Gail Sievert

Cardiac Troponin T, a Sarcomeric AKAP, Tethers Protein Kinase A at the Myofilaments

Efficient and specific phosphorylation of PKA substrates, elicited in response to β-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A-kinase anchoring protein), cardiac troponin T (cTnT). Using yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cTn exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show that PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.

Follicle-stimulating hormone interacts with exoloop 3 of the receptor.

The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of ∼330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of580KVPLITVSKAK590 was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH α but not FSH β. This is in contrast to photoaffinity labeling of FSH β by the peptide mimic of the N-terminal region of the receptor. Leu583 and Ile584 are crucial for the interaction of FSH and exoloop 3. Substitutions of these two residues enhanced the hormone binding affinity. This is due to the loss of the original side chains but not the introduction of new side chains. The Leu583 and Ile584 side chains appear to project in opposite directions. Ile584 appears to be so specific and to require flexibility and stereo specificity so that no other amino acids can fit into its place. Leu583 is less specific. The improvement in hormone binding by substitutions was offset by the severe impairment of signal generation of cAMP and/or inositol phosphate. For example, the Phe or Tyr substitution of Leu583 improved the hormone binding and cAMP induction but impaired IP induction. On the other hand, the substitutions for Ile584 and Lys590abolished the cAMP and IP induction. Our results open a logical question whether Leu583, Ile584, and Lys590 interact with the exodomain and/or the hormone. The answers will provide new insights into the mechanisms of hormone binding and signal generation.

Rem-GTPase regulates cardiac myocyte L-type calcium current

Rationale: The L-type calcium channels (LTCC) are critical for maintaining Ca2+-homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK–LTCC interactions is untested. Objective: In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca2+ current (ICa,L) via LTCC in murine cardiomyocytes. Methods and Results: Rem knockout mice (Rem−/−) were engineered, and ICa,L and Ca2+-handling properties were assessed. Rem−/− ventricular cardiomyocytes displayed increased ICa,L density. ICa,L activation was shifted positive on the voltage axis, and β-adrenergic stimulation normalized this shift compared with wild-type ICa,L. Current kinetics, steady-state inactivation, and facilitation was unaffected by Rem−/−. Cell shortening was not significantly different. Increased ICa,L density in the absence of frank phenotypic differences motivated us to explore putative compensatory mechanisms. Despite the larger ICa,L density, Rem−/− cardiomyocyte Ca2+ twitch transient amplitude was significantly less than that compared with wild type. Computer simulations and immunoblot analysis suggests that relative dephosphorylation of Rem−/− LTCC can account for the paradoxical decrease of Ca2+ transients. Conclusions: This is the first demonstration that loss of an RGK protein influences ICa,L in vivo in cardiac myocytes.

Cardiomyopathy-causing deletion K210 in cardiac troponin T alters phosphorylation propensity of sarcomeric proteins.

Ca(2+) desensitization of myofilaments is indicated as a primary mechanism for the pathogenesis of familial dilated cardiomyopathy (DCM) associated with the deletion of lysine 210 (DeltaK210) in cardiac troponin T (cTnT). DeltaK210 knock-in mice closely recapitulate the clinical phenotypes documented in patients with this mutation. Considerable evidence supports the proposition that phosphorylation of cardiac sarcomeric proteins is a key modulator of function and may exacerbate the effect of the deletion. In this study we investigate the impact of K210 deletion on phosphorylation propensity of sarcomeric proteins. Analysis of cardiac myofibrils isolated from DeltaK210 hearts identified a decrease in phosphorylation of cTnI (46%), cTnT (30%) and MyBP-C (32%) compared with wild-type controls. Interestingly, immunoblot analyses with phospho-specific antibodies show augmented phosphorylation of cTnT-Thr(203) (28%) and decreased phosphorylation of cTnI-Ser(23/24) (41%) in mutant myocardium. In vitro kinase assays indicate that DeltaK210 increases phosphorylation propensity of cTnT-Thr(203) three-fold, without changing cTnI-Ser(23/24) phosphorylation. Molecular modeling of cTnT-DeltaK210 structure reveals changes in the electrostatic environment of cTnT helix (residues 203-224) that lead to a more basic environment around Thr(203), which may explain the enhanced PKC-dependent phosphorylation. In addition, yeast two-hybrid assays indicate that cTnT-DeltaK210 binds stronger to cTnI compared with cTnT-wt. Collectively, our observations suggest that cardiomyopathy-causing DeltaK210 has far-reaching effects influencing cTnI-cTnT binding and posttranslational modifications of key sarcomeric proteins.

Rad GTPase Deletion Increases L‐type Calcium Channel Current Leading to Increased Cardiac Contraction

Background The small GTPase Rad is a negative regulator of voltage‐dependent L‐type calcium channel current (ICaL); however, the effects of Rad ablation on cardiomyocyte function are unknown. The objective of this study is to test the hypothesis that Rad‐depletion causes positive inotropic effects without inducing cardiac hypertrophy. Methods and Results Ventricular myocytes from adult Rad−/− mice were isolated and evaluated by patch‐clamp recordings for ICa,L and action potentials, Ca2+ transients, and sarcomere shortening. Maximum ICaL is elevated in Rad−/− (maximal conductance 0.35±0.04 picoSiemens/picoFarad (pS/pF) wild‐type; 0.61±0.14 pS/pF Rad−/−), decay kinetics are faster, and ICa,L activates at lower voltages (activation midpoint −7.2±0.6 wild‐type; −11.7±0.9 Rad−/−) mimicking effects of β‐adrenergic receptor stimulation. Diastolic and twitch calcium are elevated in Rad−/− (F340/380: 1.03 diastolic and 0.35 twitch for wild‐type; 1.47 diastolic and 0.736 twitch for Rad−/−) and sarcomere shortening is enhanced (4.31% wild‐type; 14.13% Rad−/−) at lower pacing frequencies. Consequentially, frequency‐dependence of Ca2+ transients is less in Rad−/−, and the frequency dependence of relaxation is also blunted. In isolated working hearts, similar results were obtained; chiefly, +dP/dt was elevated at baseline and developed pressure was relatively nonresponsive to acute β‐adrenergic receptor stimulation. In single cells, at subphysiological frequencies, nonstimulated calmodulin‐dependent protein kinase–sensitive calcium release is observed. Remarkably, Rad−/− hearts did not show hypertrophic growth despite elevated levels of diastolic calcium. Conclusions This study demonstrates that the depletion of Rad GTPase is equivalent to sympathomimetic β‐adrenergic receptor, without stimulating cardiac hypertrophy. Thus, targeting Rad GTPase is a novel potential therapeutic target for Ca2+‐homeostasis–driven positive inotropic support of the heart.

The cardiac L-type calcium channel distal carboxy terminus autoinhibition is regulated by calcium

The L-type calcium channel (LTCC) provides trigger Ca(2+) for sarcoplasmic reticulum Ca-release, and LTCC function is influenced by interacting proteins including the LTCC distal COOH terminus (DCT) and calmodulin. DCT is proteolytically cleaved and reassociates with the LTCC complex to regulate calcium channel function. DCT reduces LTCC barium current (I(Ba,L)) in reconstituted channel complexes, yet the contribution of DCT to LTCC Ca(2+) current (I(Ca,L)) in cardiomyocyte systems is unexplored. This study tests the hypothesis that DCT attenuates cardiomyocyte I(Ca,L). We measured LTCC current and Ca(2+) transients with DCT coexpressed in murine cardiomyocytes. We also heterologously coexpressed DCT and Ca(V)1.2 constructs with truncations corresponding to the predicted proteolytic cleavage site, Ca(V)1.2Δ1801, and a shorter deletion corresponding to well-studied construct, Ca(V)1.2Δ1733. DCT inhibited I(Ba,L) in cardiomyocytes, and in human embryonic kidney (HEK) 293 cells expressing Ca(V)1.2Δ1801 and Ca(V)1.2Δ1733. Ca(2+)-CaM relieved DCT block in cardiomyocytes and HEK cells. The selective block of I(Ba,L) combined with Ca(2+)-CaM effects suggested that DCT-mediated blockade may be relieved under conditions of elevated Ca(2+). We therefore tested the hypothesis that DCT block is dynamic, increasing under relatively low Ca(2+), and show that DCT reduced diastolic Ca(2+) at low stimulation frequencies but spared high frequency Ca(2+) entry. DCT reduction of diastolic Ca(2+) and relief of block at high pacing frequencies and under conditions of supraphysiological bath Ca(2+) suggests that a physiological function of DCT is to increase the dynamic range of Ca(2+) transients in response to elevated pacing frequencies. Our data motivate the new hypothesis that DCT is a native reverse use-dependent inhibitor of LTCC current.