Noninvasive analysis of contractility during identical maturations revealed two phenotypes in ventricular but not in atrial iPSC-CM.

Patient-derived induced pluripotent stem cells (iPSCs) can be differentiated into atrial and ventricular cardiomyocytes to allow for personalized drug screening. A hallmark of differentiation is the manifestation of spontaneous beating in a two-dimensional (2-D) cell culture. However, an outstanding observation is the high variability in this maturation process. We valued that contractile parameters change during differentiation serving as an indicator of maturation. Consequently, we recorded noninvasively spontaneous motion activity during the differentiation of male iPSC toward iPSC cardiomyocytes (iPSC-CMs) to further analyze similar maturated iPSC-CMs. Surprisingly, our results show that identical differentiations into ventricular iPSC-CMs are variable with respect to contractile parameters resulting in two distinct subpopulations of ventricular-like cells. In contrast, differentiation into atrial iPSC-CMs resulted in only one phenotype. We propose that the noninvasive and cost-effective recording of contractile activity during maturation using a smartphone device may help to reduce the variability in results frequently reported in studies on ventricular iPSC-CMs.NEW & NOTEWORTHY Differentiation of induced pluripotent stem cells (iPSCs) into iPSC-derived cardiomyocytes (iPSC-CMs) exhibits a high variability in mature parameters. Here, we monitored noninvasively contractile parameters of iPSC-CM during full-time differentiation using a smartphone device. Our results show that parallel maturations of iPSCs into ventricular iPSC-CMs, but not into atrial iPSC-CMs, resulted in two distinct subpopulations of iPSC-CMs. These findings suggest that our cost-effective method may help to compare iPSC-CMs at the same maturation level.

cAMP Imaging at Ryanodine Receptors Reveals ß2-Adrenoceptor Driven Arrhythmias.

[Figure: see text].

A junctional cAMP compartment regulates rapid Ca2+ signaling in atrial myocytes.

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following ß-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas ß-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas ß-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.

L-type CaV1.2 calcium channels: from in vitro findings to in vivo function.

The L-type Cav1.2 calcium channel is present throughout the animal kingdom and is essential for some aspects of CNS function, cardiac and smooth muscle contractility, neuroendocrine regulation, and multiple other processes. The L-type CaV1.2 channel is built by up to four subunits; all subunits exist in various splice variants that potentially affect the biophysical and biological functions of the channel. Many of the CaV1.2 channel properties have been analyzed in heterologous expression systems including regulation of the L-type CaV1.2 channel by Ca(2+) itself and protein kinases. However, targeted mutations of the calcium channel genes confirmed only some of these in vitro findings. Substitution of the respective serines by alanine showed that ß-adrenergic upregulation of the cardiac CaV1.2 channel did not depend on the phosphorylation of the in vitro specified amino acids. Moreover, well-established in vitro phosphorylation sites of the CaVß2 subunit of the cardiac L-type CaV1.2 channel were found to be irrelevant for the in vivo regulation of the channel. However, the molecular basis of some kinetic properties, such as Ca(2+)-dependent inactivation and facilitation, has been approved by in vivo mutagenesis of the CaV1.2α1 gene. This article summarizes recent findings on the in vivo relevance of well-established in vitro results.

Murine cardiac growth, TRPC channels, and cGMP kinase I.

Signaling via cGMP-dependent protein kinase I (cGKI) and canonical transient receptor potential (TRPC) channels appears to be involved in the regulation of cardiac hypertrophy. Recent evidence suggests that TRPC channels are targets for cGKI, and phosphorylation of these channels may mediate the antihypertrophic effects of cGMP signaling. We tested this concept by investigating the role of cGMP/cGKI signaling on angiotensin II (A II)-induced cardiac hypertrophy using a control group (Ctr), trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM mice, and trpc3(-/-)/6(-/-) × ßRM mice. ßRM mice express cGKIß only in the smooth muscle on a cGKI(-/-) background. The control group was composed of littermate mice that contained at least one wild type gene of the respective genotype. A II was infused by minipumps (7 days; 2 mg/kg/day) in Ctr, trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM, and trpc3(-/-)/6(-/-) × ßRM mice. Hypertrophy was assessed by measuring heart weight per tibia length (HW/TL) and fibrosis by staining of heart slices. A II-induced increase in HW/TL and fibrosis was absent in trpc3 (-/-) mice, whereas an increase in HW/TL and fibrosis was evident in Ctr and trpc6(-/-), minimal or absent in trpc3(-/-), moderate in ßRM, and dramatic in trpc3(-/-)/6(-/-) ßRM mice. These results suggest that TRPC3 may be necessary for A II-induced cardiac hypertrophy. On the other hand, hypertrophy and fibrosis were massively increased in ßRM mice on a TRPC3/6 × cGKI(-/-)KO background, indicating an "additive" coupling between both signaling pathways.

Phospholipase D1 is involved in α1-adrenergic contraction of murine vascular smooth muscle.

α1-Adrenergic stimulation increases blood vessel tone in mammals. This process involves a number of intracellular signaling pathways that include signaling via phospholipase C, diacylglycerol (DAG), and protein kinase C. So far, it is not certain whether signaling via phospholipase D (PLD) and PLD-derived DAG is involved in this process. We asked whether PLD participates in the α1-adrenergic-mediated signaling in vascular smooth muscle. α1-Adrenergic-induced contraction was assessed by myography of isolated aortic rings and by pressure recordings using the hindlimb perfusion model in mice. The effects of the PLD inhibitor 1-butanol (IC50 0.15 vol%) and the inactive congener 2-butanol were comparatively studied. Inhibition of PLD by 1-butanol reduced specifically the α1-adrenergic-induced contraction and the α1-adrenergic-induced pressure increase by 10 and 40% of the maximum, respectively. 1-Butanol did not influence the aortic contractions induced by high extracellular potassium, by the thromboxane analog U46619, or by a phorbol ester. The effects of 1-butanol were absent in mice that lack PLD1 (Pld1(-/-) mice) or that selectively lack the CaV1.2 channel in smooth muscle (sm-CaV1.2(-/-) mice) but still present in the heterozygous control mice. α1-Adrenergic contraction of vascular smooth muscle involves activation of PLD1, which controls a portion of the α1-adrenergic-induced CaV1.2 channel activity.

A dual-targeted drug inhibits cardiac ryanodine receptor Ca2+ leak but activates SERCA2a Ca2+ uptake.

In the heart, genetic or acquired mishandling of diastolic [Ca2+] by ryanodine receptor type 2 (RyR2) overactivity correlates with risks of arrhythmia and sudden cardiac death. Strategies to avoid these risks include decrease of Ca2+ release by drugs modulating RyR2 activity or increase in Ca2+ uptake by drugs modulating SR Ca2+ ATPase (SERCA2a) activity. Here, we combine these strategies by developing experimental compounds that act simultaneously on both processes. Our screening efforts identified the new 1,4-benzothiazepine derivative GM1869 as a promising compound. Consequently, we comparatively studied the effects of the known RyR2 modulators Dantrolene and S36 together with GM1869 on RyR2 and SERCA2a activity in cardiomyocytes from wild type and arrhythmia-susceptible RyR2R2474S/+ mice by confocal live-cell imaging. All drugs reduced RyR2-mediated Ca2+ spark frequency but only GM1869 accelerated SERCA2a-mediated decay of Ca2+ transients in murine and human cardiomyocytes. Our data indicate that S36 and GM1869 are more suitable than dantrolene to directly modulate RyR2 activity, especially in RyR2R2474S/+ mice. Remarkably, GM1869 may represent a new dual-acting lead compound for maintenance of diastolic [Ca2+].

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.

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.

Truncation of murine CaV1.2 at Asp 1904 increases CaV1.3 expression in embryonic atrial cardiomyocytes.

Cardiac CaV1.2 channels play a critical role in cardiac function. It has been proposed that the carboxyl-terminal intracellular tail of the CaV1.2 channel is the target of Ca(2+)-dependent and Ca(2+)-independent regulation of the channel. Recent studies on C-terminal truncated forms of the CaV1.2 channel reported neonatal death, reduced CaV1.2 current, and failure of ß-adrenergic stimulation of these channels in ventricular cardiomyocytes (CMs). Here, we used atrial CMs at embryonic day 18.5 that expressed a C-terminal truncated form of the CaV1.2 channel (Stop/Stop). Surprisingly, the atrial CMs showed robust L-type Ca(2+) currents which could be stimulated by forskolin, an activator of adenylyl cyclase. These currents exhibited a left-ward shift in the voltage-dependent activation curve and a reduced sensitivity to the Ca(2+) channel blocker isradipine as compared to currents in wild-type atrial CMs. RT-PCR analysis revealed normal levels of mRNA for the CaV1.2 channel but a twofold increase in the level of mRNA for the CaV1.3 channel in the Stop/Stop atrium as compared to wild-type atrium. A Western blot analysis indicated an increase of CaV1.3 protein in the Stop/Stop atrium. We suggest that, in contrast to Stop/Stop ventricular CMs, Stop/Stop atrial CMs can compensate the functional loss of the truncated CaV1.2 channel with an upregulation of the CaV1.3 channel.

Neuronal cGMP kinase I is essential for stimulation of duodenal bicarbonate secretion by luminal acid.

Brief contact of the duodenal mucosa with luminal acid elicits a long-lasting bicarbonate (HCO(3)(-)) secretory response, which is believed to be the primary protective mechanism against mucosal damage. Here, we show that cGMP-dependent protein kinase type I-knockout (cGKI(-/-)) mice are unable to respond to a physiological H(+) stimulus with a HCO(3)(-) secretory response and spontaneously develop duodenal ulcerations. Smooth muscle-selective cGKI knock-in rescued the motility disturbance but not the defective HCO(3)(-) secretion. Proton-induced HCO(3)(-) secretion was not attenuated by selective inactivation of the cGKI gene in interstitial cells of Cajal or in enterocytes, but was abolished by inactivation of cGKI in neurons (ncGKI(-/-)). cGKI was expressed in the brainstem nucleus tractus solitarius that connects the afferent with the efferent N. vagus. Accordingly, truncation of the subdiaphragmal N. vagus significantly diminished proton-induced HCO(3)(-) secretion in wild-type mice, whereas stimulation of the subdiaphragmal N. vagus elicited a similar HCO(3)(-) secretory response in cGKI(-/-), ncGKI(-/-) and wild-type mice. These findings show that protection of the duodenum from acid injury requires neuronal cGKI.

1,4-Benzothiazepines with Cyclopropanol Groups and Their Structural Analogues Exhibit Both RyR2-Stabilizing and SERCA2a-Stimulating Activities.

To discover new multifunctional agents for the treatment of cardiovascular diseases, we designed and synthesized a series of compounds with a cyclopropyl alcohol moiety and evaluated them in biochemical assays. Biological screening identified derivatives with dual activity: preventing Ca2+ leak through ryanodine receptor 2 (RyR2) and enhancing cardiac sarco-endoplasmic reticulum (SR) Ca2+ load by activation of Ca2+-dependent ATPase 2a (SERCA2a). The compounds that stabilize RyR2 at micro- and nanomolar concentrations are either structurally related to RyR-stabilizing drugs or Rycals or have structures similar to them. The novel compounds also demonstrate a good ability to increase ATP hydrolysis mediated by SERCA2a activity in cardiac microsomes, e.g., the half-maximal effective concentration (EC50) was as low as 383 nM for compound 12a, which is 1,4-benzothiazepine with two cyclopropanol groups. Our findings indicate that these derivatives can be considered as new lead compounds to improve cardiac function in heart failure.

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.

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.

A complex of Ca(V)1.2/PKC is involved in muscarinic signaling in smooth muscle.

Here we present functional and biochemical evidence for a Ca(2+) channel (Ca(V)1.2)/protein kinase C (PKC) signaling complex being a key player in muscarinic regulation of urinary bladder smooth muscle. Muscarinic stimulation induced Ca(2+) signals and concomitant contractions in detrusor muscle from mice that were dependent on functional Ca(2+) channels. These signals were still present in muscles being depolarized by 85 mM extracellular K(+). Muscarinic-induced contractions were reduced by a PKC inhibitor [bisindolylmaleimide I (BIM-I)] and a phospholipase D (PLD) inhibitor (1-butanol). A phorbol ester (PDBu) enlarged muscarinic-induced Ca(2+) signals and contractions. The effects of BIM-I and PDBu were inhibited by isradipine and/or absent in muscles from Ca(V)1.2-deficient mice. Both carbachol and PDBu increased Ca(V)1.2 channel currents in isolated bladder myocytes. Blue native-PAGE electrophoresis revealed that Ca(V)1.2, PKC, and PLD are closely associated in muscles being previously stimulated by carbachol. Immunoprecipitation using anti-Ca(V)1.2 followed by Western blotting demonstrated that Ca(V)1.2 and PKC are coupled in stimulated muscles from wild-type mice. Autoradiography on immunoprecipitates showed that Ca(V)1.2 is a substrate for PKC-mediated phosphorylation. These findings suggest that a signaling complex consisting of Ca(V)1.2, PKC, and, probably, PLD controls muscarinic-mediated phasic contraction of urinary bladder smooth muscle.

The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool.

Missense mutations in the cardiac ryanodine receptor type 2 (RyR2) characteristically cause catecholaminergic arrhythmias. Reminiscent of the phenotype in patients, RyR2-R2474S knockin mice develop exercise-induced ventricular tachyarrhythmias. In cardiomyocytes, increased mitochondrial matrix Ca2+ uptake was recently linked to non-linearly enhanced ATP synthesis with important implications for cardiac redox metabolism. We hypothesize that catecholaminergic stimulation and contractile activity amplify mitochondrial oxidation pathologically in RyR2-R2474S cardiomyocytes. To investigate this question, we generated double transgenic RyR2-R2474S mice expressing a mitochondria-restricted fluorescent biosensor to monitor the glutathione redox potential (E GSH). Electrical field pacing-evoked RyR2-WT and RyR2-R2474S cardiomyocyte contractions resulted in a small but significant baseline E GSH increase. Importantly, ß-adrenergic stimulation resulted in excessive E GSH oxidization of the mitochondrial matrix in RyR2-R2474S cardiomyocytes compared to baseline and RyR2-WT control. Physiologically ß-adrenergic stimulation significantly increased mitochondrial E GSH further in intact beating RyR2-R2474S but not in RyR2-WT control Langendorff perfused hearts. Finally, this catecholaminergic E GSH increase was significantly attenuated following treatment with the RyR2 channel blocker dantrolene. Together, catecholaminergic stimulation and increased diastolic Ca2+ leak induce a strong, but dantrolene-inhibited mitochondrial E GSH oxidization in RyR2-R2474S cardiomyocytes.

Calcium-dependent and calcium-independent inhibition of contraction by cGMP/cGKI in intestinal smooth muscle.

cGMP-dependent protein kinase I (cGKI) induces relaxation of smooth muscle via several pathways that include inhibition of intracellular Ca(2+) signaling and/or involve activation of myosin phosphatase. In the present study, we investigated these mechanisms comparatively in colon and jejunum longitudinal smooth muscle from mice. In simultaneous recordings from colon muscle, 8-bromo-cGMP (8-Br-cGMP) reduced both carbachol-induced tension and carbachol-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). These effects of 8-Br-cGMP were absent in colon from mice carrying a mutated inositol-1,4,5 trisphosphate receptor I-associated G kinase substrate (IRAG) gene or lacking cGKI. However, in jejunum, 8-Br-cGMP reduced carbachol-induced tension but did not change corresponding [Ca(2+)](i) signals. This setting was also observed in jejunum from mice carrying a mutated IRAG gene, whereas no response to 8-Br-cGMP was observed in jejunum from mice lacking cGKI. After inhibition of phosphatase activity by calyculin A, 8-Br-cGMP did not relax jejunum but still relaxed colon muscle. In Western blot analysis, 8-Br-cGMP reduced the signal for phosphorylated MYPT-1 in carbachol-stimulated jejunum but not in colon. These results suggest that cGMP/cGKI signaling differentially inhibits contraction in the muscles investigated: in jejunum, inhibition is performed without changing [Ca(2+)](i) and is dependent on phosphatase activity, whereas in colon, inhibition is mediated by inhibition of [Ca(2+)](i) signals.

Rescue of cGMP kinase I knockout mice by smooth muscle specific expression of either isozyme.

Smooth muscle expresses the Ialpha and the Ibeta isoforms of cGMP-dependent protein kinase I (cGKI). Inactivation of the murine cGKI gene prkg1 leads to multiple phenotypes and premature death at approximately 6 weeks. We reconstituted mice with the cGKIalpha or -Ibeta isozyme to test which isozyme was needed to support basic smooth muscle functions. Mice were generated by gene targeting. The cGKIalpha or the -Ibeta coding sequences were placed under the control of the SM22alpha promoter to express either isoform selectively in smooth muscle cells (SM-Ialpha or SM-Ibeta transgene). To generate smooth muscle-specific cGKIalpha or cGKIbeta rescue mice, the SM-Ialpha or SM-Ibeta transgenes were crossed on a cGKI-/- genetic background. The levels of cGKIalpha or -Ibeta expression were comparable to endogenous cGKI expression in wild-type aortic and intestinal smooth muscles. In cGKIalpha or -Ibeta rescue mice, expression of the isozymes was not detectable in non-smooth muscle tissues and cells. Median survival time of the Ialpha and Ibeta rescue mice was 52 weeks. Both isozymes mediated the 8-bromo-cGMP-induced relaxation of precontracted jejunum and aorta muscle strips. Activation of both isozymes reduced hormone- or K+-induced [Ca2+]i levels. The cGKIalpha and cGKIbeta rescue mice did not show a significant difference in intestinal passage time of BaSO4 in comparison with wild-type animals. Telemetric blood pressure measurements in conscious freely moving animals did not show differences between rescues and control mice in basal blood pressure and its regulation by DETA-NO, sodium nitroprusside, carbachol, or Y-27632. These results show that cGKI in smooth muscle is essential and that either cGKI isozyme alone can rescue basic vascular and intestinal smooth muscle functions.

Severe intestinal obstruction on induced smooth muscle-specific ablation of the transcription factor SRF in adult mice.

BACKGROUND & AIMS: SRF (Serum Response Factor), a widely expressed transcription factor, controls expression of mitogen-responsive and muscle-specific genes, thereby regulating the contractile actin microfilament. Genetic Srf deletion studies showed SRF to be indispensable for in vivo skeletal and cardiac muscle cell development. We now investigated for the first time in vivo SRF functions in smooth muscle cells of adult mice. METHODS: We conditionally deleted a floxed Srf allele (Srf(flex1)) in adult mice by inducible activation of the CreER(T2) recombinase expressed specifically in smooth muscle cells. Tamoxifen-induced CreER(T2) activity stimulated deletion of exon 1 coding sequences of Srf(flex1), thereby abolishing full-length SRF protein expression in adult smooth muscle cells of the analyzed organs: colon, bladder, and stomach. RESULTS: Smooth muscle cell-specific ablation of full-length SRF protein in adult mice showed impaired contraction of intestinal smooth muscle, resulting in defective peristalsis. Mutant mice died within 2 weeks of tamoxifen treatment, displaying clear symptoms of ileus paralyticus. Cultured primary SRF-deficient colon smooth muscle cells were viable, but displayed drastic structural alterations and elevated senescence, paralleled by degeneration of the actin microfilament and impaired expression of smooth muscle-specific genes. CONCLUSIONS: SRF plays a vital role in the contractile activity and cytoskeletal architecture of adult smooth muscle cells and is therefore essential for physiologic functions of the gastrointestinal tract in vivo. Our mouse genetic model may resemble features of human chronic intestinal pseudo-obstruction.

Control of intestinal motility by the Ca(v)1.2 L-type calcium channel in mice.

The Ca(v)1.2 L-type Ca2+ channel is the dominant voltage-activated Ca2+ channel in heart and smooth muscle. The functional significance of this channel was studied in intestinal smooth muscle from mice carrying a smooth muscle-specific, conditional inactivation of the Ca(v)1.2 gene (Ca(v)1.2SMACKO mice). Inactivation was complete within 4 wk after tamoxifen treatment and confirmed by RT-PCR, Western blot and functional analysis. Ca(v)1.2SMACKO mice show reduced feces excretion, absence of rhythmic contractions in small and large intestinal muscle and signs of paralytic ileus. Extracellular field stimulation evoked smaller contractions in jejunum muscles from Ca(v)1.2SMACKO than from CTR mice, whereas carbachol-induced contractions of similar magnitude in both muscles. The Ca2+ needed for contraction in jejunum was provided mainly by Ca(v)1.2 channels and by store-operated channels in muscles from CTR and Ca(v)1.2SMACKO mice, respectively. In conclusion, the Ca(v)1.2 channel is essential for electromechanical coupling and important for pharmaco-mechanical coupling in intestinal smooth muscle and cannot be substituted functionally by other Ca2+ entry pathways.