-adrenergic-induced increase in ICa caused larger dynamic changes in absolute ICa density. Isoproterenol increased Ca2+ transients to a comparable amplitude in DM and WT. There were no changes in myofilament Ca2+ sensitivity, or the expression levels and Ca2+ removal activities of other Ca2+-handling proteins. Nor was there evidence of cardiac remodeling up to 10 months of age. Thus, chronic inhibition of SERCA2a by ablation of phospholamban phosphorylation (abolishing its adrenergic regulation) results in a unique cellular adaptation involving greater dynamic ICa modulation. This ICa modulation may partly compensate for the loss in SERCA2a responsiveness and thereby partially normalize beta-adrenergic inotropy in DM phospholamban mice.

Effects of kappa-opioid receptor activation on myocardium.

Kappa-opioid receptor stimulation of the heart transiently increases twitch amplitude and decreases Ca2+-dependent actomyosin Mg2+-ATPase activity through an undetermined mechanism. One purpose of the present study was to determine if the increase in twitch amplitude is due to changes in myofilament Ca2+ sensitivity. We also wanted to determine if kappa-opioid receptor activation alters maximum actin-myosin ATPase activity and Ca2+ sensitivity of tension in a way consistent with protein kinase A or protein kinase C (PKC) action. Rat hearts were treated with U50,488H (a kappa-opioid receptor agonist), phenylephrine plus propranolol (alpha-adrenergic receptor stimulation), isoproterenol (a beta-adrenergic receptor agonist), or phorbol 12-myristate 13-acetate (PMA, receptor independent activator of PKC) or were untreated (control), and myofibrils were isolated. U50,488H, phenylephrine plus propranolol, and PMA all decreased maximum Ca2+-dependent actomyosin Mg2+-ATPase activity, whereas isoproterenol treatment increased maximum Ca2+-dependent actomyosin Mg2+- ATPase activity. Untreated myofibrils exposed to exogenous PKC-epsilon, but not PKC-delta, decreased maximum actomyosin Mg2+-ATPase activity. Langendorff-perfused hearts treated with U50,488H, phenylephrine plus propranolol, or isoproterenol had significantly higher ventricular ATP levels compared with control hearts. PKC inhibitors abolished the effects of U50,488H on Ca2+-dependent actomyosin Mg2+-ATPase activity and myocardial ATP levels. U50,488H and PMA treatment of isolated ventricular myocytes increased Ca2+ sensitivity of isometric tension compared with control myocytes at pH 7.0. The U50,488H-dependent increase in Ca2+ sensitivity of tension was retained at pH 6.6. Together, these findings are consistent with the hypotheses that 1) the positive inotropy associated with kappa-opioid receptor activation may be due in part to a PKC-mediated increase in myofilament Ca2+-sensitivity of tension and 2) the kappa-opioid receptor-PKC pathway is a modulator of myocardial energy status through reduction of actomyosin ATP consumption.

Effects of constitutive overexpression of insulin-like growth factor-1 on the mechanical characteristics and molecular properties of ventricular myocytes.

Recently, insulin-like growth factor-1 (IGF-1) has been claimed to positively influence the cardiac performance of the decompensated heart. On this basis, the effects of constitutive overexpression of IGF-1 on the mechanical behavior of myocytes were examined in transgenic mice in which the cDNA for the human IGF-1B was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene and in nontransgenic littermates at 2.5 months of age, the alterations in Ca2+ sensitivity of tension development, unloaded shortening velocity, and sarcomere compliance were measured in skinned myocytes. The quantities and state of phosphorylation of myofilament proteins in these enzymatically dissociated ventricular myocytes were also examined. The overexpression of IGF-1 was characterized by a nearly 15% reduction in myofilament isometric tension at submaximum Ca2+ levels in the physiological range, whereas developed tension at maximum activation was unchanged. In contrast, unloaded velocity of shortening was increased 39% in myocytes from transgenic mice. Moreover, resting tension in these cells was reduced by 24% to 33%. Myocytes from nontransgenic mice pretreated with IGF-1 failed to reveal changes in myofilament Ca2+ sensitivity and unloaded velocity of shortening. The quantities of C protein, troponin I, and myosin light chain-2 were comparable in transgenic and nontransgenic mice, but their endogenous state of phosphorylation increased 117%, 100%, and 100%, respectively. Troponin T content was not altered, and myosin isozymes were essentially 100% V1 in both groups of mice. In conclusion, constitutive overexpression of IGF-1 may influence positively the performance of myocytes by enhancing shortening velocity and cellular compliance.

Endotoxemia-induced myocardial dysfunction is not associated with changes in myofilament Ca2+ responsiveness.

Myocardial contractile function is depressed after onset of endotoxemia and is intrinsic to the ventricular myocyte. We tested the hypothesis that decreased Ca2+ responsiveness of the contractile myofilaments underlies this inotropic depression. Specifically, we evaluated the relationship between Ca2+ and unloaded cell shortening and isometric tension development of skinned guinea pig ventricular myocytes. Myocytes were isolated 4 h after intraperitoneal injection of 4 mg/kg Escherichia coli lipopolysaccharide (LPS) or saline (control; Ctl). Myofilament Ca2+ responsiveness assessed by image analysis of shortening of skinned myocytes at pH 7.0 was not different between Ctl[pCa value that resulted in half-maximal shortening (pCa50): 5.78 +/- 0.04] and LPS (pCa50: 5.72 +/- 0.02). Similarly, myofilament Ca2+ responsiveness measured by isometric tension of skinned myocytes was not different between Ctl (pCa50: 5.73 +/- 0.02) and LPS (pCa50: 5.76 +/- 0.02). Maximal tension generated by LPS myocytes (2.89 +/- 0.23 g/mm2) was significantly less (P < 0.05) than Ctl (3.75 +/- 0.34 g/mm2). However, when myocytes were isolated and skinned in the presence of protease inhibitors, maximal tension generated by LPS myocytes (3.53 +/- 0.98 g/mm2) was similar to Ctl (3.01 +/- 0.80 g/mm2). We conclude that in vivo administration of LPS resulting in endotoxemia without shock does not alter myofilament Ca2+ responsiveness of ventricular myocytes. Rather, reduced contractility is more likely a result of decreased Ca2+ availability because systolic Ca2+ transients of fura 2-loaded LPS myocytes were significantly decreased (P < 0.05) compared with Ctl myocytes.

Troponin I phosphorylation and myofilament calcium sensitivity during decompensated cardiac hypertrophy.

We have measured myocyte cell shortening, troponin-I (Tn-I) phosphorylation, Ca2+ dependence of actomyosin adenosinetriphosphatase (ATPase) activity, adenosine 3',5'-cyclic monophosphate (cAMP) levels, and myofibrillar isoform expression in the spontaneously hypertensive rat (SHR) during decompensated cardiac hypertrophy (76 wk old) and in age-matched Wistar-Kyoto rat (WKY) controls. The decreased inotropic response to beta-adrenergic stimulation previously observed in myocytes from 26-wk-old SHR was further reduced at 76 wk of age. In response to beta-adrenergic stimulation, Tn-I phosphorylation was greater in the 76-wk-old SHR than in the WKY, although cAMP-dependent protein kinase A (PKA)-dependent Tn-I phosphorylation in the SHR did not increase with progression from compensated (26 wk) to decompensated (76 wk) hypertrophy. We also observed a dissociation between the increased PKA-dependent Tn-I phosphorylation and decreased cAMP levels in the 76-wk-old SHR versus WKY during beta-adrenergic stimulation. Baseline Tn-I phosphorylation was significantly reduced in 76-wk-old SHR versus WKY and was associated with decreased basal cAMP levels and increased Ca2+ sensitivity of actomyosin ATPase activity. The change in myofilament Ca2+ sensitivity during beta-adrenergic stimulation in the 76-wk-old SHR (0.65 pCa units) was over twofold greater than in the 76-wk-old WKY (0.30 pCa units). We also determined whether embryonic troponin T isoforms were reexpressed in decompensated hypertrophy and observed significant reexpression of the embryonic cardiac troponin T isoforms in the 76-wk-old SHR. The significant decrease in Ca2+ sensitivity with beta-adrenergic stimulation in 76-wk-old SHR may contribute to the severely impaired inotropic response during decompensated hypertrophy in the SHR.

Ejection has both positive and negative effects on left ventricular isovolumic relaxation.

In isovolumically beating hearts, the speed of left ventricular (LV) relaxation is uniquely determined by peak active stress (sigma max). In contrast, such a succinct description of relaxation is lacking for the ejection beats, although ejection is generally thought to hasten relaxation. We set out to determine how ejection modifies the relaxation-sigma max relationship obtained in the isovolumically beating hearts. Experiments were performed on five isolated rabbit hearts subjected to various loading conditions. Instantaneous LV pressure and volume were recorded and converted to active stress, from which isovolumic relaxation time (Tr) was defined as the time for stress to fall from 75 to 25% of sigma max (isovolumic beats) or its end-ejection value (ejection beats). Steady-state and transient isovolumic beat and steady-state ejection beat data were used to develop a multiple regression model. This model identified stress, current beat ejection, and previous beat ejection history as independent predictor variables of Tr and fit the data well in all hearts (r2 > 0.98). Furthermore, this model could predict relaxation in transient ejection beats (r2 = 0.30 for all hearts). Whereas the coefficient for the current beat ejection was negative (i.e., negative effect or hastening relaxation), the ejection history coefficient was positive (i.e., positive effect or slowing relaxation). The sum of these two coefficients was negative, corresponding to the commonly observed net negative effect of ejection on relaxation. The expected positive inotropic effect of ejection was also observed. The dissipations of both positive inotropic and relaxation effects were slow, suggesting a nonmechanical underlying mechanism(s). We postulate that these two effects are linked and caused by ejection-mediated changes in myofilament Ca2+ sensitivity.