Pregnancy lowers the risk of breast cancer, largely attributable to alpha-fetoprotein (AFP). A sm... more Pregnancy lowers the risk of breast cancer, largely attributable to alpha-fetoprotein (AFP). A small AFP-derived peptide (AFPep) which mimics the active site of AFP has been developed and may be useful for decreasing the risk of breast cancer for women. AFPep has been shown previously to stop the growth of estrogen-dependent human breast cancer xenografts in mice and prevent carcinogen-induced breast cancer in a rat model. Since AFPep disrupts an estrogen-responsive pathway, it is essential to assess its effects on the female reproductive cycle and fertility. Ten cycling female Sprague-Dawley rats (age 81 days) were given 100 μg AFPep in saline s.c. daily for 20 days. A second group of ten rats was given 50 μg tamoxifen s.c. daily and a third group received saline only. Vaginal smears were obtained twice per day and stained to assess estrous cycle phase. After completion of estrous cycle assessment (five cycles, 21 days), rats were maintained on drug and allowed to mate. Effects on birth of offspring and maternal body weights were assessed. AFPep had no significant effect on the incidence or duration of any estrous cycle phase, and no effect on reproductive potential or maternal body mass. Tamoxifen significantly increased the length of diestrus, locking the cycle in this phase for most animals. Only half of the tamoxifen-treated rats mated, and none became pregnant. Tamoxifen significantly slowed the rate of body mass increase. In rats, AFPep has no toxicity and no effect on female reproduction. This molecule may be developed into an attractive modality for prevention of breast cancer in women.
UMI, ProQuest ® Dissertations & Theses. The world's most comprehensive collection of dis... more UMI, ProQuest ® Dissertations & Theses. The world's most comprehensive collection of dissertations and theses. Learn more... ProQuest, Second generation analogs of the anticancer peptide AFPep: Enhancement of efficacy and dose range. ...
B87 Purpose : Cyclo [EKTOVNOGN] (AFPep), a cyclic peptide derived from the active site of alpha-f... more B87 Purpose : Cyclo [EKTOVNOGN] (AFPep), a cyclic peptide derived from the active site of alpha-fetoprotein, has been shown to prevent breast cancer in animal models. This peptide is comprised of 9 amino acids in cyclic head-to-tail peptide linkage. Previous synthetic studies suggested that the pharmacophore consisted of OVNO, while replica exchange molecular dynamics suggested that the pharmacophore consisted of TOVN. We hypothesized that smaller analogs of AFPep, especially those that include OVN, would be effective anticancer agents. Experimental Design : Twelve linear AFPep analogs were assembled on PAL-PEG-PS resin beginning with the C-terminus using N-terminally protected amino acids. These peptides were either four or five amino acids long, and were designed to resemble the putative pharmacophoric region of AFPep. An unrelated five amino acid peptide (PGVGQ) was synthesized as a negative control, and AFPep was used as a positive control. These peptides were evaluated using an immature mouse uterine growth inhibition assay (for antiestrogenicity) and a T47D breast cancer cell proliferation assay (for anticancer activity). Results and Conclusions : Linear analogs of AFPep were synthesized; mass spectrometry showed that each peptide was of the correct molecular weight. Inhibition of uterine growth was as follows: TOVNO (31± 2%), TOVN (23±2%), OVNO (9 ± 4%), OVNOG (21± 4%), KTOVN (28± 5%), TPVNP (27± 1%), TPVN (29±2%), PVNP (9±2%), EKTPV (0%), EMTPV (11%), EMTOV (0%), and VNOG (0%) compared to AFPep (38±2%) and PGVGQ (0%). Inhibition of E2-stimulated T47D cell growth was as follows: TOVNO (41±10%), OVNO (10%), and OVNOG (47%) compared to AFPep (53±6%) and PGVGQ (11%). We concluded that analogs of AFPep as small as 4 or 5 amino acids are effective antiestrotrophic and anticancer agents. OVNO is of low biological activity, whereas TOVN may be, and TOVNO is, biologically active.
Background-Diabetes and obesity are associated with an increased risk of arrhythmia and sudden ca... more Background-Diabetes and obesity are associated with an increased risk of arrhythmia and sudden cardiac death. Abnormal lipid accumulation is observed in cardiomyocytes of obese and diabetic patients, which may contribute to arrhythmia, but the mechanisms are poorly understood. A transgenic mouse model of cardiac lipid overload, the PPARg cardiac overexpression mouse, has long QT and increased ventricular ectopy. Objective-We evaluated the hypothesis that the increase in ventricular ectopy during cardiac lipid overload is caused by abnormalities in calcium handling due to increased mitochondrial oxidative stress. Methods-Ventricular myocytes were isolated from adult mouse hearts to record sparks and calcium transients. Mice were implanted with heart rhythm monitors for in vivo recordings. Results-PPARg cardiomyocytes have more frequent triggered activity and increased sparks compared to control. Sparks and triggered activity are reduced by mitotempo, a mitochondrialtargeted antioxidant. This is explained by a significant increase in oxidation of RyR2. Calcium transients are increased in amplitude and SR calcium stores are increased in PPARg cardiomyocytes. Computer modeling of the cardiac action potential demonstrates that long QT contributes to increased SR calcium. Mitotempo decreased ventricular ectopy in vivo.
Summary The response of vital organs to different types of nutrition or diet is a fundamental que... more Summary The response of vital organs to different types of nutrition or diet is a fundamental question in physiology. We examined the cardiac response to 4 weeks of high-fat diet in mice, measuring cardiac metabolites and mRNA. Metabolomics showed dramatic differences after a high-fat diet, including increases in several acyl-carnitine species. The RNA-seq data showed changes consistent with adaptations to use more fatty acid as substrate and an increase in the antioxidant protein catalase. Changes in mRNA were correlated with changes in protein level for several highly responsive genes. We also found significant sex differences in both metabolomics and RNA-seq datasets, both at baseline and after high fat diet. This work reveals the response of a vital organ to dietary intervention at both metabolomic and transcriptomic levels, which is a fundamental question in physiology. This work also reveals significant sex differences in cardiac metabolites and gene expression.
Some missense gain-of-function mutations in the CACNA1C gene, encoding calcium channel Ca V 1.2, ... more Some missense gain-of-function mutations in the CACNA1C gene, encoding calcium channel Ca V 1.2, cause a life-threatening form of long QT syndrome (LQTS) named Timothy syndrome with currently no clinically effective therapeutics. Here we report that pharmacological targeting of sigma non-opioid intracellular receptor 1 (SIGMAR1) can restore electrophysiological function in induced pluripotent stem cell (iPSC)-derived cardiomyocytes generated from patients with Timothy syndrome and two common forms of LQTS, type 1 (LQTS1) and type 2 (LQTS2), caused by missense trafficking mutations in potassium channels. Electrophysiological recordings demonstrate that a Food and Drug Administration (FDA)-approved cough suppressant, dextromethorphan, can be used as an agonist of SIGMAR1 to shorten the prolonged action potential in cardiomyocytes from patients with Timothy syndrome and human cellular models of LQTS1 and LQTS2. When tested in vivo, dextromethorphan also normalized the prolonged QT intervals in a mouse model of Timothy syndrome. Overall, our study demonstrates that SIGMAR1 is a potential therapeutic target for Timothy syndrome and possibly other inherited arrhythmias such as LQTS1 and LQTS2.
Cardiac arrhythmias are responsible for many cardiovascular disease-related deaths worldwide. Whi... more Cardiac arrhythmias are responsible for many cardiovascular disease-related deaths worldwide. While arrhythmia pathogenesis is complex, there is increasing evidence for metabolic causes. Obesity, diabetes, and chronically consuming high-fat foods significantly increase the likelihood of developing arrhythmias. Although these correlations are well established, mechanistic explanations connecting a high-fat diet (HFD) to arrhythmogenesis are incomplete, although oxidative stress appears to be critical. This review investigates the metabolic changes that occur in obesity and after HFD. Potential therapies to prevent or treat arrhythmias are discussed, including antioxidants.
Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) plays an important role in pulm... more Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) plays an important role in pulmonary hypertension (PH). However, the underlying specific ion channel mechanisms remain largely unknown. Here, we report ryanodine receptor (RyR) channel activity and Ca2+ release both are increased, and association of RyR2 by FK506 binding protein 12.6 (FKBP12.6) is decreased in PASMCs from mice with chronic hypoxia (CH)-induced PH. Smooth muscle cell (SMC)-specific RyR2 knockout (KO) or Rieske iron-sulfur protein (RISP) knockdown inhibits the altered Ca2+ signaling, increased nuclear factor (NF)-κB/cyclin D1 activation and cell proliferation, and CH-induced PH in mice. FKBP12.6 KO or FK506 treatment enhances CH-induced PH, while S107 (a specific stabilizer of RyR2/FKBP12.6 complex) produces an opposite effect. In conclusion, CH causes RISP-dependent ROS generation and FKBP12.6/RyR2 dissociation, leading to PH. RISP inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blocka...
Highlights d Cardiomyocytes expressing pathogenic lamin A/C exhibit altered nuclear positioning d... more Highlights d Cardiomyocytes expressing pathogenic lamin A/C exhibit altered nuclear positioning d ERK1/2 phosphorylates FHOD1/3 and inhibits nuclear movement in fibroblasts d ERK1/2-phosphorylated FHOD1/3 loses its F-actin bundling necessary for nuclear movement d FHOD3 phosphorylation by ERK1/2
Circulation: Arrhythmia and Electrophysiology, 2019
Background:Obesity and diets high in saturated fat increase the risk of arrhythmias and sudden ca... more Background:Obesity and diets high in saturated fat increase the risk of arrhythmias and sudden cardiac death. However, the molecular mechanisms are not well understood. We hypothesized that an increase in dietary saturated fat could lead to abnormalities of calcium homeostasis and heart rhythm by a NOX2 (NADPH oxidase 2)-dependent mechanism.Methods:We investigated this hypothesis by feeding mice high-fat diets. In vivo heart rhythm telemetry, optical mapping, and isolated cardiac myocyte imaging were used to quantify arrhythmias, repolarization, calcium transients, and intracellular calcium sparks.Results:We found that saturated fat activates NOX (NADPH oxidase), whereas polyunsaturated fat does not. The high saturated fat diet increased repolarization heterogeneity and ventricular tachycardia inducibility in perfused hearts. Pharmacological inhibition or genetic deletion of NOX2 prevented arrhythmogenic abnormalities in vivo during high statured fat diet and resulted in less induci...
Mutations in LMNA encoding lamin A/C and EMD encoding emerin cause cardiomyopathy and muscular dy... more Mutations in LMNA encoding lamin A/C and EMD encoding emerin cause cardiomyopathy and muscular dystrophy. Lmna null mice develop these disorders and have a lifespan of 7–8 weeks. Emd null mice show no overt pathology and have normal skeletal muscle but with regeneration defects. We generated mice with germline deletions of both Lmna and Emd to determine the effects of combined loss of the encoded proteins. Mice without lamin A/C and emerin are born at the expected Mendelian ratio, are grossly normal at birth but have shorter lifespans than those lacking only lamin A/C. However, there are no major differences between these mice with regards to left ventricular function, heart ultrastructure or electrocardiographic parameters except for slower heart rates in the mice lacking both lamin A/C and emerin. Skeletal muscle is similarly affected in both of these mice. Lmna+/− mice also lacking emerin live to at least 1 year and have no significant differences in growth, heart or skeletal mus...
American journal of physiology. Lung cellular and molecular physiology, May 1, 2018
Hypoxia-induced pulmonary vasoconstriction (HPV) is attributed to an increase in intracellular Ca... more Hypoxia-induced pulmonary vasoconstriction (HPV) is attributed to an increase in intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs). We have reported that phospholipase C-γ1 (PLCγ1) plays a significant role in the hypoxia-induced increase in [Ca2+]i in PASMCs and attendant HPV. In this study, we intended to determine molecular mechanisms for hypoxic Ca2+ and contractile responses in PASMCs. Our data reveal that hypoxic vasoconstriction occurs in PAs, but not in mesenteric arteries (MAs). Hypoxia caused a large increase in [Ca2+]i in PASMCs, which is diminished by the PLC inhibitor U73122 and not by its inactive analog U73433. Hypoxia augments PLCγ1-dependent inositol 1,4,5-trisphosphate (IP3) generation. Exogenous ROS, hydrogen peroxide (H2O2), increases PLCγ1 phosphorylation at tyrosine-783 and IP3 production. IP3 receptor-1 (IP3R1) knock-down (KD) remarkably diminishes hypoxia- or H2O2-induced increase in [Ca2+]i. Hypoxia or H2O2 increases ...
reduced acyl-carnitine long-chain fatty acid metabolites. We also observed changes in multiple pa... more reduced acyl-carnitine long-chain fatty acid metabolites. We also observed changes in multiple pathways associated with the development of PAH, including increased markers of proliferation, inflammation, fibrosis and endothelial dysfunction. Importantly, our global metabolic profile data compare favorably with prior work carried out in humans with PAH. We conclude that despite the MCT-model not recapitulating all the structural changes associated with humans with advanced PAH, including endothelial cell proliferation and the formation of plexiform lesions, it is very similar at a metabolic level. Also these data indicate that metabolic reprogramming precedes inflammatory, proliferative and fibrotic changes in PAH lungs.
Cardiomyopathy frequently complicates sepsis and is associated with increased mortality. Increase... more Cardiomyopathy frequently complicates sepsis and is associated with increased mortality. Increased cardiac oxidative stress and mitochondrial dysfunction have been observed during sepsis, but the mechanisms responsible for these abnormalities have not been determined. We hypothesized that NADPH oxidase 2 (NOX2) activation could be responsible for sepsis-induced oxidative stress and cardiomyopathy. Treatment of isolated adult mouse cardiomyocytes with low concentrations of the endotoxin lipopolysaccharide (LPS) increased total cellular reactive oxygen species (ROS) and mitochondrial superoxide. Elevated mitochondrial superoxide was accompanied by depolarization of the mitochondrial inner membrane potential, an indication of mitochondrial dysfunction, and mitochondrial calcium overload. NOX2 inhibition decreased LPS-induced superoxide and prevented mitochondrial dysfunction. Further, cardiomyocytes from mice with genetic ablation of NOX2 did not have LPS-induced superoxide or mitochon...
Background-Diabetes and obesity are associated with an increased risk of arrhythmia and sudden ca... more Background-Diabetes and obesity are associated with an increased risk of arrhythmia and sudden cardiac death. Abnormal lipid accumulation is observed in cardiomyocytes of obese and diabetic patients, which may contribute to arrhythmia, but the mechanisms are poorly understood. A transgenic mouse model of cardiac lipid overload, the PPARg cardiac overexpression mouse, has long QT and increased ventricular ectopy. Objective-We evaluated the hypothesis that the increase in ventricular ectopy during cardiac lipid overload is caused by abnormalities in calcium handling due to increased mitochondrial oxidative stress. Methods-Ventricular myocytes were isolated from adult mouse hearts to record sparks and calcium transients. Mice were implanted with heart rhythm monitors for in vivo recordings. Results-PPARg cardiomyocytes have more frequent triggered activity and increased sparks compared to control. Sparks and triggered activity are reduced by mitotempo, a mitochondrialtargeted antioxidant. This is explained by a significant increase in oxidation of RyR2. Calcium transients are increased in amplitude and SR calcium stores are increased in PPARg cardiomyocytes. Computer modeling of the cardiac action potential demonstrates that long QT contributes to increased SR calcium. Mitotempo decreased ventricular ectopy in vivo.
Cardiomyopathy caused by lamin A/C gene mutations (LMNA cardiomyopathy) is characterized by incre... more Cardiomyopathy caused by lamin A/C gene mutations (LMNA cardiomyopathy) is characterized by increased myocardial fibrosis, which impairs left ventricular relaxation and predisposes to heart failure, and cardiac conduction abnormalities. While we previously discovered abnormally elevated extracellular signal-regulated kinase 1/2 (ERK1/2) activities in heart in LMNA cardiomyopathy, its role on the development of myocardial fibrosis remains unclear. We now showed that transforming growth factor (TGF)-β/Smad signaling participates in the activation of ERK1/2 signaling in LMNA cardiomyopathy. ERK1/2 acts on connective tissue growth factor (CTGF/CCN2) expression to mediate the myocardial fibrosis and left ventricular dysfunction. Studies in vivo demonstrate that inhibiting CTGF/CCN2 using a specific antibody decreases myocardial fibrosis and improves the left ventricular dysfunction. Together, these findings show that cardiac ERK1/2 activity is modulated in part by TGF-β Smad signaling, l...
Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The mol... more Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS) in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.
We previously showed that striated muscle-selective depletion of lamina-associated polypeptide 1 ... more We previously showed that striated muscle-selective depletion of lamina-associated polypeptide 1 (LAP1), an integral inner nuclear membrane protein, leads to profound muscular dystrophy with premature death in mice. As LAP1 is also depleted in hearts of these mice, we examined their cardiac phenotype. Striated muscle-selective LAP1 knockout mice display ventricular systolic dysfunction with abnormal induction of genes encoding cardiomyopathy related proteins. To eliminate possible confounding effects due to skeletal muscle pathology, we generated a new mouse line in which LAP1 is deleted in a cardiomyocyte-selective manner. These mice had no skeletal muscle pathology and appeared overtly normal at 20 weeks of age. However, cardiac echocardiography revealed that they developed left ventricular systolic dysfunction and cardiac gene expression analysis revealed abnormal induction of cardiomyopathy-related genes. Our results demonstrate that LAP1 expression in cardiomyocytes is required for normal left ventricular function, consistent with a report of cardiomyopathy in a human subject with mutation in the gene encoding LAP1.
American Journal of Physiology-Lung Cellular and Molecular Physiology, 2012
An increase in intracellular calcium concentration ([Ca2+]i) in pulmonary arterial smooth muscle ... more An increase in intracellular calcium concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs) induces hypoxic cellular responses in the lungs; however, the underlying molecular mechanisms remain incompletely understood. We report, for the first time, that acute hypoxia significantly enhances phospholipase C (PLC) activity in mouse resistance pulmonary arteries (PAs), but not in mesenteric arteries. Western blot analysis and immunofluorescence staining reveal the expression of PLC-γ1 protein in PAs and PASMCs, respectively. The activity of PLC-γ1 is also augmented in PASMCs following hypoxia. Lentiviral shRNA-mediated gene knockdown of mitochondrial complex III Rieske iron-sulfur protein (RISP) to inhibit reactive oxygen species (ROS) production prevents hypoxia from increasing PLC-γ1 activity in PASMCs. Myxothiazol, a mitochondrial complex III inhibitor, reduces the hypoxic response as well. The PLC inhibitor U73122, but not its inactive analog U73433, attenuates t...
Pregnancy lowers the risk of breast cancer, largely attributable to alpha-fetoprotein (AFP). A sm... more Pregnancy lowers the risk of breast cancer, largely attributable to alpha-fetoprotein (AFP). A small AFP-derived peptide (AFPep) which mimics the active site of AFP has been developed and may be useful for decreasing the risk of breast cancer for women. AFPep has been shown previously to stop the growth of estrogen-dependent human breast cancer xenografts in mice and prevent carcinogen-induced breast cancer in a rat model. Since AFPep disrupts an estrogen-responsive pathway, it is essential to assess its effects on the female reproductive cycle and fertility. Ten cycling female Sprague-Dawley rats (age 81 days) were given 100 μg AFPep in saline s.c. daily for 20 days. A second group of ten rats was given 50 μg tamoxifen s.c. daily and a third group received saline only. Vaginal smears were obtained twice per day and stained to assess estrous cycle phase. After completion of estrous cycle assessment (five cycles, 21 days), rats were maintained on drug and allowed to mate. Effects on birth of offspring and maternal body weights were assessed. AFPep had no significant effect on the incidence or duration of any estrous cycle phase, and no effect on reproductive potential or maternal body mass. Tamoxifen significantly increased the length of diestrus, locking the cycle in this phase for most animals. Only half of the tamoxifen-treated rats mated, and none became pregnant. Tamoxifen significantly slowed the rate of body mass increase. In rats, AFPep has no toxicity and no effect on female reproduction. This molecule may be developed into an attractive modality for prevention of breast cancer in women.
UMI, ProQuest ® Dissertations & Theses. The world's most comprehensive collection of dis... more UMI, ProQuest ® Dissertations & Theses. The world's most comprehensive collection of dissertations and theses. Learn more... ProQuest, Second generation analogs of the anticancer peptide AFPep: Enhancement of efficacy and dose range. ...
B87 Purpose : Cyclo [EKTOVNOGN] (AFPep), a cyclic peptide derived from the active site of alpha-f... more B87 Purpose : Cyclo [EKTOVNOGN] (AFPep), a cyclic peptide derived from the active site of alpha-fetoprotein, has been shown to prevent breast cancer in animal models. This peptide is comprised of 9 amino acids in cyclic head-to-tail peptide linkage. Previous synthetic studies suggested that the pharmacophore consisted of OVNO, while replica exchange molecular dynamics suggested that the pharmacophore consisted of TOVN. We hypothesized that smaller analogs of AFPep, especially those that include OVN, would be effective anticancer agents. Experimental Design : Twelve linear AFPep analogs were assembled on PAL-PEG-PS resin beginning with the C-terminus using N-terminally protected amino acids. These peptides were either four or five amino acids long, and were designed to resemble the putative pharmacophoric region of AFPep. An unrelated five amino acid peptide (PGVGQ) was synthesized as a negative control, and AFPep was used as a positive control. These peptides were evaluated using an immature mouse uterine growth inhibition assay (for antiestrogenicity) and a T47D breast cancer cell proliferation assay (for anticancer activity). Results and Conclusions : Linear analogs of AFPep were synthesized; mass spectrometry showed that each peptide was of the correct molecular weight. Inhibition of uterine growth was as follows: TOVNO (31± 2%), TOVN (23±2%), OVNO (9 ± 4%), OVNOG (21± 4%), KTOVN (28± 5%), TPVNP (27± 1%), TPVN (29±2%), PVNP (9±2%), EKTPV (0%), EMTPV (11%), EMTOV (0%), and VNOG (0%) compared to AFPep (38±2%) and PGVGQ (0%). Inhibition of E2-stimulated T47D cell growth was as follows: TOVNO (41±10%), OVNO (10%), and OVNOG (47%) compared to AFPep (53±6%) and PGVGQ (11%). We concluded that analogs of AFPep as small as 4 or 5 amino acids are effective antiestrotrophic and anticancer agents. OVNO is of low biological activity, whereas TOVN may be, and TOVNO is, biologically active.
Background-Diabetes and obesity are associated with an increased risk of arrhythmia and sudden ca... more Background-Diabetes and obesity are associated with an increased risk of arrhythmia and sudden cardiac death. Abnormal lipid accumulation is observed in cardiomyocytes of obese and diabetic patients, which may contribute to arrhythmia, but the mechanisms are poorly understood. A transgenic mouse model of cardiac lipid overload, the PPARg cardiac overexpression mouse, has long QT and increased ventricular ectopy. Objective-We evaluated the hypothesis that the increase in ventricular ectopy during cardiac lipid overload is caused by abnormalities in calcium handling due to increased mitochondrial oxidative stress. Methods-Ventricular myocytes were isolated from adult mouse hearts to record sparks and calcium transients. Mice were implanted with heart rhythm monitors for in vivo recordings. Results-PPARg cardiomyocytes have more frequent triggered activity and increased sparks compared to control. Sparks and triggered activity are reduced by mitotempo, a mitochondrialtargeted antioxidant. This is explained by a significant increase in oxidation of RyR2. Calcium transients are increased in amplitude and SR calcium stores are increased in PPARg cardiomyocytes. Computer modeling of the cardiac action potential demonstrates that long QT contributes to increased SR calcium. Mitotempo decreased ventricular ectopy in vivo.
Summary The response of vital organs to different types of nutrition or diet is a fundamental que... more Summary The response of vital organs to different types of nutrition or diet is a fundamental question in physiology. We examined the cardiac response to 4 weeks of high-fat diet in mice, measuring cardiac metabolites and mRNA. Metabolomics showed dramatic differences after a high-fat diet, including increases in several acyl-carnitine species. The RNA-seq data showed changes consistent with adaptations to use more fatty acid as substrate and an increase in the antioxidant protein catalase. Changes in mRNA were correlated with changes in protein level for several highly responsive genes. We also found significant sex differences in both metabolomics and RNA-seq datasets, both at baseline and after high fat diet. This work reveals the response of a vital organ to dietary intervention at both metabolomic and transcriptomic levels, which is a fundamental question in physiology. This work also reveals significant sex differences in cardiac metabolites and gene expression.
Some missense gain-of-function mutations in the CACNA1C gene, encoding calcium channel Ca V 1.2, ... more Some missense gain-of-function mutations in the CACNA1C gene, encoding calcium channel Ca V 1.2, cause a life-threatening form of long QT syndrome (LQTS) named Timothy syndrome with currently no clinically effective therapeutics. Here we report that pharmacological targeting of sigma non-opioid intracellular receptor 1 (SIGMAR1) can restore electrophysiological function in induced pluripotent stem cell (iPSC)-derived cardiomyocytes generated from patients with Timothy syndrome and two common forms of LQTS, type 1 (LQTS1) and type 2 (LQTS2), caused by missense trafficking mutations in potassium channels. Electrophysiological recordings demonstrate that a Food and Drug Administration (FDA)-approved cough suppressant, dextromethorphan, can be used as an agonist of SIGMAR1 to shorten the prolonged action potential in cardiomyocytes from patients with Timothy syndrome and human cellular models of LQTS1 and LQTS2. When tested in vivo, dextromethorphan also normalized the prolonged QT intervals in a mouse model of Timothy syndrome. Overall, our study demonstrates that SIGMAR1 is a potential therapeutic target for Timothy syndrome and possibly other inherited arrhythmias such as LQTS1 and LQTS2.
Cardiac arrhythmias are responsible for many cardiovascular disease-related deaths worldwide. Whi... more Cardiac arrhythmias are responsible for many cardiovascular disease-related deaths worldwide. While arrhythmia pathogenesis is complex, there is increasing evidence for metabolic causes. Obesity, diabetes, and chronically consuming high-fat foods significantly increase the likelihood of developing arrhythmias. Although these correlations are well established, mechanistic explanations connecting a high-fat diet (HFD) to arrhythmogenesis are incomplete, although oxidative stress appears to be critical. This review investigates the metabolic changes that occur in obesity and after HFD. Potential therapies to prevent or treat arrhythmias are discussed, including antioxidants.
Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) plays an important role in pulm... more Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) plays an important role in pulmonary hypertension (PH). However, the underlying specific ion channel mechanisms remain largely unknown. Here, we report ryanodine receptor (RyR) channel activity and Ca2+ release both are increased, and association of RyR2 by FK506 binding protein 12.6 (FKBP12.6) is decreased in PASMCs from mice with chronic hypoxia (CH)-induced PH. Smooth muscle cell (SMC)-specific RyR2 knockout (KO) or Rieske iron-sulfur protein (RISP) knockdown inhibits the altered Ca2+ signaling, increased nuclear factor (NF)-κB/cyclin D1 activation and cell proliferation, and CH-induced PH in mice. FKBP12.6 KO or FK506 treatment enhances CH-induced PH, while S107 (a specific stabilizer of RyR2/FKBP12.6 complex) produces an opposite effect. In conclusion, CH causes RISP-dependent ROS generation and FKBP12.6/RyR2 dissociation, leading to PH. RISP inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blocka...
Highlights d Cardiomyocytes expressing pathogenic lamin A/C exhibit altered nuclear positioning d... more Highlights d Cardiomyocytes expressing pathogenic lamin A/C exhibit altered nuclear positioning d ERK1/2 phosphorylates FHOD1/3 and inhibits nuclear movement in fibroblasts d ERK1/2-phosphorylated FHOD1/3 loses its F-actin bundling necessary for nuclear movement d FHOD3 phosphorylation by ERK1/2
Circulation: Arrhythmia and Electrophysiology, 2019
Background:Obesity and diets high in saturated fat increase the risk of arrhythmias and sudden ca... more Background:Obesity and diets high in saturated fat increase the risk of arrhythmias and sudden cardiac death. However, the molecular mechanisms are not well understood. We hypothesized that an increase in dietary saturated fat could lead to abnormalities of calcium homeostasis and heart rhythm by a NOX2 (NADPH oxidase 2)-dependent mechanism.Methods:We investigated this hypothesis by feeding mice high-fat diets. In vivo heart rhythm telemetry, optical mapping, and isolated cardiac myocyte imaging were used to quantify arrhythmias, repolarization, calcium transients, and intracellular calcium sparks.Results:We found that saturated fat activates NOX (NADPH oxidase), whereas polyunsaturated fat does not. The high saturated fat diet increased repolarization heterogeneity and ventricular tachycardia inducibility in perfused hearts. Pharmacological inhibition or genetic deletion of NOX2 prevented arrhythmogenic abnormalities in vivo during high statured fat diet and resulted in less induci...
Mutations in LMNA encoding lamin A/C and EMD encoding emerin cause cardiomyopathy and muscular dy... more Mutations in LMNA encoding lamin A/C and EMD encoding emerin cause cardiomyopathy and muscular dystrophy. Lmna null mice develop these disorders and have a lifespan of 7–8 weeks. Emd null mice show no overt pathology and have normal skeletal muscle but with regeneration defects. We generated mice with germline deletions of both Lmna and Emd to determine the effects of combined loss of the encoded proteins. Mice without lamin A/C and emerin are born at the expected Mendelian ratio, are grossly normal at birth but have shorter lifespans than those lacking only lamin A/C. However, there are no major differences between these mice with regards to left ventricular function, heart ultrastructure or electrocardiographic parameters except for slower heart rates in the mice lacking both lamin A/C and emerin. Skeletal muscle is similarly affected in both of these mice. Lmna+/− mice also lacking emerin live to at least 1 year and have no significant differences in growth, heart or skeletal mus...
American journal of physiology. Lung cellular and molecular physiology, May 1, 2018
Hypoxia-induced pulmonary vasoconstriction (HPV) is attributed to an increase in intracellular Ca... more Hypoxia-induced pulmonary vasoconstriction (HPV) is attributed to an increase in intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs). We have reported that phospholipase C-γ1 (PLCγ1) plays a significant role in the hypoxia-induced increase in [Ca2+]i in PASMCs and attendant HPV. In this study, we intended to determine molecular mechanisms for hypoxic Ca2+ and contractile responses in PASMCs. Our data reveal that hypoxic vasoconstriction occurs in PAs, but not in mesenteric arteries (MAs). Hypoxia caused a large increase in [Ca2+]i in PASMCs, which is diminished by the PLC inhibitor U73122 and not by its inactive analog U73433. Hypoxia augments PLCγ1-dependent inositol 1,4,5-trisphosphate (IP3) generation. Exogenous ROS, hydrogen peroxide (H2O2), increases PLCγ1 phosphorylation at tyrosine-783 and IP3 production. IP3 receptor-1 (IP3R1) knock-down (KD) remarkably diminishes hypoxia- or H2O2-induced increase in [Ca2+]i. Hypoxia or H2O2 increases ...
reduced acyl-carnitine long-chain fatty acid metabolites. We also observed changes in multiple pa... more reduced acyl-carnitine long-chain fatty acid metabolites. We also observed changes in multiple pathways associated with the development of PAH, including increased markers of proliferation, inflammation, fibrosis and endothelial dysfunction. Importantly, our global metabolic profile data compare favorably with prior work carried out in humans with PAH. We conclude that despite the MCT-model not recapitulating all the structural changes associated with humans with advanced PAH, including endothelial cell proliferation and the formation of plexiform lesions, it is very similar at a metabolic level. Also these data indicate that metabolic reprogramming precedes inflammatory, proliferative and fibrotic changes in PAH lungs.
Cardiomyopathy frequently complicates sepsis and is associated with increased mortality. Increase... more Cardiomyopathy frequently complicates sepsis and is associated with increased mortality. Increased cardiac oxidative stress and mitochondrial dysfunction have been observed during sepsis, but the mechanisms responsible for these abnormalities have not been determined. We hypothesized that NADPH oxidase 2 (NOX2) activation could be responsible for sepsis-induced oxidative stress and cardiomyopathy. Treatment of isolated adult mouse cardiomyocytes with low concentrations of the endotoxin lipopolysaccharide (LPS) increased total cellular reactive oxygen species (ROS) and mitochondrial superoxide. Elevated mitochondrial superoxide was accompanied by depolarization of the mitochondrial inner membrane potential, an indication of mitochondrial dysfunction, and mitochondrial calcium overload. NOX2 inhibition decreased LPS-induced superoxide and prevented mitochondrial dysfunction. Further, cardiomyocytes from mice with genetic ablation of NOX2 did not have LPS-induced superoxide or mitochon...
Background-Diabetes and obesity are associated with an increased risk of arrhythmia and sudden ca... more Background-Diabetes and obesity are associated with an increased risk of arrhythmia and sudden cardiac death. Abnormal lipid accumulation is observed in cardiomyocytes of obese and diabetic patients, which may contribute to arrhythmia, but the mechanisms are poorly understood. A transgenic mouse model of cardiac lipid overload, the PPARg cardiac overexpression mouse, has long QT and increased ventricular ectopy. Objective-We evaluated the hypothesis that the increase in ventricular ectopy during cardiac lipid overload is caused by abnormalities in calcium handling due to increased mitochondrial oxidative stress. Methods-Ventricular myocytes were isolated from adult mouse hearts to record sparks and calcium transients. Mice were implanted with heart rhythm monitors for in vivo recordings. Results-PPARg cardiomyocytes have more frequent triggered activity and increased sparks compared to control. Sparks and triggered activity are reduced by mitotempo, a mitochondrialtargeted antioxidant. This is explained by a significant increase in oxidation of RyR2. Calcium transients are increased in amplitude and SR calcium stores are increased in PPARg cardiomyocytes. Computer modeling of the cardiac action potential demonstrates that long QT contributes to increased SR calcium. Mitotempo decreased ventricular ectopy in vivo.
Cardiomyopathy caused by lamin A/C gene mutations (LMNA cardiomyopathy) is characterized by incre... more Cardiomyopathy caused by lamin A/C gene mutations (LMNA cardiomyopathy) is characterized by increased myocardial fibrosis, which impairs left ventricular relaxation and predisposes to heart failure, and cardiac conduction abnormalities. While we previously discovered abnormally elevated extracellular signal-regulated kinase 1/2 (ERK1/2) activities in heart in LMNA cardiomyopathy, its role on the development of myocardial fibrosis remains unclear. We now showed that transforming growth factor (TGF)-β/Smad signaling participates in the activation of ERK1/2 signaling in LMNA cardiomyopathy. ERK1/2 acts on connective tissue growth factor (CTGF/CCN2) expression to mediate the myocardial fibrosis and left ventricular dysfunction. Studies in vivo demonstrate that inhibiting CTGF/CCN2 using a specific antibody decreases myocardial fibrosis and improves the left ventricular dysfunction. Together, these findings show that cardiac ERK1/2 activity is modulated in part by TGF-β Smad signaling, l...
Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The mol... more Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS) in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.
We previously showed that striated muscle-selective depletion of lamina-associated polypeptide 1 ... more We previously showed that striated muscle-selective depletion of lamina-associated polypeptide 1 (LAP1), an integral inner nuclear membrane protein, leads to profound muscular dystrophy with premature death in mice. As LAP1 is also depleted in hearts of these mice, we examined their cardiac phenotype. Striated muscle-selective LAP1 knockout mice display ventricular systolic dysfunction with abnormal induction of genes encoding cardiomyopathy related proteins. To eliminate possible confounding effects due to skeletal muscle pathology, we generated a new mouse line in which LAP1 is deleted in a cardiomyocyte-selective manner. These mice had no skeletal muscle pathology and appeared overtly normal at 20 weeks of age. However, cardiac echocardiography revealed that they developed left ventricular systolic dysfunction and cardiac gene expression analysis revealed abnormal induction of cardiomyopathy-related genes. Our results demonstrate that LAP1 expression in cardiomyocytes is required for normal left ventricular function, consistent with a report of cardiomyopathy in a human subject with mutation in the gene encoding LAP1.
American Journal of Physiology-Lung Cellular and Molecular Physiology, 2012
An increase in intracellular calcium concentration ([Ca2+]i) in pulmonary arterial smooth muscle ... more An increase in intracellular calcium concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs) induces hypoxic cellular responses in the lungs; however, the underlying molecular mechanisms remain incompletely understood. We report, for the first time, that acute hypoxia significantly enhances phospholipase C (PLC) activity in mouse resistance pulmonary arteries (PAs), but not in mesenteric arteries. Western blot analysis and immunofluorescence staining reveal the expression of PLC-γ1 protein in PAs and PASMCs, respectively. The activity of PLC-γ1 is also augmented in PASMCs following hypoxia. Lentiviral shRNA-mediated gene knockdown of mitochondrial complex III Rieske iron-sulfur protein (RISP) to inhibit reactive oxygen species (ROS) production prevents hypoxia from increasing PLC-γ1 activity in PASMCs. Myxothiazol, a mitochondrial complex III inhibitor, reduces the hypoxic response as well. The PLC inhibitor U73122, but not its inactive analog U73433, attenuates t...
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Papers by Leroy Joseph