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Circulation Research. 2007;100:1182-1190
Published online before print March 15, 2007, doi: 10.1161/01.RES.0000264102.00706.4e
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(Circulation Research. 2007;100:1182.)
© 2007 American Heart Association, Inc.

Cellular Biology

Calcium-Independent Negative Inotropy by ß-Myosin Heavy Chain Gene Transfer in Cardiac Myocytes

Todd J. Herron, Rene Vandenboom, Ekaterina Fomicheva, Lakshmi Mundada, Terri Edwards, Joseph M. Metzger

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


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {alpha}-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 {alpha}-MyHC with ß-MyHC attenuated contractility in a dose-dependent manner, whereas calcium transients were unaffected. For example, when ß-MyHC expression accounted for {approx}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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {alpha}-MyHC and ß-MyHC, are differentially expressed in mammalian myocardium. ß-MyHC, the "slow" molecular motor, hydrolyzes ATP {approx}3 to 7 times slower than {alpha}-MyHC,1,2 the "fast" motor in the heart. Expression of the slow ß-MyHC motor increases relative to {alpha}-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 {alpha}-MyHC expression, and in the absence of thyroid hormone, by thyroidectomy or propyl-thio-uracil treatment, there is a reduction of {alpha}-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 {alpha}-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 {alpha}-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 {approx}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 {alpha}-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.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {alpha}-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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 {alpha}-MyHC, the fast myosin motor. Successful gene transfer and sarcomeric incorporation of ß-MyHC is demonstrated in Figures 1 and 2Down. Figure 1 shows Western blot data using {alpha}- 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 {alpha}-MyHC fell (Figure 1A and 1C). This provides evidence that the endogenous {alpha}-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 {approx}18% of the total myosin 1 day after gene transfer, {approx}37% of the total myosin on day 2 after gene transfer and {approx}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 ({approx}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.


Figure 1
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  		Figure 1. Western blot analysis of cardiac myosin isoforms. A, In rat cardiac myocytes, ß-MyHC expression was genetically engineered into myocytes that did not express ß-MyHC by using AdMYH7. Rabbit ventricular myocytes were used as a positive control for ß-MyHC expression. B, Relative to actin, AdMYH7-treated myocytes expressed ß-MyHC in a time-dependent manner, in which the amount of expression increased after viral transduction. C, Expression levels of the endogenous MyHC ({alpha}-MyHC) fell relative to actin following AdMYH7 transduction but did not change in control myocytes. This is evidence that the endogenous {alpha}-MyHC was replaced by AdMYH7-directed ß-MyHC protein, thus preserving the proper stoichiometry of the sarcomere; n=3 blots.


Figure 2
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  		Figure 2. Sarcomeric localization and incorporation of AdMYH7-directed ß-MyHC protein. Gene transfer efficiency was near 100% (A, right column), and control myocytes did not express detectable ß-MyHC protein (A, left column). As soon as 1 day after AdMYH7 transduction, ß-MyHC protein (green) (B) was incorporated into the sarcomere with a banding pattern identical to that of the endogenous {alpha}-MyHC (red) (C). Colocalization of the 2 signals (yellow) (D) is apparent by the yellow appearance of the overlay of the 2 images (white arrows added to point out colocalization in the sarcomeric A-band).

Using the same myosin isoform specific antibodies for indirect immunofluorescence, Figure 2A demonstrates high efficiency of AdMYH7 gene transfer ({approx}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 {alpha}- and ß-MyHC in a broad banding pattern is consistent with the length of the myosin containing A-band ({approx}1.6 µm; Figure 2D).

Functional Effects of AdMYH7 Gene Transfer
The functional effects of genetically titrating ß-MyHC into a background of {alpha}-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 {approx}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).


Figure 3
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  		Figure 3. AdMYH7 gene expression attenuates the amplitude of sarcomere shortening 1 day after gene transfer. A, Original recordings of intracellular calcium transients and sarcomere-length shortening in control (left) and AdMYH7-transduced (right) myocytes 1 day after gene transfer. B, Compared with control values, AdMYH7-directed ß-MyHC expression (18% of total myosin) attenuated sarcomere shortening by 42% (151.0±10.7, n=51 [control]; 155.5±15.8, n=29 [AdGFP[; vs 87.0±5.4 nm, n=74 [AdMYH7 transduced]) 1 day after gene transfer. C, Calcium transient amplitudes were unaltered by AdGFP (123.1±12.0 nmol/L, n=29) and AdMYH7 (132.4±4.1 nmol/L, n=80) gene transfer (control: 140.7±6.5nmol/L, n=53). *Difference from control and AdGFP (P<0.005).

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).


Figure 4
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  		Figure 4. A, Resting and peak sarcomere lengths on consecutive days after AdMYH7 gene transfer (control: day 0, n=26; day 1, n=51; day 2, n=37; day 3, n=27; AdMYH7: day 0, n=34; day 1, n=74; day 2, n=43; day 3, n=42). B, Resting and peak calcium concentrations on consecutive days after AdMYH7 gene transfer (control: day 0, n=26; day 1, n=53; day 2, n=41; day 3, n=31; AdMYH7: day 0, n=36; day 1, n=80; day 2, n=52; day 3, n=43). AdMYH7 gene transfer affected only the peak sarcomere length (*significant difference from control, with P<0.001). C, When compared with control values, sarcomere-shortening amplitude was 42% lower on day 1, 57% lower on day 2 (138.9±13.0 nm [n=37] vs 59.7±7.1 nm [n=43]) and 53% lower on day 3 (135.1±16.3 nm [n=27] vs 62.4±8.3 nm [n=42]) after AdMYH7 gene transfer. *Significant difference from control (P<0.001); {dagger}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).


Figure 5
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  		Figure 5. AdMYH7 gene transfer slows cross-bridge cycling kinetics (ktr s–1) in {alpha}-MyHC–containing rat myocytes. A, Maximal calcium activated tension (pCa 4.5) was not affected by AdMYH7. B, The maximal rate of force development (pCa 4.5) was slower in AdMYH7-transduced myocytes (3.42±0.09 sec–1 [n=10] vs 2.38±0.09 sec–1 [n=9]). *P=0.000003 (t test). C, Original force recordings showing the ktr measurement. D, Exponential fits of the data in C. Data were fit to a single exponential equation: F=Fmax[1–exp(–ktr·t)]+Fres, where F is tension at time t, Fmax is maximal tension, and ktr is the rate constant of tension redevelopment. Fres represents any residual tension present immediately after the slack–restretch maneuver.

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.


Figure 6
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  		Figure 6. AdMYH7 effects on maximal velocity of sarcomere shortening (–µm/sec) and relengthening (µm/sec). A, Superimposed representative shortening traces collected from a control and an AdMYH7-transduced myocyte on day 2 after gene transfer. B, Normalized representative shortening traces (the peak shortening was set to 1.0 for each of the traces on the left). C, Compared with control myocytes, the maximal velocity of sarcomere shortening was slower in myocytes transduced with AdMYH7. Shortening velocity was 48% slower on day 1 after gene transfer (–2.45±0.22 [n=37] vs –1.27±0.10 µm/sec [n=59]), 58% slower on day 2 after gene transfer (–1.85±0.18 [n=31] vs –0.77±0.12 µm/sec [n=35]), and 62% slower on day 3 after gene transfer (–2.34±0.27 µm/sec [n=27] vs –0.87±0.11 µm/sec [n=39]) slower on day 3 after gene transfer. D, The maximal velocity of sarcomere relengthening was also slower in myocytes treated with AdMYH7. *Significant difference from control (P<0.0005).

Genetic titration of ß-MyHC into {alpha}-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 ({approx}0% on day 0, {approx}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.


Figure 7
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  		Figure 7. Acute genetic engineering of ß-MyHC into rodent cardiac myocytes attenuates sarcomere-shortening amplitude and shortening velocity in a dose-dependent manner. A, Nonlinear regression analysis of the relationship between the amount ß-MyHC (relative to total myosin) and sarcomere-shortening amplitude. B, Nonlinear regression analysis of the relationship between the amount of ß-MyHC and maximal sarcomere-shortening velocity. The data in both A and B were well described by a decaying exponential equation (r2=0.99).

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).


Figure 8
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  		Figure 8. ß-Adrenergic responsiveness (with 10 nmol/L ISO). A, Representative sarcomere-shortening traces for control (left column) and AdMYH7 transduced (right column) showing the effect of ISO on contractility. B, Summary of the effects of ISO on shortening amplitude. C, Summary of the effects of ISO on the time to 50% relaxation (RT50) of contraction. D, ISO increased contraction amplitude (change in sarcomere-length [{Delta}SL] amplitude) to the same extent (P>0.05) in control and AdMYH7-transduced myocytes. Similarly, ISO hastened relaxation ({Delta}RT50) to the same extent in control and AdMYH7-transduced myocytes. *Difference from baseline within a group (paired t test, P<0.0005); #difference between control and AdMYH7 at baseline or after ISO treatment (independent t test, P<0.0005).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
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 {alpha}-MyHC–dominant intact cardiac myocyte contractility. Using acute genetic engineering of the cardiac molecular motor, there was time-dependent stoichiometric replacement of the endogenous {alpha}-MyHC (fast motor) with ß-MyHC (slow motor). The main contractile effects of ß-MyHC expression in {alpha}-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 {alpha}-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 {alpha}-MyHC cross-bridges). Therefore, myocytes that predominantly express {alpha}-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.


*    Acknowledgments
 
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.


*    Footnotes
 
Original received November 1, 2006; revision received March 2, 2007; accepted March 7, 2007.


*    References
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 *References
 

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