Sonya C. Bardswell

Oxidant-induced Activation of Type I Protein Kinase A Is Mediated by RI Subunit Interprotein Disulfide Bond Formation

Here we demonstrate that type I protein kinase A is redoxactive, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. This oxidative disulfide formation causes a subcellular translocation and activation of the kinase, resulting in phosphorylation of established substrate proteins. The translocation is mediated at least in part by the oxidized form of the kinase having an enhanced affinity for α-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of β-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase.

Protein Kinase D Selectively Targets Cardiac Troponin I and Regulates Myofilament Ca2+ Sensitivity in Ventricular Myocytes

Protein kinase D (PKD) is a serine/threonine kinase with emerging myocardial functions; in skinned adult rat ventricular myocytes (ARVMs), recombinant PKD catalytic domain phosphorylates cardiac troponin I at Ser22/Ser23 and reduces myofilament Ca2+ sensitivity. We used adenoviral gene transfer to determine the effects of full-length PKD on protein phosphorylation, sarcomere shortening and [Ca2+]i transients in intact ARVMs. In myocytes transduced to express wild-type PKD, the heterologously expressed enzyme was activated by endothelin 1 (ET1) (5 nmol/L), as reflected by PKD phosphorylation at Ser744/Ser748 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). The ET1-induced increase in cellular PKD activity was accompanied by increased cardiac troponin I phosphorylation at Ser22/Ser23; this measured approximately 60% of that induced by isoproterenol (10 nmol/L), which activates cAMP-dependent protein kinase (PKA) but not PKD. Phosphorylation of other PKA targets, such as phospholamban at Ser16, phospholemman at Ser68 and cardiac myosin-binding protein C at Ser282, was unaltered. Furthermore, heterologous PKD expression had no effect on isoproterenol-induced phosphorylation of these proteins, or on isoproterenol-induced increases in sarcomere shortening and relaxation rate and [Ca2+]i transient amplitude. In contrast, heterologous PKD expression suppressed the positive inotropic effect of ET1 seen in control cells, without altering ET1-induced increases in relaxation rate and [Ca2+]i transient amplitude. Complementary experiments in “skinned” myocytes confirmed reduced myofilament Ca2+ sensitivity by ET1-induced activation of heterologously expressed PKD. We conclude that increased myocardial PKD activity induces cardiac troponin I phosphorylation at Ser22/Ser23 and reduces myofilament Ca2+ sensitivity, suggesting that altered PKD activity in disease may impact on contractile function.

Distinct Sarcomeric Substrates Are Responsible for Protein Kinase D-mediated Regulation of Cardiac Myofilament Ca2+ Sensitivity and Cross-bridge Cycling

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser22/Ser23, reduces myofilament Ca2+ sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser22/Ser23 remains to be established. To determine the role of cTnI phosphorylation at Ser22/Ser23 in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser22/Ser23 are substituted by nonphosphorylatable Ala (cTnI-Ala2). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser22/Ser23 and decreased the Ca2+ sensitivity of force. In contrast, PKD had no effect on the Ca2+ sensitivity of force in myocardium from cTnI-Ala2 mice, in which Ser22/Ser23 were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala2 mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser273, Ser282, and Ser302, and revealed that PKD phosphorylates only Ser302. Furthermore, PKD phosphorylated Ser302 selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala2 mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca2+ sensitivity through cTnI phosphorylation at Ser22/Ser23 but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser302, which may mediate the latter effect.

Increased myofilament Ca2+ sensitivity and diastolic dysfunction as early consequences of Mybpc3 mutation in heterozygous knock-in mice

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca2+ sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca2+ sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca2+ transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E′/A′, and higher E/E′ ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca2+ sensitivity, faster Ca2+ transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca2+ sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca2+ transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities.

Novel role for p90 ribosomal S6 kinase in the regulation of cardiac myofilament phosphorylation.

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na+/H+ exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser282, which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser273 or Ser302, cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser282. Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser282 phosphorylation by RSK. Immunolabeling with a Ser(P)282 antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser282 phosphorylation in cMyBP-C, accompanied by significant reduction in Ca2+ sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser22/Ser23. The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser273 and Ser302. We conclude that Ser282 in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.

Normal passive viscoelasticity but abnormal myofibrillar force generation in human hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, increased ventricular stiffness and impaired diastolic filling. We investigated to what extent myocardial functional defects can be explained by alterations in the passive and active properties of human cardiac myofibrils. Skinned ventricular myocytes were prepared from patients with obstructive HCM (two patients with MYBPC3 mutations, one with a MYH7 mutation, and three with no mutation in either gene) and from four donors. Passive stiffness, viscous properties, and titin isoform expression were similar in HCM myocytes and donor myocytes. Maximal Ca2+-activated force was much lower in HCM myocytes (14 ± 1 kN/m2) than in donor myocytes (23 ± 3 kN/m2; P < 0.01), though cross-bridge kinetics (ktr) during maximal Ca2+ activation were 10% faster in HCM myocytes. Myofibrillar Ca2+ sensitivity in HCM myocytes (pCa50 = 6.40 ± 0.05) was higher than for donor myocytes (pCa50 = 6.09 ± 0.02; P < 0.001) and was associated with reduced phosphorylation of troponin-I (ser-23/24) and MyBP-C (ser-282) in HCM myocytes. These characteristics were common to all six HCM patients and may therefore represent a secondary consequence of the known and unknown underlying genetic variants. Some HCM patients did however exhibit an altered relationship between force and cross-bridge kinetics at submaximal Ca2+ concentrations, which may reflect the primary mutation. We conclude that the passive viscoelastic properties of the myocytes are unlikely to account for the increased stiffness of the HCM ventricle. However, the low maximum Ca2+-activated force and high Ca2+ sensitivity of the myofilaments are likely to contribute substantially to any systolic and diastolic dysfunction, respectively, in hearts of HCM patients.

Paradoxical resistance to myocardial ischemia and age-related cardiomyopathy in NHE1 transgenic mice: A role for ER stress?

Sarcolemmal Na(+)/H(+) exchanger (NHE) activity, which is provided by the NHE isoform 1 (NHE1), has been implicated in ischemia/reperfusion-induced myocardial injury in animal models and humans, on the basis of studies with pharmacological NHE1 inhibitors. We generated a transgenic (TG) mouse model with cardiac-specific over-expression of NHE1 to determine whether this would be sufficient to increase myocardial susceptibility to ischemia/reperfusion-induced injury. TG mouse hearts exhibited increased sarcolemmal NHE activity and normal morphology and function. Surprisingly, they also showed reduced susceptibility to ischemia/reperfusion-induced injury, as reflected by improved functional recovery and smaller infarcts. Such protection was sustained in the presence of NHE1 inhibition with zoniporide, indicating a mechanism that is independent of sarcolemmal NHE activity. Immunoblot analysis revealed accumulation of immature NHE1 protein as well as marked upregulation of both cytoprotective (78/94 kDa glucose-regulated proteins, calreticulin, protein disulfide isomerase) and pro-apoptotic (C/EBP homologous protein) components of the endoplasmic reticulum (ER) stress response in TG myocardium. With increasing age, NHE1 TG mice exhibited increased myocyte apoptosis, developed left ventricular contractile dysfunction, underwent cardiac remodelling and died prematurely. Our findings indicate that: (1) Cardiac-specific NHE1 over-expression induces the ER stress response in mouse myocardium, which may afford protection against ischemia/reperfusion-induced injury despite increased NHE activity; (2) Ageing NHE1 TG mice exhibit myocyte apoptosis, cardiac remodelling and failure, likely as a result of sustained ER stress; (3) The pluripotent effects of the ER stress response may confound studies that are based on the chronic over-expression of complex proteins in myocardium.

cMyBP-C as a promiscuous substrate: phosphorylation by non-PKA kinases and its potential significance

It is now generally accepted that phosphorylation of cMyBP-C is critically important in maintaining normal cardiac function. Although much of the work to date on phospho-regulation of cMyBP-C has focused on the role of protein kinase A (PKA, also known as cAMP-dependent protein kinase), recent evidence suggests that a number of non-PKA serine/threonine kinases, such as Ca2+/calmodulin-dependent protein kinase II, protein kinase C, protein kinase D and the 90-kDa ribosomal S6 kinase are also capable of targeting this key regulatory sarcomeric protein. This article reviews such evidence and proposes a hypothetical role for some of the pertinent signalling pathways in phospho-regulation of cMyBP-C in the setting of heart failure.

Esmolol cardioplegia: the cellular mechanism of diastolic arrest

AIMS Esmolol, an ultra-short-acting beta-blocker, acts as a cardioplegic agent at millimolar concentrations. We investigated the mechanism by which esmolol induces diastolic ventricular arrest. METHODS AND RESULTS In unpaced Langendorff-perfused rat hearts, esmolol (0.03-3 mmol/L) had a profound negative inotropic effect resulting in diastolic arrest at 1 mmol/L and above. This inhibition of contraction was maintained during ventricular pacing. At 3 mmol/L, esmolol also abolished action potential conduction. To determine the cellular mechanism for the negative inotropism, we measured contraction (sarcomere shortening) and the calcium transient (fura-2 fluorescence ratio; Ca(tr)) in electrically-stimulated rat ventricular myocytes at 23 and 34 degrees C. The decrease in contraction (by 72% at 23 degrees C, from 0.16 +/- 0.01 to 0.04 +/- 0.01 microm, P < 0.001) was similar to that of isolated hearts and was caused by a large decrease in Ca(tr) (from 0.13 +/- 0.02 to 0.07 +/- 0.02, P < 0.001). There was no additional effect on myofilament Ca(2+) sensitivity. Esmolols effects on contraction and Ca(tr) were not shared or altered by the beta-blocker, atenolol (1 mmol/L). Sarcoplasmic reticulum inhibition with thapsigargin did not alter the inhibitory effects of esmolol. Whole-cell voltage-clamp experiments revealed that esmolol inhibited the L-type calcium current (I(Ca,L)) and the fast sodium current (I(Na)), with IC(50) values of 0.45 +/- 0.05 and 0.17 +/- 0.025 mmol/L, respectively. CONCLUSION Esmolol at millimolar concentrations causes diastolic ventricular arrest by two mechanisms: at 1 mmol/L (and below), the pronounced negative inotropic effect is due largely to inhibition of L-type Ca(2+) channels; additionally, higher concentrations prevent action potential conduction, probably due to the inhibition of fast Na(+) channels.