(1) Role of PARP-SIRT1 axis of signaling in development of HF
Poly (ADP-ribose) polymerase-1 (PARP) is a prototype member of the PARP family of nuclear enzymes present in eukaryotes. It is activated by increased ROS synthesis, DNA-damage and Ca2+ overloading. PARP catalyzes transfer of successive units of ADP-ribose from nicotinamide adenine dinucleotide (NAD) to target proteins, a process called poly-ADP-ribosylation. Under homeostatic conditions, PARP participates in the regulation of many cellular processes, including DNA-repair, gene transcription, cell-cycle progression, cell-survival, chromatin remodeling and genome stability. However, over-activation of PARP threatens cell-survival as it consumes cellular NAD content to add extended chains of ADP-ribose moieties (up to 200) to target proteins. Since NAD is essential for many cellular reactions, depletion of NAD stores by PARP over-activation threatens cell-survival. One group of factors which are most affected by change in cellular NAD levels are the class-III HDACs, also called Sirtuins or SIRTs (2). The mammalian genome encodes seven sirtuin isoforms (SIRT1-SIRT3). SIRT1 is a redox-sensitive NAD dependent nuclear sirtuin. In addition to deacetylating histones, SIRT1 also deacetylates other proteins, including p53, Ku70, FOXOs and Nf-kB and inhibits their pro-apoptotic activity. Recent data obtained in our laboratory indicates that reciprocal expression of these two enzymes (PARP & SIRT1) plays a central role in myocyte cell-survival/death and the progression of cardiac hypertrophy to failure. In this project our interest is to test the hypothesis, whether PARP over activation during pathologic stress of cells reduces the SIRT1 deacetylase activity due to depletion of cellular NAD stores. These changes shift the balance from cell-survival towards cell death during hypertrophic stimulation of cardiomyocytes, resulting into gene dys-regulation and myocyte cell death, which eventually leads to chamber dilation and muscle decompensation associated with the development of pathologic hypertrophy.
Figure 1: Model illustrating the role of PARP to regulate cardiac gene expression and cell-survival: Cellular stress activates Ca2+-signaling and synthesis of reactive (R) oxygen (O) and nitrogen (N) species. These signals activate PARP, leading to poly (ADP) ribosylation of proteins by adding multiple ADP-ribose units (up to 200units) from NAD to the target protein. This reaction consumes cellular NAD stores and generates nicotinamide as a by-product. Protein de-ribosylation is carried out by another enzyme called poly (ADP) ribose glycohydrolase (PARG). In conditions of mild PARP activation, protein-poly (ADP) ribosylation helps to DNA repair process and chromatin loosening, leading to gene activation and cell-survival. However, in conditions of PARP over-activation, cell survival is threatened in many ways: (a) By inhibiting gene transcription; either directly by ribosylation of transcription factors, which adds massive negative charge on factors and thus interferes in their protein-protein interaction; or indirectly, by suppressing F-actin stabilization, which leads to repression of actin-signaling to key transcription factors, such as SRF. (b) Depletion of cellular NAD levels reduces activity of SIRT1 deactylase leading to hyper acetylation of factors such as p53, Ku70, FOXOs and NF-kB, which consequently promotes their pro-apoptotic potential. Reduction of cellular NAD content could also interfere with mitochondrial respiration leading to ATP-deficit and release of apoptotic inducing factors (AIF) (12). Question mark (?) signifies dispute in opinion whether a drop in cytosolic NAD content will also result in depletion of mitochondrial NAD stores.
(2) Activating Sirtuins to prevent adverse cardiac remodeling post MI
One of the most common forms of heart disease leading to heart failure is coronary artery disease. Patients with coronary artery disease often need to be revascularized with coronary artery bypass grafting. Despite excellent overall outcomes of this procedure, long-term survival after surgical revascularization of patients with coronary artery disease remains poor, because of ischemia to peri-infarct region and to remote territories. Dysfunction of the heart that occurs after myocardial infarction (MI) is largely due to adverse cardiac remodeling, a process associated with transformation of cardiac fibroblasts (CF) to myofibroblasts (myoFB), and excessive synthesis of extra cellular matrix (ECM) and contractile proteins like α-smooth muscle actin (α-SMA). In the heart excessive ECM production from CF generates fibrosis, myocardial stiffening, and cardiac dysfunction, a process known as maladaptive cardiac hypertrophy which is a precursor to HF. Recent studies have shown that aberrant regulation of GSK3/βcatenin and TGFβ signaling plays a central role in the initiation of the CF differentiation to myoFB. Pro-fibrotic stimuli decrease the activity of GSK3β, which results in β-catenin stabilization and its localization into nucleus, where it activates transcription of genes promoting fibroblast proliferation and transformation to myoFB. We are exploring the role sirtuins to block or minimize the evolution of pathological fibrosis in post MI models of HF.
Figure 2: Model illustrating the role of sirtuins to block induction of cardiac fibrosis: GSK3β is a constitutively active kinase which forms a destruction complex with β-catenin together with Axin and APC. GSK3β phosphorylates β-catenin which permits β-catenin degradation by proteosoms. Wnt binding to receptors [frizzled (FZ) and LRP] recruits the cytosolic protein Dishevelled (Dvl) to receptors. This event causes inhibition of GSK3β and thus permits β-catenin stabilization in the cytoplasm. GSK3β is also inhibited by Akt-dependent phosphorylation. Stabilized β-catenin enters into the nucleus and activates transcription of target genes, many of which are hallmark of myoFB differentiation. GSK3β also blocks the expression of α-SMA by inhibiting MRTF/SRF complex, a master regulator of SM gene expression. Thus, stimulation of cells by GF, cytokine and/or Wnt inhibit GSK3β leading to removal of its negative constrain on β-catenin and MRTF/SRF complex. Activation of these targets promotes CF differentiation to myoFB. Work done is our laboratory has shown that NAD treatment blocks Ras-mediated Akt activation. We are exploring several pathways to understand the possible role of sirtuins in regulating CF differentiation to MyoFB and development of Post MI cardiac fibrosis.
Figure 2: Model illustrating the role of sirtuins to block induction of cardiac fibrosis: GSK3β is a constitutively active kinase which forms a destruction complex with β-catenin together with Axin and APC. GSK3β phosphorylates β-catenin which permits β-catenin degradation by proteosoms. Wnt binding to receptors [frizzled (FZ) and LRP] recruits the cytosolic protein Dishevelled (Dvl) to receptors. This event causes inhibition of GSK3β and thus permits β-catenin stabilization in the cytoplasm. GSK3β is also inhibited by Akt-dependent phosphorylation. Stabilized β-catenin enters into the nucleus and activates transcription of target genes, many of which are hallmark of myoFB differentiation. GSK3β also blocks the expression of α-SMA by inhibiting MRTF/SRF complex, a master regulator of SM gene expression. Thus, stimulation of cells by GF, cytokine and/or Wnt inhibit GSK3β leading to removal of its negative constrain on β-catenin and MRTF/SRF complex. Activation of these targets promotes CF differentiation to myoFB. Work done is our laboratory has shown that NAD treatment blocks Ras-mediated Akt activation. We are exploring several pathways to understand the possible role of sirtuins in regulating CF differentiation to MyoFB and development of Post MI cardiac fibrosis.
(3) Blocking cardiotoxicity of anti-cancer drugs
Anti-cancer drugs often exhibit late onset toxicity to the heart despite current medical therapy. Delayed cardiomyopathy is a frequent complication with doxorubicin (Dox) therapy which is common drug used for treatment of many forms of cancer. In the past two decades considerable research efforts have been directed towards the goal of understanding the mechanism of cardio-toxicity of anticancer drugs; however, the casual mechanism of cardiomyopathy remains still unclear. It is generally believed that mitochondrial damage and overt ROS production are involved. But the use of ROS inhibitors for treating Dox cardiomyopathy has not been successful, and at present no effective therapy is available to treat an established Dox-cardiomyopathy. Cardiac transplantation remains the only ultimate solution for a patient with Dox-induced heart failure. Because decision to discontinue anti-cancer therapy for a patient is difficult, new understanding of the underlying mechanism of drug cardio toxicity is warranted. We are interested to understand the role of cardio-protective sirtuins to block Dox toxicity to the heart.
(4) Role of sirtuins in the aging associated heart and skeletal muscle diseases
We lose skeletal muscle mass as we age, and our aging heart becomes more susceptible to develop cardiomyopathy and adverse cardiac remodeling following sustained stress. There is loss of myocyte numbers and loss of contractile elements within myocytes of aging muscle tissue. The cardiac levels of sirtuins have been also reported to be reduced as we age. In this project we are interested to test the hypothesis whether sirtuins are capable of maintaining myocyte numbers of an aging heart and skeletal muscle, and whether they play a role in regulating contractile function of myocytes. We are also in the process of screening a library of pharmacological compounds with the goal of identifying isoform-specific sirtuin activators which could be used to induce endogenous sirtuin levels and to protect the heart and skeletal muscle from deteriorating with age.