Mutation of the Calmodulin Binding Motif IQ of the L-type Cav1.2 Ca2+ Channel to EQ Induces Dilated Cardiomyopathy and Death*

Background: Mutation of the IQ motif to EQ abolished in vitro CDI and CDF of the Cav1.2 channel. Results: Cardiac-specific expression of Cav1.2EQ prevents CDI and CDF, reduces ICa, and induces dilated cardiomyopathy. Conclusion: The cardiac-specific EQ mutation leads to premature death. Significance: Survival depends on the expression of a native Cav1.2 protein. Cardiac excitation-contraction coupling (EC coupling) links the electrical excitation of the cell membrane to the mechanical contractile machinery of the heart. Calcium channels are major players of EC coupling and are regulated by voltage and Ca2+/calmodulin (CaM). CaM binds to the IQ motif located in the C terminus of the Cav1.2 channel and induces Ca2+-dependent inactivation (CDI) and facilitation (CDF). Mutation of Ile to Glu (Ile1624Glu) in the IQ motif abolished regulation of the channel by CDI and CDF. Here, we addressed the physiological consequences of such a mutation in the heart. Murine hearts expressing the Cav1.2I1624E mutation were generated in adult heterozygous mice through inactivation of the floxed WT Cav1.2L2 allele by tamoxifen-induced cardiac-specific activation of the MerCreMer Cre recombinase. Within 10 days after the first tamoxifen injection these mice developed dilated cardiomyopathy (DCM) accompanied by apoptosis of cardiac myocytes (CM) and fibrosis. In Cav1.2I1624E hearts, the activity of phospho-CaM kinase II and phospho-MAPK was increased. CMs expressed reduced levels of Cav1.2I1624E channel protein and ICa. The Cav1.2I1624E channel showed “CDI” kinetics. Despite a lower sarcoplasmic reticulum Ca2+ content, cellular contractility and global Ca2+ transients remained unchanged because the EC coupling gain was up-regulated by an increased neuroendocrine activity. Treatment of mice with metoprolol and captopril reduced DCM in Cav1.2I1624E hearts at day 10. We conclude that mutation of the IQ motif to IE leads to dilated cardiomyopathy and death.

Cardiac excitation-contraction (EC) 3 coupling links the electrical excitation of the cell membrane to the mechanical contractile machinery of the heart (1,2). An important step in EC coupling is the transient rise in [Ca 2ϩ ] i caused by an influx of Ca 2ϩ through the Ca v 1.2 channel and the subsequent Ca 2ϩ release from the sarcoplasmic reticulum (SR) through ryanodine receptor 2 (RyR2) into the "fuzzy space" (3). The [Ca 2ϩ ] in the fuzzy space is further controlled by the Na ϩ -Ca 2ϩ exchanger (NCX) (4 -6). The relative high [Ca 2ϩ ] in this space inactivates the Ca v 1.2 channel by Ca 2ϩ -dependent inactivation (CDI) (1,7) and terminates thereby Ca 2ϩ entry to avoid Ca 2ϩ overload and arrhythmias (7). Ca 2ϩ triggers a second process called Ca 2ϩ -dependent facilitation (CDF). The physiological role of CDF is not entirely clear, but it may serve to offset partly reduced Ca 2ϩ channel availability at high heart rates (8,9). Both types of regulation involve direct binding of the Ca 2ϩ sensor protein calmodulin (CaM) to the Ca v 1.2 channel (7, 10 -12) and activation of Ca 2ϩ /CaM-activated protein kinase II (CaMKII) (8,11,(13)(14)(15)(16).
CaM binds to the IQ motif located at amino acids 1624 -1635 of the Ca v 1.2 carboxyl terminus (10,11,17,18). Isoleucine 1624 is essential for CaM binding. The mutation I1624E decreased the affinity of the IQ motif for CaM approximately 100-fold in vitro (11,16). As a consequence, the I/E mutation abrogated CDF and CDI of L-type Ca 2ϩ currents expressed in Xenopus oocytes (16).
To clarify the physiological significance of the I/E mutation, we created a mouse line that carried the I1624E mutation in the Ca v 1.2 gene (19). Because mice homozygous for the I/E mutation died early during embryogenesis (19), we crossed the heterozygous Ca v 1.2 ϩ/I1624E mouse to the floxed Ca v 1.2 L2/L2 * This work was supported by grants from the Deutsche Forschungsgemein-mice (20) and to mice expressing the tamoxifen-inducible Cre recombinase (MerCreMer (MCM)) under the control of the ␣-myosin heavy chain promoter (21). This line allows the cardio-specific induction of the Cre recombinase by tamoxifen injection in adult mice (21). Electrophysiological analysis of L-type Ca v 1.2 currents in cardiac myocytes (CMs) from these I/E mice revealed that the cardiac I Ca was decreased and showed no regulation by CDI and CDF (19). Furthermore, the channel showed kinetic properties that suggested that the channel had permanently adopted CDI kinetics regardless of the permeating ion (19). This finding was not expected because previously the I/E mutation was associated with a loss of the Ca 2ϩ -dependent acceleration of channel inactivation (11,16).
Analysis of the mouse line carrying the heart-specific I/E mutation was hindered by the well documented finding that the MCM-Cre mouse by itself shows a transient phenotype after activation of the Cre construct by tamoxifen (22)(23)(24). To distinguish between a potentially Cre-induced phenotype and the I/E induced alteration, we used four mouse lines: the Ca v 1.2 I1624E ϫ MCM (I/E) line, the Ca v 1.2 ϩ/L2 ϫ MCM (Ctr) line, the Ca v 1.2 ϩ/L2 (WT) line, and the Ca v 1.2 L2/Ϫ ϫ MCM (KO) line (see also Table 1). These mouse lines allowed us to differentiate phenotypes induced only by activation of the MCM-Cre protein without affecting the expression of a wildtype Ca v 1.2 protein and a phenotype induced by inactivation of the cardiac Ca v 1.2 gene.
Analysis of these mouse lines was performed at day 10 after activation of the Cre recombinase that removing the floxed WT Ca v 1.2 gene because isolated CMs from the I/E mice still showed a robust I Ca (19). The analysis revealed that hearts from I/E mice had an reduced overall contractile activity and developed dilated cardiomyopathy (DCM).

EXPERIMENTAL PROCEDURES
All substances used were of the highest purity available. The Ca v 1.2-specific antibody used in this study has been described previously (25). Amino acid numbering is according to the O. cuniculus Ca v 1.2 sequence (GenBank accession number Q01815).
Creation of I/E Mice-Generation of mice with the I1624E mutation has been described (19). The cardio-specific Ca v 1.2 mutation was induced by crossing the heterozygous Ca v 1.2 ϩ/I1624E mouse with Ca v 1.2 L2/L2 mice (20) and with mice expressing Cre under the control of the ␣-myosin heavy chain promoter (MCM) (21). The intercross of the three mouse lines resulted in production of Ca v 1.2 I1624E/L2 ϫ MCM identified as I/E, Ca v 1.2 L2/ϩ ϫ MCM (Ctr), Ca v 1.2 L2/ϩ (WT), and Ca v 1.2 L2/L2 ϫ MCM (KO) offspring at the expected Mendelian ratio. The experiments were performed with litter-matched mice aged 8 -10 weeks on a mixed C57BL6/129Sv background. The mice were injected with 2 mg of tamoxifen (Sigma) per mouse each day for 4 days. The angiotensin-converting enzyme inhibitor captopril (0.25 mg/ml) (Sigma) and the ␤-blocker metoprolol (0.5 mg/ml) (Sigma) were added to the drinking water 1 week before the first tamoxifen injection. Treatment was continued until day 10 after the first tamoxifen injection. All experiments were performed 10 days after the first tamoxifen injection. All animals were maintained and bred in the animal facility of the FOR923, Institut für Pharmakologie und Toxikologie, Technische Universität München, and had access to water and standard chow ad libitum. All procedures relating to animal care and treatment were authorized by the "Regierung von Oberbayern" and conformed to the institutional, governmental, Directive 2010/63/EU of the European Parliament guidelines and to the Care and Use of Laboratory Animals published by the US National Institutes of Health. Anesthetized mice (1.5% isoflurane) were euthanized by cervical dislocation.
Heart Weight-Mice were euthanized, and the hearts were isolated. The whole heart was briefly rinsed in PBS to remove blood. The hearts were blotted dry and weighed.
Histological Analyses-Hearts were collected at the indicated time points and fixed in 4% paraformalin in PBS. Tissues were embedded in paraffin using standard procedures. Serial sections were cut at a thickness of 12 m. The slides were stained with Masson's trichrome (Sigma) according to the manufacturer's instructions. Ventricle size and septum diameter were taken from representative sections.
Assessment of Cell Death-Heart sections were used for the quantification of cell death. The TUNEL assay (Roche Applied Science) was performed according to the manufacturer's instructions.
Echocardiography-Images were obtained using a Vevo 770 Visual Sonic scanner equipped with a 30-MHz probe (Visual Sonics Inc., Toronto, ON, Canada). The procedure was as detailed in Ref. 27. Electrophysiological Recordings-Whole cell I Ca or I Ba was measured as described in Ref. 19,26. All fits showed a correlation coefficient Ͼ0.98. The relation between I Ba and I Ca current fraction remaining 100 ms after depolarization (f 100 ) was calculated as follows: f 100 ϭ (r 100Ba /r 100Ca )-1, where f 100 is the fractional current after 100 ms, r 100Ba is the remain- ing I Ba after 100 ms, and r 100Ca is the remaining I Ca after 100 ms.
Simultaneous Calcium and Electrophysiological Recordings-Recordings were performed as described earlier (28). For assessing the EC coupling gain we followed a protocol described in Ref. 5. Recording temperature was 22°C.
Sarcomere Length and Calcium Measurements-For contraction and cell length measurements as well as global calcium recordings, we used methods described previously (29). Recording temperature was 22°C.
Statistics-Data are presented as mean Ϯ S.E. Statistical significance was tested by using a two-tailed unpaired Student's t test or a two-way ANOVA where appropriate. The null hypothesis was rejected if p Ͻ 0.05. If applicable, the number of experiments are given as n ϭ number of cells/obtained from number of animals.

RESULTS
Isoleucine 1624 of the CACNA1C gene has been mutated to glutamate using transgenic gene knock-in techniques (19). The resulting homozygote mice (genotype Ca v 1.2 I1624E on both alleles) were not viable. Therefore, we cross-bred heterozygous Ca v 1.2 ϩ/I1624E mice with mice that expressed the floxed Ca v 1.2 gene (20) and the ␣MHC-MerCreMer construct (21) allowing tissue-and time-dependent inactivation of the Ca v 1.2 gene by the tamoxifen-controlled MerCreMer recombinase. The adult I/E mice had a reduced life span and died within 3 weeks after treatment with tamoxifen (Fig. 1A). ECG recordings showed that, about 1 h before death, the beat frequency decreased continuously and became arrhythmic shortly before death. Twenty percent of the control mice (Ctr) died during the first 10 days. These mice expressed a wild-type Ca v 1.2 gene at an unaltered expression level (supplemental Fig. 1) supporting the previous notion that the MerCreMer mice show a transient phenotype after activation of the Cre recombinase (22, 23) that is not caused by a change in the Ca v 1.2 channel expression (supplemental Fig. 1). In contrast to the Ctr mice, WT mice that contained a wild-type and a floxed Ca v 1.2 gene but no Cre recombinase were not affected by the tamoxifen injections ( Fig. 1A) (for nomenclature and genotype, see Table 1).
Western blots 4 of cardiac muscle using the anti-Ca v 1.2 antibody (25) detected reduced protein levels in the ventricle of I/E mice compared with litter-matched control (Ctr) mice at day 10 ( Fig. 1, B and C). Reduced expression of the Ca v 1.2 I1624E protein was confirmed in the HEK293 expression system (supplemental Fig. 2). As expected from Western blotting, I Ca was reduced from 2.0 Ϯ 0.21 pA/pF (n ϭ 18/3) in Ctr CMs to 1.1 Ϯ 0.14 pA/pF (n ϭ 19/3) in I/E CMs at day 10 (see Fig. 4D).
For further investigations, I/E mice were studied at day 10 after the first injection of tamoxifen. 5 Already at this stage, cardiac performance was significantly reduced as indicated by the decreased fractional shortening in the living mouse (Fig. 1D) and by the impaired myocyte contractility after rest (Fig. 1E), whereas contractility was unchanged under steady-state pacing conditions (Fig. 1E). Morphological inspection of the CMs did not reveal a severe pathology in the basic sarcomere structure as visualized by ␣-actinin staining (Fig. 1F). The sarcomere length of native isolated CMs was reduced in I/E mice compared with Ctr cells in both resting and steady-state diastole (Fig. 1G).
We next analyzed the cardiac phenotype ex vivo. Inspection of the heart showed a DCM ( Fig. 2A) for I/E mice. The septum thickness of I/E hearts was decreased (Fig. 2B), whereas the ventricle size was increased (Fig. 2C) in agreement with a slightly but not significantly increased heart weight to body weight ratio (Fig. 2D). As expected for DCM (30), TUNEL staining showed an increased rate of apoptosis (Fig. 2, E and G) and changes in fibrosis (Fig. 2, F and H).
In agreement with previous studies on cardiac dilation/hypertrophy (31), the hearts with the Ca v 1.2 I/E channel displayed increased activity levels for the CaMKII (Fig. 3, A and C) and the MAP kinase (ERK1/2) (Fig. 3, B and D) pathway. As expected, the total amount of immunologically determined ERK1/2 and RyR2 protein was not changed in the Ca v 1.2 I/E compared with Ctr hearts. In cardiac hypertrophy (31), these pathways are often activated by the neuroendocrine axis, i.e. the reninangiotensin and sympathetic systems. Interestingly, we were 4 We would like to add a notice of caution here. The densitometric quantification of Western blots implies accuracy that depends on the quality of the used antibodies, on the limited tissue available, and on the blots. We agree with one of our reviewers that Western blots may suggest inaccurate conclusions. 5 Mice that contain one floxed Ca v 1.2 allele, one Ca v 1.2 I1624E allele, and the MCM-Cre construct already show at day 10 after the tamoxifen injection the electrophysiology of the mutated channel (19). This indicates to us that the WT gene product is already absent in these CMs. The Western blots always show an extensive reduced Ca v 1.  not able to detect an increased nuclear translocation of nuclear factor of activated T cells (NFAT) (Fig. 3E). DCM is mostly caused by a substantial loss of functional ventricle muscle as evidenced by the highly elevated apoptosis rate (32). We were therefore not surprised that the size of the CMs was not increased in Ca v 1.2 I/E hearts (Fig. 3F). Next, we investigated the cause of DCM in more detail. As shown previously (19), CMs expressing mutated Ca v 1.2 I/E channels have a significantly reduced I Ca loss of facilitation and no change in inactivation with Ca 2ϩ as charge carrier (19). The mutation Ca v 1.2 I1624E shortened the fast and slow inactivation time constant for I Ba to the values obtained with Ca 2ϩ as charge carrier (Fig. 4A). This change in kinetics is also observed by the f 100 value (consult "Experimental Procedures" for calculation) (Fig. 4B) (11, 16). The f 100 value decreased significantly (p Ͻ 0.003) from 1.65 Ϯ 0.38 (n ϭ 9) in Ctr CMs to 0.53 Ϯ 0.14 (n ϭ 16) in I/E CMs (Fig. 4A) and indicated that, in the presence of Ba 2ϩ , inactivation of the Ca v 1.2 I/E channel was as fast as that in the presence of Ca 2ϩ . These results confirm that the Ca v 1.2 I/E channel always has the "CDI kinetics" regardless of the permeating ion. This kinetic will not lead to a reduced Ca 2ϩ influx during depolarization and reduced Ca 2ϩ availability in the SR. In agreement, shortening of isolated Ctr and Ca v 1.2 I/E CMs (see Fig. 1E) and global Ca 2ϩ transients (Fig. 4B) was unchanged, suggesting that electrical stimulation released similar amounts of Ca 2ϩ from the SR under steady-state condition.
Despite a decreased SR Ca 2ϩ content (see Fig. 4Cb), steadystate contractility and global Ca 2ϩ transients of the CMs were unchanged between Ctr and I/E mice. This finding strongly suggested a change in EC coupling. We therefore investigated the properties of coupling between L-type Ca 2ϩ channels and RyRs by measuring EC coupling gain (Fig. 4, D and E). For this measurement, CMs were voltage clamped and repetitively depolarized (10 s at 0.5 Hz) to obtain Ca 2ϩ steady-state conditions. At the end of this prepulsing period, a test depolarization from Ϫ40 mV to membrane potential between Ϫ50 mV and ϩ50 mV was applied, and the resulting membrane currents as well as cytosolic Indo-1 Ca 2ϩ transients were recorded simul-taneously. As expected, the Ca 2ϩ current density was reduced over the entire voltage range (Fig. 4Db). Under voltage clamp conditions, the Ca 2ϩ transients apparently had an increased amplitude in the I/E CMs (Fig. 4Da). These data strongly indicated that under voltage clamp conditions the Ca 2ϩ transient was higher even though the Ca 2ϩ current density was decreased in the I/E mice, most likely by a combination of a decreased NCX activity (see above) and an increased RyR2 sensitivity (see below). To quantify this, we calculated the CICR gain expressed as the ratio of Ca 2ϩ transient amplitude and Ca 2ϩ current (Fig.  4Dc). This analysis strongly supported our notion that the CICR gain was significantly increased in the I/E cells.
To understand further the puzzling relationship between CM behavior (higher EC coupling gain) and functional parameters (e.g. decreased fractional shortening (FS)), we investigated the putative contributions of hormonal systems to the I/E phenotype. The observed DCM is partially caused by a loss of functional CMs and leads to activation of the sympathetic and renin-angiotensin system (30,31). These hormone systems increase the activity of PKA and CaMKII. As expected (36), the phosphorylation of Ser 2808 and Ser 2814 of the RyR2 was enhanced in the I/E hearts (Fig. 5, C-F), suggesting a higher sensitivity of the calcium release mechanism of the RyR2 receptor. 4 The data described so far are in good agreement with the hypothesis (30) that the phenotype of the I/E mice was in part induced by an increased activity of the neuroendocrine system. Therefore, we tested whether or not treatment of the mice with metoprolol (a cardiac ␤1-adrenoreceptor blocker) and captopril (an inhibitor of the conversion of angiotensin I to angiotensin II) improves the cardiac outcome. Treatment started 7 days before the first tamoxifen injection and reduced the dilated cardiomyopathy (Fig. 6A). We substantiated this macroscopic impression by analyzing key parameters that were aggravated in the I/E mice (see Figs. 1-3). Treatment with these inhibitors diminished or vastly reduced most changes induced by the I/E mutation: cardiac dilation was suppressed as shown by the reduced ventricle size and the septum thickness (Fig. 6B), and the CaMKII and MAPK pathways were less activated (Fig. 6C). Nevertheless, FS was still reduced in the I/E mice compared with their Ctr littermates (Fig. 6E).
An alternative possibility was that the observed properties were not due to the I/E mutation of the Ca v 1.2 channel, but were caused by the decreased incorporation of the Ca v 1.2 protein into the plasma membrane of the CMs. We therefore carefully compared the phenotype of the Ctr and I/E mice with mice containing two inactivated Ca v 1.2 alleles (Ca v 1.2 KO ) 10 days after tamoxifen injection (supplemental Fig. 3). Survival rate, Ca v 1.2 protein expression, and FS did not significantly differ between Ca v 1.2 KO and Cav1.2 I/E mice. Ca v 1.2 KO developed a similar DCM, septum thinning, and fibrosis (supplemental Figs. 4 and 5) but a higher apoptosis rate, CM size, and Z-Z distance (supplemental Figs. 4 and 5), suggesting that the reduction of the WT Cav1.2 channel had an additional negative impact on the heart. In agreement with these results, pCaMKII and pMAPK were significantly higher in Ca v 1.2 KO than in Ctr hearts (supplemental Fig. 6, A and B). Treatment of the mouse lines with captopril and metoprolol resulted in the expected  Fig. 6C). These different pharmacological sensitivities of I/E versus KO hearts further supported the notion that the I/E mutation not only reduced Ca v 1.2 expression but affected other functions by its inability to bind CaM with high affinity.

DISCUSSION
Mutation of the IQ motif to EQ in the C terminus of the Ca v 1.2 channel reduced the in vitro affinity of the channel for CaM and abrogated or abolished CDI and CDF (11,16). We generated a mouse line in which the cardiac Ca v 1.2 channel carried this mutation and showed that CDI and CDF of the Cav1.2 are absent in CMs expressing the I/E mutation (19). Under voltage clamp condition, the gain of Ca 2ϩ release was significantly affected by this mutation suggesting an "altered EC coupling." EC coupling depends on the amplitude, kinetics, and spatial features of the Ca 2ϩ signal in the microdomain of the fuzzy space (3). The [Ca 2ϩ ] in this space is shaped by the activity of the Ca v 1.2 channel, the RyR2, and the NCX exchanger. In a recent paper, Acsai et al. estimated that during SR Ca 2ϩ release [Ca 2ϩ ] in the fuzzy space reached 10 -15 M within milliseconds (37). We have no direct evidence about the [Ca 2ϩ ] concentration in the dyadic space, but the experiments of Fig. 4 suggest significant alterations of signal-FIGURE 6. Effects of metoprolol and captopril on Ctr and I/E mice at day 10. A, representative microscopic pictures (magnification, ϫ1.6) of hearts at day 10 from mice that were treated with and without metoprolol and captopril. B, upper, ventricle size in mm 2 of Ctr (n ϭ 9) and I/E (n ϭ 11) from mice treated with metoprolol and captopril, and I/E (n ϭ 8) from untreated mice. B, lower, septum diameter in mm of Ctr (n ϭ 9) and I/E (n ϭ 11) from mice treated with metoprolol and captopril, and I/E (n ϭ 8) from untreated mice. C, quantification of the Western blots for pCaMKII and CaMKII. Ctr (n ϭ 4) and I/E (n ϭ 7) from mice treated with metoprolol and captopril, and I/E (n ϭ 5) from untreated mice are shown. D, quantification of the Western blots for pMAPK and MAPK. Ctr (n ϭ 5) and I/E (n ϭ 7) from mice treated with metoprolol and captopril, and I/E (n ϭ 5) from untreated mice. E, echocardiographic assessment of cardiac function as FS in Ctr Ϫmetoprolol/captopril (n ϭ 18), Ctr ϩmetoprolol/captopril (n ϭ 10), I/E Ϫmetoprolol/captopril (n ϭ 11), and I/E ϩmetoprolol/captopril (n ϭ 10) mice. *, p Ͻ 0.05 between Ctr and I/E mice. Open columns, Ctr; black columns, I/E; hatched columns, I/E from untreated mice.
ing in this coupling space. These changes or adaptations might at least in part be brought about by the chronic activation of the sympathetic and renin-angiotensin system, leading to an increased phosphorylation of the RyR2 accompanied by a sensitization of the Ca 2ϩ release mechanism (36,38).
The phosphorylation and presumably activation of CaMKII and Erk1/2 are induced by similar factors. It has been reported that wall stress and activation of G␣ q /␣ 11 -coupled receptors such as the AII receptor activate CaMKII and the MAPK pathways that contribute to cardiac hypertrophy (for review, see Ref. 31). However, signaling through the MAPK pathway is complicated. Depending on the MAPK isozyme, translocation of NFAT to the nucleus may be inhibited or promoted. Further research is needed to analyze these pathways.
The I/E mutation had no significant effect on the structure of the CMs despite slight decreases in the cross-striation distance (Fig. 1G). This finding is strongly contrasted by the Ca v 1.2 KO mice that developed a significantly increased CM size and increase in cross-striational distance within 10 days.
Both types of mice rapidly developed dilated cardiomyopathy. In our search for the cause of this severe phenotype, we noticed a lower resting [Ca 2ϩ ] and a decreased loading of the SR. As observed in heart failure (6,39), the NCX protein was increased. The global decrease in NCX activity assessed during caffeine application might be a result of structural remodeling, often observed during cardiac diseases such as T-tubular loss during remodeling (40). Such a remodeling process will lead to a lower surface/volume ratio and thus decrease the global functional Ca 2ϩ removal through NCX. The Ca v 1.2 I/E channel was expressed at a lower rate in the heart as also observed in the HEK expression system. This reduction contributed significantly to the observed phenotype.
Measurement of the contractility of isolated CM I/E s did not show a reduction, whereas a reduced cardiac force development was present in the in vivo situation (see reduced FS). This discrepancy is most likely caused by the fact that the performance of the intact heart has to be considered as the combination of single myocyte contractility and the number of contributing myocytes. Analysis of the I/E hearts (see Fig. 2) revealed severe apoptosis of cardiac myocytes and an increased fibrosis. These findings strongly support the notion that a lower number of functional myocytes contribute to the overall force development and thus leading, despite a maintained contractility at the cellular level, to a decreased organ performance. To compensate the decreased cardiac function, the mouse increased the activity of the sympathetic and renin-angiotensin system to overcome the loss of functioning myocytes. Chronic hormonal stimulation leads to cellular loss through apoptosis and eventually to cardiac dilation as reported by several groups (for review, see Ref. 30). Similar results have been reported when the number of cardiac Ca v 1.2 channels was reduced (41). 6 A DCM phe-notype was also observed after certain inflammatory, metabolic, or toxic insults which result in a significant loss of working myocardium (42).
The DCM of the I/E mice was caused by an initially reduced influx of Ca 2ϩ during depolarization. This reduction was caused not only by a change in channel kinetics but also by a reduced expression of the Ca v 1.2 I/E protein leading to decreased peak I Ca . A recent publication suggested that activated CaMKII represses cardiac transcription of the Ca v 1.2 gene (43) and prevents CM hypertrophy (1,31) as found in this mouse model. In humans, DCM has been associated with either a "defective force transmission" or a "defective force generation" (44). As discussed above, the DCM associated with the Ca v 1.2 I/E mutation qualifies for the group caused by a defective force generation because the Ca 2ϩ content of the SR is inadequate to provide an adequate cardiac output. Similar considerations apply to the phenotype of the total Ca v 1.2 KO mice, suggesting that part of the phenotype observed may be attributed to a general loss of the Ca v 1.2 channel protein resulting in apoptosis.
Upon deletion of the wild-type allele, expression of the Ca v 1.2 I/E gene led to a reduced Ca 2ϩ influx resulting in a smaller global Ca 2ϩ transient by reduced fractional Ca 2ϩ release and contractility of myocytes. To compensate this, the tonus of the various neurohormonal systems increased leading to an increased EC coupling gain, transiently compensated (i.e. "normalized") contractility and cardiac hypertrophy (1,31,45). During the course of chronic increased neurohormonal stimulation, myocyte loss by apoptosis begins, and the heart enters a vicious circle of increased hormonal levels, transiently compensated contractility, and higher apoptotic loss of myocytes until compensation fails and the heart goes into DCM. Interfering with the signaling of some of these neuroendocrine factors reduced the development of DCM significantly, but could not affect apoptosis and the reduction in whole heart force development. These findings support the notion that force development and cardiac hypertrophy can be triggered by independent pathways as suggested by the work of many research groups (see 1,22,31,45).