The glitter of gold: biolistic transfection of fresh adult cardiac myocytes. Focus on “Normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes by a biolistic method”

examining a protein outside of its native context is like studying shark behavior in an aquarium. One can learn a lot about how the fish swims, but appreciating how it tracks and captures its prey in the ocean requires imagination. Heterologous expression systems, the fish bowls of cell biology, provide a window into function within a tightly controlled environment. As they often do not faithfully recapitulate a protein's native milieu, they may fail to reveal key aspects of regulation or function. Adrenergic potentiation of the cardiac Ca²⁺ current, a cornerstone of the fight-or-flight response, was described more than 40 years ago (11), yet this phenomenon still cannot be reproduced in heterologous expression systems and the underlying mechanisms remain hotly debated (9). Although recent advances in gene expression strategies have rendered primary cells more pliable to the controlled manipulations offered by heterologous expression systems, certain cell types remain intractable. Among these, adult ventricular cardiomyocytes pose particular challenges. Thus, the report by Dou et al. (3), which describes the efficient expression of functional Kv1.5 K⁺ channels with a “gene gun,” represents a potentially important advance in dissecting the roles of cardiac proteins in their native context.

The impediments in the use of adult ventricular cardiomyocytes are twofold. First, they cannot be efficiently transfected by methods typically used with immortalized cell lines. Adult ventricular cardiomyocytes are terminally differentiated cells and do not divide, so liposomal or chemical strategies for transfection are ineffective. Second, adult cardiac myocytes are difficult to maintain in culture without dedifferentiation and associated loss of myocyte-specific morphological characteristics (7).

Most investigators have therefore used viral vector-mediated gene transfer. Adenovirus was used first in the 1990s and proved to be very efficient in rat (8), rabbit (4), and mouse (14) adult ventricular myocytes. Although there is question as to whether adenoviral-mediated gene transfer disrupts cellular physiology by promoting dedifferentiation (13), this technique has enjoyed substantial success. For example, Zhou et al. (14) were able to express either β₁- or β₂-adrenergic receptors and rescue contractile and calcium current responses to β-adrenergic receptor stimulation in β₁/β₂-receptor double-knockouts. Lentivirus has also been used effectively and offers the advantage of having about twofold greater transgene capacity relative to that of adenoviruses (2). A shortcoming, however, is a very low transduction efficiency. Recently, calcium phosphate coprecipitation has been demonstrated to increase efficiency of lentiviral transduction in cardiomyocytes (12). Unfortunately, even with greater transduction efficiency, low transgene expression and the relative difficulty in generating the viruses remain impediments.

Given these obstacles to studying cardiac cellular physiology, the report by Dou et al. showing successful transfection of freshly isolated adult rat ventricular myocytes with a biolistic (short for biological ballistics) method using a gene gun represents a potentially important advance. The authors' objective was to characterize the subcellular localization of Kv1.5 K⁺ channels, which underlie the ultrarapid delayed rectifier K⁺ current, I_Kur (6). These channels have garnered considerable attention because their restricted expression in humans to atrial (and not ventricular) myocytes makes them an attractive target for pharmacological treatment of atrial fibrillation, the most common cardiac arrhythmia. In rats, however, Kv1.5 is also expressed in ventricular myocytes (5). Dou et al. addressed the debate regarding whether the apparent localization of Kv1.5 to the intercalated disc is an artifact from “sticking” of anti-Kv1.5 antibodies to that structure. Transfection of functional channels into ventricular myocytes [by using a lipofectamine protocol previously described for atrial cardiomyocytes (1)] was hampered by the requirement for serum in the posttransfection media and the prolonged time required for gene expression, which led to rounding up of the cardiomyocytes and an inability to track the subcellular localization of the channel.

As an alternative, Dou et al. adapted the gene gun approach. This biolistic method, which uses high-speed propulsions of subcellular particles (typically gold) coated with DNA, has been successfully employed in neuronal tissue sections and primary neuronal culture systems, both of which share with ventricular myocytes the difficulty of transfection into terminally differentiated cells (10). Previous attempts to use this technique for ventricular myocytes failed to produce surviving, transfected myocytes—an experience that Dou et al. initially replicated. Systematic alterations to the standard protocol (e.g., omitting polyvinylpyrrolidone during preparation of the cartridges, and optimization of both gold particle size and bombardment pressure), however, yielded a transfection efficiency of almost 30% of surviving myocytes in less than 24 hours, and the transfected cells remained viable and retained their morphological characteristics. In cells transfected with labeled Kv1.5, Dou et al. confirmed the localization of these channels at intercalated disks and demonstrated an increased, sustained K⁺ current component sensitive to 100 μM 4-aminopyridine, a Kv1.5 blocker. While these results establish that Kv1.5 channels are, indeed, localized to intercalated disks and, through use of expressed, truncated channels, identify channel determinants for proper trafficking to the plasma membrane, the most interesting result may be the demonstration of an effective and simple way to transfect genes into cardiac ventricular myocytes.

Although transfection by gene gun may not be suitable for all applications, it offers significant advantages over viral transduction for certain experimental questions. For example, the opportunity to use plasmid DNA obviates the need for generating viruses, which should save considerable time and effort that could then be applied to experimental studies, such as more thorough and creative testing of multiple constructs and mutants (see Table 1 for a comparison of transgene expression techniques). Moreover, the maintenance of the normal rod-shaped myocyte morphology after transfection suggests that information gained from gene gun transfections will provide valuable insights into the function of cardiac myocytes.

Table 1.

Comparison of methods for transgene expression in terminally differentiated cells

Advantages	Disadvantages
Chemical transfection*
• Well characterized in primary neuronal culture	• Possible alteration of cell physiology
• Plasmid DNA, no lengthy production methods	• Toxicity
• Safety	• Dedifferentiation in cardiomyocytes
Adenoviral transduction
• High efficiency	• Potential dedifferentiation of cells
• Robust gene expression	• Limit of transgene size
• No integration into the genome	• Length of time for generation (4–6 wk)
Lentiviral transduction
• Trophic for nondividing cells	• Low efficiency
• Twofold greater transgene capacity compared with adenovirus	• Transduction efficiency sensitive to culture conditions
• Relies on normal cell function for its survival	• Length of time of generation (2–3 wk)
Biolistic transfection
• Plasmid DNA, faster preparation of transgene	• Low efficiency, not ideal for biochemical studies
• Fast expression (within 24 h)	• High rate of cell death from bombardment
• No overt physiologic effects on cells	• Reproducibility

^*Calcium phosphate or lipofectamine.

Despite these bright spots, there are some areas in which this technique might lack luster. As reported by Dou et al., the gene gun bombardment method kills 90% of the viable myocytes, and of the 10% surviving, only ∼30% are transfected. Thus, this technique may be especially suited for single-cell experiments, e.g., imaging and electrophysiology as performed in this study, in which the ability to perform analysis in transfected myocytes transcends the need for highly efficient transfection; in contrast, the relatively low level of transfection will render biochemical analyses challenging. Dou et al. reported an interesting change to the standard protocol for preparing the cartridges by omitting polyvinylpyrrolidone. Polyvinylpyrrolidone acts as an adhesive for the gold particles to the plastic during cartridge preparation. Most in vitro protocols call for a low concentration of polyvinylpyrrolidone and thus perhaps its omission is critical to successful biolistic gene transfer in cardiomyocytes.

As for all new techniques, it will be essential to determine whether the success reported by Dou et al. is reproducible by other groups. Even so, while use of the gene gun may not yet supply the silver bullet missing from the myocyte researcher's armamentarium, this report by Dou et al. shines with promise.

GRANTS

This work was supported by National Institutes of Health Grants T32-GM007171 (to J. J. Amenta) and R01-HL071165 and R01-HL088089 (to G. S. Pitt).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.