The Hyperpolarization-Activated HCN4 Channel is Important for Proper Maintenance of Oscillatory Activity in the Thalamocortical System.

Hyperpolarization-activated cation channels are involved, among other functions, in learning and memory, control of synaptic transmission and epileptogenesis. The importance of the HCN1 and HCN2 isoforms for brain function has been demonstrated, while the role of HCN4, the third major neuronal HCN subunit, is not known. Here we show that HCN4 is essential for oscillatory activity in the thalamocortical (TC) network. HCN4 is selectively expressed in various thalamic nuclei, excluding the thalamic reticular nucleus. HCN4-deficient TC neurons revealed a massive reduction of Ih and strongly reduced intrinsic burst firing, whereas the current was normal in cortical pyramidal neurons. In addition, evoked bursting in a thalamic slice preparation was strongly reduced in the mutant mice probes. HCN4-deficiency also significantly slowed down thalamic and cortical oscillations during active wakefulness. Taken together, these results establish that thalamic HCN4 channels are essential for the production of rhythmic intrathalamic oscillations and determine regular TC oscillatory activity during alert states.

Roles of cGMP-dependent protein kinase I (cGKI) and PDE5 in the regulation of Ang II-induced cardiac hypertrophy and fibrosis.

Conflicting results have been reported for the roles of cGMP and cGMP-dependent protein kinase I (cGKI) in various pathological conditions leading to cardiac hypertrophy and fibrosis. A cardioprotective effect of cGMP/cGKI has been reported in whole animals and isolated cardiomyocytes, but recent evidence from a mouse model expressing cGKIß only in smooth muscle (ßRM) but not in cardiomyocytes, endothelial cells, or fibroblasts has forced a reevaluation of the requirement for cGKI activity in the cardiomyocyte antihypertrophic effects of cGMP. In particular, ßRM mice developed the same hypertrophy as WT controls when subjected to thoracic aortic constriction or isoproterenol infusion. Here, we challenged ßRM and WT (Ctr) littermate control mice with angiotensin II (AII) infusion (7 d; 2 mg ⋅ kg(-1) ⋅ d(-1)) to induce hypertrophy. Both genotypes developed cardiac hypertrophy, which was more pronounced in Ctr animals. Cardiomyocyte size and interstitial fibrosis were increased equally in both genotypes. Addition of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, in the drinking water had a small effect in reducing myocyte hypertrophy in WT mice and no effect in ßRM mice. However, sildenafil substantially blocked the increase in collagen I, fibronectin 1, TGFß, and CTGF mRNA in Ctr but not in ßRM hearts. These data indicate that, for the initial phase of AII-induced cardiac hypertrophy, lack of cardiomyocyte cGKI activity does not worsen hypertrophic growth. However, expression of cGKI in one or more cell types other than smooth muscle is necessary to allow the antifibrotic effect of sildenafil.

Mutation of the calmodulin binding motif IQ of the L-type Ca(v)1.2 Ca2+ channel to EQ induces dilated cardiomyopathy and death.

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 Ca(2+)/calmodulin (CaM). CaM binds to the IQ motif located in the C terminus of the Ca(v)1.2 channel and induces Ca(2+)-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 Ca(v)1.2(I1624E) mutation were generated in adult heterozygous mice through inactivation of the floxed WT Ca(v)1.2(L2) 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 Ca(v)1.2(I1624E) hearts, the activity of phospho-CaM kinase II and phospho-MAPK was increased. CMs expressed reduced levels of Ca(v)1.2(I1624E) channel protein and I(Ca). The Ca(v)1.2(I1624E) channel showed "CDI" kinetics. Despite a lower sarcoplasmic reticulum Ca(2+) content, cellular contractility and global Ca(2+) 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 Ca(v)1.2(I1624E) hearts at day 10. We conclude that mutation of the IQ motif to IE leads to dilated cardiomyopathy and death.

Deletion of the C-terminal phosphorylation sites in the cardiac ß-subunit does not affect the basic ß-adrenergic response of the heart and the Ca(v)1.2 channel.

Phosphorylation of the cardiac ß subunit (Ca(v)ß(2)) of the Ca(v)1.2 L-type Ca(2+) channel complex has been proposed as a mechanism for regulation of L-type Ca(2+) channels by various protein kinases including PKA, CaMKII, Akt/PKB, and PKG. To test this hypothesis directly in vivo, we generated a knock-in mouse line with targeted mutation of the Ca(v)ß(2) gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; ßStop mouse). This mutation prevented translation of the Ca(v)ß(2) C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac ßStop mice were born at Mendelian ratio, had a normal life expectancy, and normal basal L-type I(Ca). The regulation of the L-type current by stimulation of the ß-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). ßStop mice were cross-bred with mice expressing the Ca(v)1.2 gene containing the mutation S1928A (SAßStop) or S1512A and S1570A (SFßStop) in the C terminus of the α(1C) subunit. The ß-adrenergic regulation of the cardiac I(Ca) was unaltered in these mouse lines. In contrast, truncation of the Ca(v)1.2 at Asp(1904) abolished ß-adrenergic up-regulation of I(Ca) in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Ca(v)ß(2), Ser(1928), Ser(1512), and Ser(1570) of the Ca(v)1.2 protein is functionally not involved in the adrenergic regulation of the murine cardiac Ca(v)1.2 channel.

Truncation of murine CaV1.2 at Asp-1904 results in heart failure after birth.

The carboxyl-terminal intracellular tail of the L-type Ca(2+) channel CaV1.2 modulates various aspects of channel activity.For example, deletion of the carboxyl-terminal sequence at Ser-1905 increased CaV1.2 currents in an expression model. To verify this finding in an animal model, we inserted three stop codons at the corresponding Asp-1904 in the murine CaV1.2 gene. Mice homozygous for the Stop mutation (Stop/Stop mice)were born at a Mendelian ratio but died after birth. Stop/Stop hearts showed reduced beating frequencies and contractions.Surprisingly, Stop/Stop cardiomyocytes displayed reduced IBa and a minor expression of the CaV1.2Stop protein. In contrast,expression of the CaV1.2Stop protein was normal in pooled smooth muscle samples from Stop/Stop embryos. As the CaV1.2 channel exists in a cardiac and smooth muscle splice variant, HK1 and LK1, respectively, we analyzed the consequences of the deletion of the carboxyl terminus in the respective splice variant using the rabbit CaV1.2 clone expressed in HEK293 cells.HEK293 cells transfected with the HK1Stop channel showed a reduced IBa and CaV1.2 expression. Treatment with proteasome inhibitors increased the expression of HK1Stop protein and IBa in HEK293 cells and in Stop/Stop cardiomyocytes indicating that truncation of CaV1.2 containing the cardiac exon 1a amino terminus results in proteasomal degradation of the translated protein. In contrast, HEK293 cells transfected with the LK1Stop channel had normal IBa and CaV1.2 expression. These findings indicate that absence of the carboxyl-terminal tail differentially determines the fate of the cardiac and smooth muscle splice variant of the CaV1.2 channel in the mouse.

Facilitation and Ca2+-dependent inactivation are modified by mutation of the Ca(v)1.2 channel IQ motif.

The heart muscle responds to physiological needs with a short-term modulation of cardiac contractility. This process is determined mainly by properties of the cardiac L-type Ca(2+) channel (Ca(v)1.2), including facilitation and Ca(2+)-dependent inactivation (CDI). Both facilitation and CDI involve the interaction of calmodulin with the IQ motif of the Ca(v)1.2 channel, especially with Ile-1624. To verify this hypothesis, we created a mouse line in which Ile-1624 was mutated to Glu (Ca(v)1.2(I1624E) mice). Homozygous Ca(v)1.2(I1624E) mice were not viable. Therefore, we inactivated the floxed Ca(v)1.2 gene of heterozygous Ca(v)1.2(I1624E) mice by the α-myosin heavy chain-MerCreMer system. The resulting I/E mice were studied at day 10 after treatment with tamoxifen. Electrophysiological recordings in ventricular cardiomyocytes revealed a reduced Ca(v)1.2 current (I(Ca)) density in I/E mice. Steady-state inactivation and recovery from inactivation were modified in I/E versus control mice. In addition, voltage-dependent facilitation was almost abolished in I/E mice. The time course of I(Ca) inactivation in I/E mice was not influenced by the use of Ba(2+) as a charge carrier. Using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid as a chelating agent for intracellular Ca(2+), inactivation of I(Ca) was slowed down in control but not I/E mice. The results show that the I/E mutation abolishes Ca(2+)/calmodulin-dependent regulation of Ca(v)1.2. The Ca(v)1.2(I1624E) mutation transforms the channel to a phenotype mimicking CDI.

Facilitation of murine cardiac L-type Ca(v)1.2 channel is modulated by calmodulin kinase II-dependent phosphorylation of S1512 and S1570.

Activity-dependent means of altering calcium (Ca(2)(+)) influx are assumed to be of great physiological consequence, although definitive tests of this assumption have only begun to emerge. Facilitation and inactivation offer two opposing, activity-dependent means of altering Ca(2+) influx via cardiac Ca(v)1.2 calcium channels. Voltage- and frequency-dependent facilitation of Ca(v)1.2 has been reported to depend on Calmodulin (CaM) and/or the activity of Calmodulin kinase II (CaMKII). Several sites within the cardiac L-type calcium channel complex have been proposed as the targets of CaMKII. Here, we generated mice with knockin mutations of alpha(1)1.2 S1512 and S1570 phosphorylation sites [sine facilitation (SF) mice]. Homocygote SF mice were viable and reproduced in a Mendelian ratio. Voltage-dependent facilitation in ventricular cardiomyocytes carrying the SF mutation was decreased from 1.58- to 1.18-fold. The CaMKII inhibitor KN-93 reduced facilitation to 1.28 in control cardiomyocytes. SF mutation negatively shifted the voltage-dependent inactivation and slowed recovery from inactivation, thereby making fewer channels available for activation. Telemetric ECG recordings at different heart rates showed that QT time decreased significantly more in SF than in control mice at higher rates. Our results strongly support the notion that CaMKII-dependent phosphorylation of Cav1.2 at S1512 and S1570 mediates Ca(2+) current facilitation in the murine heart.

Unchanged beta-adrenergic stimulation of cardiac L-type calcium channels in Ca v 1.2 phosphorylation site S1928A mutant mice.

Phosphorylation of serine 1928 (Ser(1928)) of the cardiac Ca(v)1.2 subunit of L-type Ca(2+) channels has been proposed as the mechanism for regulation of L-type Ca(2+) channels by protein kinase A (PKA). To test this directly in vivo, we generated a knock-in mouse with targeted mutation of Ser(1928) to alanine. This mutation did not affect basal L-type current characteristics or regulation of the L-type current by PKA and the beta-adrenergic receptor, whereas the mutation abolished phosphorylation of Ca(v)1.2 by PKA. Therefore, our data show that PKA phosphorylation of Ser(1928) of Ca(v)1.2 is not functionally involved in beta-adrenergic stimulation of Ca(v)1.2-mediated Ca(2+) influx into the cardiomyocyte.