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Cardiovascular Research 2002 55(2):300-308; doi:10.1016/S0008-6363(02)00406-6
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Copyright © 2002, European Society of Cardiology

Intracellular β-blockade: overexpression of G{alpha}i2 depresses the β-adrenergic response in intact myocardium

Paul M.L Janssena,b,*, Wolfgang Schillingera, J.Kevin Donahueb, Oliver Zeitza, Shahriyar Emamia, Stephan E Lehnarta, Joachim Weilc, Thomas Eschenhagenc, Gerd Hasenfussa and Juergen Prestlea

aDepartment Cardiology and Pneumology, University of Göttingen, Göttingen, Germany
bInstitute of Molecular Cardiobiology, Johns Hopkins University School of Medicine, 844 Ross Building, 720 Rutland Ave., Baltimore, MD 21205, USA
cInstitute of Pharmacology and Toxicology, University of Erlangen, Erlangen, Germany

* Corresponding author. Present address: Dept. of Physiology and Cell Biology, Ohio State University, 302 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210-1218, USA. Tel.: +1-614-247-7838; fax: +1-614-292-4888 janssen.10{at}osu.edu

Received 21 December 2001; accepted 11 March 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Increased levels of inhibitory G proteins have been observed in heart failure, but their physiological relevance in mediating the reduced β-adrenergic response is largely unknown. Methods: To evaluate the functional consequences of G{alpha}i2 overexpression, we studied myocardial contraction in intact isometric contracting cardiac rabbit trabeculae and isolated myocytes after adenovirus-mediated gene transfer of G{alpha}i2. Results: Neither G{alpha}i2 nor lacZ (control) overexpression altered baseline contractile force. After 72 h of continuous contractions, developed force (Fdev) increased after addition of 1 µM isoproterenol by 28.5±9.7 mN/mm2 in the control group, which was unchanged from the initial response at t = 0 h (23.7±3.8 mN/mm2). In sharp contrast, in preparations transfected with AdG{alpha}i2, the response to isoproterenol was significantly attenuated (5.9±2.0 vs. 27.6±4.2 mN/mm2, t = 72 vs. 0 h, respectively, P<0.01). In a primary culture of transfected isolated myocytes from a nearly identical baseline, isoproterenol increased cell shortening by 3.1±0.6% in the lacZ transfected myocytes, but only by 1.3±0.5% in G{alpha}i2 transfected myocytes (t = 72 h, P<0.01). In G{alpha}i2 transfected myocytes, pertussis toxin restored β-adrenergic responsiveness, indicating specificity of attenuation by the transgene. Conclusions: Overexpression of G{alpha}i2 attenuates the positive inotropic effects of β-adrenergic stimulation in myocardium. In addition, the method we developed allows investigation of a causal link between altered protein expression and subsequent alterations in contractile function in a physiological relevant in vitro manner.

KEYWORDS Adrenergic (ant)agonists; Contractile function; e-c coupling; G-proteins; Gene expression; Inotropic agents


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
A reduced β-adrenergic response is well documented in human heart failure and may result from alterations in the protein expression pattern including a down-regulation of β-adrenergic receptor density, up-regulation of inhibitory G protein (Gi), upregulation of RGS3 and RGS4, upregulation of GRK2, and reduced adenylyl cyclase activity [1–9]. When multiple alterations are present simultaneously (e.g. as in human heart failure), it is very difficult to determine the role played by the individual components to develop an understanding of the overall failure phenotype.

Adenoviral-mediated gene transfer has emerged as a useful tool to study the functional consequences of specific gene overexpression [10–14]. It has already been demonstrated that expression of gene products relevant to the failing heart can improve cardiac function. In isolated animal and failing human myocytes, adenovirus-mediated gene delivery of the SR Ca2+-ATPase improves contractility [11], and gene-transfer of K+ channels shortens action potential in failing myocytes [15]. In addition, a recently developed trabecula culture system [16–18] allows for the evaluation of protein expression in direct conjunction with physiological relevant assessment of cardiac contraction in vitro.

To study the relevance of upregulated levels of G{alpha}i2 the overexpression (as a proof-of-principle that represents upregulation) of G{alpha}i2 was used to provide further insight into the importance of this protein in human heart failure. Our results show that overexpression of G{alpha}i2 alone leads to inhibition of the β-adrenergic response; this finding may give further insight into the importance of this protein in human heart failure.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Preparation and ex vivo transfection of rabbit hearts
Adult female Chinchilla–Bastard rabbits (1.5–2.5 kg, n = 28) received heparin anticoagulation (1000 Units i.v.) prior to anesthesia (sodium thiopental, 50 mg/kg i.v.). Hearts were rapidly excised and retrogradely perfused by a modified Langendorff perfusion technique with oxygenated Krebs–Henseleit (K–H) buffer for 5 min at 30–40 ml/min. Transfection was done after pretreatment with low Ca2+ and serotonin (to increase transfection rate) [19] by 7.2x108 pfu/ml of recombinant adenovirus for 10 min at 37 °C with a controlled flow rate of 30 ml/min [20], resulting in a transfection efficiency of >90% of the myocytes. We have previously shown that this transfection protocol does not impact on contractile function of the trabecula over time [18]. Procedures regarding care and use of animals were performed in accordance with institutional guidelines and conforms with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Adenovirus vectors
AdlacZ encoding for β-galactosidase (E. coli) and AdG{alpha}i2 encoding for the inhibitory G-protein G{alpha}i2 (rat) were under control of the human cytomegalovirus immediate early promoter, in an adenovirus vector. High titer adenovirus-stocks were prepared and tested for replication competent adenoviruses [21]. Adenoviral titers were determined by plaque assays [18–20]. Although we have previously [18] shown that no deleterious effects of transgene transfection is observed in our system, we used the lacZ virus rather than sham transfection as a control to circumvent non-specific effects of transgene overexpression.

2.3 Muscle preparation, experimental set-up, and protocol
Thin, uniform myocardial trabeculae and small papillary muscles from the free wall and septum of the right ventricle were dissected [16]. Average dimensions of the preparations (n = 60) were 2.70±0.12 mm long, 399±20 µm thick, and 497±22 µm wide. Preparations were mounted in a closed sterile chamber (Scientific Instruments GmbH, Heidelberg, Germany) between a force transducer and a micromanipulator and equilibrated in M-199 cell-culture medium. All experiments were executed under isometric conditions (diastolic force set to 2–3 mN/mm2) for 72 h, 1 Hz stimulation frequency, 37 °C, at pH 7.4. Solution and gas mixture were refreshed at least every 12 h. Experiments were excluded when bacterial contamination or technical problems occurred, or when rundown (>40%) of Fdev during the first 3 h occurred, or in which baseline force at t = 0 h was <2 mN/mm2.

To investigate the response of β-adrenergic stimulation, concentration–response curves (10–9–10–6 M) of isoproterenol were measured. This protocol was done after contractile parameters had stabilized (t = 0 h), and thereafter repeated every 24 h.

2.4 Myocyte isolation, experimental set-up and protocol
After dissection of the heart as described above, myocytes were harvested using the protease/collagenase perfusion method, and placed in primary culture [22,23], and plated onto laminin coated plates at 0.5x105 rod-shaped cells/cm2. Either AdG{alpha}i2 or AdlacZ was added at a multiplicity of infection (MOI) of 10. After 2 h unattached cells and residual virus was removed. Shortening of myocytes was measured at 1 Hz stimulation frequency, 37 °C, in circulating K–H solution containing 1.75 mM Ca2+. Prior to the shortening measurements, myocyte dishes were blinded to avoid bias for myocyte selection by the investigator. An additional protocol with pertussis toxin (50 nmol/ml added 24 h prior to measurement) was performed to confirm G{alpha}i2 specific effects of the inhibiting action on β-adrenergic stimulation. The 48-h post-transfection myocyte shortening and full-dose (1 µM) isoproterenol response were measured in G{alpha}i2 transfected myocytes with and without PT. In this protocol two myocytes were excluded from analysis because of unstable behavior; these two myocytes also happened to contract weakly (<4% of diastolic length), reflecting possible damage. Because there was one in each group, it did not affect outcome of the results.

2.5 Detection of transgene expression
After 72 h of continuous contractions multicellular preparations were fixed in situ in 4% formalin and imbedded in paraffin. Thin, 10-µm sections were stained with using a monoclonal primary antibodies against lacZ β-galactosidase (clone GAL-13, Sigma; 1:1000) or G{alpha}i2 (clone L5.6, NeoMarkers; 1:250), and peroxidase-coupled secondary antibodies using the HistostainTM-plus kit (Zymed, CA, USA). For Western immunoblot protein analysis, protein lysates of cultured myocytes (30 µg of total protein) were subjected to SDS–PAGE (without urea) and transferred to nitrocellulose membranes by electroblotting. Immunodetection was performed using monoclonal mouse antibodies (G{alpha}i2, clone L5.6, 1:400; and β-Gal, clone GAL-13; 1:2000). Visualization of immunoreactive bands was performed with peroxidase-labeled secondary antibodies using an enhanced chemiluminescense detection kit (ECL, Amersham).

2.6 Data analysis and statistics
Multicellular preparation data were analyzed using two-factor repeated measures ANOVA: We tested subjects (muscle), with factors virus (AdlacZ or AdG{alpha}i2), and time (0, 24, 48, and 72 h), Student–Newman–Keuls’ post-hoc test was applied for multiple comparisons between means. For statistical analysis, only the preparations with complete data sets were included (n = 8/group). Myocyte shortening experiments were analyzed similarly. Statistical significance was determined by Student's t-test for paired or unpaired data where applicable. Data are presented as means±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Transgene expression
Preparations that maintained a contractile response for 72 h were stained for transgene expression. Transgenes were homogeneously expressed throughout the preparations (Fig. 1). In contrast to non-infected preparations (Fig. 1AA), AdlacZ transfected preparations showed high level expression of the lacZ transgene (Fig. 1AB). Preparations transfected with AdlacZ showed low-level G{alpha}i2 staining (Fig. 1AC), and overexpression of G{alpha}i2 is clearly seen in the AdG{alpha}i2 transfected preparations (Fig. 1AD). Western blotting of transfected myocyte cultures revealed a high level expression of the transgene 48 h post-transfection. The expression of the G{alpha}i2 increases at increasing MOI (Fig. 1B). The upper, constant band was visible in absence of any primary antibody as well, and therefore must have resulted from non-specific binding of the secondary antibody.


Figure 1
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  		Fig. 1 Left panel (A): (A) Non-transfected muscle stained for lacZ expression indicates absence of false-positive staining; (B) lacZ transfected trabecula stained for lacZ shows that nearly all myocytes are transfected; (C) lacZ transfected trabecula stained for G{alpha}i2 expression indicating the low-level, native expression of G{alpha}i2; (D) G{alpha}i2 transfected muscle stained for G{alpha}i2 demonstrating widespread expression of the transgene. Inserts of the panels depict sections without primary antibody incorporation (negative control). Right panel (B): Western blot depicting overexpression of G{alpha}i2 in isolated myocytes. At increasing multiplicity of infection (MOI) more transgene product is formed. Bar indicates 50 µM.

 
3.2 Baseline contractile parameters of multicellular preparations
There were no significant differences in baseline measurements between the AdlacZ and the AdG{alpha}i2 groups. The initial developed force (Fdev) of all initially started experiments was 4.6±0.7 mN/mm2 in the lacZ group (n = 30) and 5.8±0.7 mN/mm2 in the AdG{alpha}i2 group (n = 30, P = NS). The diastolic force (Fdia) at t = 0 h was 2.4±0.2 and 2.3±0.3 mN/mm2 for lacZ- and G{alpha}i2 transfected preparations and twitch timing parameters were also similar in both groups. Both groups showed fluctuations in force development over time. In the muscles that contracted for 72 h, after 48 h of continuous contractions, Fdev had increased from 7.6±2.0 to 10.7±1.9 mN/mm2 in the lacZ group (n = 8) and from 7.3±1.8 to 10.6±3.3 mN/mm2 in the G{alpha}i2 group (P = NS). After 72 h, Fdev returned close to the initial value in the lacZ and there was a slight, but non-significant increase in the G{alpha}i2 group. Although initial twitch timing parameters were similar in both groups, both time and virus affected these. In both groups, after 24 h time to peak tension (TTP) and time from peak tension to 50% relaxation (RT50%) had increased (Table 1). From 24 to 72 h both these parameters remained constant in the lacZ group, but increased further at both 48 and 72 h in the G{alpha}i2 transfected muscles.


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  		Table 1 Effect of myocardial contraction in intact isometric-contracting cardiac rabbit trabeculae and isolated myocytes after adenovirus-mediated gene transfer of G{alpha}i2

 
3.3 β-Adrenergic response in multicellular preparations
In lacZ transfected muscles, isoproterenol significantly increased developed force at t = 0 (Fig. 2C). Over the next 72 h, this response remained unchanged. Every 24 h, starting at t = 0, an isoproterenol dose–response curve was measured, the resulting increase in Fdev can be seen by the peaks in force every 24 h (Fig. 2A). In Fig. 2C, the average response to the highest concentration (1 µM) of isoproterenol is given. In sharp contrast, both the example of a representative G{alpha}i2 transfected muscle (panel B) and the average response of this group (panel D) show that over time the isoproterenol response became severely attenuated. In the G{alpha}i2 group, at t = 48 and 72 h the response was significantly lower than in the lacZ group (P<0.05), and was also significantly lower (P<0.05 at t = 48, P<0.01 at t = 72 h) than its initial response at t = 0. In this G{alpha}i2 group, addition of calcium (to 5 mM) at t = 72 h could further increase force at 1 µM isoproterenol, indicating that the contractile reserve is preserved, but that this reserve can not be accessed with β-adrenergic stimulation.


Figure 2
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  		Fig. 2 (A) Developed force of an individual AdlacZ transfected trabecula. Peaks arise from the response to concentration–response curves of isoproterenol (Iso); top of the peak is response to 1 µM and this response remain stable over 72 h. (B) Force tracing of a single trabecula transfected with G{alpha}i2. The Iso response is progressively attenuated. (C) Average response of lacZ transfected preparations. Numbers in columns represent number of preparations. (D) Average response of G{alpha}i2 transfected preparations. * P<0.05 vs. lacZ group, same day; & P<0.05 vs. t = 0 h.

 
Every 24 h, a dose–response curve of isoproterenol (1 nM–1 µM) was assessed. As can be seen from Fig. 3, the maximal response became attenuated in the G{alpha}i2 group, but not in the lacZ group. Plotted to their respective maximal response (panel B and C), it can be clearly seen that EC50 for isoproterenol response was unaltered over time for both lacZ and G{alpha}i2 groups.


Figure 3
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  		Fig. 3 (A) Dose–response curve assessed at t = 0, 24, 48, and 72 h in both groups, absolute response. In G{alpha}i2 overexpressing muscles, the maximal response becomes attenuated over time. (B) Plotted relative to the maximum response, it can be seen that no significant changes occur in the EC50 in AdlacZ transfected muscles. (C) No significant change in EC50 was observed in AdG{alpha}i2 transfected muscles.

 
Twitch timing parameters (Table 1) shortened with isoproterenol exposure. At t = 0 h, 1 µM isoproterenol shortened TTP in lacZ transfected muscles from 143±6 to 111±5 ms (P<0.01, n = 8) and in G{alpha}i2 transfected muscles from 130±4 to 105±3 ms (P<0.01). Over time, the response in lacZ muscles remained similar. Isoproterenol shortened RT50% to 67±3% at t = 0, and to 67±3, 65±2, and to 67±4% at t = 24, 48 and 72 h, respectively. In G{alpha}i2 transfected muscles, this was not the case; RT50% shortened significantly less over time (59±4, 70±2, 79±4, and 84±5% at t = 0, 24, 48, and 72 h, respectively, P<0.05). Results for TTP were similar.

3.4 Isolated myocyte shortening
From 11 hearts, isolated myocytes were transfected with AdG{alpha}i2 or AdlacZ. Two days post-transfection, baseline shortening was slightly higher (4.89±0.22%, n = 72) in G{alpha}i2 transfected myocytes compared to lacZ-transfected myocytes (4.21±0.21%, n = 72, P<0.05). At 72 h post-transfection, a small difference was still present but not significant (Table 1). Other contractile parameters studied were not different between the two groups neither at day 2 nor at day 3 post-transfection.

Similar to the experiments on the multicellular preparations, overexpression of G{alpha}i2 resulted in an attenuation of the β-adrenergic response in isolated myocytes. After 2 days in primary culture, addition of 1 µM isoproterenol had a more pronounced effect on AdlacZ infected myocytes (shortening increased from 3.71±0.33 to 7.24±0.51%, n = 21) than on AdG{alpha}i2 infected myocytes (4.58±0.45 to 5.77±0.71%, n = 20, P<0.05). Thus, lacZ transfected myocytes responded to isoproterenol by an average increase in shortening of 99±16% and G{alpha}i2 transfected myocytes by only 32±16% (P<0.05). From the individual concentration response curves data (from day 2) were transformed to reflect the relative response to isoproterenol (0 at base, 1 at 10–6 M), depicted in Fig. 4. The EC50 was 1.2±0.2x10–8 M in lacZ transfected myocytes, and 7.2±1.4x10–8 M in G{alpha}i2 transfected myocytes. Day 3 data from G{alpha}i2 myocytes (see Table 1) did not allow for accurate assessment of individual EC50 values because of the very low response to isoproterenol. EC50 in lacZ overexpressing myocytes was 2.6±0.4x10–8 M. Although in freshly isolated myocytes both baseline shortening (7.5±0.5% of cell length) and the isoproterenol response (increase by 7.7±0.9% of cell length) were much larger compared to either day 2 or day 3 after transfection (P<0.01), such observation (i.e. decreasing contractile function over time) is always observed in primary cultures of adult myocytes.


Figure 4
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  		Fig. 4 Average values for baseline shortening and Iso response for lacZ (open symbols) and G{alpha}i2 (closed symbols) transfected myocytes at t = 48 h.

 
3.5 Pertussis toxin treatment
Pertussis toxin treated myocytes showed a larger increase in isoproterenol induced shortening as non-treated cells. Calculated from the individual experiments, in the pertussis experiments, G{alpha}i2 transfected non-treated cells, β-stimulation increases shortening by 43.7% (from 5.59±0.44 to 8.09±0.45%) compared to 42.9% in the initial group of experiments, this magnitude is nearly identical. Pertussis toxin-treated G{alpha}i2-overexpressing cells increased shortening by 76.9% (5.58±0.30 to 9.69±0.83%), and this was not different from lacZ transfected myocytes initially studied (93.3%, P = 0.081). Thus, pertussis toxin (largely) restores the isoproterenol effect. This confirms specificity of G{alpha}i2 induced attenuation of the β-adrenergic response.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
This study reports attenuation of the β-adrenergic inotropic response on a subcellular level by overexpression of the inhibitory G-protein G{alpha}i2. In G{alpha}i2 overexpressing preparations, the response of the β-adrenergic stimulant isoproterenol was greatly reduced. As more gene-product was formed over time in G{alpha}i2 overexpressing muscles, the increase and acceleration in force development after addition of isoproterenol became more attenuated, indicating a dose-dependency. An additional aspect of this study is the demonstration of the ability to successfully transfected intact cardiac tissue ex vivo with a functional relevant gene and to detect contractile differences and investigation of causality between altered protein expression and altered contractile function in a physiologically meaningful in vitro setting.

4.1 Inhibition of the β-adrenergic response
The data presented here indicate that overexpression of the inhibitory G-protein G{alpha}i2 reproduces the altered β-adrenergic response. Although increased levels of G{alpha}i2 have already been observed in human end-stage heart failure [2,4], this is paralleled by the down-regulated β-adrenergic receptor density [1] and upregulated β-receptor kinase [5]. The occurrence of these parallel processes precludes determination of both causality and impact on contractile function. The same complication may potentially hamper conclusions drawn from animal models. In a rat model, it has recently been shown that increased Gi protein levels attenuate the β-adrenergic response [24], but again β-adrenoceptor density was reduced. Additionally, in contrast to the human heart, rat myocardium possesses a large number of so-called ‘spare receptors’ [25], and the role for decreased receptor density is therefore potentially underestimated in such models. We observed a progressive loss of β-adrenergic response over time in preparations overexpressing G{alpha}i2, this in contrast to preparations expressing LacZ that upheld the β-adrenergic response over time, while neither group showed changes in the EC50. The fact that LacZ-transfected muscles displayed a constant β-adrenergic response over time in close agreement to our prior observation in this model that long term culture does not affect the β-adrenergic response [16,18], warrants the deduction that an altered response must be due to the transgene. The increased loss of response over time is very likely due to the increased expression of the transgene; the time-course of expression correlates well with an earlier report describing transgene production after adenoviral gene transfer [26]. Although it remains unclear whether G{alpha}i2 upregulation is part of the vicious cycle of the blunted positive inotropic effect after β-adrenergic stimulation or whether it represents a protective mechanism to attenuate the effect of adrenergic overstimulation, we showed that specific overexpression of G{alpha}i2 suffices to severely blunt the β-adrenergic response. Pertussis toxin treatment of G{alpha}i2 transfected myocytes confirmed that the attenuating effects of overexpression of G{alpha}i2 on the β-adrenergic response was a specific transgene induced attenuation. As underlying mechanism, it is clear that the reduction in β-adrenergic response is coupled to processes involving the adenylyl cyclase pathway, as recently reported in abstract form by Rau et al. [27].

β-Adrenergic blockade has in the last years emerged as a key strategy to treat heart failure [28]. Thus, the upregulation of G{alpha}i2 as observed in heart failure is likely to be a positive compensatory response rather than involved in the worsening of cardiac function. Although gene therapy is currently not an option for the treatment of heart failure, this study indicates a possible target is intracellular β-blockade via upregulation of G{alpha}i2. With the development of less immune response provoking vectors and cardiac specific promoters, one could envision a cardiac specific β-blockade, without a significant impact on vasculature β-adrenergic blockade. Although this could be achieved via β1-selective antagonists, gene-therapy may be used to focus the attenuation even more locally; it has recently been shown that targetted overexpression of G{alpha}i2 in the AV node attenuates atrial fibrillation [8]. Of note, it has recently been shown that RGS3 and RGS4, proteins that are involved in regulating G proteins like G{alpha}i2 is also increased in heart failure [9]. Thus, evidence is mounting that controlling the expression and function of G{alpha}i2 may open up potential therapeutic strategies for the treatment of heart failure. Further studies are needed to address in detail the signaling changes involved in the down-regulated β-response, as well as coupling of over-expressed G{alpha}i2 to β-receptors.

4.2 Use of cultured multicellular preparations as a tool
Previous studies have mostly used an isolated primary myocyte culture to study the effects of adenoviral gene transfer on unloaded shortening behavior as a parameter of contractile function. However, the functional study of adenovirus-infected cultured cardiac myocytes is limited by their progressive dedifferentiation and loss of contractility over the expression time [29,30] and by technical difficulties to qualitatively evaluate and to extrapolate contractile function of isolated cells to the more physiologic multicellular myocardial architecture accomplishing loaded contractions. This was also evident from our data; in the myocyte measurements we observed that after day 2, but not after day 3, the myocytes transfected with G{alpha}i2 had a slight increase baseline shortening. However, it is known that shortening in adult rat myocytes declines per se within the first 48 h by 30–50% [30] very comparable to the average decline of 40% observed in this study. These results imply that in myocytes we cannot unambiguously determine whether G{alpha}i2 increases contractility, or whether it represents an attenuated loss of shortening compared to LacZ transfected myocytes over time. Also, it is not clear whether other components that impact on the β-adrenergic response are up- or downregulated in isolated primary myocyte culture. This may even be likely, because isoproterenol-induced increases in cell shortening were overproportionally (compared to the decline in baseline shortening) reduced compared to freshly isolated myocytes. Thus, the progressive decline in myocyte function over time per se may hamper unambiguous interpretation of the data, including the observed shift in sensitivity that was observed at day 2 (but not at day 3).

The culture of myocardial preparations used in this study may potentially have several advantages over the culture of isolated cardiac myocytes. It represents (i) the more complex situation of gene delivery into a differentiated multicellular architecture [12,19,20], (ii) the more physiologic situation of loaded contractions [31,32], (iii) possibility of factors (e.g. NO, endothelin) secreted by one cell type influencing the function of other cell types in the heart, and (iv) the stable contractile behavior, protein synthesis and non-dedifferentiating cellular integrity of electrically stimulated myocardial preparations [16–18]. In addition, the recent demonstration of preserved contractile function after gene-transfer [18] and the long-term culture of human trabeculae up to 6 days [17] suggests the possibility of using this gene transfer protocol to investigate pathophysiological alterations in the failing human heart.

Although multicellular preparations have distinct advantages over isolated myocytes in the study of contractile function after gene-transfer, the culture of myocardial trabeculae has its own limitations. We have observed fluctuations in contractile force under baseline conditions, as reported before [16,18]. Although the G{alpha}i2 overexpressing preparations tended to increase in developed force over time compared to the lacZ group, these differences did not reach statistical significance. Twitch timing however did slow down over time, we even observed a difference between G{alpha}i2 and lacZ transfected preparations. In G{alpha}i2 preparations, the time to peak tension slowed more than in the lacZ group. Although this may be due experimental variability, it could possibly be due to the depressed adenylyl cyclase activity; if a baseline activity would be present, and inhibited in the preparations after overexpression of G{alpha}i2. This could also possibly explain the observed (but non-significant) changes in developed force in the G{alpha}i2 group. However, more data would be needed to unambiguously discriminate between these two possibilities. A second limitation of the trabecula culture technique is the technical difficulty and success rate of the experiments. Not always are suitable preparations found in a heart, and over time in the experimental set-up problems occur (mainly contamination issues, which are increased if the chambers need to be repeatedly opened to inject drugs and exchange media) that terminate experiments prematurely. Although this drop-out can be limited to only 15–30% for baseline studies [16–18], for more complex studies this drop-out amounts to around 50% (at 48 h), and up to 75% for 72-h experiments. Still, the fact remains that preparations (in experiments devoid of contamination/technical problems) on average do not decrease in contractile force even after 3 days in the system. This is in sharp contrast to isolated myocytes; after 3 days in primary culture the average fractional shortening drops to only 40% of their initial value, hampering unambiguous interpretation of the data so obtained.

4.3 Summary
In summary, we show in this proof-of-principle study that up-regulating levels of G{alpha}i2 induces a severe attenuation of the β-adrenergic response in intact myocardium, and potentially opens up the possibility for an intracellular level β-blockade. The second aspect of our investigation is that we now show for the first time that the method of culturing multicellular preparations can be used to directly study the link between altered protein expression with changes in cardiac contractile properties under near physiological conditions.

Time for primary review 25 days.


   Acknowledgements
 
We wish to thank Drs Koch, Peppel and Lefkowitz of Duke University for their help in constructing the G{alpha}i2 adenovirus. We acknowledge the support of the Deutsche Forschungsgemeinschaft, HA 1233/3-2.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Bristow M.R., Ginsburg R., Minobe W., et al. Decreased catecholamine sensitivity and beta-adrenergic-receptor density in failing human hearts. New Engl J Med (1982) 307:205–211.[Abstract]
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