Cardiac myosin isoforms exhibit differential rates of MgADP release and MgATP binding detected by myocardial viscoelasticity.

We measured myosin crossbridge detachment rate and the rates of MgADP release and MgATP binding in mouse and rat myocardial strips bearing one of the two cardiac myosin heavy chain (MyHC) isoforms. Mice and rats were fed an iodine-deficient, propylthiouracil diet resulting in ~100% expression of ß-MyHC in the ventricles. Ventricles of control animals expressed ~100% α-MyHC. Chemically-skinned myocardial strips prepared from papillary muscle were subjected to sinusoidal length perturbation analysis at maximum calcium activation pCa 4.8 and 17°C. Frequency characteristics of myocardial viscoelasticity were used to calculate crossbridge detachment rate over 0.01 to 5mM [MgATP]. The rate of MgADP release, equivalent to the asymptotic value of crossbridge detachment rate at high MgATP, was highest in mouse α-MyHC (111.4±6.2s(-1)) followed by rat α-MyHC (65.0±7.3s(-1)), mouse ß-MyHC (24.3±1.8s(-1)) and rat ß-MyHC (15.5±0.8s(-1)). The rate of MgATP binding was highest in mouse α-MyHC (325±32 mM(-1) s(-1)) then mouse ß-MyHC (152±23 mM(-1) s(-1)), rat α-MyHC (108±10 mM(-1) s(-1)) and rat ß-MyHC (55±6 mM(-1) s(-1)). Because the events of MgADP release and MgATP binding occur in a post power-stroke state of the myosin crossbridge, we infer that MgATP release and MgATP binding must be regulated by isoform- and species-specific structural differences located outside the nucleotide binding pocket, which is identical in sequence for these four myosins. We postulate that differences in the stiffness profile of the entire myosin molecule, including the thick filament and the myosin-actin interface, are primarily responsible for determining the strain on the nucleotide binding pocket and the subsequent differences in the rates of nucleotide release and binding observed among the four myosins examined here.

Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain.

Cardiac myosin binding protein-C (cMyBP-C) has 11 immunoglobulin or fibronectin-like domains, C0 through C10, which bind sarcomeric proteins, including titin, myosin and actin. Using bacterial expressed mouse N-terminal fragments (C0 through C3) in an in vitro motility assay of myosin-generated actin movement and the laser trap assay to assess single molecule actin-binding capacity, we determined that the first N-terminal 17 amino acids of the cMyBP-C motif (the linker between C1 and C2) contain a strong, stereospecific actin-binding site that depends on positive charge due to a cluster of arginines. Phosphorylation of 4 serines within the motif decreases the fragments' actin-binding capacity and actomyosin inhibition. Using the laser trap assay, we observed individual cMyBP-C fragments transiently binding to a single actin filament with both short (~20 ms) and long (~300 ms) attached lifetimes, similar to that of a known actin-binding protein, α-actinin. These experiments suggest that cMyBP-C N-terminal domains containing the cMyBP-C motif tether actin filaments and provide one mechanism by which cMyBP-C modulates actomyosin motion generation, i.e. by imposing an effective viscous load within the sarcomere.

Erythropoietin induces positive inotropic and lusitropic effects in murine and human myocardium.

Initial clinical studies indicate a potential beneficial effect of erythropoietin (EPO) in patients with anemia and heart failure. Here, we investigate the direct contractile effects of erythropoietin on myocardial tissue. Treatment with EPO (50U/mL) using excitable murine and human left ventricular muscle preparations resulted in a 37% and 62% increase in twitch tension, respectively (P<0.05). Isolated murine cardiomyocytes exposed to EPO demonstrated a 41% increase in peak sarcomere shortening (P=0.012). Using compounds that specifically stimulate a non-erythropoietic EPO receptor yielded similar increases in contractile dynamics. Cardiomyocyte Ca(2+)dynamics showed an 18% increase in peak calcium in EPO treated cardiomyocytes over controls (P=0.03). Studies in muscle strips skinned after EPO treatment demonstrated a phosphorylation dependant increase in the viscous modulus as well as an increase in oscillatory work. The EPO mediated increase in peak sarcomere shortening was abrogated by PI3-K blockade via wortmannin and by non-isozyme specific PKC blockade by chelerythrine. Finally, EPO treatment resulted in an increase in PKCε in the particulate cellular fraction, indicating activation of this isoform. EPO exhibits direct positive inotropic and lusitropic effects in cardiomyocytes and ventricular muscle preparation. These effects are mediated through PI3-K and PKCε isoform signaling to directly affect both calcium release dynamics and myofilament function.

Resistance training alters skeletal muscle structure and function in human heart failure: effects at the tissue, cellular and molecular levels.

Reduced skeletal muscle function in heart failure (HF) patients may be partially explained by altered myofilament protein content and function. Resistance training increases muscle function, although whether these improvements are achieved by correction of myofilament deficits is not known. To address this question, we examined 10 HF patients and 14 controls prior to and following an 18 week high-intensity resistance training programme. Evaluations of whole muscle size and strength, single muscle fibre size, ultrastructure and tension and myosin-actin cross-bridge mechanics and kinetics were performed. Training improved whole muscle isometric torque in both groups, although there were no alterations in whole muscle size or single fibre cross-sectional area or isometric tension.Unexpectedly, training reduced the myofibril fractional area of muscle fibres in both groups. This structural change manifested functionally as a reduction in the number of strongly bound myosin-actin cross-bridges during Ca²âº activation. When post-training single fibre tension data were corrected for the loss of myofibril fractional area, we observed an increase in tension with resistance training. Additionally, training corrected alterations in cross-bridge kinetics (e.g. myosin attachment time) in HF patients back to levels observed in untrained controls. Collectively, our results indicate that improvements in myofilament function in sedentary elderly with and without HF may contribute to increased whole muscle function with resistance training. More broadly, these data highlight novel cellular and molecular adaptations in muscle structure and function that contribute to the resistance-trained phenotype.

Zinc-induced cardiomyocyte relaxation in a rat model of hyperglycemia is independent of myosin isoform.

It has been reported previously that diabetic cardiomyopathy can be inhibited or reverted with chronic zinc supplementation. In the current study, we hypothesized that total cardiac calcium and zinc content is altered in early onset diabetes mellitus characterized in part as hyperglycemia (HG) and that exposure of zinc ion (Zn2+) to isolated cardiomyocytes would enhance contraction-relaxation function in HG more so than in nonHG controls. To better control for differential cardiac myosin isoform expression as occurs in rodents after ß-islet cell necrosis, hypothyroidism was induced in 16 rats resulting in 100% ß-myosin heavy chain expression in the heart. ß-Islet cell necrosis was induced in half of the rats by streptozocin administration. After 6 wks of HG, both HG and nonHG controls rats demonstrated similar myofilament performance measured as thin filament calcium sensitivity, native thin filament velocity in the myosin motility assay and contractile velocity and power. Extracellular Zn2+ reduced cardiomyocyte contractile function in both groups, but enhanced relaxation function significantly in the HG group compared to controls. Most notably, a reduction in diastolic sarcomere length with increasing pacing frequencies, i.e., incomplete relaxation, was more pronounced in the HG compared to controls, but was normalized with extracellular Zn2+ application. This is a novel finding implicating that the detrimental effect of HG on cardiomyocyte Ca2+ regulation can be amelioration by Zn2+. Among the many post-translational modifications examined, only phosphorylation of ryanodine receptor (RyR) at S-2808 was significantly higher in HG compared to nonHG. We did not find in our hypothyroid rats any differentiating effects of HG on myofibrillar protein phosphorylation, lysine acetylation, O-linked N-acetylglucosamine and advanced glycated end-products, which are often implicated as complicating factors in cardiac performance due to HG. Our results suggest that the relaxing effects of Zn2+ on cardiomyocyte function are more pronounced in the HG state due an insulin-dependent effect of enhancing removal of cytosolic Ca2+ via SERCA2a or NCX or by reducing Ca2+ influx via L-type channel or Ca2+ leak through the RyR. Investigations into the effects of Zn2+ on these mechanisms are now underway.

Blood gene expression signatures associate with heart failure outcomes.

Gene expression signatures in blood correlate with specific diseases. Such signatures may serve as valuable diagnostic and prognostic tools in disease management. Blood gene expression signatures associated with heart failure may be applied to predict prognosis, monitor disease progression, and optimize treatment. Blood gene expression profiles were generated for 71 subjects with heart failure and 15 controls without heart failure, using the Affymetrix GeneChip U133Plus2.0. Survival analysis identified 197 "mortality genes" that were significantly associated with patient outcome. Functional categorization showed that genes associated with T cell receptor signaling were most significantly overpresented. Cluster analysis of these T cell receptor signaling genes significantly categorized heart failure patients into three risk groups (P = 0.031) that were distinct from the three risk groups categorized by New York Heart Association (NYHA) Classification (P = 0.0002). By combining the analysis of clinical assessment (NYHA class) with T cell receptor signaling gene expression, we proposed a model that demonstrated an even greater differentiation of patients at risk (P = 0.0001). In this discovery study, we identified blood expression signatures associated with heart failure patient outcomes. Characterization of these mortality genes helped identify a set of T cell receptor signaling genes that may be of utility in predicting survival of heart failure patients. These data raise the possibility of prospectively risk stratifying patients with heart failure by integrating blood gene expression signatures with current clinical assessment.

Ventricular resynchronization by implementation of direct his bundle pacing in a patient with congenital complete AV block and newly diagnosed cardiomyopathy.

Congenital complete atrioventricular block (CCAVB) is usually due to failure of AV nodal conduction with preservation of the His Purkinje system, typically present at birth. While most patients with CCAVB ultimately require pacemaker therapy to restore physiologic heart rates, recent studies have suggested that chronic right ventricular (RV) pacing in patients with CCAVB can have detrimental effects on cardiac structure and function, and may account for a 7-10% incidence of congestive heart failure in these patients. Since the His Purkinje system is preserved in CCAVB, this patient population could be uniquely well served by direct His bundle pacing (DHBP) which would be expected to restore physiologic activation of both ventricles. We present a case of a young woman who presented with RV pacing-induced cardiomyopathy who responded dramatically to DHBP.

Human actin mutations associated with hypertrophic and dilated cardiomyopathies demonstrate distinct thin filament regulatory properties in vitro.

Two cardiomyopathic mutations were expressed in human cardiac actin, using a Baculovirus/insect cell system; E99K is associated with hypertrophic cardiomyopathy whereas R312H is associated with dilated cardiomyopathy. The hypothesis that the divergent phenotypes of these two cardiomyopathies are associated with fundamental differences in the molecular mechanics and thin filament regulation of the underlying actin mutation was tested using the in vitro motility and laser trap assays. In the presence of troponin (Tn) and tropomyosin (Tm), beta-cardiac myosin moved both E99K and R312H thin filaments at significantly (p<0.05) slower velocities than wild type (WT) at maximal Ca(++). At submaximal Ca(++), R312H thin filaments demonstrated significantly increased Ca(++) sensitivity (pCa(50)) when compared to WT. Velocity as a function of ATP concentration revealed similar ATP binding rates but slowed ADP release rates for the two actin mutants compared to WT. Single molecule laser trap experiments performed using both unregulated (i.e. actin) and regulated thin filaments in the absence of Ca(++) revealed that neither actin mutation significantly affected the myosin's unitary step size (d) or duration of strong actin binding (t(on)) at 20 microM ATP. However, the frequency of individual strong-binding events in the presence of Tn and Tm, was significantly lower for E99K than WT at comparable myosin surface concentrations. The cooperativity of a second myosin head binding to the thin filament was also impaired by E99K. In conclusion, E99K inhibits the activation of the thin filament by myosin strong-binding whereas R312H demonstrates enhanced calcium activation.

Chronic heart failure decreases cross-bridge kinetics in single skeletal muscle fibres from humans.

Skeletal muscle function is impaired in heart failure patients due, in part, to loss of myofibrillar protein content, in particular myosin. In the present study, we utilized small-amplitude sinusoidal analysis for the first time in single human skeletal muscle fibres to measure muscle mechanics, including cross-bridge kinetics, to determine if heart failure further impairs contractile performance by altering myofibrillar protein function. Patients with chronic heart failure (n = 9) and controls (n = 6) were recruited of similar age and physical activity to diminish the potentially confounding effects of ageing and muscle disuse. Patients showed decreased cross-bridge kinetics in myosin heavy chain (MHC) I and IIA fibres, partially due to increased myosin attachment time (t(on)). The increased t(on) compensated for myosin protein loss previously found in heart failure patients by increasing the fraction of the total cycle time myosin is bound to actin, resulting in a similar number of strongly bound cross-bridges in patients and controls. Accordingly, isometric tension did not differ between patients and controls in MHC I or IIA fibres. Patients also had decreased calcium sensitivity in MHC IIA fibres and alterations in the viscoelastic properties of the lattice structure of MHC I and IIA fibres. Collectively, these results show that heart failure alters skeletal muscle contraction at the level of the myosin-actin cross-bridge, leading to changes in muscle mechanics which could contribute to impaired muscle function. Additionally, we uncovered a unique kinetic property of MHC I fibres, a potential indication of two distinct populations of cross-bridges, which may have important physiological consequences.

Combined immunoelectron microscopic and computer-assisted image analyses to detect advanced glycation end-products in human myocardium.

Advanced glycation end-products (AGEs) result from oxidation-reduction reactions that ensue when a sugar becomes adducted to a protein. AGEs cause various complications of diabetes mellitus (DM). Experimental and clinical evidence suggest that AGEs also contribute to the complications of hypertension (HTN). Little is known about the abundance and localization of AGEs in human myocardium. In a few light microscopic studies, the AGE carboxymethyl lysine (CML) has been immunolabeled and localized virtually exclusively to the walls of small arteries. To more precisely delineate the abundance and localization of CML, we developed an immunoelectron microscopic (IEM) detection method using anti-CML monoclonal antibody 6D12 in conjunction with computer-assisted image analysis. Antibody was pre-absorbed with purified AGE-bovine serum albumin to assure specificity. Antigen-antibody (ag-ab) complexes were individually identified with protein A-conjugated colloidal gold and counted with an automated system. We applied this method in 21 patients (pts) undergoing epicardial biopsy during coronary bypass grafting (CBG) [20 M, 1 F; mean age 65 +/- 7.4 (+/- SEM) years]. Seven pts had neither DM nor HTN, seven had HTN, and seven had DM + HTN. In contrast to the prior light microscopic studies, we detected CML scattered throughout the cardiomyocyte in all pts, but in widely varying amounts. Ag-ab complexes were abundant in sections through myofilaments (mean count 23.6 +/- 9.2 per microm(2), range 9.4-48) and even more so in mitochondria (mean count 34.4 +/- 11.9 per microm(2), range 14.1-68.2, P < 0.001 vs. myofilaments). CML was also detected in vascular endothelial cells. There were no statistically significant differences based on presence or absence of HTN or DM. In conclusion, our IEM method is the first to provide detailed delineation of the localization and abundance of CML in myocardium. CML is very prevalent in CBG pts, suggesting that AGEs could play a role in abnormal cardiomyocyte function, including altered energy metabolism.

Cardiac myosin binding protein-C modulates actomyosin binding and kinetics in the in vitro motility assay.

The modulatory role of whole cardiac myosin binding protein-C (cMyBP-C) on myosin force and motion generation was assessed in an in vitro motility assay. The presence of cMyBP-C at an approximate molar ratio of cMyBP-C to whole myosin of 1:2, resulted in a 25% reduction in thin filament velocity (P<0.002) with no effect on relative isometric force under maximally activated conditions (pCa 5). Cardiac MyBP-C was capable of inhibiting actin filament velocity in a concentration-dependent manner using either whole myosin, HMM or S1, indicating that the cMyBP-C does not have to bind to myosin LMM or S2 subdomains to exert its effect. The reduction in velocity by cMyBP-C was independent of changes in ionic strength or excess inorganic phosphate. Co-sedimentation experiments demonstrated S1 binding to actin is reduced as a function of cMyBP-C concentration in the presence of ATP. In contrast, S1 avidly bound to actin in the absence of ATP and limited cMyBP-C binding, indicating that cMyBP-C and S1 compete for actin binding in an ATP-dependent fashion. However, based on the relationship between thin filament velocity and filament length, the cMyBP-C induced reduction in velocity was independent of the number of cross-bridges interacting with the thin filament. In conclusion, the effects of cMyBP-C on velocity and force at both maximal and submaximal activation demonstrate that cMyBP-C does not solely act as a tether between the myosin S2 and LMM subdomains but likely affects both the kinetics and recruitment of myosin cross-bridges through its direct interaction with actin and/or myosin head.

Quantification of protein phosphorylation by liquid chromatography-mass spectrometry.

The identification and quantification of specific phosphorylation sites within a protein by mass spectrometry has proved challenging when measured from peptides after protein digestion because each peptide has a unique ionization efficiency that alters with modification, such as phosphorylation, and because phosphorylation can alter cleavage by trypsin, shifting peptide distribution. In addition, some phosphorylated peptides generated by tryptic digest are small and hydrophilic and, thus, are not retained well on commonly used C18 columns. We have developed a novel C-terminal peptide (2)H-labeling derivatization strategy and a mass balance approach to quantify phosphorylation. We illustrate the application of our method using electrospray ionization liquid chromatography-mass spectrometry by quantifying phosphorylation of troponin I with protein kinase A and protein kinase C. The method also improves the retention and elution of hydrophilic peptides. The method defines phosphorylation without having to measure the phosphorylated peptides directly or being affected by variable miscleavage. Measurement of phosphorylation is shown to be linear (relative standard error <5%) with a detection limit of <10%.

Skeletal muscle contractile protein function is preserved in human heart failure.

Skeletal muscle weakness is a common finding in patients with chronic heart failure (CHF). This functional deficit cannot be accounted for by muscle atrophy alone, suggesting that the syndrome of heart failure induces a myopathy in the skeletal musculature. To determine whether decrements in muscle performance are related to alterations in contractile protein function, biopsies were obtained from the vastus lateralis muscle of four CHF patients and four control patients. CHF patients exhibited reduced peak aerobic capacity and knee extensor muscle strength. Decrements in whole muscle strength persisted after statistical control for muscle size. Thin filaments and myosin were isolated from biopsies and mechanically assessed using the in vitro motility assay. Isolated skeletal muscle thin-filament function, however, did not differ between CHF patients and controls with respect to unloaded shortening velocity, calcium sensitivity, or maximal force. Similarly, no difference in maximal force or unloaded shortening velocity of isolated myosin was observed between CHF patients and controls. From these results, we conclude that skeletal contractile protein function is unaltered in CHF patients. Other factors, such as a decrease in total muscle myosin content, are likely contributors to the skeletal muscle strength deficit of heart failure.

Pro-Inflammatory Biomarkers in Stable Versus Acutely Decompensated Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Underlying inflammation has been increasingly recognized in heart failure with a preserved ejection fraction (HFpEF). In this study we tested the hypothesis that pro-inflammatory biomarkers are elevated in patients with acutely decompensated HFpEF (AD-HFpEF) compared with patients with stable HFpEF (S-HFpEF). METHODS AND RESULTS: Using a post hoc analysis the serum biomarkers tumor necrosis factor-alpha, high-sensitivity C-reactive protein interleukin 6 and pentraxin 3 (PTX3) and clinical, demographic, echocardiographic-Doppler and clinical outcomes data were analyzed in HFpEF patients enrolled in NHLBI Heart Failure Research Network clinical trials which enrolled patients with either AD-HFpEF or S-HFpEF. Compared to S-HFpEF, AD-HFpEF patients had higher levels of PTX3 (3.08 ng/mL versus 1.27 ng/mL, P<0.0001), interleukin-6 (4.14 pg/mL versus 1.71 pg/mL, P<0.0001), tumor necrosis factor-alpha (11.54 pg/mL versus 8.62 pg/mL, P=0.0015), and high-sensitivity C-reactive protein (11.90 mg/dL versus 3.42 mg/dL, P<0.0001). Moreover, high-sensitivity C-reactive protein, interleukin-6 and PTX3 levels were significantly higher in AD-HFpEF compared with S-HFpEF patients admitted for decompensated HF within the previous year. PTX3 was positively correlated with left atrial volume index (r=0.41, P=0.0017) and left ventricular mass (r=0.26, P=0.0415), while tumor necrosis factor-alpha was inversely correlated with E/A ratio (r=-0.31, P=0.0395). CONCLUSIONS: Levels of pro-inflammatory biomarkers are strikingly higher in AD-HFpEF compared with S-HFpEF patients. PTX3 and tumor necrosis factor-alpha are correlated with echocardiographic-Doppler evidence of diastolic dysfunction. Taken together these data support the concept that a heightened pro-inflammatory state has a pathophysiologic role in the development of AD-HFpEF.

Abundance, localization, and functional correlates of the advanced glycation end-product carboxymethyl lysine in human myocardium.

Advanced glycation end-products (AGEs) play a role in the pathophysiology of diabetes mellitus (DM) and possibly hypertension (HTN). In experimental DM, AGEs accumulate in myocardium. Little is known about AGEs in human myocardium. We quantified abundance, localization, and functional correlates of the AGE carboxymethyl lysine (CML) in left ventricular (LV) myocardium from patients undergoing coronary bypass grafting (CBG). Immunoelectron microscopy was used to quantify CML in epicardial biopsies from 98 patients (71 M, 27 F) with HTN, HTN + DM or neither (controls), all with normal LV ejection fraction. Myofilament contraction-relaxation function was measured in demembranated myocardial strips. Echocardiography was used to quantify LV structure and function. We found that CML was abundant within cardiomyocytes, but minimally associated with extracellular collagen. CML counts/µm2 were 14.7% higher in mitochondria than the rest of the cytoplasm (P < 0.001). There were no significant sex or diagnostic group differences in CML counts [controls 45.6 ± 3.6/µm2 (±SEM), HTN 45.8 ± 3.6/µm2, HTN + DM 49.3 ± 6.2/µm2; P = 0.85] and no significant correlations between CML counts and age, HgbA1c or myofilament function indexes. However, left atrial volume was significantly correlated with CML counts (r = 0.41, P = 0.004). We conclude that in CBG patients CML is abundant within cardiomyocytes but minimally associated with collagen, suggesting that AGEs do not directly modify the stiffness of myocardial collagen. Coexistent HTN or HTN + DM do not significantly influence CML abundance. The correlation of CML counts with LAV suggests an influence on diastolic function independent of HTN, DM or sex whose mechanism remains to be determined.

C-terminal truncation of cardiac troponin I causes divergent effects on ATPase and force: implications for the pathophysiology of myocardial stunning.

Myocardial stunning is a form of reversible myocardial ischemia/reperfusion injury associated with systolic and diastolic contractile dysfunction. In the isolated rat heart model, myocardial stunning is characterized by specific C-terminal proteolysis of the myofilament protein, troponin I (cTnI) that yields cTnI1-193. To determine the effect of this particular C-terminal truncation of cTnI, without the confounding factor of other stunning-induced protein modifications, a series of solution biochemical assays has been undertaken using the human homologue of mouse/rat cTnI1-193, cTnI1-192. Affinity chromatography and actin sedimentation experiments detected little, or no, difference between the binding of cTnI (cTnI1-209) and cTnI1-192 to actin-tropomyosin, troponin T, or troponin C. Both cTnI and cTnI1-192 inhibit the actin-tropomyosin-activated ATPase activity of myosin subfragment 1 (S1), and this inhibition is released by troponin C in the presence of Ca2+. However, cTnI1-192, when reconstituted as part of the troponin complex (cTn1-192), caused a 54+/-11% increase in the maximum Ca2+-activated actin-tropomyosin-S1 ATPase activity, compared with troponin reconstituted with cTnI (cTn). Furthermore, cTn1-192 increased Ca2+ sensitivity of both the actin-tropomyosin-activated S1 ATPase activity and the Ca2+-dependent sliding velocity of reconstituted thin filaments, in an in vitro motility assay, compared with cTn. In an in vitro force assay, the actin-tropomyosin filaments bearing cTn1-192 developed only 76+/-4% (P<0.001) of the force obtained with filaments composed of reconstituted cTn. We suggest that cTnI proteolysis may contribute to the pathophysiology of myocardial stunning by altering the Ca2+-sensing and chemomechanical properties of the myofilaments.

Altered myocardial thin-filament function in the failing Dahl salt-sensitive rat heart: amelioration by endothelin blockade.

BACKGROUND: Dahl salt-sensitive rats fed a high-salt diet develop compensated left ventricular hypertrophy followed by a transition to myocardial failure. We previously reported an increase in a troponin T isoform (TnT3) and a decrease in TnT phosphorylation in failing Dahl salt-sensitive rat hearts compared with low-salt controls. The present study was undertaken to determine whether the thin filament plays a role in depression of the contractile machinery in this model. METHODS AND RESULTS: Native thin filaments (NTFs) were isolated intact from rats with compensated left ventricular hypertrophy and failing hearts and compared with age-matched controls. NTF velocity was measured as a function of free calcium in the in vitro motility assay. Maximal velocity was similar in all groups. However, NTFs from failing hearts demonstrated a reduction in calcium sensitivity compared with controls, as reflected in the pCa50 (5.88+/-0.05 versus 6.22+/-0.05, respectively, P<0.001). No difference in thin-filament motility (pCa50, V(max)) was observed in rats with compensated left ventricular hypertrophy compared with controls. Protein kinase A treatment of NTFs from control and failing hearts had no effect on thin-filament calcium sensitivity. However, the endothelin receptor blocker bosentan prevented the reduction in thin-filament calcium sensitivity found in failing hearts. CONCLUSIONS: The thin filament is a key modulator of contractile performance in the transition to failure in the Dahl salt-sensitive rat model. The alteration in thin-filament function may be mediated by an endothelin-triggered pathway potentially affecting protein kinase C signaling.

Thin-filament-based modulation of contractile performance in human heart failure.

BACKGROUND: The contribution of the sarcomere's thin filament to the contractile dysfunction of human cardiomyopathy is not well understood. METHODS AND RESULTS: We have developed techniques to isolate and functionally characterize intact (native) thin filaments obtained from failing and nonfailing human ventricular tissue. By use of in vitro motility and force assays, native thin filaments from failing ventricular tissue exhibited a 19% increase in maximal velocity but a 27% decrease in maximal contractile force compared with nonfailing myocardium. Native thin filaments isolated from human myocardium after left ventricular assist device support demonstrated a 37% increase in contractile force. Dephosphorylation of failing native thin filaments resulted in a near-normalization of thin-filament function, implying a phosphorylation-mediated mechanism. Tissue expression of the protein kinase C isoforms alpha, beta1, and beta2 was increased in failing human myocardium and reduced after left ventricular assist device support. CONCLUSIONS: These novel findings demonstrate that (1) the thin filament is a key modulator of contractile performance in the failing human heart, (2) thin-filament function is restored to near normal levels after LVAD support, and (3) the alteration of thin-filament function in failing human myocardium is mediated through phosphorylation, most likely through activation of protein kinase C.