Published online before print March 15, 2007, doi: 10.1161/01.RES.0000264102.00706.4e
© 2007 American Heart Association, Inc.
Cellular Biology |
Calcium-Independent Negative Inotropy by ß-Myosin Heavy Chain Gene Transfer in Cardiac Myocytes
From the Department of Molecular & Integrative Physiology (T.J.H., R.V., E.F., L.M., T.E., J.M.M.) and Department of Internal Medicine–Cardiology (T.J.H. and J.M.M.), University of Michigan, Ann Arbor. Current address for R.V.: Brock University, St. Catharines, Ontario, Canada.
Correspondence to Todd J. Herron, PhD, Department of Molecular & Integrative Physiology, 1301 E Catherine St, University of Michigan, Ann Arbor, MI 48109-0622. E-mail toddherr{at}umich.edu
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Abstract |
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Increased relative expression of the slow molecular motor of the heart (ß-myosin heavy chain [MyHC]) is well known to occur in many rodent models of cardiovascular disease and in human heart failure. The direct effect of increased relative ß-MyHC expression on intact cardiac myocyte contractility, however, is unclear. To determine the direct effects of increased relative ß-MyHC expression on cardiac contractility, we used acute genetic engineering with a recombinant adenoviral vector (AdMYH7) to genetically titrate ß-MyHC protein expression in isolated rodent ventricular cardiac myocytes that predominantly expressed
-MyHC (fast molecular motor). AdMYH7-directed ß-MyHC protein expression and sarcomeric incorporation was observed as soon as 1 day after gene transfer. Effects of ß-MyHC expression on myocyte contractility were determined in electrically paced single myocytes (0.2 Hz, 37°C) by measuring sarcomere shortening and intracellular calcium cycling. Gene transfer-based replacement of -MyHC with ß-MyHC attenuated contractility in a dose-dependent manner, whereas calcium transients were unaffected. For example, when ß-MyHC expression accounted for 
18% of the total sarcomeric myosin, the amplitude of sarcomere-length shortening (nanometers, nm) was depressed by 42% (151.0±10.7 [control] versus 87.0±5.4 nm [AdMYH7 transduced]); and genetic titration of ß-MyHC, leading to 38% ß-MyHC content, attenuated shortening by 57% (138.9±13.0 versus 59.7±7.1 nm). Maximal isometric cross-bridge cycling rate was also slower in AdMYH7-transduced myocytes. Results indicate that small increases of ß-MyHC expression (18%) have Ca2+ transient-independent physiologically relevant effects to decrease intact cardiac myocyte function. We conclude that ß-MyHC is a negative inotrope among the cardiac myofilament proteins.
Key Words: adenovirus • contractility • gene transfer • intracellular calcium • muscle contraction • myosin • ventricular myocytes
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Introduction |
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Myosin heavy chain (MyHC), the molecular motor of the heart, converts chemical energy from ATP hydrolysis into mechanical work during each heart beat. Two functionally distinct MyHC isoforms, namely
-MyHC and ß-MyHC, are differentially expressed in mammalian myocardium. ß-MyHC, the "slow" molecular motor, hydrolyzes ATP 3 to 7 times slower than -MyHC,1,2 the "fast" motor in the heart. Expression of the slow ß-MyHC motor increases relative to -MyHC in rodent models of cardiovascular disease, including diabetes,3 hypothyroidism, cardiac hypertrophy,4 and in aging.5–7 Therefore ß-MyHC motor expression is generally accepted as a biomarker of cardiac disease in rodents. In humans, there is increasing evidence that the relative content of ß-MyHC is increased in the diseased heart.8–11 It is unclear, however, whether increased relative ß-MyHC expression contributes directly to intact cardiac muscle dysfunction in the absence of other changes in excitation/contraction coupling mechanisms that can occur concurrently in the diseased heart.12
There have been several experimental strategies used to investigate the functional significance of the motor protein isoform profile in heart muscle. The most frequently used model has been the rodent hypothyroid animal model. Thyroid hormone is a potent inducer of -MyHC expression, and in the absence of thyroid hormone, by thyroidectomy or propyl-thio-uracil treatment, there is a reduction of -MyHC expression and a concomitant increase of ß-MyHC expression.13,14 Studies using hypothyroid rodents have provided evidence that increased ß-MyHC expression depresses contractile function of single cardiac myocytes15–18 and in the whole heart.19,20 However, a limitation of the hypothyroid model is that in concert with alterations in the MyHC isoform profile, the expression of key calcium-handling proteins, including the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a), is also affected.21,22 This presents a problem for interpretation of functional data because SERCA2a activity itself is well known to markedly affect cardiac performance.23–25 To address this confounding issue, some studies have used the permeabilized (skinned) myocyte preparation: a preparation that eliminates any contribution of physiological calcium handling to myocyte functional studies.16,17 These studies have demonstrated that mechanical properties of single myocytes (eg, power output and unloaded shortening velocity) are attenuated by increased relative expression of ß-MyHC. Although highly valuable, these studies still leave unclear the direct contribution of MyHC isoforms in the more physiologically relevant setting of the membrane intact myocyte where physiological excitation/contraction coupling is fully operable.
More recently, transgenic animal models have been developed to manipulate MyHC isoform expression in the mammalian heart in vivo.26–28 Under baseline conditions, the near-full replacement of -MyHC with ß-MyHC in the hearts of transgenic mice surprisingly had no detectable effects on echocardiography derived shortening fraction, an in vivo measure of systolic function.28 Heart rates were reportedly slower in the transgenic mice, and this may have contributed to normalize the shortening fraction measurements. Isolated permeabilized muscle preparations from these mice, however, did show negative effects on contractile performance at baseline. This raises the question of whether forced genetic transition from -MyHC to ß-MyHC may have caused other adaptations (perhaps to intracellular calcium handling) in the mouse heart to compensate for effects of ß-MyHC in vivo. This is not difficult to conceptualize, considering that these transgenic mice would have sustained 30 billion contractile cycles in vivo before the experimental assessment that is typically performed in adult mice. Therefore, in transgenic animal models, it may be difficult to distinguish primary effects caused by forced ß-MyHC expression from secondary compensatory changes that may occur.29 In this light, a question that can be posed is whether the direct effects of MyHC isoform manipulation on intact cardiac muscle performance are fully understood? For example, these intriguing transgenic studies did not assess intracellular Ca2+ transients, leaving open the issue of whether compensatory alterations in Ca2+ handling may have altered or masked a direct effect of ß-MyHC on performance.
Here we have used acute gene transfer technology in adult cardiac myocytes in vitro to determine the primary effects of ß-MyHC expression on the function of single membrane intact cardiac myocytes. The premise was to simplify interpretation by obviating organ level compensatory adaptations that can occur in animal model studies (as discussed above).29 Because the myocytes under study are quiescent during genetic conversion from -MyHC to ß-MyHC, the results obtained should directly relate to primary effects of MyHC manipulation. This is supported by previous in vitro gene transfer studies of other sarcomeric proteins showing no detected effects on other contractile or regulatory proteins.30,31 Importantly, under the experimental conditions used here, the MyHC isoform expression profile has been shown to be stable in primary culture of rat cardiac myocytes.32 Because there is a dynamic and essential connection between sarcomeric function and intracellular Ca2+ handling in cardiac muscle, we conducted parallel studies on contractile and Ca2+ transient performance at physiological temperature. To more directly assess myosin function, we also measured maximal calcium activated isometric tension and cross-bridge cycling kinetics in single membrane permeabilized cardiac myocytes. We report for the first time that acute gene transfer of ß-MyHC has negative inotropic effects on cardiac myocyte contractility independent of effects on intracellular calcium homeostasis.
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Materials and Methods |
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An expanded Materials and Methods section can be found in the online data supplement at http://circres.ahajournals.org.
Adenovirus Production
The AdMax system (Microbix) was used to generate recombinant adenoviral vectors to express ß-MyHC or GFP.31,33
Ventricular Myocyte Isolation, Primary Culture, and Gene Transfer
Ventricular cardiac myocytes were isolated from adult rats and cultured, and gene transfer was performed as described previously.31,33,34 The procedures used in this study were in agreement with the guidelines of the Internal Review Board of the University of Michigan Committee on the Use and Care of Animals. Veterinary care was provided by the University of Michigan Unit for Laboratory Animal Medicine.
ß-MyHC Protein Expression Analysis
Western Blotting
Myocytes were harvested in Laemmli sample buffer, and proteins were separated by SDS-PAGE. Separated proteins were transferred to nitrocellulose membrane and probed with monoclonal antibodies specific to -MyHC or ß-MyHC.
Indirect Immunofluorescence
Sarcomeric localization of AdMYH7-directed ß-MyHC expression was determined by immunohistochemistry and fluorescent confocal imaging as described previously.34
Contractility Measurements in Single Cardiac Myocytes
Sarcomere shortening and relengthening of field-stimulated myocytes was used as an index of contractility and intracellular calcium transients were measured using fura-2 acetoxymethyl ester (2µmol/L).
Isometric cross-bridge cycling kinetics were measured using single-membrane permeabilized (1% Triton) myocytes attached to a force transducer and length controller as previously described.35
Statistics
Two-tailed independent t tests were used to determine significant differences between groups of data (P<0.05). Paired t tests were performed to determine differences before and after application of 10 nmol/L isoproterenol (ISO) (P<0.05). All data are expressed as mean±SEM. The effects of genetic titration of ß-MyHC on sarcomere-shortening amplitude and the velocity of sarcomere shortening were examined by non linear regression analysis. These data were fit to a single, three parameter decaying exponential (r2=0.99) using the equation: y=y0+ae–bx.
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Results |
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AdMYH7 Gene Transfer and Myosin Isoform Expression
A recombinant adenovirus, AdMYH7, was generated to accomplish synchronous and efficient gene transfer of ß-MyHC, the slow myosin motor, in rat ventricular cardiac myocytes that expressed predominantly
-MyHC, the fast myosin motor. Successful gene transfer and sarcomeric incorporation of ß-MyHC is demonstrated in Figures 1 and 2
. Figure 1 shows Western blot data using - and ß-MyHC–specific antibodies. For quantitative analysis (Figure 1B and 1C), specific immunoreactivity for each myosin isoform was normalized to the actin signal for each lane. In control myocytes, there was no detectable expression of ß-MyHC by Western blot analysis (Figure 1A and 1B), whereas in AdMYH7-transduced myocytes ß-MyHC expression could be detected from day 1 after gene transfer. Following gene transfer with AdMYH7, ß-MyHC protein levels continued to rise (Figure 1A and 1B), whereas the levels of endogenous -MyHC fell (Figure 1A and 1C). This provides evidence that the endogenous -MyHC was systematically replaced by ß-MyHC following gene transfer. Such stoichiometric maintenance of the cardiac sarcomere is consistent with previous studies involving other myofilament proteins.30,31 Quantitatively, ß-MyHC protein accounted for 18% of the total myosin 1 day after gene transfer, 37% of the total myosin on day 2 after gene transfer and 40% of the total myosin on day 3 after gene transfer. This degree of replacement is somewhat accelerated based on that predicted from the biochemical turnover rate of endogenous MyHC in cardiac muscle (5.4 days36). The cardiac myocyte rigidly controls overall stoichiometry of the sarcomeric protein pool,37 and the efficient gene transfer of MyHC may accelerate this process.
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Using the same myosin isoform specific antibodies for indirect immunofluorescence, Figure 2A demonstrates high efficiency of AdMYH7 gene transfer (100%), which is consistent with sarcomere gene transfer studies.31,33,38,39 Higher magnification and confocal imaging (Figure 2B through 2D) demonstrates proper sarcomeric localization of AdMYH7-directed ß-MyHC protein in the sarcomeric A-band 1 day after AdMYH7 gene transfer. Colocalization (yellow bands) of - and ß-MyHC in a broad banding pattern is consistent with the length of the myosin containing A-band (1.6 µm; Figure 2D).
Functional Effects of AdMYH7 Gene Transfer
The functional effects of genetically titrating ß-MyHC into a background of -MyHC were determined in living cardiac myocytes isolated from adult rats. Sarcomere shortening and relengthening of field-stimulated myocytes (0.2 Hz) were used as indices of intact myocyte contractility. Calcium transients in these myocytes were also recorded using fura-2 acetoxymethyl ester. Typical recordings of calcium transients and sarcomere-length changes in stimulated myocytes are shown in Figure 3A. The calcium transients were unaffected by AdMYH7 gene transfer (Figure 3A and 3C); however, the amplitude of sarcomere shortening (Figure 3A and 3B) was attenuated by 42% (151.0±10.7 nm, control versus 87.0±5.4 nm AdMYH7 transduced) 1 day after gene transfer when ß-MyHC represented 18% of the total myosin. In a control set of experiments (time matched), AdGFP gene transfer had no significant effect on the amplitude of sarcomere-length shortening when compared with nontransduced rat myocytes (Figure 3B).
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Resting sarcomere length is an important determinant of contractility40,41; therefore, next we plotted the resting and peak sarcomere lengths for control and AdMYH7-transduced myocytes at each time point after gene transfer (days 0 to 3; Figure 4A). The effect of ß-MyHC to attenuate contraction amplitude was not attributable to any effect on resting sarcomere length (Figure 4A). Rather, AdMYH7-transduced myocytes did not shorten to the same extent as evidenced by differences in the peak sarcomere length (Figure 4A). Resting and peak calcium concentrations were not different on any day after gene transfer (Figure 4B). This difference in peak sarcomere length translated to depressed sarcomere-shortening amplitudes on each day following AdMYH7 gene transfer (Figure 4C). The effect of AdMYH7 to depress shortening amplitude was dose dependent; the depressant effect increased from day 1 to 3 (Figure 4C).
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significant difference from AdMYH7 transduced on day 1 after gene transfer (P<0.05).In control experiments, we used ventricular myocytes from adult rabbits, which predominantly express the ß-MyHC isoform (see Figure 1). Gene transfer of AdMYH7 to isolated rabbit myocytes did not have any significant effect on contractile performance (Figure I in the online data supplement).
Next we sought to determine whether the negative effect of AdMYH7 on intact myocyte contractility might be attributable to attenuated myosin force development or to slower myosin cross-bridge cycling. Therefore, maximal calcium activated force development and cross-bridge cycling kinetics were measured in single myocytes (Figure 5). Maximal tension development was not different (Figure 5A), but cross-bridge cycling kinetics (ktr) were slower in AdMYH7-transduced myocytes (Figure 5B through 5D).
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We next assessed the effect of ß-MyHC gene transfer on the velocities of unloaded sarcomere shortening and relengthening (Figure 6). Figure 6A shows superimposed sarcomere-length traces from a control (solid line) and AdMYH7-transduced (dashed line). Compared with control myocytes, the velocity of unloaded sarcomere shortening was significantly slower in myocytes transduced with AdMYH7 at each time point following gene transfer. Maximal shortening velocity was 48% slower on day 1 after gene transfer, 58% slower on day 2 after gene transfer, and 62% slower on day 3 after gene transfer when compared with control values (Figure 6C). Maximal relengthening velocity was 50% slower on day 1, 60% slower on day 2, and 59% slower on day 3 following AdMYH7 gene transfer (Figure 6D). The traces in Figure 6B were transformed to show normalized shortening (peak of shortening was set to 1.0). The times to half-maximal shortening, half-maximal relaxation, and 90% relaxation, however, were not different between control and AdMYH7-transduced myocytes at any time point in culture (supplemental Table I). The lack of effect on absolute times to specific relative points of contraction is likely attributable to the blunted amplitude of contraction in the AdMYH7 myocytes.
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Genetic titration of ß-MyHC into -MyHC–containing rat cardiac myocytes attenuated shortening amplitude and velocity in a dose-dependent manner (Figure 7A). For example, sarcomere-length shortening amplitude was progressively attenuated (193.0±14.1 nm [day 0]>87.0±5.4 nm [day 1]>59.7±7.1 nm [day 2]) as the relative expression of ß-MyHC increased (0% on day 0, 18% on day 1, and 37% on day 2) after AdMYH7 gene transfer. Figure 7B shows that maximal shortening velocity was similarly affected in a dose-dependent manner by genetic titration of ß-MyHC.
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Next, we tested whether AdMYH7 gene transfer affected ß-adrenergic responsiveness of single myocytes by using ISO (Figure 8 and supplemental Figure II). ISO increased sarcomere-length shortening by the same magnitude in control (137.6±14.0 nm, n=8; 161.5±16.2 nm at baseline and 299.1±9.8 nm after ISO) and AdMYH7-transduced myocytes (122.4±26.2 nm, n=7 57.7±14.6 nm at baseline and 180.1±22.5 nm after ISO). Although ISO increased shortening amplitude by roughly the same amount (Figure 8D), the peak amplitude after ISO treatment was still greater in control myocytes than in AdMYH7-transduced myocytes. Likewise, maximal shortening velocity was faster in both control and AdMYH7-transduced myocytes following ISO treatment, but control myocytes still shortened at a faster rate than AdMYH7-transduced myocytes (control=–5.66±0.49 and AdMYH7=–3.03±0.47 µm/s). Relaxation kinetics were also similarly faster after ISO treatment in the 2 groups (Figure 8D).
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SL] amplitude) to the same extent (P>0.05) in control and AdMYH7-transduced myocytes. Similarly, ISO hastened relaxation ( |
Discussion |
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We have used, for the first time to our knowledge, a recombinant adenovirus (AdMYH7) to study the direct effects of ß-MyHC gene (MYH7) expression on
-MyHC–dominant intact cardiac myocyte contractility. Using acute genetic engineering of the cardiac molecular motor, there was time-dependent stoichiometric replacement of the endogenous -MyHC (fast motor) with ß-MyHC (slow motor). The main contractile effects of ß-MyHC expression in -MyHC–dominant myocytes were attenuation of sarcomere-shortening amplitude and depression of myosin cross-bridge cycling rate and sarcomere shortening and relengthening velocities in the absence of effects on intracellular Ca2+ transients. These results held even after ß-adrenergic stimulation of the myocytes. Neither gene transfer alone (AdGFP), nor MYH7 gene transfer to ß-MyHC–dominant rabbit myocytes affected contractility. We take these results as direct evidence that ß-MyHC represents a Ca2+-independent negative inotropic element in the cardiac sarcomere. A recombinant adenovirus has been used previously to study the structural effects of mutant ß-MyHC expression on sarcomere assembly and myofibrillar organization.42 Marian et al42 used acute gene transfer to study the effects of the hypertrophic cardiomyopathy–associated R403Q mutation of human MYH7 on cardiac sarcomere structure. They found that whereas normal ß-MyHC had no effect on sarcomere structure (consistent with our results presented here; Figure 2), sarcomere disruption was found in nearly half of the feline cardiac myocytes transduced with the R403Q mutant myosin. The functional effect of myosin gene transfer, however, was not explored in that study. In our study, MYH7 gene transfer demonstrated near 100% efficiency at the level of the isolated adult cardiac myocytes (Figure 2). Our results provide insight into the utility of acute gene transfer of myosin molecular motors for efficiently studying myosin structure–function relationships in the context of intact adult cardiac myocytes.
Mechanistically, functional observations made here could be explained by a differential effect of MyHC isoforms on the kinetics and extent of thin-filament activation, which may also affect myofilament calcium sensitivity. Although myocardial contraction is initiated by Ca2+ binding to cardiac troponin C on the thin filament, the activation of force and the kinetics of force development and muscle shortening are also attributable to activating effects of myosin cross-bridges binding to actin and further activating the thin filament.43–45 In the electrically paced cardiac myocyte, fast-cycling cross-bridges may be expected to activate the thin filament at a faster rate and to a greater extent than slow-cycling cross-bridges. For example, a greater number of fast cycling -MyHC cross-bridges may be expected to bind actin, activate the thin filament, proceed through the conformational changes of the myosin power stroke, detach from actin, reprime, and continue again through the cross-bridge cycle a greater number of times than slow-cycling ß-MyHC cross-bridges during a fixed duration of calcium activation (ie, the calcium transient, which was unaffected by gene transfer here). This possibility is supported by a previous study that reported depressed calcium sensitivity of isometric tension in myocytes that expressed ß-MyHC independent of alterations of thin filament protein expression.17 Alternatively, the depressed amplitude of shortening reported here in AdMYH7-treated myocytes may be attributable to the presence of slow-cycling cross-bridges independent of effects on calcium sensitivity. In addition, a recent in vitro motility study has suggested that changes in myosin isoform expression and kinetics can alter myofilament function independently of thin-filament calcium sensitivity.46 Because ß-MyHC exhibits a prolonged attachment to actin per ATP molecule47 and a reduced cycling rate caused by slower ADP release,48 it is possible that slow ß-MyHC cross-bridges become compressed during shortening and impede the cycling of other cross-bridges49 (eg, fast cycling -MyHC cross-bridges). Therefore, myocytes that predominantly express -MyHC (3 to 7 times faster ATPase activity) may be expected to shorten to a greater extent than myocytes that express ß-MyHC (slower ATPase activity) during a fixed period of calcium activation (ie, the physiological calcium transient). This is also supported by our finding that increased relative ß-MyHC expression slows the overall cross-bridge cycling rate of single cardiac myocytes (Figure 5). These effects may be exaggerated here because the human ß-MyHC molecule is a significantly slower molecular motor than the rat cardiac ß-MyHC isoform.50
Our results may also offer insight into the molecular basis of cardiac contractile dysfunction that has been observed in animal models of cardiovascular disease, including diabetes,3 hypothyroidism,47,51 cardiac hypertrophy,4 and in aging.5–7 All of these models are associated with increased relative expression of ß-MyHC in the cardiac ventricles, but other regulatory proteins are also affected in all of these models. Thus it has been difficult to assign a specific role to altered MyHC isoform expression in intact myocytes. One study concluded that diminished calcium transient amplitude alone, independent of myosin isoform expression, may account for the reduced contractility of cardiac muscle isolated from hypothyroid ferrets.51 To eliminate these potentially confounding secondary adaptations, here we have used acute gene transfer to specifically increase the relative expression of ß-MyHC.
In summary, we report that ß-MyHC gene transfer directly attenuates sarcomere shortening, whereas calcium transients are unaffected. These results suggest that increased relative ß-MyHC expression alone, as a consequence of pathophysiology or genetic engineering, directly causes depressed cardiac contractile function. Genetic engineering of the MyHC isoform profile may represent a new approach to modify a Ca2+ independent inotropy of the heart in vivo.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH fellowship HL080880 (to T.J.H.) and NIH grant HL60048 (to J.M.M.). This work used the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center, funded by National Institute of Diabetes, Digestive, Kidney Diseases grant NIH5P60 DK20572.
Disclosures
None.
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Footnotes |
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Original received November 1, 2006; revision received March 2, 2007; accepted March 7, 2007.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1. Harris DE, Work SS, Wright RK, Alpert NR, Warshaw DM. Smooth, cardiac, and skeletal muscle myosin force and motion generation assessed by cross-bridge mechanical interactions in vitro. J Muscle Res Cell Motil. 1994; 15: 11–19.[CrossRef][Medline] [Order article via Infotrieve]
2. VanBuren P, Harris DE, Alpert NR, Warshaw DM. Cardiac V1 And V3 myosins differ in their hydrolytic and mechanical activities in vitro. Circ Res. 1995; 77: 439–444.
3. Rundell VLM, Geenen DL, Buttrick PM, de Tombe PP. Depressed cardiac tension cost in experimental diabetes is due to altered myosin heavy chain isoform expression. Am J Physiol Heart Circ Physiol. 2004; 287: H408–H413.
4. Mercadier JJ, Lompre AM, Wisnewsky C, Samuel JL, Bercovici J, Swynghedauw B, Schwartz K. Myosin isoenzymic changes in several models of rat cardiac hypertrophy. Circ Res. 1981; 49: 525–532.
5. Fitzsimons DP, Patel JR, Moss RL. Aging-dependent depression in the kinetics of force development in rat skinned myocardium. Am J Physiol Heart Circ Physiol. 1999; 276: H1511–H1519.
6. Wahr PA, Michele DE, Metzger JM. Effects of aging on single cardiac myocyte function in Fischer 344 x Brown Norway rats. Am J Physiol Heart Circ Physiol. 2000; 279: H559–H565.
7. Carnes CA, Geisbuhler TP, Reiser PJ. Age-dependent changes in contraction and regional myocardial myosin heavy chain isoform expression in rats. J Appl Physiol. 2004; 97: 446–453.
8. Lowes BD, Minobe W, Abraham WT, Rizeq MN, Bohlmeyer TJ, Quaife RA, Roden RL, Dutcher DL, Robertson AD, Voelkel NF, Badesch DB, Groves BM, Gilbert EM, Bristow MR. Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium. J Clin Invest. 1997; 100: 2315–2324.[Medline] [Order article via Infotrieve]
9. Lowes BD, Gilbert EM, Abraham WT, Minobe WA, Larrabee P, Ferguson D, Wolfel EE, Lindenfeld J, Tsvetkova T, Robertson AD, Quaife RA, Bristow MR. Myocardial gene expression in dilated cardiomyopathy treated with beta-blocking agents. The N Engl J Med. 2002; 346: 1357–1365.
10. Miyata S, Minobe W, Bristow MR, Leinwand LA. Myosin heavy chain isoform expression in the failing and nonfailing human heart. Circ Res. 2000; 86: 386–390.
11. Nakao K, Minobe W, Roden R, Bristow MR, Leinwand LA. Myosin heavy chain gene expression in human heart failure. J Clin Invest. 1997; 100: 2362–2370.[Medline] [Order article via Infotrieve]
12. Kubalova Z, Terentyev D, Viatchenko-Karpinski S, Nishijima Y, Gyorke I, Terentyeva R, da Cunha DNQ, Sridhar A, Feldman DS, Hamlin RL, Carnes CA, Gyorke S. Abnormal intrastore calcium signaling in chronic heart failure. Proc Natl Acad Sci U S A. 2005; 102: 14104–14109.
13. Izumo S, Nadal-Ginard B, Mahdavi V. All members of the MHC multigene family respond to thyroid hormone in a highly tissue-specific manner. Science. 1986; 231: 597–600.
14. Haddad F, Bodell PW, Qin AX, Giger JM, Baldwin KM. Role of antisense RNA in coordinating cardiac myosin heavy chain gene switching. J Biol Chem. 2003; 278: 37132–37138.
15. Herron TJ, McDonald KS. Small Amounts of {alpha}-myosin heavy chain isoform expression significantly increase power output of rat cardiac myocyte fragments. Circ Res. 2002; 90: 1150–1152.
16. Herron TJ, Korte FS, McDonald KS. Loaded shortening and power output in cardiac myocytes are dependent on myosin heavy chain isoform expression. Am J Physiol Heart Circ Physiol. 2001; 281: H1217–H1222.
17. Metzger JM, Wahr PA, Michele DE, Albayya F, Westfall MV. Effects of myosin heavy chain isoform switching on Ca2+-activated tension development in single adult cardiac myocytes. Circ Res. 1999; 84: 1310–1317.
18. Fitzsimons DP, Patel JR, Moss RL. Role of myosin heavy chain composition in kinetics of force development and relaxation in rat myocardium. J Physiol (Lond). 1998; 513: 171–183.
19. Korte FS, Herron TJ, Rovetto MJ, McDonald KS. Power output is linearly related to myosin heavy chain content in rat skinned myocytes and isolated working hearts. Am J Physiol Heart Circ Physiol. 2005; 289: H801–H812.
20. Tang YD, Kuzman JA, Said S, Anderson BE, Wang X, Gerdes AM. Low thyroid function leads to cardiac atrophy with chamber dilatation, impaired myocardial blood flow, loss of arterioles, and severe systolic dysfunction. Circulation. 2005; 112: 3122–3130.
21. Sayen MR, Rohrer DK, Dillmann WH. Thyroid hormone response of slow and fast sarcoplasmic reticulum Ca2+ ATPase mRNA in striated muscle. Mol Cell Endocrinol. 1992; 87: 87–93.[CrossRef][Medline] [Order article via Infotrieve]
22. Carr AN, Kranias EG. Thyroid hormone regulation of calcium cycling proteins. Thyroid. 2002; 12: 453–457.[CrossRef][Medline] [Order article via Infotrieve]
23. Arai M, Alpert NR, MacLennan DH, Barton P, Periasamy M. Alterations in sarcoplasmic reticulum gene expression in human heart failure. A possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ Res. 1993; 72: 463–469.
24. Hasenfuss G, Reinecke H, Studer R, Meyer M, Pieske B, Holtz J, Holubarsch C, Posival H, Just H, Drexler H. Relation between myocardial function and expression of sarcoplasmic reticulum Ca(2+)-ATPase in failing and nonfailing human myocardium. Circ Res. 1994; 75: 434–442.
25. Teucher N, Prestle J, Seidler T, Currie S, Elliott EB, Reynolds DF, Schott P, Wagner S, Kogler H, Inesi G, Bers DM, Hasenfuss G, Smith GL. Excessive sarcoplasmic/endoplasmic reticulum Ca2+-ATPase expression causes increased sarcoplasmic reticulum Ca2+ uptake but decreases myocyte shortening. Circulation. 2004; 110: 3553–3559.
26. Tardiff JC, Hewett TE, Factor SM, Vikstrom KL, Robbins J, Leinwand LA. Expression of the beta (slow)-isoform of MHC in the adult mouse heart causes dominant-negative functional effects. Am J Physiol Heart Circ Physiol. 2000; 278: H412–H419.
27. James J, Martin L, Krenz M, Quatman C, Jones F, Klevitsky R, Gulick J, Robbins J. Forced expression of {alpha}-myosin heavy chain in the rabbit ventricle results in cardioprotection under cardiomyopathic conditions. Circulation. 2005; 111: 2339–2346.
28. Krenz M, Robbins J. Impact of beta-myosin heavy chain expression on cardiac function during stress. J Am Coll Cardiol. 2004; 44: 2390–2397.
29. Michele DE, Metzger JM. Contractile dysfunction in hypertrophic cardiomyopathy: elucidating primary defects of mutant contractile proteins by gene transfer. Trends Cardiovasc Med. 2000; 10: 177–182.[CrossRef][Medline] [Order article via Infotrieve]
30. Metzger JM, Michele DE, Rust EM, Borton AR, Westfall MV. Sarcomere thin filament regulatory isoforms. Evidence of a Dominant Effect of Slow Skeletal Troponin I on Cardiac Contraction. J Biol Chem. 2003; 278: 13118–13123.
31. Westfall MV, Rust EM, Metzger JM. Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function. Proc Natl Acad Sci U S A. 1997; 94: 5444–5449.
32. Rust EM, Westfall MV, Metzger JM. Stability of the contractile assembly and calcium activated tension in adenovirus infected adult cardiac myocytes. Mol Cell Biochem. 1998; 181: 143–155.[CrossRef][Medline] [Order article via Infotrieve]
33. Westfall MV, Rust EM, Albayya F, Metzger JM. Adenovirus-mediated myofilament gene transfer into adult cardiac myocytes. Methods Cell Biol. 1997; 52: 307–322.[Medline] [Order article via Infotrieve]
34. Rust EM, Albayya FP, Metzger JM. Identification of a contractile deficit in adult cardiac myocytes expressing hypertrophic cardiomyopathy-associated mutant troponin T proteins. J Clin Invest. 1999; 103: 1459–1467.[Medline] [Order article via Infotrieve]
35. Metzger JM. Myosin binding-induced cooperative activation of the thin filament in cardiac myocytes and skeletal muscle fibers. Biophys J. 1995; 68: 1430–1442.[Medline] [Order article via Infotrieve]
36. Martin AF, Rabinowitz M, Blough R, Prior G, Zak R. Measurements of half-life of rat cardiac myosin heavy chain with leucyl-tRNA used as precursor pool. J Biol Chem. 1977; 252: 3422–3429.
37. Gulick J, Hewett TE, Klevitsky R, Buck SH, Moss RL, Robbins J. Transgenic remodeling of the regulatory myosin light chains in the mammalian heart. Circ Res. 1997; 80: 655–664.
38. Coutu P, Metzger JM. Genetic manipulation of calcium-handling proteins in cardiac myocytes. I. Experimental studies. Am J Physiol Heart Circ Physiol. 2005; 288: H601–H612.
39. Wahr PA, Michele DE, Metzger JM. Parvalbumin gene transfer corrects diastolic dysfunction in diseased cardiac myocytes. Proc Natl Acad Sci U S A. 1999; 96: 11982–11985.
40. Fukuda N, Sasaki D, Ishiwata S, Kurihara S. Length dependence of tension generation in rat skinned cardiac muscle: role of titin in the Frank-Starling mechanism of the heart. Circulation. 2001; 104: 1639–1645.
41. Vahl CF, Timek T, Bonz A, Fuchs H, Dillman R, Hagl S. Length dependence of calcium- and force-transients in normal and failing human myocardium. J Mol Cell Cardiol. 1998; 30: 957–966.[CrossRef][Medline] [Order article via Infotrieve]
42. Marian AJ, Yu QT, Mann DL, Graham FL, Roberts R. Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes. Circ Res. 1995; 77: 98–106.
43. Tobacman LS. Thin Filament-Mediated Regulation of Cardiac Contraction. Annu Rev Physiol. 1996; 58: 447–481.[CrossRef][Medline] [Order article via Infotrieve]
44. Fitzsimons DP, Patel JR, Campbell KS, Moss RL. Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers. J Gen Physiol. 2001; 117: 133–148.
45. Fitzsimons DP, Patel JR, Moss RL. Cross-bridge interaction kinetics in rat myocardium are accelerated by strong binding of myosin to the thin filament. J Physiol (Lond). 2001; 530: 263–272.
46. Schoffstall B, Brunet NM, Williams S, Miller VF, Barnes AT, Wang F, Compton LA, McFadden LA, Taylor DW, Seavy M, Dhanarajan R, Chase PB. Ca2+ sensitivity of regulated cardiac thin filament sliding does not depend on myosin isoform. J Physiol (Lond). 2006; 577: 935–944.
47. Holubarsch C, Goulette RP, Litten RZ, Martin BJ, Mulieri LA, Alpert NR. The economy of isometric force development, myosin isoenzyme pattern and myofibrillar ATPase activity in normal and hypothyroid rat myocardium. Circ Res. 1985; 56: 78–86.
48. Siemankowski RF, Wiseman MO, White HD. ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle. Proc Natl Acad Sci U S A. 1985; 82: 658–662.
49. Huxley AF. Muscle structure and theories of contraction. Prog Biophys Biophys Chem. 1957; 7: 255–318.[Medline] [Order article via Infotrieve]
50. Hasenfuss G, Mulieri LA, Blanchard EM, Holubarsch C, Leavitt BJ, Ittleman F, Alpert NR. Energetics of isometric force development in control and volume-overload human myocardium. Comparison with animal species. Circ Res. 1991; 68: 836–846.
51. MacKinnon R, Gwathmey JK, Allen PD, Briggs GM, Morgan JP. Modulation by the thyroid state of intracellular calcium and contractility in ferret ventricular muscle. Circ Res. 1988; 63: 1080–1089.
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