Reduced Cardiac L-Type Ca2+ Current in Cavβ2−/− Embryos Impairs Cardiac Development and Contraction With Secondary Defects in Vascular Maturation
Cardiac myocyte contraction depends on transmembrane L-type Ca2+ currents and the ensuing release of Ca2+ from the sarcoplasmic reticulum. Here we show that these L-type Ca2+ currents are essential for cardiac pump function in the mouse at developmental stages where the functional significance of the heart becomes imperative to blood flow and to the continuing growth and survival of the embryo. Disruption of the Cavβ2 gene, which encodes for the predominant ancillary β subunit of cardiac Ca2+ channels, resulted in diminished L-type Ca2+ currents in cardiomyocytes of embryonic day 9.5 (E9.5). This led to a functionally compromised heart, causing defective remodeling of intra- and extraembryonic blood vessels and embryonic death following E10.5. The defects in vascular remodeling were also observed when the Cavβ2 gene was selectively targeted in cardiomyocytes, demonstrating that they are secondary to cardiac failure rather than a result of the lack of Cavβ2 proteins in the vasculature. Partial rescue of the Ca2+ channel currents by a Ca2+ channel agonist significantly postponed embryonic death in Cavβ2−/− mice. Taken together, these data strongly support the essential role of L-type Ca2+ channel activity in cardiomyocytes for normal heart development and function and that this is a prerequisite for proper maturation of the vasculature.
Depolarization of the plasma membrane during the cardiac action potential causes the activation of high voltage-gated L-type Ca2+ channels.1 The ensuing Ca2+ influx across the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptors through a process called Ca2+-induced Ca2+ release (CICR).2,3 The resulting Ca2+ transients initiate myocyte contraction. Although this scenario is well understood in neonatal and adult cardiomyocytes, surprisingly little is known with respect to the developing cardiomyocytes, especially at very early embryonic stages.
High-voltage-activated (HVA) L-type Ca2+ channels are complexes of a pore-forming α1 subunit (CaV1) of ≈190 to 230 kDa associated with auxiliary β subunits (CaVβ), α2δ subunits, and in some cases a γ subunit.4 In the heart, the principal α1 subunit is encoded by the CaV1.2 gene.5 However, expression of this gene is not necessary for spontaneous beating and rhythmic activity of the heart and embryonic development until embryonic day 12.5 (E12.5).6 Nevertheless, CaV1.2−/− embryos die before E14.5 for unknown reasons.6 CaV1.2−/− cardiomyocytes have been shown to retain L-type Ca2+ channels, and it has been assumed that other CaV1 subunits act as a substitute of CaV1.2 in spontaneous contraction during cardiac development.7 A function for CaV1.18 and CaV1.4 has not been reported in the heart, but CaV1.3 appears to be important for the generation of spontaneous diastolic depolarization in sinoatrial node cells of the adult heart,9,10 although CaV1.3−/− mice are viable and fertile.
Molecular cloning has identified altogether 10 CaVα1 and 4 CaVβ subunits, and a comparison of the expression pattern of CaVα1 and CaVβ subunits indicates that there is certainly not an exclusive association between particular pairs of CaVα1 and CaVβ subunits.4,11–13 The CaVβ subunits bind to the main pore-forming CaVα1 subunits and as ancillary cytoplasmic proteins modulate the Ca2+ channel function.14 Among other effects, they shift the voltage dependence of activation in the hyperpolarizing direction, so that the channels open at less-depolarized potentials, and they are also required to enhance the number of functional channels at the plasma membrane.14
We made use of this latter property of CaVβ proteins to analyze the contribution of L-type Ca2+ channel activity independently of the underlying CaV1 pore protein to early murine cardiac function and the developing embryo. By generating mouse lines in which the predominant murine cardiac CaVβ gene, CaVβ2, was disrupted, we efficiently diminished L-type Ca2+ channel currents in cardiac myocytes. We deleted the CaVβ2 gene because mice deficient in CaVβ1,15 CaVβ3,12,16 and CaVβ417 all survived embryonic stages and did not show any significant cardiac phenotype. Our results show that L-type Ca2+ channel activity is expressed robustly in the wild-type murine heart as early as E9.5 and plays a dominant role for the progression of cardiac looping, cardiomyocyte contraction, blood flow, and blood vessel maturation. The impact of Ca2+ channel activity on blood vessel maturation depends on the cardiac performance because disruption of the CaVβ2 gene in the entire embryo or tissue specific only in cardiomyocytes provoked similar vascular defects.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org. All animal experiments were carried out in accordance with German legislation on protection of animals.
CaVβ2 Gene Targeting and Generation of Mouse Lines
Cre-loxP-mediated recombination was used to generate mouse lines carrying a null allele (CaVβ2+/−) or a floxed allele (CaVβ2+/flox), respectively. CaVβ2+/− mice were crossed with tgGFP mice18 to improve the visualization of early cardiac morphogenesis. For cardiomyocyte-restricted disruption of the Cavβ2 gene, CaVβ2+/− mice were crossed with MLC2a-Cre animals (MLC2atg/0).19 Mice with a cardiomyocyte-restricted inactivation of the CaVβ2 gene (CaVβ2−/flox/MLC2atg/0) were obtained by further crossing with CaVβ2flox/flox mice. Cardiomyocyte restricted expression of the MLC2a-Cre transgene was demonstrated by crossing with the Z/EG reporter strain.20
Antibodies and Western Blots
Western blots were performed using anti-CaVβ antibodies.11–13 Results were confirmed using additionally generated rabbit polyclonal antibodies 424 and 425 (anti-mouse CaVβ2), 828 (anti-mouse CaVβ3), and 830 (anti-mouse CaVβ4). We confirmed specificity of antibodies using microsomal membrane protein fractions from wild-type mice and mice deficient in CaVβ2 (this study), CaVβ3,12,13 and CaVβ4.17
Histology and Whole-Mount Immunostaining
Paraformaldehyde-fixed embryos and yolk sacs were stained with an endothelial cell-specific marker platelet endothelial cell adhesion molecule (PECAM) (rat anti-mouse PECAM, Pharmingen) and a goat anti-rabbit IgG conjugated with biotin.
Electrophysiology and Ca2+ Imaging
Ca2+ channel currents using Ca2+ or Ba2+ as charge carrier were recorded using the whole-cell configuration of the patch-clamp technique.21 Ca2+ transients in E10.5 cardiomyocytes were recorded after loading with 2 μmol/L Fura-2/acetoxymethyl ester (Fura-2 AM) with a fluorescence video imaging configuration (T.I.L.L. Photonics).
CaVβ2 Gene Expression Is Essential for Embryonic Survival, Cardiac Rhythmic Activity, and Contractility and Blood Vessel Maturation
CaVβ2 proteins are expressed in the developing mouse heart,22 and their expression in the developing heart tube begins at approximately E8.5 (Figure 1A). By a Cre-loxP-dependent strategy (Figure I in the online data supplement), we deleted a substantial part of the SH3 domain of CaVβ2 as well as the guanylate kinase domain (supplemental Figure VI) and obtained CaVβ2-deficient embryos (CaVβ2−/−), which died following E10.5 (Figure 1B) and were already reabsorbed on E11.5. Following E10.5, only viable wild-type and heterozygous embryos were identified. The CaVβ2+/− mice were born healthy and did not exhibit any obvious abnormalities during adulthood. Western blot analysis with various anti-CaVβ antibodies demonstrated that CaVβ2 expression was reduced in CaVβ2+/− hearts and confirmed that CaVβ2−/− E10.5 hearts no longer expressed the CaVβ2 protein (Figure 1C), whereas Cavβ3 expression was unchanged and CaVβ1 and CaVβ4 are not detectable (not shown).
Microscopic analysis of 127 CaVβ2−/− embryos harboring a green fluorescence protein (GFP) transgene expressed in cardiomyocytes (CaVβ2−/−/tgGFP) for improved visualization of cardiac development displayed no abnormalities compared with control CaVβ2+/+/tgGFP littermates until E9.5 (Figure 1D). The size of the wild-type embryos was more than doubled between E9.5 and 10.5 (Figure 1D), whereas significant retardation of CaVβ2−/− embryos including poor growth of the facial and brain primordial tissue was observed at E10.5 (Figure 1D). In addition, a massive pericardial effusion frequently associated with hemorrhages develops at E10.5 (Figure 1D). Closer inspection of CaVβ2−/− embryos at E10.5 (Figure 1E) revealed abnormalities in the progression of looping architecture with amorphous heart tubes lying distorted within the pericardial cavity, and a stenosis was frequently seen between the emerging right ventricle and the outflow tract. Ultrastructural analysis revealed no obvious differences in myofibrillar structures and cell-cell contacts (Figure 1F). However, a decrease in myocardial wall thickness was observed in most CaVβ2−/− tissue sections with numerous disruptions of the epicardial and endocardial layers (Figure 1F, bottom).
In embryonic heart, CaVβ2 is homogeneously expressed within the atrial and ventricular myocardium, including the atrioventricular canal and the outflow tract.22 The phenotype of the CaVβ2−/− embryos indicates that CaVβ2 is required for normal cardiac and embryonic development, which depends increasingly on cardiac pump function and circulatory delivery of oxygen, rather than diffusion at these stages. We, therefore, studied the cardiac performance of CaVβ2−/− E10.5 embryos in vivo by video microscopy. Wild-type embryos exhibited coordinated fillings of the cardiac inflow tract and subsequent peristaltic ejection of blood into the dorsal aorta and the circulation (see supplemental Movie E10-5_Wt). In contrast, CaVβ2−/− hearts show only barely noticeable tremors without any noticeable ejection volume (see supplemental Movie E10-5_Cacnb2-ko_01/02). Moreover, the frequencies of spontaneous heart beats (Figure 1G) were drastically reduced in explanted E10.5 CaVβ2−/− hearts (63±2.88 bpm; n=9) compared with controls (120±3.15 bpm; n=11), whereas no difference was observed at E9.5. Although inhibition of L-type Ca2+ currents can lead to negative chronotropic effects, the reduced heart rate of E10.5 CaVβ2−/− hearts might also be secondary to the morphological aberrations present at this developmental stage (Figure 1E).
The impairment of cardiac pump function during development is frequently associated with impaired angiogenesis, which coincides with the onset of blood flow and is influenced by thereby evoked fluid forces.23 In yolk sacs, where blood vessel formation is observed first during embryonic development, a delineation of blood vessels occurred in CaVβ2+/+ (Figure 2A) and CaVβ2+/− littermate controls (data not shown) starting on E9.5 and blood vessels showed a hierarchical arborization with diameters tapering from large to small as expected (Figure 2A and 2B). In contrast, no blood vessels were detectable in Cavβ2−/− embryos using stereomicroscopic analysis (Figure 2A), but whole-mount immunostaining (Figure 2B) revealed that vasculogenesis had occurred and the vascular plexus was formed also in CaVβ2−/− tissues. Initiation of capillary sprouting had also taken place in CaVβ2−/− yolk sacs (Figure 2B, arrowheads), but remodeling from primary capillary plexus into treelike mature blood vessels had been arrested, and a honeycomb-like network of tubules with enlarged and uniform diameters had occurred in both the extra- and intraembryonic vasculature (Figure 2B through 2D). Furthermore, vessels in Cavβ2−/− yolk sacs were barely filled with hemangioblasts (Figure 2B), consistent with poor cardiac pump function, and the formation of vitelline vessels could not be detected. However, CaVβ2 transcripts were only barely detectable in E10.5 wild-type yolk sacs (supplemental Figure IIA), and no CaVβ2 proteins were identified (Figure 2E). Because we could not identify expression of CaVβ2 proteins in the yolk sac, we hypothesized that the disturbed vascular maturation in CaVβ2−/− embryos might be a consequence of impaired cardiac pump function, rather than attributable to the lack of Cavβ2 proteins in the vasculature itself.
L-type Ca2+ Channel Activity and CICR in Very Early Embryonic Cardiomyocytes
At least in cells from neonatal or adult hearts, the cardiac performance essentially relies on the operation of voltage activated Ca2+ channels and the CICR mechanism. At E9.5 both low-voltage-activated (LVA) and HVA Ca2+ channels are already functional. LVA currents evoked by our protocol did not differ significantly among cells of the 3 genotypes (Figure 3A and supplemental Figure IIIA). HVA currents could be readily detected in E9.5 cardiomyocytes either with Ca2+ (1.8 mmol/L; supplemental Table I) or Ba2+ (10 mmol/L) as charge carrier (at 0 mV: ICa, −6.27±0.7 pA/pF [n=21]; IBa, −29.3±2.6 pA/pF [n=16]). These currents did not differ significantly in wild-type and Cavβ2+/− cardiomyocytes (supplemental Figure IIIA and IIIB; ICa, −5.5±0.51 pA/pF [n=34]; IBa, −33.8±5.8 pA/pF [n=12]) but were significantly larger by ≈1.4-fold in E10.5 cells compared with E9.5 cells (see supplemental Table I).
In Cavβ2−/− cells, the density of HVA Ca2+ currents was reduced to approximately one-fourth to one-third (Figure 3B and supplemental Figure IIIB), whereas mean cell capacitance was the same for wild-type and mutant cells (wild type, 29.8±1.2 pF [n=106]; Cavβ2+/−, 27.4±1.1 pF [n=101]; Cavβ2−/−, 31.4±1.8 pF [n=87]; P>0.05). In addition, the peak of the whole-cell current was typically at 0 mV in control and slightly shifted to more positive voltages in Cavβ2−/− cells. Accordingly, the activation midpoints in the normalized curves of Figure 3C and of supplemental Figure IIIC, and the potentials for half-activation fitted with a Boltzmann equation24 were significantly shifted to more depolarized potentials. A similar depolarizing shift in the voltage dependence of Ca2+ channel activation was observed in sensory neurones where the Cavβ3−/− gene was inactivated12 or when HVA CaVα1 subunits were expressed without any CaVβ subunit.14
HVA Ca2+ currents in embryonic cardiomyocytes are sensitive to dihydropyridines. Application of 10 μmol/L (±)-isradipine blocked the currents in cardiomyocytes from E10.5 wild-type (Figure 3D; ICa density, −9.9±1.2 pA/pF in the absence and −0.3±0.2 pA/pF in the presence of isradipine; n=10) and Cavβ2−/− (Figure 3D; ICa density, −2.0±0.5 pA/pF in the absence and 0.1±0.1 pA/pF in the presence of isradipine; n=8) mice. Similar results were obtained using cardiomyocytes isolated at E9.5.
We analyzed electrically induced Ca2+ transients in E10.5 cardiomyocytes (Figure 3E through 3J). In Cavβ2−/− cells, the amplitude of electrically induced Ca2+ transients was reduced by more than 80% (Figure 3E and 3F). In contrast, caffeine-evoked Ca2+ responses were unchanged in both amplitude and decay time constant τ (Figure 3G through 3I), indicating that the Ca2+ content of the SR and Ca2+ extrusion from the cytosol is unchanged in Cavβ2−/− cardiomyocytes. Figure 3J illustrates that the overall Ca2+ transients comprised components from both transmembrane Ca2+ influx and Ca2+ release from the SR. For this, electrically induced Ca2+ transients were recorded before and immediately after caffeine application. We found a marked reduction in the Ca2+ transient amplitude probably attributable to the reduced or absent contribution of SR Ca2+ release in the presence of caffeine. The rightmost traces in Figure 3J illustrate electrically induced Ca2+ transients after washout of caffeine. During this period, the SR has reloaded to the control steady-state Ca2+ levels for both wild-type and Cavβ2−/− myocytes. These data strongly suggest that the SR Ca2+ uptake function was not significantly impaired by the disruption of the Cavβ2 gene and support the conclusion that changes of overall Ca2+ transients can mainly be ascribed to the reduced voltage-dependent transmembrane Ca2+ influx. A decreased Ca2+ influx, then, resulted in markedly reduced overall Ca2+ transient and, consecutively, contraction of the myocytes, and pumping of the entire heart was diminished.
Vascularization Defects Are Secondary to Cardiac Failure
The inactivation of the CaVβ2 gene in tissues other than heart could still contribute to the observed lethal phenotype. To test this possibility, the CaVβ2 gene was inactivated in cardiomyocytes by using the transgenic mouse line MLC2atg/0, which carries the Cre recombinase gene under the myosin light chain 2a promoter.19 Recombination induced by the MLC2a-Cre transgene was demonstrated as early as E8.5, when formation of the linear heart tube is initiated, and was restricted to atrial and ventricular cardiomyocytes (supplemental Figure IV). Embryos with a cardiac-restricted disruption of the CaVβ2 gene (CaVβ2−/flox/MLC2atg/0) could be identified until E13.5 (Figure 4A and 4B), but a large number of E11.5 and E12.5 CaVβ2−/flox/MLC2atg/0 embryos already showed striking morphological destruction incompatible with survival (not shown). Additionally, in CaVβ2−/flox/MLC2atg/0 embryos extra- and intraembryonic blood vessels seemed to be arrested in a premature stage with a uniform and enlarged diameter and lacked hierarchical branching (Figure 4C and 4D), which is strikingly similar to the defects observed in CaVβ2−/− embryos (Figure 2).
The CaVβ2 protein was not detectable in the E13.5 CaVβ2−/flox/MLC2atg/0 hearts (Figure 4E), whereas it was present in the hearts from controls, which carry at least 1 functional CaVβ2 allele. In addition, the Cavβ3 protein but no Cavβ1 and Cavβ4 proteins were detectable in the heart at E13.5. Although Cre-mediated excision induced by the MLC2a-Cre transgene is highly effective and is initiated at least from E8.5 onward (supplemental Figure IV), low amounts of CaVβ2 transcripts were occasionally detectable by RT-PCR in 1 of 4 hearts isolated from E13.5 CaVβ2−/flox/MLC2atg/0 embryos (supplemental Figure IIB and IIC). Most probably, these CaVβ2 transcripts are encoded by alleles that have already been transcribed before Cre-mediated excision or arise from noncardiomyocytes present in the heart, such as vascular smooth muscle cells, neurons, endothelial cells, or fibroblasts. The translated protein levels are, however, too low to be detectable by our antibodies but sufficient to postpone somewhat the CaVβ2−/− phenotype. Similar to these results, Wettschureck et al19 also reported an incomplete penetrance of the MLC2a-Cre transgene, resulting in a variable phenotype most likely attributable to individual differences in the time course of Cre expression and/or Cre-mediated recombination. The low level of Cavβ2 transcript expression in yolk sac (supplemental Figure IIA) was not affected by the cardiac-specific targeting (supplemental Figure IIB and IIC).
Whole-cell voltage-activated Ca2+ channel currents (ICa) were recorded in CaVβ2−/flox/MLC2atg/0 E13.5 cardiomyocytes and in the various control cells (Figure 4F). As in the CaVβ2−/− cells, the current density (−3.0±0.2 pA/pF, n=52) was significantly reduced in CaVβ2−/flox/MLC2atg/0 cardiomyocytes compared with controls, and the potentials for half-activation were shifted to depolarized potentials (CaVβ2−/flox/MLC2atg/0 cells, −12.6±0.6 mV [n=52]; for example, CaVβ2+/flox/MLC2atg/0 control cells, −16.9±0.7 mV [n=36]). Also, the beating frequency of explanted CaVβ2−/flox/MLC2atg/0 E13.5 hearts was reduced significantly (Figure 4G).
Partial Rescue of Embryonic Lethality by L-Type Ca2+ Channel Agonist
The residual Ca2+ channel current was still dihydropyridine sensitive in Cavβ2−/− cells (Figure 3D). In the presence of (−)-BayK 8644 (1 μmol/L), the IBa was increased ≈1.8-fold from −29.3±2.6 pA/pF at 0 mV (n=17) to −51.4±10.1 pA/pF at −10 mV (n=8) in wild-type cells (Figure 5A and 5B) and ≈2.2-fold from −11.7±1.6 pA/pF at +10 mV (n=14) to −27.3±3.8 pA/pF at 0 mV (n=9) in CaVβ2-deficient cells (Figure 5A and 5C). The peaks of the whole-cell currents were typically shifted to less-positive voltages. This increase in current density could be sufficient to rescue the lethal phenotype and also to alleviate the detrimental consequences of cardiac failure in CaVβ2−/− embryos. Therefore, (−)-BayK 8644 was applied to the pregnant mice of heterozygous intercrosses. Inspection of E10.5, E11.5, and E12.5 embryos revealed that living CaVβ2−/− embryos could now be identified until E11.5 at the anticipated Mendelian ratio (Figure 5D). Without treatment, a survival of CaVβ2−/− embryos at E11.5 has never been observed (Figure 1B). Thus, the survival of CaVβ2−/− embryos can be prolonged in the presence of a Ca2+ channel agonist, supporting the overall conclusion that the described CaVβ2−/− phenotype was caused by a reduced L-type current density in cardiomyocytes resulting from the lack of CaVβ2. Unfortunately, the Ca2+ channel agonist (−)-BayK 8644 induces lethal self-biting behavior,25 which limited considerably the applicable doses and the duration of treatment in mice.
Little information is available on the developmental expression and the biophysical and pharmacological properties of cardiac L-type Ca2+ channels during the course of early embryonic development, especially at E9.5 and E10.5, when the heart starts to beat regularly. Therefore, we first characterized L-type Ca2+ currents in E9.5 and E10.5 embryonic cardiomyocytes (Figure 3 and supplemental Figure III). The L-type currents were sensitive to dihydropyridines, but, in agreement with previous studies,26,27 we did not detect sensitivity to catecholamine or cAMP stimulation (supplemental Figure V). The L-type current density increased from E9.5 to E10.5, probably because of the augmentation of the L-type Ca2+ channel CaV1.1, CaV1.2, and CaV1.3 subunits, which are present throughout the fetal murine heart development.7 The transmembrane Ca2+ influx through the L-type channels contributes to overall Ca2+ transients (Figure 3), indicating that CICR is functioning in E10.5 cardiomyocytes and underlies contraction of these cells.
The role of HVA Ca2+ channels in the development of cardiac morphology and function is unclear. Here we show that in early embryonic stages L-type Ca2+ channel activity is an essential determinant of cardiomyocyte contraction and cardiac pump function, which in turn is required for cardiac morphogenesis, oxygen supply to peripheral organs, and blood vessel maturation. By disrupting the gene of the ancillary Ca2+ channel subunit CaVβ2, a significant reduction of L-type current density was observed in cardiac myocytes as early as E9.5. Concomitantly, the Ca2+ transient was diminished as was cardiac contractility and heart beat frequency. The function of the SR was, however, not changed in CaVβ2−/− cardiomyocytes, indicating that the observed effects were mainly confined to the voltage-activated Ca2+ influx pathway without additional alterations of other Ca2+ transport mechanisms, such as Na+/Ca2+ exchange or Ca2+ release from the SR.
In mammals, embryonic survival and tissue integrity are largely dependent on cardiac pump function and blood flow, and our results demonstrate that L-type channel activity is an essential determinant of these processes. The reduction of L-type Ca2+ channel activity by disruption of the CaVβ2 gene led to embryonic heart failure associated with pericardial effusion and, finally, embryonic death after E10.5. Consistent with our results, inactivation of the CaVβ2 gene was reported to lead to preterm lethality, but embryogenesis and L-type Ca2+ channels were not analyzed at all in that study.28 Ineffective Ca2+ entry into myocytes might perturb cell growth and integrity at the transcriptional level but the size of cardiomyocytes, the myofibrillar structure, and cell-cell contacts appeared unchanged indicating a normal morphology. Considering the reduction in heart beat frequency and the virtual absence of coordinated blood ejection, the embryonic death can be traced to cardiac pump dysfunction. Compared with the normal myofibrillar structure in CaVβ2−/− cardiomyocytes, myofibrillogenesis is strongly affected in cardiomyocytes lacking either the Ca2+-binding protein calreticulin, in which bradykinin-induced elevations of intracellular Ca2+ concentration is almost completely absent,29 or the sodium/calcium exchanger (NCX1), which lack both spontaneous Ca2+ transients and contraction.30 In these examples, a role for Ca2+ as a trophic factor regulating gene expression and cardiomyocyte differentiation was suggested, but given the intact morphology of CaVβ2−/− cardiomyocytes, there is no indication that such mechanisms might be of major relevance when L-type Ca2+ channel-mediated Ca2+ entry is reduced in the absence of Cavβ2 proteins.
As a consequence of impaired cardiac contraction, the hemodynamic forces are altered, which cause additional functional disturbances including impaired maturation of extra- and intraembryonic vasculature and reduced oxygen supply in CaVβ2−/− embryos. One could expect expression of CaVβ2 in embryonic blood vessels and the observed vascular defects being a result of Cavβ2 gene disruption in this tissue. However, CaVβ2 transcripts were only barely discernible in wild-type yolk sac, and we could not detect expression of the CaVβ2 protein in this tissue (Figure 2E). In addition, mouse embryos that died after cardiomyocyte-restricted inactivation of the CaVβ2 gene share strikingly similar cardiac and vascular aberrations with embryos that died after ubiquitous inactivation of the CaVβ2 gene. Although initial vascular plexus had been formed and capillary formation exists in wild-type and mutated mouse lines, the development into a mature vascular network was lacking in both mutant mouse lines. These results agree with the finding that the capability to form vessel tubes and sprouts is a cell autonomous property of vascular progenitor cells, whereas vascular maturation and remodeling depends on blood flow evoked by a cardiac contraction-induced pressure gradient. Defects in vascular maturation associated with cardiac failure have been described,30 but the causative role of embryonic heart malfunction on the developing vasculature by cell specific inactivation of the gene in cardiomyocytes as described in this study has not yet been reported.
Final support for the conclusion that L-type Ca2+ channel activity is essential for embryonic development and survival resulted from the partial rescue of the observed phenotype by (−)-BayK 8644 acting as an agonist on Ca2+ channel activity. The residual L-type Ca2+ channel activity in CaVβ2−/− cardiomyocytes was sensitive to dihydropyridines, and application of (−)-BayK 8644 to pregnant mice protracts death in Cavβ2−/− embryos. Taken together, our results indicate a dominant role of L-type Ca2+ channel activity in cardiomyocytes for CICR, cardiac contraction, and heart pump function and that the functioning early embryonic heart depends on transmembrane Ca2+ influx via HVA L-type Ca2+ channels.
We thank Stephanie Buchholz, Kerstin Fischer, Susanne Stolz, and Christine Jung for expert technical assistance and Dr Adolfo Cavalié for helpful discussions.
Sources of Funding
This work was supported by the Deutsche Forschungsgemeinschaft (M.F. and V.F.), Fonds der Chemischen Industrie (V.F.), and Forschungsausschuss der Universität des Saarlandes (M.F., V.F., and P.L.).
Original received April 4, 2006; revision received July 13, 2006; accepted August 22, 2006.
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