cMyBPC phosphorylation alters response to heart failure drug

JGP study shows that the phosphorylation state of cMyBPC modulates the ability of omecamtiv mecarbil to enhance myocardial force generation.

The small molecule omecamtiv mecarbil (OM) is a cardiac-specific myosin activator that is currently undergoing clinical trials for the treatment of heart failure with reduced ejection fraction. In this issue of JGP, Mamidi et al. demonstrate that OM’s ability to increase cardiac force production is altered by the phosphorylation state of cardiac myosin-binding protein C (cMyBPC), a target of β-adrenergic signaling that is often dysregulated in late-stage heart failure patients (1).(Left to right) Ranganath Mamidi, Joshua Holmes, Julian Stelzer, and colleagues reveal that the effects of the heart failure drug OM are modulated by the phosphorylation state of the contractile protein cMyBPC. For example, OM’s ability to increase force generation is significantly blunted in mouse myocardial preparations expressing phosphoablated (SA) rather than WT cMyBPC due to changes in myosin cross-bridge kinetics.OM enhances myocardial force generation by increasing the number of strongly bound myosin cross-bridges (2), partly by slowing ADP release and cross-bridge detachment (3). Though the drug has progressed to phase 3 clinical trials, little is known about how its effects may be influenced by pathophysiological changes in other sarcomeric proteins, such as cMyBPC, that regulate myosin cross-bridges and force production.During exercise or other physiological stresses, adrenaline stimulates the phosphorylation of cMyBPC by PKA, thereby accelerating cross-bridge kinetics and myocardial contractility to meet the increased demand for cardiac output (4). In late-stage heart failure patients, however, β-adrenergic signaling is dysregulated and cMyBPC phosphorylation is greatly reduced. “We wanted to test how the phosphorylation state of cMyBPC would effect OM treatment,” explains Julian Stelzer, a professor at Case Western Reserve University.Stelzer’s team, including cofirst authors Ranganath Mamidi and Joshua Holmes, prepared myocardial tissue from both WT mice and mice expressing a cMyBPC mutant that lacks the three main PKA phosphorylation sites. The researchers treated the preparations with OM and found that the ablation of cMyBPC phosphorylation significantly blunted OM’s ability to increase force production (1).Dephosphorylated cMyBPC is thought to stabilize the super-relaxed state of myosin, in which the head domains are folded back toward the filament backbone and are less available to form active cross-bridges (5). Stelzer and colleagues have previously shown that ablating cMyBPC phosphorylation slows cross-bridge kinetics (6).“This is exacerbated by the addition of OM,” Stelzer says. “It creates an even slower system that limits cross-bridge recruitment, and those that are recruited can’t really be detached.” This may reduce the effectiveness of OM in end-stage heart failure patients with low levels of cMyBPC phosphorylation.In contrast, phosphorylation of cMyBPC by PKA usually accelerates myosin cross-bridge kinetics. However, when Stelzer and colleagues treated their myocardial preparations with both PKA and OM, mimicking the scenario of an early-stage heart failure patient exercising or experiencing stress, the effects of the drug dominated the effects of the kinase.“OM did not allow any acceleration and, in fact, slowed cross-bridge kinetics even further, completely negating the effect of PKA on contractility,” Stelzer says. This could mean that early-stage patients on OM are unable to increase their cardiac output during exercise, elevating the risk of ischemia.New iterations of OM are already being explored as potential next-generation treatments for heart failure. Stelzer says that it will be important to investigate how these drugs interact with cMyBPC and other components of the contractile machinery. In the meantime, Stelzer’s laboratory is focused on developing novel therapeutic approaches involving the direct manipulation of cMyBPC phosphorylation.     

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 β-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of −5 mV, the I_Ca,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser¹⁶ 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 Ca²⁺ sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation.     

The HCM-causing Y235S cMyBPC mutation accelerates contractile function by altering C1 domain structure

Mutations in cardiac myosin binding protein C (cMyBPC) are a major cause of hypertrophic cardiomyopathy (HCM). In particular, a single amino acid substitution of tyrosine to serine at residue 237 in humans (residue 235 in mice) has been linked to HCM with strong disease association. Although cMyBPC truncations, deletions and insertions, and frame shift mutations have been studied, relatively little is known about the functional consequences of missense mutations in cMyBPC. In this study, we characterized the functional and structural effects of the HCM-causing Y235S mutation by performing mechanical experiments and molecular dynamics simulations (MDS). cMyBPC null mouse myocardium was virally transfected with wild-type (WT) or Y235S cMyBPC (KO^Y235S). We found that Y235S cMyBPC was properly expressed and incorporated into the cardiac sarcomere, suggesting that the mechanism of disease of the Y235S mutation is not haploinsufficiency or poison peptides. Mechanical experiments in detergent-skinned myocardium isolated from KO^Y235S hearts revealed hypercontractile behavior compared to KO^WT hearts, evidenced by accelerated cross-bridge kinetics and increased Ca²⁺ sensitivity of force generation. In addition, MDS revealed that the Y235S mutation causes alterations in important intramolecular interactions, surface conformations, and electrostatic potential of the C1 domain of cMyBPC. Our combined in vitro and in silico data suggest that the Y235S mutation directly disrupts internal and surface properties of the C1 domain of cMyBPC, which potentially alters its ligand-binding interactions. These molecular changes may underlie the mechanism for hypercontractile cross-bridge behavior, which ultimately results in the development of cardiac hypertrophy and in vivo cardiac dysfunction.     

Human heart failure is accompanied by altered protein kinase A subunit expression and post-translational state

β-Adrenergic receptor blockade reduces total mortality and all-cause hospitalizations in patients with heart failure (HF). Nonetheless, β-blockade does not halt disease progression, suggesting that cAMP-dependent protein kinase (PKA) signaling downstream of β-adrenergic receptor activation may persist through unique post-translational states. In this study, human myocardial tissue was used to examine the state of PKA subunits. As expected, total myosin binding protein-C phosphorylation and Ser23/24 troponin I phosphorylation significantly decreased in HF. Examination of PKA subunits demonstrated no change in type II regulatory (RIIα) or catalytic (Cα) subunit expression, although site specific RIIα (Ser96) and Cα (Thr197) phosphorylation were increased in HF. Further, the expression of type I regulatory subunit (RI) was increased in HF. Isoelectric focusing of RIα demonstrated up to three variants, consistent with reports that Ser77 and Ser83 are in vivo phosphorylation sites. Western blots with site-specific monoclonal antibodies showed increased Ser83 phosphorylation in HF. 8-fluo-cAMP binding by wild type and phosphomimic Ser77 and Ser83 mutant RIα proteins demonstrated reduced Kd for the double mutant as compared to WT RIα. Therefore, failing myocardium displays altered expression and post-translational modification of PKA subunits that may impact downstream signaling.     

PKCepsilon increases phosphorylation of the cardiac myosin binding protein C at serine 302 both in vitro and in vivo

Cardiac myosin binding protein C (cMyBPC) phosphorylation is essential for normal cardiac function. Although PKC was reported to phosphorylate cMyBPC in vitro, the relevant PKC isoforms and functions of PKC-mediated cMyBPC phosphorylation are unknown. We recently reported that a transgenic mouse model with cardiac-specific overexpression of PKCepsilon (PKCepsilon TG) displayed enhanced sarcomeric protein phosphorylation and dilated cardiomyopathy. In the present study, we have investigated cMyBPC phosphorylation in PKCepsilon TG mice. Western blotting and two-dimensional gel electrophoresis demonstrated a significant increase in cMyBPC serine (Ser) phosphorylation in 12-month-old TG mice compared to wild type (WT). In vitro PKCepsilon treatment of myofibrils increased the level of cMyBPC Ser phosphorylation in WT mice to that in TG mice, whereas treatment of TG myofibrils with PKCepsilon showed only a minimal increase in cMyBPC Ser phosphorylation. Three peptide motifs of cMyBPC were identified as the potential PKCepsilon consensus sites including a 100% matched motif at Ser302 and two nearly matched motifs at Ser811 and Ser1203. We treated synthetic peptides corresponding to the sequences of these three motifs with PKCepsilon and determined phosphorylation by mass spectrometry and ELISA assay. PKCepsilon induced phosphorylation at the Ser302 site but not at the Ser811 or Ser1203 sites. A S302A point mutation in the Ser302 peptide abolished the PKCepsilon-dependent phosphorylation. Taken together, our data show that the Ser302 on mouse cMyBPC is a likely PKCepsilon phosphorylation site both in vivo and in vitro and may contribute to the dilated cardiomyopathy associated with increased PKCepsilon activity.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α₂-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α₂-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α₂-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α₂-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α₂-adrenergic receptor stimulation.     

?-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca²⁺ and Mg²⁺ and the role of these changes in SR Ca²⁺ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N₂ to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca²⁺] <1 µM, ß-adrenergic stimulation increased luminal Ca²⁺ activation of single RyR channels, decreased luminal Mg²⁺ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg²⁺. At cytoplasmic [Ca²⁺] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg²⁺ and Ca²⁺ inhibition of RyRs. The K_a and maximum levels of cytoplasmic Ca²⁺ activation site were not affected by ß-adrenergic stimulation.Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca²⁺ binding to the luminal Ca²⁺ site and decreasing its affinity for luminal Mg²⁺ and 2) decreasing affinity of the low-affinity Ca²⁺/Mg²⁺ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.     

Lack of β2‐adrenoceptors aggravates heart failure‐induced skeletal muscle myopathy in mice

Skeletal myopathy is a hallmark of heart failure (HF) and has been associated with a poor prognosis. HF and other chronic degenerative diseases share a common feature of a stressed system: sympathetic hyperactivity. Although beneficial acutely, chronic sympathetic hyperactivity is one of the main triggers of skeletal myopathy in HF. Considering that β₂‐adrenoceptors mediate the activity of sympathetic nervous system in skeletal muscle, we presently evaluated the contribution of β₂‐adrenoceptors for the morphofunctional alterations in skeletal muscle and also for exercise intolerance induced by HF. Male WT and β₂‐adrenoceptor knockout mice on a FVB genetic background (β₂KO) were submitted to myocardial infarction (MI) or SHAM surgery. Ninety days after MI both WT and β₂KO mice presented to cardiac dysfunction and remodelling accompanied by significantly increased norepinephrine and epinephrine plasma levels, exercise intolerance, changes towards more glycolytic fibres and vascular rarefaction in plantaris muscle. However, β₂KO MI mice displayed more pronounced exercise intolerance and skeletal myopathy when compared to WT MI mice. Skeletal muscle atrophy of infarcted β₂KO mice was paralleled by reduced levels of phosphorylated Akt at Ser 473 while increased levels of proteins related with the ubiquitin‐–proteasome system, and increased 26S proteasome activity. Taken together, our results suggest that lack of β₂‐adrenoceptors worsen and/or anticipate the skeletal myopathy observed in HF.     

Increased Cardiac Myocyte PDE5 Levels in Human and Murine Pressure Overload Hypertrophy Contribute to Adverse LV Remodeling

Background

The intracellular second messenger cGMP protects the heart under pathological conditions. We examined expression of phosphodiesterase 5 (PDE5), an enzyme that hydrolyzes cGMP, in human and mouse hearts subjected to sustained left ventricular (LV) pressure overload. We also determined the role of cardiac myocyte-specific PDE5 expression in adverse LV remodeling in mice after transverse aortic constriction (TAC).

Methodology/Principal Findings

In patients with severe aortic stenosis (AS) undergoing valve replacement, we detected greater myocardial PDE5 expression than in control hearts. We observed robust expression in scattered cardiac myocytes of those AS patients with higher LV filling pressures and BNP serum levels. Following TAC, we detected similar, focal PDE5 expression in cardiac myocytes of C57BL/6NTac mice exhibiting the most pronounced LV remodeling. To examine the effect of cell-specific PDE5 expression, we subjected transgenic mice with cardiac myocyte-specific PDE5 overexpression (PDE5-TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dt_max and dP/dt_min with prolonged tau (P<0.05 for all). Greater cardiac dysfunction in PDE5-TG was associated with reduced myocardial cGMP and SERCA2 levels, and higher passive force in cardiac myocytes in vitro.

Conclusions/Significance

Myocardial PDE5 expression is increased in the hearts of humans and mice with chronic pressure overload. Increased cardiac myocyte-specific PDE5 expression is a molecular hallmark in hypertrophic hearts with contractile failure, and represents an important therapeutic target.     

Beta3-adrenergic eNOS stimulation in left ventricular murine myocardium

This study investigates mechanisms underlying beta3-adrenergic activation of the endothelial nitric oxide synthase (eNOS) in myocardial tissue of wild-type (WT) and beta3-adrenoceptor knockout (beta3-KNO) mice, in the absence and presence of BRL 37344 (BRL), the preferential beta3-adrenoceptor selective agonist. Nitric oxide (NO)-liberation was measured after the application of BRL (10 micromol/L), using fluorescence dye diaminofluorescein (DAF), in left ventricular cardiac preparations. Phosphorylation of eNOSSer1177, eNOSThr495, eNOSSer114, and eNOS translocation, and alterations of 8-isoprostaglandin F2alpha (a parameter for reactive oxygen radical generation), after application of BRL (10 micromol/L), were studied using immunohistochemical stainings in isolated, electrically stimulated (1 Hz) right atrial (RA) and left ventricular (LV) myocardium. An increased NO release after BRL application (10 micromol/L) was observed in the RA and LV myocardial tissue of WT mice, but not in beta3-KNO mice. This NO liberation in WT mice was paralleled by an increased eNOSSer1177, but not eNOSThr495, phosphorylation. A cytosolic eNOS translocation was observed after the application of BRL (10 micromol/L) only in the RA myocardial tissue of WT mice. A BRL (10 micromol/L)-dependent increase in eNOSSer114 phosphorylation was observed only in the LV myocardial tissue of WT mice; this was paralleled by an increase in 8-isoprostaglandin F2alpha. In murine myocardium, 3 beta3-adrenoceptor-dependent activation pathways for eNOS exist (i.e., a translocation and phosphorylation of eNOSSer1177 and eNOSSer114). These pathways are used in a regional-dependent manner. beta3-adrenergic oxygen-derived free radical production might be important in situations of enhanced beta3-adrenoceptor activation, as has been described in human heart failure.     

Calpain activation mediates microgravity-induced myocardial abnormalities in mice via p38 and ERK1/2 MAPK pathways

The human cardiovascular system has adapted to function optimally in Earth''s 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1. Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47^phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47^phox, and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47^phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47^phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.     

Desert hedgehog is a mammal-specific gene expressed during testicular and ovarian development in a marsupial

Background

Sphingosine-1-phosophate (S1P) is a biologically active sphingolipid metabolite that influences cellular events including differentiation, proliferation, and migration. S1P acts through five distinct cell surface receptors designated S1P_1-5R, with S1P₁R having the highest expression level in the developing heart. S1P₁R is critical for vascular maturation, with its loss leading to embryonic death by E14.5; however, its function during early cardiac development is not well known. Our previous studies demonstrated that altered S1P levels adversely affects atrioventricular (AV) canal development in vitro, with reduced levels leading to cell death and elevated levels inhibiting cell migration and endothelial to mesenchymal cell transformation (EMT).

Results

We determined, by real-time PCR analysis, that S1P₁R was expressed at least 10-fold higher than other S1P receptors in the developing heart. Immunohistochemical analysis revealed S1P₁R protein expression in both endothelial and myocardial cells in the developing atrium and ventricle. Using AV canal cultures, we observed that treatment with either FTY720 (an S1P_1,3,4,5R agonist) or KRP203 (an S1P₁R-specific agonist) caused similar effects on AV canal cultures as S1P treatment, including induction of cell rounding, inhibition of cell migration, and inhibition of EMT. In vivo, morphological analysis of embryonic hearts at E10.5 revealed that S1P₁R-/- hearts were malformed with reduced myocardial tissue. In addition to reduced myocardial tissue, E12.5 S1P₁R-/- hearts had disrupted morphology of the heart wall and trabeculae, with thickened and disorganized outer compact layer and reduced fibronectin (FN) deposition compared to S1P₁R+/+ littermates. The reduced myocardium was accompanied by a decrease in cell proliferation but not an increase in apoptosis.

Conclusions

These data indicate that S1P₁R is the primary mediator of S1P action in AV canal cultures and that loss of S1P₁R expression in vivo leads to malformed embryonic hearts, in part due to reduced fibronectin expression and reduced cell proliferation.     

CaMKII‐dependent ryanodine receptor phosphorylation mediates sepsis‐induced cardiomyocyte apoptosis

Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis‐induced apoptosis. Wild‐type (WT) CASP mice hearts showed an increase in apoptosis respect to WT‐Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3‐I) were protected against sepsis‐induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca²⁺ release, prevented apoptosis in WT‐CASP. To examine whether CaMKII‐dependent RyR2 phosphorylation mediates diastolic Ca²⁺ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation‐dependent enhancement in diastolic SR Ca²⁺ release leading to mitochondrial Ca²⁺ overload, mitochondrial Ca²⁺ retention capacity was reduced in mitochondria isolated from WT‐CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene‐treated mice. We conclude that in sepsis, CaMKII‐dependent RyR2 phosphorylation results in diastolic Ca²⁺ release from SR which leads to mitochondrial Ca²⁺ overload and apoptosis.     

Defect of Mitotic Vimentin Phosphorylation Causes Microophthalmia and Cataract via Aneuploidy and Senescence in Lens Epithelial Cells

Vimentin, a type III intermediate filament (IF) protein, is phosphorylated predominantly in mitosis. The expression of a phosphorylation-compromised vimentin mutant in T24 cultured cells leads to cytokinetic failure, resulting in binucleation (multinucleation). The physiological significance of intermediate filament phosphorylation during mitosis for organogenesis and tissue homeostasis was uncertain. Here, we generated knock-in mice expressing vimentin that have had the serine sites phosphorylated during mitosis substituted by alanine residues. Homozygotic mice (VIM^SA/SA) presented with microophthalmia and cataracts in the lens, whereas heterozygotic mice (VIM^WT/SA) were indistinguishable from WT (VIM^WT/WT) mice. In VIM^SA/SA mice, lens epithelial cell number was not only reduced but the cells also exhibited chromosomal instability, including binucleation and aneuploidy. Electron microscopy revealed fiber membranes that were disorganized in the lenses of VIM^SA/SA, reminiscent of similar characteristic changes seen in age-related cataracts. Because the mRNA level of the senescence (aging)-related gene was significantly elevated in samples from VIM^SA/SA, the lens phenotype suggests a possible causal relationship between chromosomal instability and premature aging.     

PKA Phosphorylation of Cardiac Troponin I Modulates Activation and Relaxation Kinetics of Ventricular Myofibrils

Protein kinase A (PKA) phosphorylation of myofibril proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus (Ser-23/24) of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. The effect of PKA-mediated phosphorylation on the magnitude of contraction has been studied in some detail, but little is known about how it modulates the kinetics of thin filament activation and myofibril relaxation as Ca²⁺ levels vary. Troponin C (cTnC) interaction with cTnI (C-I interaction) is a critical step in contractile activation that can be modulated by cTnI phosphorylation. We tested the hypothesis that altering C-I interactions by PKA, or by cTnI phosphomimetic mutations (S23D/S24D-cTnI), directly affects thin filament activation and myofilament relaxation kinetics. Rat ventricular myofibrils were isolated and endogenous cTn was exchanged with either wild-type cTnI, or S23D/S24D-cTnI recombinant cTn. Contractile mechanics were monitored at maximum and submaximal Ca²⁺ concentrations. PKA treatment of wild-type cTn or exchange of cTn containing S23D/S24D-cTnI resulted in an increase in the rate of early, slow phase of relaxation (k_REL,slow) and a decrease in its duration (t_REL,slow). These effects were greater for submaximal Ca²⁺ activated contractions. PKA treatment also reduced the rate of contractile activation (k_ACT) at maximal, but not submaximal Ca²⁺, and reduced the Ca²⁺ sensitivity of contraction. Using a fluorescent probe coupled to cTnC (C35S-IANBD), the Ca²⁺-cTn binding affinity and C-I interaction were monitored. Ca²⁺ binding to cTn (pCa₅₀) was significantly decreased when cTnI was phosphorylated by PKA (ΔpCa₅₀ = 0.31). PKA phosphorylation of cTnI also weakened C-I interaction in the presence of Ca²⁺. These data suggest that weakened C-I interaction, via PKA phosphorylation of cTnI, may slow thin filament activation and result in increased myofilament relaxation kinetics, the latter of which could enhance early phase diastolic relaxation during β-adrenergic stimulation.     

PKCβII inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro‐inflammatory responses

Protein kinase C βII (PKCβII) levels increase in the myocardium of patients with end‐stage heart failure (HF). Also targeted overexpression of PKCβII in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKCβII in HF development. Using a post‐myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKCβII on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKCβII selective inhibitor (βIIV5‐3 conjugated to TAT_47–57 carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT_47–57 carrier peptide alone). Formalin‐fixed mid‐ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two‐fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKCβII inhibitor. Further, a 90% decrease in active TGFβ1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKCβII attenuates cardiac remodelling mediated by the TGF‐SMAD signalling pathway. Therefore, sustained selective inhibition of PKCβII in a post‐MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.     

Effects of HCM cTnI Mutation R145G on Troponin Structure and Modulation by PKA Phosphorylation Elucidated by Molecular Dynamics Simulations

Cardiac troponin (cTn) is a key molecule in the regulation of human cardiac muscle contraction. The N-terminal cardiac-specific peptide of the inhibitory subunit of troponin, cTnI (cTnI_1-39), is a target for phosphorylation by protein kinase A (PKA) during β-adrenergic stimulation. We recently presented evidence indicating that this peptide interacts with the inhibitory peptide (cTnl_137–147) when S23 and S24 are phosphorylated. The inhibitory peptide is also the target of the point mutation cTnI-R145G, which is associated with hypertrophic cardiomyopathy (HCM), a disease associated with sudden death in apparently healthy young adults. It has been shown that both phosphorylation and this mutation alter the cTnC-cTnI (C-I) interaction, which plays a crucial role in modulating contractile activation. However, little is known about the molecular-level events underlying this modulation. Here, we computationally investigated the effects of the cTnI-R145G mutation on the dynamics of cTn, cTnC Ca²⁺ handling, and the C-I interaction. Comparisons were made with the cTnI-R145G/S23D/S24D phosphomimic mutation, which has been used both experimentally and computationally to study the cTnI N-terminal specific effects of PKA phosphorylation. Additional comparisons between the phosphomimic mutations and the real phosphorylations were made. For this purpose, we ran triplicate 150 ns molecular dynamics simulations of cTnI-R145G Ca²⁺-bound cTnC_1-161-cTnI_1-172-cTnT_236-285, cTnI-R145G/S23D/S24D Ca²⁺-bound cTnC_1-161-cTnI_1-172-cTnT_236-285, and cTnI-R145G/PS23/PS24 Ca²⁺-bound cTnC_1-161-cTnI_1-172-cTnT_236-285, respectively. We found that the cTnI-R145G mutation did not impact the overall dynamics of cTn, but stabilized crucial Ca²⁺-coordinating interactions. However, the phosphomimic mutations increased overall cTn fluctuations and destabilized Ca²⁺ coordination. Interestingly, cTnI-R145G blunted the intrasubunit interactions between the cTnI N-terminal extension and the cTnI inhibitory peptide, which have been suggested to play a crucial role in modulating troponin function during β-adrenergic stimulation. These findings offer a molecular-level explanation for how the HCM mutation cTnI-R145G reduces the modulation of cTn by phosphorylation of S23/S24 during β-adrenergic stimulation.     

Ablation of Ventricular Myosin Regulatory Light Chain Phosphorylation in Mice Causes Cardiac Dysfunction in Situ and Affects Neighboring Myofilament Protein Phosphorylation

There is little direct evidence on the role of myosin regulatory light chain phosphorylation in ejecting hearts. In studies reported here we determined the effects of regulatory light chain (RLC) phosphorylation on in situ cardiac systolic mechanics and in vitro myofibrillar mechanics. We compared data obtained from control nontransgenic mice (NTG) with a transgenic mouse model expressing a cardiac specific nonphosphorylatable RLC (TG-RLC(P-). We also determined whether the depression in RLC phosphorylation affected phosphorylation of other sarcomeric proteins. TG-RLC(P-) demonstrated decreases in base-line load-independent measures of contractility and power and an increase in ejection duration together with a depression in phosphorylation of myosin-binding protein-C (MyBP-C) and troponin I (TnI). Although TG-RLC(P-) displayed a significantly reduced response to β₁-adrenergic stimulation, MyBP-C and TnI were phosphorylated to a similar level in TG-RLC(P-) and NTG, suggesting cAMP-dependent protein kinase signaling to these proteins was not disrupted. A major finding was that NTG controls were significantly phosphorylated at RLC serine 15 following β₁-adrenergic stimulation, a mechanism prevented in TG-RLC(P-), thus providing a biochemical difference in β₁-adrenergic responsiveness at the level of the sarcomere. Our measurements of Ca²⁺ tension and Ca²⁺-ATPase rate relations in detergent-extracted fiber bundles from LV trabeculae demonstrated a relative decrease in maximum Ca²⁺-activated tension and tension cost in TG-RLC(P-) fibers, with no change in Ca²⁺ sensitivity. Our data indicate that RLC phosphorylation is critical for normal ejection and response to β₁-adrenergic stimulation. Our data also indicate that the lack of RLC phosphorylation promotes compensatory changes in MyBP-C and TnI phosphorylation, which when normalized do not restore function.Phosphorylation of sarcomeric proteins tunes the intensity and dynamics of cardiac contraction and relaxation independent of membrane Ca²⁺ fluxes to meet physiologic demands (1, 2). We focus here on ventricular myosin regulatory light chain, which is phosphorylated in vivo (3–5) but whose functional role in control of cardiac dynamics has remained unclear. The identification of RLC² mutations linked to familial hypertrophic cardiomyopathy (6) underscores the importance of understanding its action as a regulator of contraction. Functionally, in vitro cardiac RLC phosphorylation by MLCK produces a sensitizing shift in the force-Ca²⁺ relation in skinned fibers (7–11). Moreover, studies show that RLC phosphorylation manifests as a gradient across the wall of the heart, which may be important for both normalizing wall stress and for generation of torsion about the long axis of the ejecting heart (12–14). Yet there remains a lack of understanding of the in situ functional effects of RLC phosphorylation and whether phosphorylation of RLC influences other sarcomeric sites as substrates for kinases and phosphatases.Understanding the precise mechanisms by which phosphorylation of RLC affects function of ejecting ventricles is particularly important, because mechanisms downstream of Ca²⁺ fluxes at the level of the sarcomere appear to dominate ejection and to sustain ventricular elastance (15). Myosin motors are important in this, and RLC is well positioned at the S1-S2 junction to modulate myosin heavy chain directly by fine-tuning lever arm motion and indirectly by interacting with the essential light chain, the thick filament backbone, and MyBP-C (16, 17). Accordingly, the hypothesis underlying this study was that ablation of N-terminal RLC phosphorylation would elicit a depression in ventricular ejection and compensatory changes in phosphorylation of sarcomeric proteins neighboring RLC.To understand the role of RLC phosphorylation in the ejection phase of the cardiac cycle, we determined in situ pressure-volume functions in ejecting, auxotonically loaded ventricles expressing either wild type RLC (NTG) or a nonphosphorylatable RLC (TG-RLC(P-)) (10). Our experiments provide novel data demonstrating the importance of RLC phosphorylation in systolic pump function and provide new insights into how a lack of phosphorylation of RLC induces a redistribution of charge among myofilament proteins. Furthermore, our data demonstrate an enigmatic blunting of TG-RLC(P-) functional response to β₁-adrenergic simulation despite a normal TnI and MyBP-C phosphorylation profile. RLC serine 15 phosphorylation increased significantly in NTG controls but was not permitted in TG-RLC(P-) (RLC S14/15/19/A), suggesting that a change in RLC phosphorylation following β₁-adrenergic simulation may be critical for eliciting a normal response.     

XB130 Deficiency Affects Tracheal Epithelial Differentiation during Airway Repair

The repair and regeneration of airway epithelium is important for maintaining homeostasis of the respiratory system. XB130 is an adaptor protein involved in the regulation of cell proliferation, survival and migration. In the human trachea, XB130 is expressed on the apical site of ciliated epithelial cells. We hypothesize that XB130 may play a role in epithelial repair and regeneration after injury. Xb130 knockout (KO) mice were generated, and a mouse isogenic tracheal transplantation model was used. Adult Xb130 KO mice did not show any significant anatomical and physiological phenotypes in comparison with their wild type (WT) littermates. The tracheal epithelium in Xb130 KO mice, however, was significantly thicker than that in WT mice. Severe ischemic epithelial injury was observed immediately after the tracheal transplantation, which was followed by epithelial cell flattening, proliferation and differentiation. No significant differences were observed in terms of initial airway injury and apoptosis. However, at Day 10 after transplantation, the epithelial layer was significantly thicker in Xb130 KO mice, and associated with greater proliferative (Ki67+) and basal (CK5+) cells, as well as thickening of the connective tissue and fibroblast layer between the epithelium and tracheal cartilages. These results suggest that XB130 is involved in the regulation of airway epithelial differentiation, especially during airway repair after injury.     

Specific loss of LHCII phosphorylation in the Lemna mutant 1073 lacking the cytochrome b6/f complex

The thylakoid protein kinase(s) activity of Lemna perpusilla strain 6746 (wild type, WT) and the cytochrome (cyt) b₆/f-less mutant 1073 was compared. Isolated thylakoids of both WT and mutant phosphorylated the polypeptides of 9–15, 29, 32–34 and 40–45 kDa. This kinase(s) activity was light-dependent and could be elicited by addition of duroquinol in the dark. Thylakoids from both WT and mutant phosphorylated histone III-S at comparable rates. However, the redox-controlled phosphorylation of the LHCII polypeptide which could be demonstrated in vitro and in vivo in the WT thylakoids could not be detected under any experimental condition in the cyt b₆/f-less thylakoids. Halogenated quinone analogues known to inhibit reduction of the cyt b₆/f complex inhibited both the electron flow and duroquinol-activated LHCII phosphorylation, but had no effect on the duroquinol-dependent phosphorylation of the other thylakoid polypeptides. These results indicate that the Lemna thylakoids contain at least two redox-activated protein kinase(s). A quinone-binding site is involved in the activation of the LHCII kinase system which is rendered inactive in the absence of the cyt b₆/f complex.