Patricia Rodriguez

Inducible Expression of Active Protein Phosphatase-1 Inhibitor-1 Enhances Basal Cardiac Function and Protects Against Ischemia/Reperfusion Injury

Ischemic heart disease, which remains the leading cause of morbidity and mortality in the Western world, is invariably characterized by impaired cardiac function and disturbed Ca2+ homeostasis. Because enhanced inhibitor-1 (I-1) activity has been suggested to preserve Ca2+ cycling, we sought to define whether increases in I-1 activity in the adult heart may ameliorate contractile dysfunction and cellular injury in the face of an ischemic insult. To this end, we generated an inducible transgenic mouse model that enabled temporally controlled expression of active I-1 (T35D). Active I-1 expression in the adult heart elicited significant enhancement of contractile function, associated with preferential phospholamban phosphorylation and enhanced sarcoplasmic reticulum Ca2+-transport. Further phosphoproteomic analysis revealed alterations in proteins associated with energy production and protein synthesis, possibly to support the increased metabolic demands of the hyperdynamic hearts. Importantly, on ischemia/reperfusion-induced injury, active I-1 expression augmented contractile function and recovery. Further examination revealed that the infarct region and apoptotic as well as necrotic injuries were significantly attenuated by enhanced I-1 activity. These cardioprotective effects were associated with suppression of the endoplasmic reticulum stress response. The present findings indicate that increased I-1 activity in the adult heart enhances Ca2+ cycling and improves mechanical recovery, as well as cell survival after an ischemic insult, suggesting that active I-1 may represent a potential therapeutic strategy in myocardial infarction.

Small Heat Shock Protein 20 Interacts With Protein Phosphatase-1 and Enhances Sarcoplasmic Reticulum Calcium Cycling

Background: Heat shock proteins (Hsp) are known to enhance cell survival under various stress conditions. In the heart, the small Hsp20 has emerged as a key mediator of protection against apoptosis, remodeling, and ischemia/reperfusion injury. Moreover, Hsp20 has been implicated in modulation of cardiac contractility ex vivo. The objective of this study was to determine the in vivo role of Hsp20 in the heart and the mechanisms underlying its regulatory effects in calcium (Ca) cycling. Methods and Results: Hsp20 overexpression in intact animals resulted in significant enhancement of cardiac function, coupled with augmented Ca cycling and sarcoplasmic reticulum Ca load in isolated cardiomyocytes. This was associated with specific increases in phosphorylation of phospholamban (PLN) at both Ser16 and Thr17, relieving its inhibition of the apparent Ca affinity of SERCA2a. Accordingly, the inotropic effects of Hsp20 were abrogated in cardiomyocytes expressing nonphosphorylatable PLN (S16A/T17A). Interestingly, the activity of type 1 protein phosphatase (PP1), a known regulator of PLN signaling, was significantly reduced by Hsp20 overexpression, suggesting that the Hsp20 stimulatory effects are partially mediated through the PP1–PLN axis. This hypothesis was supported by cell fractionation, coimmunoprecipitation, and coimmunolocalization studies, which revealed an association between Hsp20, PP1, and PLN. Furthermore, recombinant protein studies confirmed a physical interaction between AA 73 to 160 in Hsp20 and AA 163 to 330 in PP1. Conclusions: Hsp20 is a novel regulator of sarcoplasmic reticulum Ca cycling by targeting the PP1–PLN axis. These findings, coupled with the well-recognized cardioprotective role of Hsp20, suggest a dual benefit of targeting Hsp20 in heart disease.

Identification of a novel phosphorylation site in protein phosphatase inhibitor-1 as a negative regulator of cardiac function.

Human and experimental heart failure is characterized by increases in type-1 protein phosphatase activity, which may be partially attributed to inactivation of its endogenous regulator, protein phosphatase inhibitor-1. Inhibitor-1 represents a nodal integrator of two major second messenger pathways, adenosine 3′,5′-cyclic monophosphate (cAMP) and calcium, which mediate its phosphorylation at threonine 35 and serine 67, respectively. Here, using recombinant inhibitor-1 wild-type and mutated proteins, we identified a novel phosphorylation site in inhibitor-1, threonine 75. This phosphoamino acid was phosphorylated in vitro by protein kinase Cα independently and to the same extent as serine 67, the previous protein kinase Cα-identified site. Generation of specific antibodies for the phosphorylated and dephosphorylated threonine 75 revealed that this site is phosphorylated in rat and dog hearts. Adenoviral-mediated expression of the constitutively phosphorylated threonine 75 inhibitor-1 in isolated myocytes was associated with specific stimulation of type-1 protein phosphatase activity and marked inhibition of the sarcoplasmic calcium pump affinity for calcium, resulting in depressed contractility. Thus, phosphorylation of inhibitor-1 at threonine 75 represents a new mechanism of cardiac contractility regulation, partially through the alteration of sarcoplasmic reticulum calcium transport activity.

A human polymorphism of protein phosphatase-1 inhibitor-1 is associated with attenuated contractile response of cardiomyocytes to β-adrenergic stimulation

Aberrant β‐adrenergic signaling and de pressed calcium homeostasis, associated with an imbal ance of protein kinase A and phosphatase‐1 activities, are hallmarks of heart failure. Phosphatase‐1 is re strained by its endogenous inhibitor, protein phospha tase inhibitor‐1 (PPI‐1). We assessed 352 normal sub jects, along with 959 patients with heart failure and identified a polymorphism in PPI‐1 (G147D) exclu sively in black subjects. To determine whether the G147D variant could affect cardiac function, we in fected adult cardiomyocytes with adenoviruses expressing D147 or wild‐type (G147) PPI‐1. Under basal con ditions, there were no significant differences in fractional shortening or contraction or relaxation rates. However, the enhancement of contractile parameters after isoproterenol stimulation was significantly blunted in D147 compared with G147 and control myocytes. Similar findings were observed in calcium kinetics. The attenuated β‐agonist response was associ ated with decreased (50%) phosphorylation of phos‐ pholamban (PLN) at serine 16, whereas phosphorylation of troponin I and ryanodine receptor was unaltered. These findings suggest that the human G147D PPI‐1 can attenuate responses of cardiomyo cytes to β‐adrenergic agonists by decreasing PLN phos phorylation and therefore may contribute to deterio rated function in heart failure.— Chen, G., Zhou, X., Nicolaou, P., Rodriguez, P., Song, G., Mitton, B., Pathak, A., Zachariah, A., Fan, G.‐C., Dorn, G. A., II., Kranias, E. G. A human polymorphism of protein phosphatase‐1 inhibitor‐1 is associated with attenuated contractile response of cardiomyocytes to β‐adrenergic stimulation. FASEB J. 22, 1790–1796 (2008)

Phosphorylation of Glutathione-S-transferase by Protein Kinase C-α Implications for Affinity-Tag Purification

Expression and purification of proteins as fusions with glutathione S-transferase (GST) is a standard and widely employed system. In more than 2500 published studies, GST has been used to facilitate the purification of recombinant proteins, assess protein–protein interactions, and establish protein function. In this report, we provide evidence that GST can be phosphorylated in vitro by protein kinase C-α (PKC-α) at Ser-93. Therefore, since GST itself may be a target for a number of catalytic enzymes, failure to remove the GST tag from the recombinant protein may lead to inaccurate conclusions.

Deletion of delta-like 1 homologue accelerates fibroblast–myofibroblast differentiation and induces myocardial fibrosis

AIMS Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process. METHODS AND RESULTS Using Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardio-fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is mediated by TGF-β1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-β1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-β1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR-370 mediated Dlk1s regulation of cardiac fibroblast-myofibroblast differentiation by directly targeting TGFβ-R2/Smad-3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process. CONCLUSION These findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differentiation by interfering with TGFβ/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFβ signalling leads to chronic fibrosis.

The Small Heat Shock Protein 20 (Hsp20) Interacts with Protein Phosphatase-1 and Enhances Sarcoplasmic Reticulum Ca-Cycling

Background: Heat shock proteins (Hsp) are known to enhance cell survival under various stress conditions. In the heart, the small Hsp20 has emerged as a key mediator of protection against apoptosis, remodeling, and ischemia/reperfusion injury. Moreover, Hsp20 has been implicated in modulation of cardiac contractility ex vivo. The objective of this study was to determine the in vivo role of Hsp20 in the heart and the mechanisms underlying its regulatory effects in calcium (Ca) cycling. Methods and Results: Hsp20 overexpression in intact animals resulted in significant enhancement of cardiac function, coupled with augmented Ca cycling and sarcoplasmic reticulum Ca load in isolated cardiomyocytes. This was associated with specific increases in phosphorylation of phospholamban (PLN) at both Ser16 and Thr17, relieving its inhibition of the apparent Ca affinity of SERCA2a. Accordingly, the inotropic effects of Hsp20 were abrogated in cardiomyocytes expressing nonphosphorylatable PLN (S16A/T17A). Interestingly, the activity of type 1 protein phosphatase (PP1), a known regulator of PLN signaling, was significantly reduced by Hsp20 overexpression, suggesting that the Hsp20 stimulatory effects are partially mediated through the PP1–PLN axis. This hypothesis was supported by cell fractionation, coimmunoprecipitation, and coimmunolocalization studies, which revealed an association between Hsp20, PP1, and PLN. Furthermore, recombinant protein studies confirmed a physical interaction between AA 73 to 160 in Hsp20 and AA 163 to 330 in PP1. Conclusions: Hsp20 is a novel regulator of sarcoplasmic reticulum Ca cycling by targeting the PP1–PLN axis. These findings, coupled with the well-recognized cardioprotective role of Hsp20, suggest a dual benefit of targeting Hsp20 in heart disease.

Small Heat Shock Protein 20 Interacts With Protein Phosphatase-1 and Enhances Sarcoplasmic Reticulum Calcium CyclingNovelty and Significance

Background: Heat shock proteins (Hsp) are known to enhance cell survival under various stress conditions. In the heart, the small Hsp20 has emerged as a key mediator of protection against apoptosis, remodeling, and ischemia/reperfusion injury. Moreover, Hsp20 has been implicated in modulation of cardiac contractility ex vivo. The objective of this study was to determine the in vivo role of Hsp20 in the heart and the mechanisms underlying its regulatory effects in calcium (Ca) cycling. Methods and Results: Hsp20 overexpression in intact animals resulted in significant enhancement of cardiac function, coupled with augmented Ca cycling and sarcoplasmic reticulum Ca load in isolated cardiomyocytes. This was associated with specific increases in phosphorylation of phospholamban (PLN) at both Ser16 and Thr17, relieving its inhibition of the apparent Ca affinity of SERCA2a. Accordingly, the inotropic effects of Hsp20 were abrogated in cardiomyocytes expressing nonphosphorylatable PLN (S16A/T17A). Interestingly, the activity of type 1 protein phosphatase (PP1), a known regulator of PLN signaling, was significantly reduced by Hsp20 overexpression, suggesting that the Hsp20 stimulatory effects are partially mediated through the PP1–PLN axis. This hypothesis was supported by cell fractionation, coimmunoprecipitation, and coimmunolocalization studies, which revealed an association between Hsp20, PP1, and PLN. Furthermore, recombinant protein studies confirmed a physical interaction between AA 73 to 160 in Hsp20 and AA 163 to 330 in PP1. Conclusions: Hsp20 is a novel regulator of sarcoplasmic reticulum Ca cycling by targeting the PP1–PLN axis. These findings, coupled with the well-recognized cardioprotective role of Hsp20, suggest a dual benefit of targeting Hsp20 in heart disease.