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Toll-like Receptor 2 Mediates Cardiac Adaptive Response to Pressure Overload
S146 Journal of Cardiac Failure Vol. 18 No. 10S October 2012
YIA Competitions (Basic: 1-5; Clinical: 1-5; Co-medical: 1-2) YIA-BA-1 The Identification of Mtus1 Splicing Variant as a Novel Inhibitory Factor Against Cardiac Hypertrophy SHIN ITO, MASANORI ASAKURA, MASAFUMI KITAKAZE Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute; Suita, Japan Alternative splicing has been linked to the pathogenesis of several diseases, although its role in cardiovascular diseases has not been fully elucidated. A global analysis of splicing variants in murine pressure-overload hearts found mitochondrial tumor suppressor 1 gene (Mtus1) as a novel spliced gene related to cardiac hypertrophy. One of the splice variants of Mtus1, Mtus1A, was specifically up-regulated even though the total expression of the Mtus1 gene remained unchanged. The Mtus1A protein was predominantly observed in the myocardium and the expression levels of Mtus1A correlated with the extent of left ventricular (LV) wall thickness using various murine cardiac hypertrophic models. In cardiomyocytes, adenoviral overexpression of Mtus1A inhibited phenylephrine-induced phosphorylation of both MEK and ERK, resulting in a decrease in both cell size and protein synthesis. By contrast, knockdown of Mtus1A using siRNA led to an increase in both cell size and protein synthesis. We also generated cardiac-specific Mtus1A transgenic mice (TG). We observed LV wall thinning, a smaller cross-sectional area of cardiomyocytes (wild type 342 mm2 vs. TG 263 mm2, P!0.05), LV dysfunction (fractional shortening, wild type 54% vs. TG 41%, P!0.05), and a reduced hypertrophic response to phenylephrine. We concluded that the Mtus1 gene is specifically spliced in pressure-overloaded hearts and that Mtus1A is a novel inhibitory factor against cardiac hypertrophy and contributes to heart failure.
YIA-BA-2 Induction of Cardiomyocyte-like Cells in Infarct Hearts by Gene Transfer of Gata4, Mef2c and Tbx5 KOHEI INAGAWA1,2, KAZUTAKA MIYAMOTO1,2, RIE WADA2, NAOTO MURAOKA1,2, HIROYUKI YAMAKAWA1,2, TAKETAROU SADAHIRO1,2, TOMOHIKO UMEI2, KEIICHI FUKUDA1, MASAKI IEDA1,2 1 The Department of Cardiology, Keio University School of Medicine, Tokyo, Japan, 2 The Department of Clinical and Molecular Cardiovascular Research, Keio University School of Medicine, Tokyo, Japan Rationale: After myocardial infarction !MIO, cell death in the myocardium initiates fibrosis and scar formation. We found that a combination of three cardiac transcription factors, Gata4, Mef2c and Tbx5 !GMTO reprograms cardiac fibroblasts directly into functional cardiomyocytes in vitro. Objective: To investigate whether viral gene transfer of GMT into mouse hearts can induce cardiomyocyte generation after MI. Methods and Results: We used retroviral vectors for gene transfer into mouse hearts after coronary artery ligation. Only proliferating non-cardiomyocytes were infected by retrovirus, indicated by GFP fluorescence. Retroviral GFP expression was diminished after 10 days of gene transfer in immuno-competent mice, but stable till 3 months in immuno-deficient mouse. To determine whether GMT can induce cardiomyocyte generation in vivo, a pool of GMT retroviruses was injected into the immuno-deficient mouse hearts after MI. 1% of the retrovirus infected cells expressed cardiac protein, a-actinin, and around 80% of the a-actinin+ cells did not have clear sarcomere structures. To transduce all three genes efficiently into cells in vivo, we generated a single polycistronic retrovirus of GMT separataed by 2A “self-cleaving” peptides. A polycistronic GMT retrovirus induced cardiac conversion in vivo and 30% of the a-actinin+ cells had sarcomeric structures. Cardiac Troponin T was also expressed in the induced cardiomyocytes. Conclusions: Gene transfer of GMT into adult mouse hearts induced cardiomyocyte generation after MI.
YIA-BA-3 Activation of Brain AT1 Receptors Caused by Increased Circulating Angiotensin II is Critically Involved in Left Ventricular Diastolic Dysfunction KEISUKE SHINOHARA1, YOSHITAKA HIROOKA2, TAKUYA KISHI3, KENJI SUNAGAWA1 1 Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan, 2Department of Advanced Cardiovascular Regulation and Therapeutics, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan, 3Department of Advanced Therapeutics for Cardiovascular Diseases, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan Background: Sympathoexcitation is an important factor for progression of heart failure (HF). An increase in circulating angiotensin II (Ang II) may cause the central sympathoexcitation acting on subfornical organ (SFO), a primary sensor for circulating Ang II. We hypothesized that the increase in circulating Ang II to the same level as that in HF affects cardiac function through the central
sympathoexcitation via activating Ang II type 1 receptors (AT1R) in SFO and rostral ventrolatreal medulla (RVLM) which is the cardiovascular center in the brainstem. Methods and Results: In Sprague-Dawley rats, the subcutaneous infusion of Ang II (100 ng/kg/min for 14 days) increased circulating Ang II to the same level as reported in ischemic-HF model rats. Compared with vehicle infusion, Ang II infusion increased AT1R expression level in RVLM as well as SFO with sympathoexcitation. We evaluated the cardiac function by echocardiography and the conductance catheter. Ang II infusion induced left ventricular (LV) hypertrophy and LV diastolic dysfunction (EDP: 5.060.3 vs 2.760.3 mmHg; -dP/dtmax: -75106202 vs -86746299 mmHg/s; EDPVR slope: 0.04060.003 vs 0.02460.002; n54-8, p!0.05), although it did not change the parameters of LV systolic function. Chronic intracerebroventricular infusion of AT1R blocker, losartan, attenuated these Ang II-induced changes. Conclusion: Activation of AT1R in SFO and RVLM is critically involved in circulating Ang II-induced sympathoexcitation leading to LV diastolic dysfunction.
YIA-BA-4 Aberrant Calcium Handling Inhibits Functional Maturation and Excitation in Factors-based Human Cardiomyocytes Differentiation SUGURU TARUI1, JUNKO KOBAYASHI1, MASATAKA HIRATA1, KEN TAKAHASHI2, GENTARO IRIBE2, KEIJI NARUSE2, SHINGO KASAHARA1, SHUNJI SANO1, HIDEMASA OU3 1 Department of Cardiovascular Surgery, Okayama University, Okayama, Japan, 2 Department of Cardiovascular Physiology, Okayama University, Okayama, Japan, 3 Department of Regenerative Medicine, Center for Innovative Clinical Medicine, Okayama University Hospital, Okayama, Japan Rationale: Recent breakthrough in direct cardiac reprogramming in mice provided a new technology towards regenerative medicine; however, the underlying mechanisms controlling human cardiomyogenesis remain unknown. Here, we investigate the role of calcium-dependent cardiac maturation during the process of factors-based human cardiomyocytes differentiation. Methods and Results: Human cardiac progenitor cells (CPCs) were isolated and transduced with lentiviral vectors encoding Tbx5, GATA4, and Mef2C (TGM). Differentiated cardiomyocytes were identified by alpha-MHC promoter-driven fluorescence and mitochondrial content. Calcium oscillation imaging, FACS analysis using signal-regulatory protein alpha (SIRPA), and cardiac structural/ion channel regulatory proteins were examined to verify cardiac maturation. We found that TGM transduction in CPCs significantly augmented SIRPA and mitochondrial expressing cell population; however, FACS analysis showed that functionally mature cardiomyocytes expressing alpha-MHC and cTnT were modest. Although TGM induction markedly upregulated the expressions of sodium and potassium channels modulators include SCN5A, Kir2.2, HERG, KvLQT1, and Kv4.3, increased calcium oscillation frequency was only functionally coupled with the upregulation of ryanodine receptors and L-type calcium-channel alpha 1C. Electrical stimulation had no effects on calcium oscillation on TGM-induced CPCs, as also shown by reduced expressions of sarcolemmal calcium-cycling regulatory proteins, NCX1, SERCA2, and inositol 1,4,5-triphosphate receptors. Conclusions: Our data suggest that factors-based cardiac-lineage induction in human CPCs had critical deficits towards functional maturation and excitation, consistent with a reduction in calcium handling proteins.
YIA-BA-5 Toll-like Receptor 2 Mediates Cardiac Adaptive Response to Pressure Overload YASUTOMI HIGASHIKUNI1, RYOZO NAGAI2, MASATAKA SATA3 1 Department of Cardiovascular Medicine, The University of Tokyo, Tokyo, Japan, 2 Jichi Medical University, Shimotsuke, Japan, 3Department of Cardiovascular Medicine, The University of Tokushima, Tokushima, Japan Background: Toll-like receptor 2 (TLR2) recognizes endogenous ligands which induce inflammatory response called homeostatic inflammation. Recently, homeostatic inflammation has been shown to contribute to the pathophysiology of various diseases. However, the role of homeostatic inflammation mediated by TLR2 in pressure overload-induced cardiac hypertrophy remains unclear. Methods and Results: Pressure overload was induced in 8- to 12-week-old wild-type (WT) and TLR2 knock-out (KO) mice by transverse aortic constriction (TAC). At 2 weeks after TAC, KO mice showed reduced cardiac hypertrophy and fibrosis with more left ventricular dilatation and impaired systolic function compared with WT mice. Bone marrow transplantation experiment revealed that TLR2 expressed in the heart, not in blood cells, plays an important role in cardiac adaptive response to pressure overload. In vitro experiments demonstrated that Pam3CSK4, a specific TLR2 agonist, induces cardiomyocyte hypertrophy, and fibroblast and vascular endothelial cell proliferation in nuclear factor kB (NF-kB) and interleukin 1b (IL-1b)-dependent manner. Systemic administration of a NF-kB inhibitor or a IL-1b neutralization antibody to WT mice resulted in impaired cardiac adaptive response to pressure overload. We also found that extracellular heat shock protein 70 is involved in cardiac adaptive response to pressure overload in TLR2-dependent manner. Conclusions: TLR2 mediates cardiac adaptive response to pressure overload. Thus, modulation of TLR2 signaling may provide a novel strategy for treating cardiac hypertrophy and heart failure.
YIA Competitions (Basic: 1-5; Clinical: 1-5; Co-medical: 1-2) YIA-BA-1 The Identification of Mtus1 Splicing Variant as a Novel Inhibitory Factor Against Cardiac Hypertrophy SHIN ITO, MASANORI ASAKURA, MASAFUMI KITAKAZE Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute; Suita, Japan Alternative splicing has been linked to the pathogenesis of several diseases, although its role in cardiovascular diseases has not been fully elucidated. A global analysis of splicing variants in murine pressure-overload hearts found mitochondrial tumor suppressor 1 gene (Mtus1) as a novel spliced gene related to cardiac hypertrophy. One of the splice variants of Mtus1, Mtus1A, was specifically up-regulated even though the total expression of the Mtus1 gene remained unchanged. The Mtus1A protein was predominantly observed in the myocardium and the expression levels of Mtus1A correlated with the extent of left ventricular (LV) wall thickness using various murine cardiac hypertrophic models. In cardiomyocytes, adenoviral overexpression of Mtus1A inhibited phenylephrine-induced phosphorylation of both MEK and ERK, resulting in a decrease in both cell size and protein synthesis. By contrast, knockdown of Mtus1A using siRNA led to an increase in both cell size and protein synthesis. We also generated cardiac-specific Mtus1A transgenic mice (TG). We observed LV wall thinning, a smaller cross-sectional area of cardiomyocytes (wild type 342 mm2 vs. TG 263 mm2, P!0.05), LV dysfunction (fractional shortening, wild type 54% vs. TG 41%, P!0.05), and a reduced hypertrophic response to phenylephrine. We concluded that the Mtus1 gene is specifically spliced in pressure-overloaded hearts and that Mtus1A is a novel inhibitory factor against cardiac hypertrophy and contributes to heart failure.
YIA-BA-2 Induction of Cardiomyocyte-like Cells in Infarct Hearts by Gene Transfer of Gata4, Mef2c and Tbx5 KOHEI INAGAWA1,2, KAZUTAKA MIYAMOTO1,2, RIE WADA2, NAOTO MURAOKA1,2, HIROYUKI YAMAKAWA1,2, TAKETAROU SADAHIRO1,2, TOMOHIKO UMEI2, KEIICHI FUKUDA1, MASAKI IEDA1,2 1 The Department of Cardiology, Keio University School of Medicine, Tokyo, Japan, 2 The Department of Clinical and Molecular Cardiovascular Research, Keio University School of Medicine, Tokyo, Japan Rationale: After myocardial infarction !MIO, cell death in the myocardium initiates fibrosis and scar formation. We found that a combination of three cardiac transcription factors, Gata4, Mef2c and Tbx5 !GMTO reprograms cardiac fibroblasts directly into functional cardiomyocytes in vitro. Objective: To investigate whether viral gene transfer of GMT into mouse hearts can induce cardiomyocyte generation after MI. Methods and Results: We used retroviral vectors for gene transfer into mouse hearts after coronary artery ligation. Only proliferating non-cardiomyocytes were infected by retrovirus, indicated by GFP fluorescence. Retroviral GFP expression was diminished after 10 days of gene transfer in immuno-competent mice, but stable till 3 months in immuno-deficient mouse. To determine whether GMT can induce cardiomyocyte generation in vivo, a pool of GMT retroviruses was injected into the immuno-deficient mouse hearts after MI. 1% of the retrovirus infected cells expressed cardiac protein, a-actinin, and around 80% of the a-actinin+ cells did not have clear sarcomere structures. To transduce all three genes efficiently into cells in vivo, we generated a single polycistronic retrovirus of GMT separataed by 2A “self-cleaving” peptides. A polycistronic GMT retrovirus induced cardiac conversion in vivo and 30% of the a-actinin+ cells had sarcomeric structures. Cardiac Troponin T was also expressed in the induced cardiomyocytes. Conclusions: Gene transfer of GMT into adult mouse hearts induced cardiomyocyte generation after MI.
YIA-BA-3 Activation of Brain AT1 Receptors Caused by Increased Circulating Angiotensin II is Critically Involved in Left Ventricular Diastolic Dysfunction KEISUKE SHINOHARA1, YOSHITAKA HIROOKA2, TAKUYA KISHI3, KENJI SUNAGAWA1 1 Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan, 2Department of Advanced Cardiovascular Regulation and Therapeutics, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan, 3Department of Advanced Therapeutics for Cardiovascular Diseases, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan Background: Sympathoexcitation is an important factor for progression of heart failure (HF). An increase in circulating angiotensin II (Ang II) may cause the central sympathoexcitation acting on subfornical organ (SFO), a primary sensor for circulating Ang II. We hypothesized that the increase in circulating Ang II to the same level as that in HF affects cardiac function through the central
sympathoexcitation via activating Ang II type 1 receptors (AT1R) in SFO and rostral ventrolatreal medulla (RVLM) which is the cardiovascular center in the brainstem. Methods and Results: In Sprague-Dawley rats, the subcutaneous infusion of Ang II (100 ng/kg/min for 14 days) increased circulating Ang II to the same level as reported in ischemic-HF model rats. Compared with vehicle infusion, Ang II infusion increased AT1R expression level in RVLM as well as SFO with sympathoexcitation. We evaluated the cardiac function by echocardiography and the conductance catheter. Ang II infusion induced left ventricular (LV) hypertrophy and LV diastolic dysfunction (EDP: 5.060.3 vs 2.760.3 mmHg; -dP/dtmax: -75106202 vs -86746299 mmHg/s; EDPVR slope: 0.04060.003 vs 0.02460.002; n54-8, p!0.05), although it did not change the parameters of LV systolic function. Chronic intracerebroventricular infusion of AT1R blocker, losartan, attenuated these Ang II-induced changes. Conclusion: Activation of AT1R in SFO and RVLM is critically involved in circulating Ang II-induced sympathoexcitation leading to LV diastolic dysfunction.
YIA-BA-4 Aberrant Calcium Handling Inhibits Functional Maturation and Excitation in Factors-based Human Cardiomyocytes Differentiation SUGURU TARUI1, JUNKO KOBAYASHI1, MASATAKA HIRATA1, KEN TAKAHASHI2, GENTARO IRIBE2, KEIJI NARUSE2, SHINGO KASAHARA1, SHUNJI SANO1, HIDEMASA OU3 1 Department of Cardiovascular Surgery, Okayama University, Okayama, Japan, 2 Department of Cardiovascular Physiology, Okayama University, Okayama, Japan, 3 Department of Regenerative Medicine, Center for Innovative Clinical Medicine, Okayama University Hospital, Okayama, Japan Rationale: Recent breakthrough in direct cardiac reprogramming in mice provided a new technology towards regenerative medicine; however, the underlying mechanisms controlling human cardiomyogenesis remain unknown. Here, we investigate the role of calcium-dependent cardiac maturation during the process of factors-based human cardiomyocytes differentiation. Methods and Results: Human cardiac progenitor cells (CPCs) were isolated and transduced with lentiviral vectors encoding Tbx5, GATA4, and Mef2C (TGM). Differentiated cardiomyocytes were identified by alpha-MHC promoter-driven fluorescence and mitochondrial content. Calcium oscillation imaging, FACS analysis using signal-regulatory protein alpha (SIRPA), and cardiac structural/ion channel regulatory proteins were examined to verify cardiac maturation. We found that TGM transduction in CPCs significantly augmented SIRPA and mitochondrial expressing cell population; however, FACS analysis showed that functionally mature cardiomyocytes expressing alpha-MHC and cTnT were modest. Although TGM induction markedly upregulated the expressions of sodium and potassium channels modulators include SCN5A, Kir2.2, HERG, KvLQT1, and Kv4.3, increased calcium oscillation frequency was only functionally coupled with the upregulation of ryanodine receptors and L-type calcium-channel alpha 1C. Electrical stimulation had no effects on calcium oscillation on TGM-induced CPCs, as also shown by reduced expressions of sarcolemmal calcium-cycling regulatory proteins, NCX1, SERCA2, and inositol 1,4,5-triphosphate receptors. Conclusions: Our data suggest that factors-based cardiac-lineage induction in human CPCs had critical deficits towards functional maturation and excitation, consistent with a reduction in calcium handling proteins.
YIA-BA-5 Toll-like Receptor 2 Mediates Cardiac Adaptive Response to Pressure Overload YASUTOMI HIGASHIKUNI1, RYOZO NAGAI2, MASATAKA SATA3 1 Department of Cardiovascular Medicine, The University of Tokyo, Tokyo, Japan, 2 Jichi Medical University, Shimotsuke, Japan, 3Department of Cardiovascular Medicine, The University of Tokushima, Tokushima, Japan Background: Toll-like receptor 2 (TLR2) recognizes endogenous ligands which induce inflammatory response called homeostatic inflammation. Recently, homeostatic inflammation has been shown to contribute to the pathophysiology of various diseases. However, the role of homeostatic inflammation mediated by TLR2 in pressure overload-induced cardiac hypertrophy remains unclear. Methods and Results: Pressure overload was induced in 8- to 12-week-old wild-type (WT) and TLR2 knock-out (KO) mice by transverse aortic constriction (TAC). At 2 weeks after TAC, KO mice showed reduced cardiac hypertrophy and fibrosis with more left ventricular dilatation and impaired systolic function compared with WT mice. Bone marrow transplantation experiment revealed that TLR2 expressed in the heart, not in blood cells, plays an important role in cardiac adaptive response to pressure overload. In vitro experiments demonstrated that Pam3CSK4, a specific TLR2 agonist, induces cardiomyocyte hypertrophy, and fibroblast and vascular endothelial cell proliferation in nuclear factor kB (NF-kB) and interleukin 1b (IL-1b)-dependent manner. Systemic administration of a NF-kB inhibitor or a IL-1b neutralization antibody to WT mice resulted in impaired cardiac adaptive response to pressure overload. We also found that extracellular heat shock protein 70 is involved in cardiac adaptive response to pressure overload in TLR2-dependent manner. Conclusions: TLR2 mediates cardiac adaptive response to pressure overload. Thus, modulation of TLR2 signaling may provide a novel strategy for treating cardiac hypertrophy and heart failure.