Eva Denise Martin

New Therapeutic Targets in Cardiology p38 Alpha Mitogen-Activated Protein Kinase for Ischemic Heart Disease

The p38 mitogen-activated protein kinases (p38s) are members of a key signaling pathway that responds to varied stresses, including those that contribute to heart failure. This review will focus on the ways in which p38 can be manipulated based on its mechanisms of activation and structure and how this knowledge has led to current cardiovascular clinical trials. Phosphorylation is the process through which extracellular signals are communicated to the interior of the cell and it is catalyzed by kinases. The protein kinases transfer the terminal phosphate group from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue. This posttranslational modification can transform the function of the substrate protein by changing its binding partners, subcellular location, stability, and/or activity. In turn, these transformations have an impact on diverse fundamental cellular processes to alter transcription, translation, metabolism, contractility, growth, death, and/or differentiation. The 518 known protein kinases occupy a relatively high proportion (1.7%) of the human proteome of which an even higher proportion (≈30%) is modified by phosphorylation.1 The dysregulation of kinases is a common feature of many cancers, because growth signals can be inappropriately amplified by mutations that render kinases constitutively active. Consequently, drugs to inhibit kinases are the most successful and rapidly growing of the recent advances in cancer therapy. This success has occurred despite initial concerns of the constraints imposed by the high degree of homology between the ATP binding sites of different protein kinases and of the need to compete with millimolar concentrations of ATP. Initial success was achieved with imatinib (Gleevec) that targets an abnormal fusion protein kinase formed in the translocation event that creates the Philadelphia Chromosome in chronic myeloid leukemia.2 Another early success was the targeting of the human epidermal growth factor receptor 2 tyrosine kinase that is abnormally active …

Mechanism and consequence of the autoactivation of p38α mitogen-activated protein kinase promoted by TAB1

p38α mitogen-activated protein kinase (p38α) is activated by a variety of mechanisms, including autophosphorylation initiated by TGFβ-activated kinase 1 binding protein 1 (TAB1) during myocardial ischemia and other stresses. Chemical-genetic approaches and coexpression in mammalian, bacterial and cell-free systems revealed that mouse p38α autophosphorylation occurs in cis by direct interaction with TAB1(371–416). In isolated rat cardiac myocytes and perfused mouse hearts, TAT-TAB1(371–416) rapidly activates p38 and profoundly perturbs function. Crystal structures and characterization in solution revealed a bipartite docking site for TAB1 in the p38α C-terminal kinase lobe. TAB1 binding stabilizes active p38α and induces rearrangements within the activation segment by helical extension of the Thr-Gly-Tyr motif, allowing autophosphorylation in cis. Interference with p38α recognition by TAB1 abolishes its cardiac toxicity. Such intervention could potentially circumvent the drawbacks of clinical pharmacological inhibitors of p38 catalytic activity.

p38 MAPK in cardioprotection – are we there yet?

PKs transfer a phosphate from ATP to the side‐chain hydroxyl group of a serine, threonine or tyrosine residue of a substrate protein. This in turn can alter that proteins function; modulating fundamental cellular processes including, metabolism, transcription, growth, division, differentiation, motility and survival. PKs are subdivided into families based on homology. One such group are the stress‐activated kinases, which as the name suggests, are activated in response to cellular stresses such as toxins, cytokines, mechanical deformation and osmotic stress. Members include the p38 MAPK family, which is composed of α, β, γ and δ, isoforms which are encoded by separate genes. These kinases transduce extracellular signals and coordinate the cellular responses needed for adaptation and survival. However, in cardiovascular and other disease states, these same systems can trigger maladaptive responses that aggravate, rather than alleviate, the disease. This situation is analogous to adrenergic, angiotensin and aldosterone signalling in heart failure, where inhibition is beneficial despite the importance of these hormones to homeostasis. The question is whether similar benefits could accrue from p38 inhibition? In this review, we will discuss the structure and function of p38, the history of p38 inhibitors and their use in preclinical studies. Finally, we will summarize the results of recent cardiovascular clinical trials with p38 inhibitors.

Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response

The Frank–Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16—a site important for diastolic relaxation. We find that hearts of Cys42Ser PKGIα knock-in (KI) mice, which are resistant to PKGIα oxidation, have diastolic dysfunction and a diminished ability to couple ventricular filling with cardiac output on a beat-to-beat basis. Intracellular calcium dynamics of ventricular myocytes isolated from KI hearts are altered in a manner consistent with impaired relaxation and contractile function. We conclude that oxidation of PKGIα during myocardial stretch is crucial for diastolic relaxation and fine-tunes the Frank–Starling response.

Quantifying the Release of Biomarkers of Myocardial Necrosis from Cardiac Myocytes and Intact Myocardium

BACKGROUND Myocardial infarction is diagnosed when biomarkers of cardiac necrosis exceed the 99th centile, although guidelines advocate even lower concentrations for early rule-out. We examined how many myocytes and how much myocardium these concentrations represent. We also examined if dietary troponin can confound the rule-out algorithm. METHODS Individual rat cardiac myocytes, rat myocardium, ovine myocardium, or human myocardium were spiked into 400-μL aliquots of human serum. Blood was drawn from a volunteer after ingestion of ovine myocardium. High-sensitivity assays were used to measure cardiac troponin T (cTnT; Roche, Elecsys), cTnI (Abbott, Architect), and cardiac myosin-binding protein C (cMyC; EMD Millipore, Erenna®). RESULTS The cMyC assay could only detect the human protein. For each rat cardiac myocyte added to 400 μL of human serum, cTnT and cTnI increased by 19.0 ng/L (95% CI, 16.8-21.2) and 18.9 ng/L (95% CI, 14.7-23.1), respectively. Under identical conditions cTnT, cTnI, and cMyC increased by 3.9 ng/L (95% CI, 3.6-4.3), 4.3 ng/L (95% CI, 3.8-4.7), and 41.0 ng/L (95% CI, 38.0-44.0) per μg of human myocardium. There was no detectable change in cTnI or cTnT concentration after ingestion of sufficient ovine myocardium to increase cTnT and cTnI to approximately 1 × 108 times their lower limits of quantification. CONCLUSIONS Based on pragmatic assumptions regarding cTn and cMyC release efficiency, circulating species, and volume of distribution, 99th centile concentrations may be exceeded by necrosis of 40 mg of myocardium. This volume is much too small to detect by noninvasive imaging.

In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy

ABSTRACT In yeast, Tom22, the central component of the TOMM (translocase of outer mitochondrial membrane) receptor complex, is responsible for the recognition and translocation of synthesized mitochondrial precursor proteins, and its protein kinase CK2-dependent phosphorylation is mandatory for TOMM complex biogenesis and proper mitochondrial protein import. In mammals, the biological function of protein kinase CSNK2/CK2 remains vastly elusive and it is unknown whether CSNK2-dependent phosphorylation of TOMM protein subunits has a similar role as that in yeast. To address this issue, we used a skeletal muscle-specific Csnk2b/Ck2β-conditional knockout (cKO) mouse model. Phenotypically, these skeletal muscle Csnk2b cKO mice showed reduced muscle strength and abnormal metabolic activity of mainly oxidative muscle fibers, which point towards mitochondrial dysfunction. Enzymatically, active muscle lysates from skeletal muscle Csnk2b cKO mice phosphorylate murine TOMM22, the mammalian ortholog of yeast Tom22, to a lower extent than lysates prepared from controls. Mechanistically, CSNK2-mediated phosphorylation of TOMM22 changes its binding affinity for mitochondrial precursor proteins. However, in contrast to yeast, mitochondrial protein import seems not to be affected in vitro using mitochondria isolated from muscles of skeletal muscle Csnk2b cKO mice. PINK1, a mitochondrial health sensor that undergoes constitutive import under physiological conditions, accumulates within skeletal muscle Csnk2b cKO fibers and labels abnormal mitochondria for removal by mitophagy as demonstrated by the appearance of mitochondria-containing autophagosomes through electron microscopy. Mitophagy can be normalized by either introduction of a phosphomimetic TOMM22 mutant in cultured myotubes, or by in vivo electroporation of phosphomimetic Tomm22 into muscles of mice. Importantly, transfection of the phosphomimetic Tomm22 mutant in muscle cells with ablated Csnk2b restored their oxygen consumption rate comparable to wild-type levels. In sum, our data show that mammalian CSNK2-dependent phosphorylation of TOMM22 is a critical switch for mitophagy and reveal CSNK2-dependent physiological implications on metabolism, muscle integrity and behavior.

The TAB1-p38α complex aggravates myocardial injury and can be targeted by small molecules

Inhibiting MAPK14 (p38α) diminishes cardiac damage in myocardial ischemia. During myocardial ischemia, p38α interacts with TAB1, a scaffold protein, which promotes p38α autoactivation; active p38α (pp38α) then transphosphorylates TAB1. Previously, we solved the X-ray structure of the p38α-TAB1 (residues 384–412) complex. Here, we further characterize the interaction by solving the structure of the pp38α-TAB1 (residues 1–438) complex in the active state. Based on this information, we created a global knock-in (KI) mouse with substitution of 4 residues on TAB1 that we show are required for docking onto p38α. Whereas ablating p38α or TAB1 resulted in early embryonal lethality, the TAB1-KI mice were viable and had no appreciable alteration in their lymphocyte repertoire or myocardial transcriptional profile; nonetheless, following in vivo regional myocardial ischemia, infarction volume was significantly reduced and the transphosphorylation of TAB1 was disabled. Unexpectedly, the activation of myocardial p38α during ischemia was only mildly attenuated in TAB1-KI hearts. We also identified a group of fragments able to disrupt the interaction between p38α and TAB1. We conclude that the interaction between the 2 proteins can be targeted with small molecules. The data reveal that it is possible to selectively inhibit signaling downstream of p38α to attenuate ischemic injury.

22 Identifying the Substrates of P38 MAPK alpha in the Heart

Activation of p38 mitogen activated protein kinase alpha during myocardial ischaemia results in myocardial damage. Identifying and targeting the relevant substrates downstream of p38alpha may potentially provide a therapeutic target to limit myocardial injury. We aim to identify the substrates of p38alpha particularly during myocardial ischaemia. We have adopted the analogue sensitive kinase strategy, as developed by the Shokat lab, of mutating the kinase to allow it to use expanded forms of ATP that wild type kinases are not capable of using. In addition, these ATP analogues are modified with a gamma thiophosphate instead of the gamma phosphate that is transferred during catalysis. This transferred thiophosphate acts as a label and allows for identification and isolation of the substrates of the kinase. We have characterised an analogue sensitive form of p38 and confirmed it to have a similar substrate specificity to wild type p38alpha. We have successfully optimized the conditions for labelling p38alpha substrates in mouse heart homogenates exposed to the analogue sensitive kinase and the expanded form of ATP. We isolated and used MS to identify the substrates and the sites of phosphorylation. 61 proteins with phosphorylation at p38 consensus sequences were identified and we are currently in the process of validating these results. In conclusion we have optimized the conditions for identification and isolation of substrates of p38 alpha in the mouse heart. In the future, we will use the identification of these substrates to elucidate the mechanism through which p38alpha mediates its effect during myocardial ischaemia.