- Email: [email protected]
TLR4-Mediated Signals Converge on the PGC-1 Family of Nuclear Receptor Coactivators to Control Myocardial Metabolism and Function
The 13th Annual Scientific Meeting
HFSA
S1
Jay N. Cohn New Investigator Award: Basic Science 001
003
TLR4-Mediated Signals Converge on the PGC-1 Family of Nuclear Receptor Coactivators to Control Myocardial Metabolism and Function Joel D. Schilling1, Teresa Leone2, Ling Lai2, Nanda Sambandam1, Daniel P. Kelly2; 1 Internal Medicine-Cardiology, Washington University School of Medicine, St. Louis, MO; 2Burnham Institute for Medical Research, Orlando, FL
Modulating Neuregulin/ErbB4 Signaling to Promote Cardiomyocyte Replacement Kevin Bersell1, Shima Arab1, Bernhard Haring1,2, Bernhard Kuhn1,2; 1Cardiology, Children’s Hospital Boston, Boston, MA; 2Pediatrics, Harvard Medical School, Boston, MA
Introduction: Heart failure (HF) is associated with myocardial inflammation and dysregulated metabolism. Toll-like Receptor 4 (TLR4) is activated by inflammatory stimuli such as lipopolysaccharide (LPS) leading to cytokine production and LV dysfunction. TLR4 has also been implicated in the pathogenesis of insulin resistance (IR), obesity, and HF. PGC-1a/b are inducible, cardiac enriched, transcriptional co-activators that regulate metabolism via interactions with nuclear receptors such as PPARa and ERRa. HF is associated with reduced PGC-1 signals. Hypothesis: TLR4-mediated inflammation modulates myocardial metabolism through PGC-1 co-activators. Results: Using an isolated working heart system to assess myocardial fuel utilization, we found that LPS decreased fatty acid oxidation (FAO) and glycolytic rates by 30% and 45%, respectively. LPS also triggered myocyte triglyceride (TG) accumulation, consistent with the observed decrease FAO capacity. PGC-1a/b, which serve as key regulators of FAO gene expression in heart, were rapidly downregulated by LPS. PGC-1 gene targets involved in FAO and mitochondrial respiration were also downregulated in a coordinated manner following LPS stimulation. In contrast, TLR4-/- mice did not exhibit LPS-mediated PGC-1 downregulation or myocyte TG accumulation. Using a myocyte specific, tetracycline inducible PGC-1b transgenic mouse (tet-on PGC-1b), PGC-1b induction prior to LPS administration reversed the effects of LPS on cardiac FAO and mitochondrial gene expression without altering cytokine levels. Myocardial TG accumulation induced by LPS was also significantly attenuated by forced expression of PGC-1b. Importantly, LPS-mediated LV dysfunction was rescued in tet-on PGC1b mice (LV fractional shortening 40% vs. 25%), demonstrating the physiologic importance of these metabolic alterations. Conclusions: TLR4 activation leads to reduced expression of PGC-1a/b in the heart, which in turn triggers a cascade of metabolic changes characterized by decreased FAO capacity, myocyte TG accumulation, and LV dysfunction. Restoration of PGC-1b levels in the heart reverses the metabolic and functional alterations caused by this inflammatory insult. These data describe a novel PGC-1-mediated regulatory crosstalk mechanism between inflammatory signals and metabolism in the heart, a pathway that is relevant to the pathogenesis of HF.
Introduction: The goal of regenerative therapy for heart failure is to replace cardiomyocytes. Whether differentiated cardiomyocytes can contribute to myocardial regeneration is a controversial question of great biomedical importance. Here we address whether and how differentiated cardiomyocytes can be induced to proliferate. Hypothesis: We hypothesized that the growth factor neuregulin1 (NRG1) and its tyrosine kinase receptor ErbB4 control post-natal proliferation of differentiated cardiomyocytes. Methods: We applied NRG1 systemically by intraperitoneal injection into three different lines of mice: a line that allowed for inducible genetic inactivation of ErbB4, a transgenic line over-expressing ErbB4, and wild type mice (C57BL6). We used uptake of BrdU, a thymidine analog, as readout of DNA synthesis and staining for phosphorylated histone H3 and aurora B kinase as readouts of karyokinesis and cytokinesis, respectively. Results: Inactivation of ErbB4 reduced postnatal cardiomyocyte proliferation, resulting in 19% less cardiomyocyte nuclei compared to littermate controls. Increasing ErbB4 expression enhanced proliferation, resulting in 16% more cardiomyocyte nuclei. Injecting NRG1 in adult wild-type animals increased cycling of differentiated cardiomyocytes. NRG1 induced mononucleated, but not binucleated cardiomyocytes to divide. Using two different thymidine analogs, we demonstrate that under NRG1 stimulation, differentiated cardiomyocytes entered the cell cycle twice and completed the cell cycle at least once. Using sparse genetic labeling of differentiated cardiomyocytes, we determined that NRG1 induced proliferation of individual cardiomyocytes, resulting in a 35-fold increase in the number of genetically labeled clusters of four or more differentiated cardiomyocytes in vivo. To divide, differentiated cardiomyocytes completely disassembled their contractile apparatus in the cleavage furrow. Using genetic fate mapping of differentiated cardiomyocytes, we demonstrate that undifferentiated progenitor cells did not contribute to NRG1-induced cardiomyocyte proliferation. Conclusions: Differentiated mononucleated cardiomyocytes have proliferative potential. ErbB4 is required and NRG1 or ErbB4 are sufficient to induce proliferation of differentiated cardiomyocytes. Thus, controlling NRG1/ErbB4 signaling may provide a promising strategy to promote myocardial regeneration.
002 Tetrahydrobiopterin Rescues b3-/- Mice from Cardiac Hypertrophy and Impaired Function Induced by Pressure Overload Jordan S. Leyton-Mange1, Xiaulin Niu1, Ronghua Yang1, Karen L. Miller1, An L. Moens2, Ashley B. Hale3, Djahida Bedja1, Kathleen L. Gabrielson1, Keith M. Channon3, David A. Kass1, Lili A. Barouch1; 1Dept of Medicine, Johns Hopkins School of Medicine, Baltimore, MD; 2Dept of Cardiology, Maastricht Univ, Maastrich, Netherlands; 3Dept of Cardiovasc Med, Univ of Oxford, Oxford, United Kingdom b3-Adrenergic receptor signaling in the mammalian cardiovascular system has a depressant effect on ventricular contractility, antagonizing the stimulatory role traditionally ascribed to b1/b2-AR’s. Via NOS-dependent increases in NO availability, b3-AR activation may induce an adaptive braking response to excessive catecholamine stimulation present in failing hearts. We have recently presented data revealing impaired cardiac functional compensation and increases in NOS-derived superoxide in b3-/- mice after pressure overload with transverse aortic constriction (TAC). The reducing agent and essential NOS cofactor, tetrahydrobiopterin (BH4), maintains functional coupling and NO-generating catalytic activity. We therefore hypothesized that treatment with BH4 would preserve cardiac function and reduce hypertrophy in b3-/- mice after TAC. Compared to WT mice, 3 weeks of TAC in b3-/- mice produced more marked cardiac hypertrophy as well as impaired function as assessed by echocardiography. These changes were accompanied by an increase in NOS-dependent superoxide production of 39.0% in hearts of TAC’d b3-/- mice over baseline levels, while NOS-dependent superoxide production was undetectable in WT mice. Total cardiac superoxide production in the TAC’d b3-/- hearts trended towards an increase (29.0%) as well, though this was not statistically significantly different from WT hearts or from baseline values. In addition, nNOS and iNOS protein levels were increased in response to TAC in b3-/- mice, though overall cardiac NOS-activity was unchanged, as assessed by arginine-to-citrulline conversion. BH4 treatment completely inhibited the increase in NOS-dependent superoxide production and rescued b3-/- mice from cardiac hypertrophy and impaired fractional shortening when compared to WT. Measurement of BH4 levels revealed no absolute differences between strains, though the BH4/(BH2þB) ratio was slightly lower in b3-/- mice and was unchanged with TAC. These results implicate NOS-derived superoxide in the diminished cardiac compensation after TAC in mice lacking the b3-AR, and that reduction of this superoxide reduces hypertrophy and preserves myocardial systolic function.
004 Survival and Cardiac Remodeling after Myocardial Infarction Are Critically Dependent on the Host Innate Immune Interleukin-1 Receptor Associated Kinase-4 (IRAK-4) Signaling: A Regulator of Bone Marrow-Derived Dendritic Cells Yuichiro Maekawa1, Nobuo Mizue3, Annie Chan2, Yu Shi2, Youan Liu2, Chen Manyin2, Fayez Dawood2, Geoffrey de Coute2, Guo Hua Li2, Jeffrey A. Medin3, Peter P. Liu2,4; 1Cardiology, Keio University School of Medicine, Tokyo, Japan; 2 Toronto General Hospital, University Health Network, Toronto, ON, Canada; 3 Ontario Cancer Institute, University Health Network, Toronto, ON, Canada; 4 Institute of Circulatory and Respiratory Health, Canadian Institutes of Health Research, Toronto, ON, Canada Background: The Innate immune system greatly contributes to the inflammatory process after myocardial infarction (MI). Interleukin-1 receptor associated kinase-4 (IRAK-4), downstream to Toll/Interleukin-1 receptor signaling, has an essential role in regulating the innate immune response. The present study was designed to determine the mechanism by which IRAK-4 is responsible for the cardiac inflammatory process, which consequently affects the left ventricular (LV) remodeling following MI. Methods and Results: Experimental MI was created in IRAK-4-/- and wildtype (WT) mice by left coronary ligation. Mice with a targeted deletion of IRAK4 had an improved survival rate at 4 weeks post-MI. IRAK-4-deficient mice also demonstrated attenuated cardiac dilation and decreased inflammation in the infarcted myocardium, which was associated with less pro-inflammatory and Th1 cytokines expression mediated by suppression of nuclear factor-kB and c-Jun N-terminal kinase activation. IRAK-4-/- mice had fewer infiltrations of CD45þ leukocytes and CD11cþ dendritic cells (DCs), inhibition of apoptosis and reduced fibrosis and nitric oxide production. Cardiac DCs in IRAK-4-/- mice were relatively immature or functionally naı¨ve, as they demonstrated less cytokine and costimulatory molecule gene expression, whereas cardiac DCs in WT mice were mature and functional following MI. Furthermore, IRAK-4-/- DCs have less mobilization capacity. Transfer of WT-derived bone marrow DCs into IRAK-4-/- mice for functional dendritic cell reconstitution negated the survival advantage and reduced cardiac dilation observed with IRAK-4-/mice at 28 days after MI. Conclusions: Deletion of IRAK-4 has favorable effects on survival and LV remodeling post myocardial infarction through modification of the host inflammatory process by blunting the detrimental bone marrow DCs mobilization after myocardial ischemia.
HFSA
S1
Jay N. Cohn New Investigator Award: Basic Science 001
003
TLR4-Mediated Signals Converge on the PGC-1 Family of Nuclear Receptor Coactivators to Control Myocardial Metabolism and Function Joel D. Schilling1, Teresa Leone2, Ling Lai2, Nanda Sambandam1, Daniel P. Kelly2; 1 Internal Medicine-Cardiology, Washington University School of Medicine, St. Louis, MO; 2Burnham Institute for Medical Research, Orlando, FL
Modulating Neuregulin/ErbB4 Signaling to Promote Cardiomyocyte Replacement Kevin Bersell1, Shima Arab1, Bernhard Haring1,2, Bernhard Kuhn1,2; 1Cardiology, Children’s Hospital Boston, Boston, MA; 2Pediatrics, Harvard Medical School, Boston, MA
Introduction: Heart failure (HF) is associated with myocardial inflammation and dysregulated metabolism. Toll-like Receptor 4 (TLR4) is activated by inflammatory stimuli such as lipopolysaccharide (LPS) leading to cytokine production and LV dysfunction. TLR4 has also been implicated in the pathogenesis of insulin resistance (IR), obesity, and HF. PGC-1a/b are inducible, cardiac enriched, transcriptional co-activators that regulate metabolism via interactions with nuclear receptors such as PPARa and ERRa. HF is associated with reduced PGC-1 signals. Hypothesis: TLR4-mediated inflammation modulates myocardial metabolism through PGC-1 co-activators. Results: Using an isolated working heart system to assess myocardial fuel utilization, we found that LPS decreased fatty acid oxidation (FAO) and glycolytic rates by 30% and 45%, respectively. LPS also triggered myocyte triglyceride (TG) accumulation, consistent with the observed decrease FAO capacity. PGC-1a/b, which serve as key regulators of FAO gene expression in heart, were rapidly downregulated by LPS. PGC-1 gene targets involved in FAO and mitochondrial respiration were also downregulated in a coordinated manner following LPS stimulation. In contrast, TLR4-/- mice did not exhibit LPS-mediated PGC-1 downregulation or myocyte TG accumulation. Using a myocyte specific, tetracycline inducible PGC-1b transgenic mouse (tet-on PGC-1b), PGC-1b induction prior to LPS administration reversed the effects of LPS on cardiac FAO and mitochondrial gene expression without altering cytokine levels. Myocardial TG accumulation induced by LPS was also significantly attenuated by forced expression of PGC-1b. Importantly, LPS-mediated LV dysfunction was rescued in tet-on PGC1b mice (LV fractional shortening 40% vs. 25%), demonstrating the physiologic importance of these metabolic alterations. Conclusions: TLR4 activation leads to reduced expression of PGC-1a/b in the heart, which in turn triggers a cascade of metabolic changes characterized by decreased FAO capacity, myocyte TG accumulation, and LV dysfunction. Restoration of PGC-1b levels in the heart reverses the metabolic and functional alterations caused by this inflammatory insult. These data describe a novel PGC-1-mediated regulatory crosstalk mechanism between inflammatory signals and metabolism in the heart, a pathway that is relevant to the pathogenesis of HF.
Introduction: The goal of regenerative therapy for heart failure is to replace cardiomyocytes. Whether differentiated cardiomyocytes can contribute to myocardial regeneration is a controversial question of great biomedical importance. Here we address whether and how differentiated cardiomyocytes can be induced to proliferate. Hypothesis: We hypothesized that the growth factor neuregulin1 (NRG1) and its tyrosine kinase receptor ErbB4 control post-natal proliferation of differentiated cardiomyocytes. Methods: We applied NRG1 systemically by intraperitoneal injection into three different lines of mice: a line that allowed for inducible genetic inactivation of ErbB4, a transgenic line over-expressing ErbB4, and wild type mice (C57BL6). We used uptake of BrdU, a thymidine analog, as readout of DNA synthesis and staining for phosphorylated histone H3 and aurora B kinase as readouts of karyokinesis and cytokinesis, respectively. Results: Inactivation of ErbB4 reduced postnatal cardiomyocyte proliferation, resulting in 19% less cardiomyocyte nuclei compared to littermate controls. Increasing ErbB4 expression enhanced proliferation, resulting in 16% more cardiomyocyte nuclei. Injecting NRG1 in adult wild-type animals increased cycling of differentiated cardiomyocytes. NRG1 induced mononucleated, but not binucleated cardiomyocytes to divide. Using two different thymidine analogs, we demonstrate that under NRG1 stimulation, differentiated cardiomyocytes entered the cell cycle twice and completed the cell cycle at least once. Using sparse genetic labeling of differentiated cardiomyocytes, we determined that NRG1 induced proliferation of individual cardiomyocytes, resulting in a 35-fold increase in the number of genetically labeled clusters of four or more differentiated cardiomyocytes in vivo. To divide, differentiated cardiomyocytes completely disassembled their contractile apparatus in the cleavage furrow. Using genetic fate mapping of differentiated cardiomyocytes, we demonstrate that undifferentiated progenitor cells did not contribute to NRG1-induced cardiomyocyte proliferation. Conclusions: Differentiated mononucleated cardiomyocytes have proliferative potential. ErbB4 is required and NRG1 or ErbB4 are sufficient to induce proliferation of differentiated cardiomyocytes. Thus, controlling NRG1/ErbB4 signaling may provide a promising strategy to promote myocardial regeneration.
002 Tetrahydrobiopterin Rescues b3-/- Mice from Cardiac Hypertrophy and Impaired Function Induced by Pressure Overload Jordan S. Leyton-Mange1, Xiaulin Niu1, Ronghua Yang1, Karen L. Miller1, An L. Moens2, Ashley B. Hale3, Djahida Bedja1, Kathleen L. Gabrielson1, Keith M. Channon3, David A. Kass1, Lili A. Barouch1; 1Dept of Medicine, Johns Hopkins School of Medicine, Baltimore, MD; 2Dept of Cardiology, Maastricht Univ, Maastrich, Netherlands; 3Dept of Cardiovasc Med, Univ of Oxford, Oxford, United Kingdom b3-Adrenergic receptor signaling in the mammalian cardiovascular system has a depressant effect on ventricular contractility, antagonizing the stimulatory role traditionally ascribed to b1/b2-AR’s. Via NOS-dependent increases in NO availability, b3-AR activation may induce an adaptive braking response to excessive catecholamine stimulation present in failing hearts. We have recently presented data revealing impaired cardiac functional compensation and increases in NOS-derived superoxide in b3-/- mice after pressure overload with transverse aortic constriction (TAC). The reducing agent and essential NOS cofactor, tetrahydrobiopterin (BH4), maintains functional coupling and NO-generating catalytic activity. We therefore hypothesized that treatment with BH4 would preserve cardiac function and reduce hypertrophy in b3-/- mice after TAC. Compared to WT mice, 3 weeks of TAC in b3-/- mice produced more marked cardiac hypertrophy as well as impaired function as assessed by echocardiography. These changes were accompanied by an increase in NOS-dependent superoxide production of 39.0% in hearts of TAC’d b3-/- mice over baseline levels, while NOS-dependent superoxide production was undetectable in WT mice. Total cardiac superoxide production in the TAC’d b3-/- hearts trended towards an increase (29.0%) as well, though this was not statistically significantly different from WT hearts or from baseline values. In addition, nNOS and iNOS protein levels were increased in response to TAC in b3-/- mice, though overall cardiac NOS-activity was unchanged, as assessed by arginine-to-citrulline conversion. BH4 treatment completely inhibited the increase in NOS-dependent superoxide production and rescued b3-/- mice from cardiac hypertrophy and impaired fractional shortening when compared to WT. Measurement of BH4 levels revealed no absolute differences between strains, though the BH4/(BH2þB) ratio was slightly lower in b3-/- mice and was unchanged with TAC. These results implicate NOS-derived superoxide in the diminished cardiac compensation after TAC in mice lacking the b3-AR, and that reduction of this superoxide reduces hypertrophy and preserves myocardial systolic function.
004 Survival and Cardiac Remodeling after Myocardial Infarction Are Critically Dependent on the Host Innate Immune Interleukin-1 Receptor Associated Kinase-4 (IRAK-4) Signaling: A Regulator of Bone Marrow-Derived Dendritic Cells Yuichiro Maekawa1, Nobuo Mizue3, Annie Chan2, Yu Shi2, Youan Liu2, Chen Manyin2, Fayez Dawood2, Geoffrey de Coute2, Guo Hua Li2, Jeffrey A. Medin3, Peter P. Liu2,4; 1Cardiology, Keio University School of Medicine, Tokyo, Japan; 2 Toronto General Hospital, University Health Network, Toronto, ON, Canada; 3 Ontario Cancer Institute, University Health Network, Toronto, ON, Canada; 4 Institute of Circulatory and Respiratory Health, Canadian Institutes of Health Research, Toronto, ON, Canada Background: The Innate immune system greatly contributes to the inflammatory process after myocardial infarction (MI). Interleukin-1 receptor associated kinase-4 (IRAK-4), downstream to Toll/Interleukin-1 receptor signaling, has an essential role in regulating the innate immune response. The present study was designed to determine the mechanism by which IRAK-4 is responsible for the cardiac inflammatory process, which consequently affects the left ventricular (LV) remodeling following MI. Methods and Results: Experimental MI was created in IRAK-4-/- and wildtype (WT) mice by left coronary ligation. Mice with a targeted deletion of IRAK4 had an improved survival rate at 4 weeks post-MI. IRAK-4-deficient mice also demonstrated attenuated cardiac dilation and decreased inflammation in the infarcted myocardium, which was associated with less pro-inflammatory and Th1 cytokines expression mediated by suppression of nuclear factor-kB and c-Jun N-terminal kinase activation. IRAK-4-/- mice had fewer infiltrations of CD45þ leukocytes and CD11cþ dendritic cells (DCs), inhibition of apoptosis and reduced fibrosis and nitric oxide production. Cardiac DCs in IRAK-4-/- mice were relatively immature or functionally naı¨ve, as they demonstrated less cytokine and costimulatory molecule gene expression, whereas cardiac DCs in WT mice were mature and functional following MI. Furthermore, IRAK-4-/- DCs have less mobilization capacity. Transfer of WT-derived bone marrow DCs into IRAK-4-/- mice for functional dendritic cell reconstitution negated the survival advantage and reduced cardiac dilation observed with IRAK-4-/mice at 28 days after MI. Conclusions: Deletion of IRAK-4 has favorable effects on survival and LV remodeling post myocardial infarction through modification of the host inflammatory process by blunting the detrimental bone marrow DCs mobilization after myocardial ischemia.