Hypercontractile cardiac phenotype in mice overexpressing the regulatory subunit PR72 of protein phosphatase 2A.

Background: The activity, localization, and substrate specificity of the protein phosphatase 2A (PP2A) heterotrimer are controlled by various regulatory B subunits. PR72 belongs to the B'' gene family and has been shown to be upregulated in human heart failure. However, little is known about the functions of PR72 in the myocardium. Methods: To address this issue, we generated a transgenic mouse model with heart-specific overexpression of PP2A-PR72. Biochemical and physiological methods were used to determine contractility, Ca2+ cycling parameters, and protein phosphorylation. Results: A 2.5-fold increase in PR72 expression resulted in moderate cardiac hypertrophy. Maximal ventricular pressure was increased in catheterized transgenic mice (TG) compared to wild-type (WT) littermates. This was accompanied by an increased shortening of sarcomere length and faster relaxation at the single-cell level in TG. In parallel with these findings, the peak amplitude of Ca2+ transients was increased, and the decay in intracellular Ca2+ levels was shortened in TG compared to WT. The changes in Ca2+ cycling in TG were also evident from an increase in the full duration and width at half maximum of Ca2+ sparks. Consistent with the contractile data, phosphorylation of phospholamban at threonine-17 was higher in TG hearts. The lower expression of the Na+/Ca2+ exchanger may also contribute to the hypercontractile state in transgenic myocardium. Conclusion: Our results suggest that PP2A-PR72 plays an important role in regulating cardiac contractile function and Ca2+ cycling, indicating that the upregulation of PR72 in heart failure is an attempt to compensate functionally.

Impaired myocellular Ca2+ cycling in protein phosphatase PP2A-B56α KO mice is normalized by ß-adrenergic stimulation.

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B'-family in the heart. Its role in regulating the cardiac response to ß-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B' subunits (ß and γ) and higher phosphorylation of sarcoplasmic reticulum Ca2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca2+ current was reduced and the inactivation kinetics of ICaL were prolonged in KO cardiomyocytes. However, we show ß-adrenergic stimulation resulted in a comparable peak amplitude of Ca2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca2+ spark frequencies might facilitate the normalized Ca2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca2+ signaling and contractility in response to ß-adrenergic stimulation in the myocardium.

Inward Rectifier K+ Currents Contribute to the Proarrhythmic Electrical Phenotype of Atria Overexpressing Cyclic Adenosine Monophosphate Response Element Modulator Isoform CREM-IbΔC-X.

BACKGROUND Transgenic mice (TG) with heart-directed overexpresion of the isoform of the transcription factor cyclic adenosine monophosphate response element modulator (CREM), CREM-IbΔC-X, display spontaneous atrial fibrillation (AF) and action potential prolongation. The remodeling of the underlying ionic currents remains unknown. Here, we investigated the regulatory role of CREM-IbΔC-X on the expression of K+ channel subunits and the corresponding K+ currents in relation to AF onset in TG atrial myocytes. METHODS AND RESULTS ECG recordings documented the absence or presence of AF in 6-week-old (before AF onset) and 12-week-old TG (after AF onset) and wild-type littermate mice before atria removal to perform patch clamp, contractility, and biochemical experiments. In TG atrial myocytes, we found reduced repolarization reserve K+ currents attributed to a decrease of transiently outward current and inward rectifier K+ current with phenotype progression, and of acetylcholine-activated K+ current, age independent. The molecular determinants of these changes were lower mRNA levels of Kcnd2/3, Kcnip2, Kcnj2/4, and Kcnj3/5 and decreased protein levels of K+ channel interacting protein 2 (KChIP2 ), Kir2.1/3, and Kir3.1/4, respectively. After AF onset, inward rectifier K+ current contributed less to action potential repolarization, in line with the absence of outward current component, whereas the acetylcholine-induced action potential shortening before AF onset (6-week-old TG mice) was smaller than in wild-type and 12-week-old TG mice. Atrial force of contraction measured under combined vagal-sympathetic stimulation revealed increased sensitivity to isoprenaline irrespective of AF onset in TG. Moreover, we identified Kcnd2, Kcnd3, Kcnj3, and Kcnh2 as novel CREM-target genes. CONCLUSIONS Our study links the activation of cyclic adenosine monophosphate response element-mediated transcription to the proarrhythmogenic electrical remodeling of atrial inward rectifier K+ currents with a role in action potential duration, resting membrane stability, and vagal control of the electrical activity.

Induction of ICER is superseded by smICER, challenging the impact of ICER under chronic beta-adrenergic stimulation.

ICER (inducible cAMP early repressor) isoforms are transcriptional repressors encoded by the Crem (cAMP responsive element modulator) gene. They were linked to the regulation of a multitude of cellular processes and pathophysiological mechanisms. Here, we show for the first time that two independent induction patterns for CREM repressor isoforms exist in the heart, namely for ICER and smICER (small ICER), which are induced in response to ß-adrenergic stimulation in a transient- and saturation-like manner, respectively. This time-shifted induction pattern, driven by two internal promoters in the Crem gene, leads to the predominant transcription of smIcer after prolonged ß-adrenergic stimulation. Using an ICER knockout mouse model with preserved smICER induction, we show that the transient-like induction of Icer itself has minor effects on gene regulation, cardiac hypertrophy or contractile function in the heart. We conclude that the functions previously linked to ICER may be rather attributed to smICER, also beyond the cardiac background.

A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart.

Whereas myosin 18B (Myo18B) is known to be a critical sarcomeric protein, the function of myosin 18A (Myo18A) is unclear, although it has been implicated in cell motility and Golgi shape. Here, we show that homozygous deletion (homozygous tm1a, tm1b, or tm1d alleles) of Myo18a in mouse is embryonic lethal. Reminiscent of Myo18b, Myo18a was highly expressed in the embryo heart, and cardiac-restricted Myo18a deletion in mice was embryonic lethal. Surprisingly, using Western blot analysis, we were unable to detect the known isoforms of Myo18A, Myo18Aα and Myo18Aß, in mouse heart using a custom C-terminal antibody. However, alternative anti-Myo18A antibodies detected a larger than expected protein, and RNA-Seq analysis indicated that a novel Myo18A transcript is expressed in mouse ventricular myocytes (and human heart). Cloning and sequencing revealed that this cardiac isoform, denoted Myo18Aγ, lacks the PDZ-containing N terminus of Myo18Aα but includes an alternative N-terminal extension and a long serine-rich C terminus. EGFP-tagged Myo18Aγ expressed in ventricular myocytes localized to the level of A-bands in sarcomeres, and Myo18a knockout embryos at day 10.5 exhibited disorganized sarcomeres with wavy thick filaments. We additionally generated myeloid-restricted Myo18a knockout mice to investigate the role of Myo18A in nonmuscle cells, exemplified by macrophages, which express more Myo18Aß than Myo18Aα, but no defects in cell shape, motility, or Golgi shape were detected. In summary, we have identified a previously unrecognized sarcomere component, a large novel isoform (denoted Myo18Aγ) of Myo18A. Thus, both members of class XVIII myosins are critical components of cardiac sarcomeres.

Chronic ß-adrenergic stimulation reverses depressed Ca handling in mice overexpressing inhibitor-2 of protein phosphatase 1.

RATIONALE: A higher expression/activity of type 1 serine/threonine protein phosphatase 1 (PP1) may contribute to dephosphorylation of cardiac regulatory proteins triggering the development of heart failure. OBJECTIVE: Here, we tested the putatively protective effects of PP1 inhibitor-2 (I2) overexpression using a heart failure model induced by chronic ß-adrenergic stimulation. METHODS AND RESULTS: Transgenic (TG) and wild-type (WT) mice were subjected to isoprenaline (ISO) or isotonic NaCl solution supplied via osmotic minipumps for 7 days. I2 overexpression was associated with a depressed PP1 activity. Basal contractility was unchanged in catheterized mice and isolated cardiomyocytes between TGNaCl and WTNaCl. TGISO mice exhibited more fibrosis and a higher expression of hypertrophy marker proteins as compared to WTISO. After acute administration of ISO, the contractile response was accompanied by a higher sensitivity in TGISO as compared to WTISO. In contrast to basal contractility, the peak amplitude of [Ca]i and SR Ca load were reduced in TGNaCl as compared to WTNaCl. These effects were normalized to WT levels after chronic ISO stimulation. Cardiomyocyte relaxation and [Ca]i decay kinetics were hastened in TGISO as compared to WTISO, which can be explained by a higher phospholamban phosphorylation at Ser16. Chronic catecholamine stimulation was followed by an enhanced expression of GSK3ß, whereas the phosphorylation at Ser9 was lower in TG as compared to the corresponding WT group. This resulted in a higher I2 phosphorylation that may reactivate PP1. CONCLUSION: Our findings suggest that the basal desensitization of ß-adrenergic signaling and the depressed Ca handling in TG by inhibition of PP1 is restored by a GSK3ß-dependent phosphorylation of I2.

Homozygous CREM-IbΔC-X Overexpressing Mice Are a Reliable and Effective Disease Model for Atrial Fibrillation.

Background: Atrial fibrillation (AF) is a significant cause of morbidity and mortality with foreseeably increasing prevalence. While large animal models of the disease are well established but resource intensive, transgenic AF mouse models are not yet widely used to develop or validate novel therapeutics for AF. Hemizygous mice with a cardiomyocyte-specific overexpression of the human cAMP response element modulator (CREM) isoform IbΔC-X spontaneously develop AF on grounds of an arrhythmogenic substrate consisting of alterations in structure, conduction, and calcium handling. Objective: We investigated if homozygous expression of the CREM-IbΔC-X transgene in mice alters the time course of AF development, and if homozygous CREM-IbΔC-X transgenics could be suitable as a disease model of AF. Methods: Southern Blot, quantitative real-time PCR, and immunoblotting were used to identify and verify homozygous transgenics. Cardiac gravimetry, quantitative real-time RT-PCR, histology, survival analysis, and repeated ECG recordings allowed assessment of phenotypic development and effects of antiarrhythmic drugs. Results: Homozygous animals could be identified by Southern blot and quantitative PCR, showing a strong trend to increased transgenic protein expression. In homozygous animals, atrial hypertrophy appeared earlier and more pronounced than in hemizygous animals, going along with an earlier onset of spontaneous AF, while no increased early mortality was observed. Application of a rate-controlling drug (esmolol) led to the expected result of a decreased heart rate. Application of a rhythm-controlling drug (flecainide) showed effects on heart rate variability, but did not lead to a definitive conversion to sinus rhythm. Conclusion: We suggest homozygous CREM-IbΔC-X overexpressing mice as a reliable model of early onset, rapidly progressive AF.

Characterization of the Genetic Program Linked to the Development of Atrial Fibrillation in CREM-IbΔC-X Mice.

BACKGROUND: Reduced expression of genes regulated by the transcription factors CREB/CREM (cAMP response element-binding protein/modulator) is linked to atrial fibrillation (AF) susceptibility in patients. Cardiomyocyte-directed expression of the inhibitory CREM isoform CREM-IbΔC-X in transgenic mice (TG) leads to spontaneous-onset AF preceded by atrial dilatation and conduction abnormalities. Here, we characterized the altered gene program linked to atrial remodeling and development of AF in CREM-TG mice. METHODS AND RESULTS: Atria of young (TGy, before AF onset) and old (TGo, after AF onset) TG mice were investigated by mRNA microarray profiling in comparison with age-matched wild-type controls (WTy/WTo). Proteomic alterations were profiled in young mice (8 TGy versus 8 WTy). Annotation of differentially expressed genes revealed distinct differences in biological functions and pathways before and after onset of AF. Alterations in metabolic pathways, some linked to altered peroxisome proliferator-activated receptor signaling, muscle contraction, and ion transport were already present in TGy. Electron microscopy revealed significant loss of sarcomeres and mitochondria and increased collagen and glycogen deposition in TG mice. Alterations in electrophysiological pathways became prominent in TGo, concomitant with altered gene expression of K+-channel subunits and ion channel modulators, relevant in human AF. CONCLUSIONS: The most prominent alterations of the gene program linked to CREM-induced atrial remodeling were identified in the expression of genes related to structure, metabolism, contractility, and electric activity regulation, suggesting that CREM transgenic mice are a valuable experimental model for human AF pathophysiology.

Annexin A4 is a novel direct regulator of adenylyl cyclase type 5.

Annexin A4 (AnxA4), a Ca(2+)- and phospholipid-binding protein, is up-regulated in the human failing heart. In this study, we examined the impact of AnxA4 on ß-adrenoceptor (ß-AR)/cAMP-dependent signal transduction. Expression of murine AnxA4 in human embryonic kidney (HEK)293 cells dose-dependently inhibited cAMP levels after direct stimulation of adenylyl cyclases (ACs) with forskolin (FSK), as determined with an exchange protein activated by cAMP-Förster resonance energy transfer (EPAC-FRET) sensor and an ELISA (control vs. +AnxA4: 1956 ± 162 vs. 1304 ± 185 fmol/µg protein; n = 8). Disruption of the anxA4 gene led to a consistent increase in intracellular cAMP levels in isolated adult mouse cardiomyocytes, with heart-directed expression of the EPAC-FRET sensor, stimulated with FSK, and as determined by ELISA, also in mouse cardiomyocytes stimulated with the ß-AR agonist isoproterenol (ISO) (anxA4a(+/+) vs. anxA4a(-/-): 5.1 ± 0.3 vs. 6.7 ± 0.6 fmol/µg protein) or FSK (anxA4a(+/+) vs. anxA4a(-/-): 1891 ± 238 vs. 2796 ± 343 fmol/µg protein; n = 9-10). Coimmunoprecipitation experiments in HEK293 cells revealed a direct interaction of murine AnxA4 with human membrane-bound AC type 5 (AC5). As a functional consequence of AnxA4-mediated AC inhibition, AnxA4 inhibited the FSK-induced transcriptional activation mediated by the cAMP response element (CRE) in reporter gene studies (10-fold vs. control; n = 4 transfections) and reduced the FSK-induced phosphorylation of the CRE-binding protein (CREB) measured on Western blots (control vs. +AnxA4: 150 ± 17% vs. 105 ± 10%; n = 6) and by the use of the indicator of CREB activation caused by phosphorylation (ICAP)-FRET sensor, indicating CREB phosphorylation. Inactivation of AnxA4 in anxA4a(-/-) mice was associated with an increased cardiac response to ß-AR stimulation. Together, these results suggest that AnxA4 is a novel direct negative regulator of AC5, adding a new facet to the functions of annexins.

Cardiac function is regulated by B56α-mediated targeting of protein phosphatase 2A (PP2A) to contractile relevant substrates.

Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of ß-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of -5 mV, the ICa,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser(16) was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca(2+) sensitivity and increased basal contractility in TG hearts. These effects were reversed by ß-adrenergic stimulation.

Protein phosphatase 2A is regulated by protein kinase Cα (PKCα)-dependent phosphorylation of its targeting subunit B56α at Ser41.

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser(41) of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca(2+) release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser(41). This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca(2+) homeostasis.

A novel intronic promoter of the Crem gene induces small ICER (smICER) isoforms.

The transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) regulate gene transcription in response to elevated cAMP levels. The Crem isoform inducible cAMP early repressor (Icer) is transcribed by the internal promoter P2 as a critical regulator of multiple cellular processes. Here, we describe a novel inducible Crem isoform, small Icer (smIcer), regulated by a newly identified promoter (P6). ChIP revealed binding of CREB to P6 in human and mouse myocardium. P6 activity was induced by constitutively active CREB or stimulation of adenylyl cyclase. In mice, smIcer mRNA was ubiquitously expressed and transiently induced by ß-adrenoceptor stimulation e.g., in heart and lung. SmICER repressed both basal and cAMP-induced activities of P6 and P2 promoters. Stimulation of adenylyl cyclase induced P2 and P6 in cell type-specific manner. Alternative translational start sites resulted in three different smICER proteins, linked to increased apoptosis sensitivity. In conclusion, the Crem gene provides two distinct and mutually controlled mechanisms of a cAMP-dependent induction of transcriptional repressors. Our results suggest not only that smICER is a novel regulator of cAMP-mediated gene regulation, but also emphasize that biological effects that have been ascribed solely to ICER, should be revised with regard to smICER.

Water fleas (Daphnia magna) provide a separate ventilatory mechanism for their brood.

The planktonic filter feeder Daphnia magna depends on a steady oxygen supply by convection. In the ventral carapace chamber, this convection is established by the feeding current which is generated by the movement of the thoracic limbs. The present study revealed that this movement can cause an additional flow of medium which passes through the brood chamber of the animal. To visualise this current, ink or fluorescent microspheres were released by a microcapillary near the posterior opening of the brood chamber. The tracks of these probes were monitored by video microscopy. Digital motion analysis was used for the determination of flow velocity and flow rate. Ambient medium entered the brood chamber at the abdomen of the animal and moved then to the anterior end of the brood chamber before entering the ventral carapace chamber. Two horizontal lamellae, which are attached at both sides of the trunk and project laterally to contact the carapace walls, almost completely separate the dorsal brood chamber from the ventral carapace chamber. Water can only pass these barriers through small depressions in these lamellae at the level of the 3rd and 4th appendages. Female daphnids with embryos at late developmental stages showed more rapid water currents in the brood chamber than those with younger embryos. Moreover, animals showed higher flow rates when exposed to hypoxic conditions. As the oxygen uptake rate of older embryos is approximately three times higher than that of younger embryos, the enhanced brood chamber current could improve the oxygen availability for both the mother and its brood under conditions of reduced oxygen availability.