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1 Institute of Molecular Cardiology, Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York
2 Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio
3 Department of Physiology and Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, Utah
4 Institute of Molecular Cardiology, Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, New York
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ion channel; gene regulation; ion transport; cardiac myocyte
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Neither of these principles is an inviolate law; rather, they are thought to reflect the most common mechanisms used in the evolution of developmental systems. The applicability of these ideas to the evolution of physiological systems either has been brought into question (16) or has been uncertain (6). There is, in principle, no reason why these same concepts should not apply to the evolution of physiological systems, and we have chosen to address this issue by studying the evolution of the electrophysiological properties of mammalian cardiac ventricular myocytes.
A primary constraint on the electrophysiological properties of the mammalian heart is the scaling of body weight. Heart weight scales directly with body weight, and there are no significant changes in the morphology of either the heart or the cardiac myocytes (21, 30). Similarly, several fundamental physiological properties of the cardiovascular system, including mean arterial pressure and minimum diastolic pressure (11, 17), are highly constrained and remain relatively invariant across mammalian phylogeny. These constrained physiological properties reflect the core function of the system: to maintain arterial blood pressure at a level sufficient to ensure adequate organ perfusion, particularly for critical organs such as the brain.
In contrast, many of the electrophysiological properties of the heart show systematic changes with body weight. These include heart rate (17, 38, 41), action potential duration (the rate of action potential repolarization), action potential morphology, and the rate of calcium ion reuptake (3). The electrophysiological properties of the heart vary in a systematic fashion in order to compensate for the changes in the physical properties of the vasculature and the heart produced by scaling of body and heart weight. These concerted changes in electrophysiological function act to maintain the constrained physiological properties, such as mean arterial blood pressure, largely unchanged across mammalian phylogeny and are a critical factor in the ability of mammals to assume such a large range of body sizes (17, 48).
This report focuses on the electrophysiological properties of left ventricular myocytes, the primary function of which is the maintenance of arterial blood pressure. This function is both relatively simple and well understood within the context of the whole animal physiology. Ventricular myocytes are also one of the most intensively studied and best understood systems at the level of cellular physiology, with a correspondingly detailed knowledge of the molecular biology of ion channel and transport function (3, 24), and, as such, they provide an unparalleled system in which to study the evolution of electrophysiological traits. We describe the mechanisms that produce changes in one specific trait, action potential morphology, and examine the relative importance of regulatory and structural evolution in the scaling of action potential duration and calcium reuptake.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Choice of species.
Mammalian species that are common experimental models for cardiac electrophysiological studies were chosen for study. The eight species used in the study encompass a broad range of body weights and show the typical allometric relationship between body size and heart rate or action potential duration observed for most terrestrial mammals (Fig. 1, A and B). These species were human (Homo sapiens), canine (Canis familiaris), ferret (Mustela putorius furo), rabbit (Oryctolagus cuniculus), guinea pig (Cavia porcellus), rat (Rattus norvegicus), Chinese hamster (Cricetus griseus), and mouse (Mus musculus).
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Complementary DNAs were prepared as previously described (32). Three independent primer pairs for each gene were used for mRNA quantitation by real-time PCR, which was performed with the SYBR Green QuantiTect PCR Kit (Qiagen). Experimental samples were analyzed in triplicate. Expression values for a given gene were the average of results from three independent sets of eight RNA samples. Real-time PCR products were sequenced to confirm that the amplicons were from the mRNA of interest.
Isolation and sequencing of genomic DNA regions from hamster and guinea pig.
Bacterial artificial chromosome (BAC) clones encompassing the Kv2.1 proximal promoter regions were identified in BAC libraries (CHORI) with a nonradioactive probe labeled with digoxigenin-11-dUTP (DIG-11-dUTP alkali-labile, Roche). Probe sequences were based on cDNA sequences or conserved regions from multiple species, and positive clones were detected with anti-DIG-AP, Fab fragments (Roche), and CDP-Star (Roche). Specific DNA fragments of interest were isolated from positive BAC clones (BACPAC Resources Center) by a combination of restriction mapping and Southern blotting, and subfragments were subcloned into pBluescript for sequencing. DNA sequences were submitted to GenBank (accession nos. EU643795 and EU643796).
Subcloning of proximal promoter regions.
Comparisons of Kv2.1 and Kv4.2 proximal promoter sequences were performed with the Vista alignment program (12), and conserved regions were used as landmarks to select orthologous sequences from the two genes, although these were not identical in length (see Supplemental Material).1
For both genes the selected sequences terminated immediately before the initiator methionine in the first exon. DNA fragments for the Kv2.1 (mouse 1,601 bp, hamster 1,680 bp, guinea pig 1,786 bp, human 1,891 bp) and Kv4.2 (mouse 2,590 bp, human 2,567 bp) genes were subcloned from BAC clones into a luciferase reporter plasmid (pGL2, Promega).
Rat neonatal myocyte transfection, culture, and luciferase assay.
Neonatal rat cardiomyocytes were isolated and cultured as described previously (51). Transfection was performed with the Rat Cardiomyocyte Nucleofector Kit (Amaxa) in a Nucleofector I device (Amaxa). Each sample included an internal control Renilla luciferase plasmid (phRL-SV40, 1,000-fold lower concentration than test plasmids). Negative (pGL2-basic) and positive (pGL2-control) controls were also included in each experiment. After electroporation the cells were plated onto fibronectin-coated 12-well plates and cultured at 37°C in 5% CO2 for 48 h. Cell survival was 
35%. Luciferase assays were performed with the Dual Luciferase Reporter Assay Kit (Promega). Firefly and Renilla luciferase activities were measured with a Lumat luminometer (Berthold).
Myocyte electrophysiology.
Preparation of guinea pig and canine myocytes was performed as described previously (10, 44), and mouse ventricular myocytes were isolated by the same method as that used for guinea pig. For the recording of Ca2+ currents, isolated ventricular myocytes were maintained at room temperature and perfused with a Na+- and K+-free solution that contained (in mM) 137 TEA-Cl, 5.4 CsCl, 2 CaCl2, 1 MgCl2, 5 HEPES, 10 glucose, and 3 4-aminopyridine (pH = 7.4). Glass pipettes were filled with solution containing (in mM) 115 Cs-aspartate, 20 CsCl, 11 EGTA, 10 HEPES, 2.5 MgCl2, and 2 Mg-ATP (pH = 7.2) and had a resistance of 1.5–2.5 M
. After the membrane was ruptured, cells were clamped at –60 mV for 10 min to allow dialysis of the intracellular solution and stabilization of the Ca2+ currents before measurement of Ca2+ currents began.
Perforated patch-clamp recordings were used for action potential recordings and the dynamic clamp studies. Glass pipettes were backfilled with a pipette solution containing (in mM) 110 K-aspartate, 20 KCl, 8 NaCl, 10 HEPES, 2.5 MgCl2, and 0.1 CaCl2 and 240 mg/ml amphotericin B (pH adjusted to 7.2 with KOH). Cells were studied once stable series resistances <7 M were achieved. All action potential and contraction experiments were performed at 34°C.
Dynamic clamp experiments were performed as previously described (10, 44). A modified version of the Windows-based DynClamp software was used in the dynamic clamp studies (27). Voltage sampling of the dynamic clamp software and output of the current injection command were through an Axon Digidata 2100 A/D board. The transient outward potassium current (Ito) was defined as a rapidly and fully inactivating outward current, formulated as described previously (10).
Myocyte contraction was imaged with a charge-coupled device camera, and cell length and shortening were measured with a video edge detector (Crescent Electronics).
Expression of KCNH2 and KCNQ1 channels.
The human KCNH2 and KCNQ1 cDNA clones have been described previously (36, 37). Full-length guinea pig cDNA clones were derived with a combination of rapid amplification of cDNA ends (RACE) and standard PCR as described previously (40) (accession nos. EF204534 and EF204535). Channel expression and recording with Xenopus oocytes were performed as described previously (36, 37). The decaying phase of tail current traces was fitted to one [KCNH2 or slow delayed rectifier potassium current (IKs)] or two (KCNQ1) exponential functions. Peak tail current amplitudes were normalized to the maximum value, and the resulting data were fitted to a Boltzmann function.
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RESULTS |
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The scaling of heart rate and action potential duration are similar but not perfectly matched. There is a modest increase in the fraction of the cardiac cycle taken up by the action potential in smaller mammals, so that this value displays a weak dependence on body mass (Fig. 1C). This is consistent with the observation that the diastolic interval decreases as a fraction of the cardiac cycle in smaller mammals (29). Because the coronary blood supply to the left ventricle is only active during diastole, there is a strong constraint on the minimum fraction of the cardiac cycle that must be devoted to diastole in order to ensure adequate perfusion of the myocardium, which must be one important factor maintaining the relatively tight linkage between heart rate and action potential duration.
Discontinuities in scaling of ventricular action potential morphology.
Although ventricular action potential duration scales over a large range of mammalian body weights without any obvious discontinuities (Fig. 1B), at the cellular level there are marked discontinuities in the electrophysiological mechanisms underlying action potential repolarization in the ventricles of mammals of different weights. In species the size of guinea pigs and larger, action potential repolarization depends primarily on three potassium currents: the IKs and the rapid delayed rectifier potassium current (IKr), which have relatively slow kinetics of activation, and the inward rectifier potassium current (IK1) (24). In smaller species, repolarization is primarily dependent on two relatively large and rapidly activating potassium currents, Ito and the ultrarapid potassium current (IKur). These differences in repolarization mechanism are reflected in the morphology of the ventricular action potential.
In large and intermediate-sized mammals, such as canine and guinea pig, action potentials have a "spike and dome" morphology, whereas action potentials in small mammals, such as the mouse, have a "triangular" morphology, which is distinguished by the lack of a prominent plateau phase (Fig. 2A). The presence of a large Ito in the myocytes of small mammals precludes the development of the high plateau phase observed in larger animals. The critical role of Ito expression levels in determining action potential morphology can be shown experimentally by adding a large Ito to either guinea pig or canine myocytes with dynamic clamp (gray traces in Fig. 2A). After addition of this current, the action potential assumes a morphology that is similar to that of the mouse action potential.
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A second channel type that also contributes to IKur, the Kv1.5 channel, was tested, but expression of Kv1.5 mRNA was poorly correlated with expression of the channel, suggesting that regulation of this channel is posttranscriptional or dependent on a yet-to-be identified accessory subunit or that the mRNA is expressed at significant levels in cells other than myocytes in the ventricle wall (Refs. 5, 47; Rosati and McKinnon, unpublished data). The Kv4.2 
-subunit makes the primary contribution to Ito expression in small rodents (13). In larger animals expression of Ito is highly variable, is relatively small when present (34), and has little or no effect on action potential duration in these species (44). The small Ito found in these species is encoded by the Kv4.3 gene (9), which displays highly variable species-dependent expression patterns, with no correlation to body size (data not shown).
Comparison of mouse, hamster, guinea pig, and human Kv2.1 proximal promoter function.
In principle, the changes in Kv2.1 mRNA expression in different species (Fig. 2B) can be produced by changes in either the cis- or trans-regulatory elements controlling gene expression. To address this issue the transcriptional activity of the proximal promoter regions of the mouse, hamster, guinea pig, and human Kv2.1 genes were compared in cultured rat myocytes.
There are clear differences in the transcriptional activity of the Kv2.1 proximal promoters from different species that closely reflect the differences in mRNA expression (Fig. 2D). This result suggests that the differences in Kv2.1 mRNA expression observed in vivo are mediated by changes in the functional properties of cis-regulatory elements found in the Kv2.1 gene.
Given the highly distributed nature of mammalian gene regulation, it is notable how well the different in vivo expression patterns of the Kv2.1 gene are retained by the proximal promoter regions when expressed in vitro. In addition to the large difference between the mouse/hamster and guinea pig/human expression levels, the guinea pig/human promoters are essentially turned "off" in cardiac myocytes in vitro, as they are in vivo.
Comparison of mouse and human Kv4.2 proximal promoter function.
There were also clear differences in the transcriptional activity of the mouse and human Kv4.2 proximal promoter regions (Fig. 2E). For the Kv4.2 gene, although there are large differences between the mouse and human constructs, expression of the human promoter is significantly above the background levels seen in vivo, and expression relative to the Kv2.1 constructs was also anomalously high. Both results suggest that the Kv4.2 promoter construct lacks one or more repressor elements found in the native gene.
Function and regulation of slowly activating repolarizing currents.
The changes in Ito and IKur expression are clear examples of regulatory evolution. The scaling of action potential duration in larger species could, however, involve structural evolution modifying the function of one or more of the other currents that are required for action potential generation in these animals. Guinea pig and larger species depend predominantly on two voltage-gated potassium currents, IKs and IKr, for action potential repolarization. Therefore, of all the channels involved in this process, these channels would seem to be the most obvious candidates for structural evolution. The -subunits of the IKs and IKr channels are encoded by the KCNQ1 and KCNH2 genes, respectively. If, for example, the activation rate of the KCNQ1 channel scaled between human and guinea pig, this change in channel function could contribute significantly to the decrease in action potential duration observed in guinea pigs.
Because it is difficult to accurately record the kinetic properties of IKs and IKr in large mammals because of the relatively small size of the currents, the properties of the guinea pig and human KCNQ1 and KCNH2 channels were compared with a heterologous expression system. No significant difference in either the rate or voltage dependence of KCNQ1 channel activation was observed, either when the channel was coexpressed with its normal auxiliary subunit, KCNE1 (Fig. 3, A and B), or when it was expressed alone (data not shown). The only detectable difference between the channels from the two species was an 20% slower deactivation rate for the guinea pig channel compared with the human channels. This small difference was observed when the channels were expressed alone and when they were coexpressed with KCNE1. For IKr, no detectable differences were found in comparisons of the kinetic properties of human and guinea pig KCNH2 channels, and the two currents were indistinguishable (Fig. 3, C–E).
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-subunits are likely to contribute to the regulation of these currents.
Function and regulation of L-type calcium current.
One other channel for which changes in kinetic properties could have an impact on action potential duration, particularly in species with a spike and dome action potential morphology, is the L-type calcium current (ICa,L). The kinetic properties of ICa,L were determined with voltage-clamp recordings in three species, mouse, guinea pig, and canine, and the biophysical properties of the currents were found to be quite similar (Fig. 4A). The normalized current-voltage (I-V) curves for the three species were very similar (Fig. 4B). The steady-state inactivation curves were also similar, although the mouse curve was shifted 4 mV more negative relative to the guinea pig curve, which overlapped with the canine curve (Fig. 4C). The rate of inactivation for the mouse and guinea pig currents was best described by two time constants, which were indistinguishable for the two species (Fig. 4D). The relatively small canine current could not be reliably fitted with two time constants. In this case, the single fitted time constant was intermediate between those of mouse and guinea pig. Qualitatively, there is very little difference in the inactivation properties of the three currents (Fig. 4A).
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Expression of Cav1.2 (CACNA1C) mRNA, which encodes the -subunit of the predominant L-type calcium channel in ventricular myocytes (31, 42), was relatively constant (Fig. 4F), with the changes between species being significantly less than was seen for ICa,L density. Expression of several cardiac calcium channel auxiliary subunit genes, β2, 2
-1, and 2-2, which have been shown to affect calcium current expression (1, 42), showed no consistent dependence on body weight for the species tested (data not shown). Therefore, the mechanism by which ICa,L expression is modified between different species remains uncertain.
Interactions between ICa,L density and maintenance of excitation-contraction coupling.
Given that ICa,L shows systematic changes in magnitude with body size that cannot be explained by regulation of action potential duration, it is likely that the changes in ICa,L magnitude reflect constraints imposed by the maintenance of excitation-contraction coupling. This possibility was examined in canine ventricular myocytes with action potential clamp (Fig. 5). A normal canine action potential waveform elicits a robust contraction in canine myocytes, as expected. In contrast, a mouse action potential waveform fails completely to elicit a contraction in these cells. To determine whether the failure of the mouse action potential to elicit a contraction in canine myocytes was due simply to the smaller size of ICa,L in canine myocytes, ICa,L was increased by one of two independent methods: an increase in external calcium concentration or treatment with the calcium channel activator BAY K 8644. In both cases, the increased magnitude of ICa,L restored excitation-contraction coupling, suggesting that constraints related to the maintenance of excitation-contraction coupling drive the increased ICa,L density in mouse myocytes.
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These body mass-dependent changes in calcium handling appear to be determined at the level of transcription. Expression of SERCA2 (ATP2A2) mRNA, which encodes the SR Ca2+-ATPase pump, scales steeply with body weight, with a scaling factor similar to that of inverse action potential duration (Fig. 6A). Expression of the NCX1 gene, which encodes the Na+/Ca2+ exchanger, is essentially independent of body size, although there is some species-dependent variation in expression levels (Fig. 6B). This result is consistent with the observation that the level of Na+/Ca2+ exchanger activity does not vary systematically with body mass (39).
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Regulation of sodium channel.
There is a modest dependence of SCN5A (Nav1.5) mRNA expression on body weight (Fig. 6C), and there is a significant difference in the average expression values for the three smallest species, which express large Ito and IKur currents, compared with the five largest species (P < 0.01). Because sodium channel expression appears to be mediated predominantly by transcriptional regulation (4, 26), this may produce an increase in sodium current expression in these smaller species. The relatively fast activation times of the large Ito and IKur result in partial overlap of these currents with the activation period of the sodium current, and the increased SCN5A expression may represent a compensatory mechanism, possibly to maintain action potential peak height (25).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Evolution of action potential morphology.
Mammalian ventricular myocytes can display one of two different action potential morphologies: either a spike and dome morphology or a triangular morphology. The spike and dome action potential morphology, which is observed in most mammals, is evolutionarily ancient. The IKs and IKr channels, which produce action potential repolarization in these species, are also the major repolarizing currents in the hearts of nonmammalian vertebrates, which have similar spike and dome action potential morphologies (20, 46). Expression of either Ito or IKur has not been described in the ventricular myocytes of nonmammalian vertebrates. The triangular waveform appears to be an innovation that is restricted to small rodents with very rapid heart rates and reflects the acquisition of a novel trait in these species. The shift to the triangular waveform morphology is produced by the increased expression of two rapidly activating potassium currents, Ito and IKur. The experimental evidence presented in this report suggests that the switch in this particular physiological trait is due primarily to regulatory evolution modifying the expression of the Kv4.2 and Kv2.1 genes, with little or no role for structural evolution in determining this trait. It has been shown previously that artificially increasing Kv4 -subunit mRNA expression is, by itself, sufficient to produce the expected change in action potential morphology (18, 53).
The analysis of Kv4.2 and Kv2.1 proximal promoter function described in this report suggests that the evolution of cis-regulatory function is likely to be the predominant factor contributing to the changes in Kv4.2 and Kv2.1 gene expression seen in small mammals. We cannot, however, exclude a role for changes in transcription factor network function without a complete analysis of the expression levels of the transcription factors that are important for regulating expression of these genes, which currently remain, in large part, unknown.
Evolution of action potential duration.
The evolution of action potential duration is a complex phenomenon, involving a relatively large number of different genes (shown in part in Table 1). In addition, the trait grades smoothly across the entire mammalian phylogeny and requires a complex set of changes including graded changes in the expression of multiple genes, in addition to the "on" or "off" changes in Kv2.1 and Kv4.2 gene expression. The complex nature of this evolution is illustrated by the paradoxical changes in calcium channel expression, with expression levels of this current being primarily constrained by the role of this current in excitation-contraction coupling rather than in regulating action potential duration.
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-subunit genes (8, 23, 52). Comparisons of the deduced amino acid sequences of the channels and transporters that contribute to the electrophysiological phenotype of ventricular myocytes are shown in Table 1. For those genes that are functionally important in all mammals, there is no evidence for significant changes in the functional properties of their protein products. The function of the cardiac sodium channel current (INa) is generally assumed to be invariant across mammalian phylogeny (25), and the function of the inward rectifier (IK1) is unchanged from similar currents found in simple invertebrates (14). For the calcium current, as described in this report, there are minor changes in the biophysical function of the channel, but these changes do not contribute in any obvious way to the changes in action potential duration. The sequence of the calcium channel -subunit is highly conserved for such a large protein containing several loosely structured regions (Table 1), and the minor changes in the biophysical function of this channel may reflect changes in the stoichiometry of the accessory subunits associated with the main subunit. For the Ca2+-ATPase, its key functional property, the specific activity of the pump, is unchanged across mammalian phylogeny (15, 19, 43, 45), whereas its expression levels change significantly. As described in this report, there was no significant change in the function of the IKs and IKr channels, which control action potential repolarization in larger mammals. The increase in KCNQ1 and KCNH2 mRNA expression is consistent with the increased current expression in guinea pig myocytes (22), although it is likely that other channel subunits also regulate the expression of these currents. Of the channels that are important for action potential repolarization in small mammals, only the Kv1.5 gene is <94% identical in mouse-human sequence comparisons. The relevant comparison for these channels, however, is within the subset of small mammals where these channels are expressed, and in this case sequence identity is much higher (Table 1).
Taken together, the evidence suggests that regulatory evolution is predominant in the evolution of action potential duration in the ventricular myocytes of mammals. A similar conclusion has been made for the evolution of a nonmammalian electrophysiological system, the giant axon of squid (35). This conclusion does not exclude the possibility that one or more channels show some gene-encoded change in functional properties, but clearly the major thrust of evolutionarily mediated modifications is at the level of channel expression.
Whether the graded changes in expression seen for multiple genes, including KCNQ1, KCNH2, SCN5A, and SERCA2, primarily reflect cis-regulatory evolution, as seems most likely for the Kv2.1 and Kv4.2 genes, remains an open question. There are quantitative changes in transcription factor gene expression in the ventricular myocytes from different mammalian species (33), possibly reflecting evolution of the cardiac regulatory network within the mammalian lineage. Determining whether changes in the transcription factor network contribute to the graded changes in ion channel and transporter gene expression will require a more detailed understanding of the regulation of the relevant genes.
Conclusions.
The evolution of ventricular myocyte electrophysiology appears to conform, in broad outline, to the principles derived from the study of developmental system evolution (6, 7, 49). In particular, differences in the functional properties of ion channels and transporters appear to be relatively small compared with the multiple, relatively large changes in channel and transporter expression levels. As demonstrated in this report, action potential repolarization and calcium handling evolve in mammals primarily by changes in the level of expression of the relevant ion channels and transporters, in general agreement with the concept that regulatory evolution is the predominant mechanism underlying the evolution of complex multicellular organisms. Although this issue is confused in the physiological and modeling literature, because multiple and shifting combinations of gene products can contribute to single physiologically identified currents, future efforts in this area should benefit from a recognition of the relative unity of the molecular underpinnings of mammalian cardiac electrophysiology.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The online version of this article contains supplemental material. 
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50 pA/pF) with dynamic clamp is shown in gray. B and C: comparison of Kv2.1 (KCNB1; B) and Kv4.2 (KCND2; C) mRNA expression in the left ventricular wall of 8 mammalian species. Values are means ± SD (n = 3). a.u., Arbitrary units. D: comparison of the functional properties of mouse, hamster, guinea pig, and human Kv2.1 proximal promoter regions. E: comparison of the functional properties of mouse and human Kv4.2 proximal promoter regions. For promoter analysis, orthologous regions of genomic DNA (see MATERIALS AND METHODS) were subcloned into a luciferase reporter gene construct, and gene activity was determined by assaying luciferase activity following transfection into cultured rat neonatal cardiac myocytes. Data values are expressed relative to the background level of luciferase activity following transfection of the control plasmid (pGL2-basic). Values are means ± SD (n = 3–6).
) for human and guinea pig KCNH2 channels. B, D, and E: values are means ± SE; 
, human; 
, guinea pig. F and G: comparison of KCNQ1 (F) and KCNH2 (G) gene expression in the left ventricular free wall of guinea pig and larger species. Values are means ± SD (n = 3).
