Proceedings of the National Academy of Sciences, 2006
Thyroid hormone (TH) is critical for cardiac development and heart function. In heart disease, TH... more Thyroid hormone (TH) is critical for cardiac development and heart function. In heart disease, TH metabolism is abnormal, and many biochemical and functional alterations mirror hypothyroidism. Although TH therapy has been advocated for treating heart disease, a clear benefit of TH has yet to be established, possibly because of peripheral actions of TH. To assess the potential efficacy of TH in treating heart disease, type 2 deiodinase (D2), which converts the prohormone thyroxine to active triiodothyronine (T3), was expressed transiently in mouse hearts by using the tetracycline transactivator system. Increased cardiac D2 activity led to elevated cardiac T3 levels and to enhanced myocardial contractility, accompanied by increased Ca 2+ transients and sarcoplasmic reticulum (SR) Ca 2+ uptake. These phenotypic changes were associated with up-regulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) 2a expression as well as decreased Na + /Ca 2+ exchanger, β-myosin heavy chain,...
Proceedings of the National Academy of Sciences of the United States of America, Jan 15, 2015
Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to pro... more Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (PDE3A), protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B(-/-) heart was reversed by blocking cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling. Compared with WT mitochondria, PDE3B(-/-) mitochondria were enriched in antiapoptotic Bcl-2, produced less reactive oxygen species, and more frequently contacted transverse tubules where PDE3B was localized with caveolin-3. Moreover, a PDE3B(-/-) mitochondrial fraction containing connexin-43 and caveolin-3 was ...
Journal of molecular and cellular cardiology, 2014
Cardiac sarcolemmal syntaxin (Syn)-1A interacts with sulfonylurea receptor (SUR) 2A to inhibit AT... more Cardiac sarcolemmal syntaxin (Syn)-1A interacts with sulfonylurea receptor (SUR) 2A to inhibit ATP-sensitive potassium (KATP) channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a ubiquitous endogenous inositol phospholipid, known to bind Kir6.2 subunit to open KATP channels, has recently been shown to directly bind Syn-1A in plasma membrane to form Syn-1A clusters. Here, we sought to determine whether the interaction between Syn-1A and PIP2 interferes with the ability of Syn-1A to bind SUR2A and inhibit KATP channel activity. We found that PIP2 dose-dependently reduced SUR2A binding to GST-Syn-1A by in vitro pulldown assays. FRET studies in intact cells using TIRFM revealed that increasing endogenous PIP2 levels led to increased Syn-1A (-EGFP) cluster formation and a severe reduction in availability of Syn-1A molecules to interact with SUR2A (-mCherry) molecules outside the Syn-1A clusters. Correspondingly, electrophysiological studies employing SUR2A/Kir6.2-expressing HEK cell...
Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca2+ release fro... more Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca2+ release from the sarcoplasmic reticulum (SR) by L-type Ca channels, whose activity is strongly influenced by action potential (AP) profile. The contribution of Ca2+ entry via the Na+/Ca2+ exchanger (NCX) to trigger SR Ca2+ release during ECC in response to an AP remains uncertain. To isolate the contribution of NCX
American journal of physiology. Cell physiology, Jan 15, 2014
We have previously shown that ischemic preconditioning (IPC) protection against necrosis in whole... more We have previously shown that ischemic preconditioning (IPC) protection against necrosis in whole hearts and in both fresh and cultured cardiomyocytes, as well as the improved regulatory volume decrease to hypoosmotic swelling in cardiomyocytes, is abrogated through Cl(-) channel blockade, pointing to a role for enhanced cell volume regulation in IPC. To further define this cardioprotective mechanism, cultured rabbit ventricular cardiomyocytes were preconditioned either by 10-min simulated ischemia (SI) followed by 10-min simulated reperfusion (SR), by 10-min exposure/10-min washout of remote IPC (rIPC) plasma dialysate (from rabbits subjected to repetitive limb ischemia), or by adenoviral transfection with the constitutively active PKC-ε gene. These interventions were done before cardiomyocytes were subjected to either 60- or 75-min SI/60-min SR to assess cell necrosis (by trypan blue staining), 30-min SI to assess ischemic cell swelling, or 30-min hypoosmotic (200 mosM) stress to ...
Journal of the American College of Cardiology, Jan 17, 2011
Our objective was to test the hypothesis that there is a significant diurnal variation for the th... more Our objective was to test the hypothesis that there is a significant diurnal variation for the therapeutic benefit of angiotensin-converting enzyme (ACE) inhibitors on pressure-overload cardiovascular hypertrophy. Physiological and molecular processes exhibit diurnal rhythms that may affect efficacy of disease treatment (chronotherapy). Evidence suggests that the heart primarily remodels during sleep. Although a growing body of clinical and epidemiological evidence suggests that the timing of therapy, such as ACE inhibition, alters diurnal blood pressure patterns in patients with hypertension, the benefits of chronotherapy on myocardial and vascular remodeling have not been studied. We examined the effects of the short-acting ACE inhibitor, captopril, on the structure and function of cardiovascular tissue subjected to pressure overload by transverse aortic constriction (TAC) in mice. Captopril (15 mg/kg intraperitoneally) or placebo was administered at either murine sleep time or wa...
Heart rate is controlled by the opposing activities of sympathetic and parasympathetic inputs to ... more Heart rate is controlled by the opposing activities of sympathetic and parasympathetic inputs to pacemaker myocytes in the sinoatrial node (SAN). Parasympathetic activity on nodal myocytes is mediated by acetylcholine- dependent stimulation of M2 muscarinic receptors and activation of Gi/o signaling. Although regulators of G protein signaling (RGS) proteins are potent inhibitors of Gi/o signaling in many tissues, the RGS
Endoplasmic reticulum (ER) resident protein 44 (ERp44) is a member of the protein disulfide isome... more Endoplasmic reticulum (ER) resident protein 44 (ERp44) is a member of the protein disulfide isomerase family, is induced during ER stress, and may be involved in regulating Ca(2+) homeostasis. However, the role of ERp44 in cardiac development and function is unknown. The aim of this study was to investigate the role of ERp44 in cardiac development and function in mice, zebrafish, and embryonic stem cell (ESC)-derived cardiomyocytes to determine the underlying role of ERp44. We generated and characterized ERp44(-/-) mice, ERp44 morphant zebrafish embryos, and ERp44(-/-) ESC-derived cardiomyocytes. Deletion of ERp44 in mouse and zebrafish caused significant embryonic lethality, abnormal heart development, altered Ca(2+) dynamics, reactive oxygen species generation, activated ER stress gene profiles, and apoptotic cell death. We also determined the cardiac phenotype in pressure overloaded, aortic-banded ERp44(+/-) mice: enhanced ER stress activation and increased mortality, as well as ...
Abstract—Cytokine and extracellular matrix (ECM) homeostasis,are distinct systems that are each d... more Abstract—Cytokine and extracellular matrix (ECM) homeostasis,are distinct systems that are each dysregulated in heart failure. Here we show,that tissue inhibitor of metalloproteinase (TIMP)-3 is a critical regulator of both systems in a mouse,model of left ventricular (LV) dilation and dysfunction. Timp-3,-AB mice. This study provides a basis for anti-TNF and MMP inhibitor combination,therapy in heart disease. (Circ Res. 2005;97:0-0.) Key
Cytokine and extracellular matrix (ECM) homeostasis are distinct systems that are each dysregulat... more Cytokine and extracellular matrix (ECM) homeostasis are distinct systems that are each dysregulated in heart failure. Here we show that tissue inhibitor of metalloproteinase (TIMP)-3 is a critical regulator of both systems in a mouse model of left ventricular (LV) dilation and dysfunction. Timp-3 Ϫ/Ϫ mice develop precipitous LV dilation and dysfunction reminiscent of dilated cardiomyopathy (DCM), culminating in early onset of heart failure by 6 weeks, compared with wild-type aortic-banding (AB). Timp-3 deficiency resulted in increased TNF␣ converting enzyme (TACE) activity within 6 hours after AB leading to enhanced tumor necrosis factor-␣ (TNF␣) processing. In addition, TNF␣ production increased in timp-3 Ϫ/Ϫ -AB myocardium. A significant elevation in gelatinase and collagenase activities was observed 1 week after AB, with localized ECM degradation in timp-3 Ϫ/Ϫ -AB myocardium. Timp-3 Ϫ/Ϫ / tnf␣ Ϫ/Ϫ mice were generated and subjected to AB for comparative analyses with timp-3 Ϫ/Ϫ -AB mice. This revealed the critical role of TNF␣ in the early phase of LV remodeling, de novo expression of Matrix metalloproteinases (MMP)-8 in the absence of TNF␣, and highlighted the importance of interstitial collagenases (MMP-2, MMP-13, and MT1-MMP) for cardiac ECM degradation. Ablation of TNF␣, or limiting MMP activity with a synthetic MMP inhibitor (PD166793), each partially attenuated LV dilation and cardiac dysfunction in timp-3 Ϫ/Ϫ -AB mice. Notably, combining TNF␣ ablation with MMP inhibition completely rescued heart disease in timp-3 Ϫ/Ϫ -AB mice. This study provides a basis for anti-TNF␣ and MMP inhibitor combination therapy in heart disease. (Circ Res. 2005;97:380-390.) Key Words: left ventricular dilation and dysfunction Ⅲ extracellular matrix Ⅲ tissue inhibitor of metalloproteinase-3 Ⅲ matrix metalloproteinase Ⅲ tumor necrosis factor-␣
J-wave ECG patterns are associated with an increased risk of sudden arrhythmic death, and experim... more J-wave ECG patterns are associated with an increased risk of sudden arrhythmic death, and experimental evidence supports a transient outward current (Ito)-mediated mechanism of J-wave formation. This study aimed to determine the frequency of genetic mutations in genes encoding the Ito in patients with J waves on ECG. Comprehensive mutational analysis was performed on Ito-encoding KCNA4, KCND2, and KCND3 genes, as well as the previously described J-wave-associated KCNJ8 gene, in 51 unrelated patients with ECG evidence defining a J-wave syndrome. Only patients with a resuscitated cardiac arrest or type 1 Brugada ECG pattern were included for analysis. A rare genetic mutation of the KCND2 gene, p.D612N, was identified in a single patient. Co-expression of mutant and wild-type KCND2 with KChIP2 in HEK293 cells demonstrated a gain-of-function phenotype, including an increase in peak Ito density of 48% (P<0.05) in the heterozygous state. Using computer modeling, this increase in Ito re...
Anti-(Tityus serrulatus + Tityus bahiensis) and anti-Tityus discrepans venom polyclonal antisera ... more Anti-(Tityus serrulatus + Tityus bahiensis) and anti-Tityus discrepans venom polyclonal antisera were used to investigate whether antigenic differences exist between the venoms of the Brazilian T. serrulatus and the Venezuelan T. discrepans scorpions. Both antisera recognised the toxin-containing electrophoretic fractions of their cognate venoms and also those from Tityus zulianus and Tityus trinitatis venoms on Western blots. The anti-T. discrepans antiserum reacted only weakly with T. serrulatus toxic polypeptides. The effect of T. serrulatus alpha- or beta-toxins on rat skeletal muscle Na+ channels expressed in Xenopus laevis oocytes was abolished by pre-incubating the venom with anti-(T. serrulatus + T. bahiensis) serum but not with anti-T. discrepans serum. Nor did the Brazilian or the Venezuelan sera prevent the reduction in K+ currents by T. serrulatus venom in X. laevis oocytes expressing the rat brain delayed rectifying Shaker K+ channel (Kv1.2). These results indicate that toxins from T. serrulatus and T. discrepans venoms, which primarily target mammalian Na+ channels, are antigenically distinct, although they probably share common epitopes. Our results also suggest that Na+ channel-active toxins are the immunodominant antigens of the T. serrulatus venom.
1. Cardiac hypertrophy and prolongation of the cardiac action potential are hallmark features of ... more 1. Cardiac hypertrophy and prolongation of the cardiac action potential are hallmark features of heart disease. We examined the molecular mechanisms and the functional consequences of this action potential prolongation on calcium handling in right ventricular myocytes obtained from rats 8 weeks following ligation of the left anterior descending coronary artery (post-myocardial infarction (MI) myocytes). 2. Compared with myocytes from sham-operated rats (sham myocytes), post-MI myocytes showed significant reductions in transient outward K+ current (Ito) density (sham 19.7 +/- 1.1 pA pF-1 versus post-MI 11.0 +/- 1.3 pA pF-1; means +/- s.e.m.), inward rectifier K+ current density (sham -13.7 +/- 0.6 pA pF-1 versus post-MI -10.3 +/- 0.9 pA pF-1) and resting membrane potential (sham -84.4 +/- 1.3 mV versus post-MI -74.1 +/- 2.6 mV). Depressed Ito amplitude correlated with significant reductions in Kv4.2 and Kv4.3 mRNA and Kv4.2 protein levels. Kv1.4 mRNA and protein levels were increased and coincided with the appearance of a slow component of recovery from inactivation for Ito. 3. In current-clamp recordings, post-MI myocytes showed a significant increase in [Ca2+]i transient amplitude compared with sham myocytes. Using voltage-clamp depolarizations, no intrinsic differences in Ca2+ handling by the sarcoplasmic reticulum or in L-type Ca2+ channel density (ICa,L) were detected between the groups. 4. Stimulation of post-MI myocytes with an action potential derived from a sham myocyte reduced the [Ca2+] transient amplitude to the sham level and vice versa. 5. The net Ca2+ influx per beat via ICa,L was increased about 2-fold in myocytes stimulated with post-MI action potentials compared with sham action potentials. 6. Our findings demonstrate that reductions in K+ channel expression in post-MI myocytes prolong action potential duration resulting in elevated Ca2+ influx and [Ca2+]i transients.
Proceedings of the National Academy of Sciences, 2008
At the end of every heartbeat, cardiac myocytes must relax to allow filling of the heart. Impaire... more At the end of every heartbeat, cardiac myocytes must relax to allow filling of the heart. Impaired relaxation is a significant factor in heart failure, but all pathways regulating the cardiac relaxation apparatus are not known. Haploinsufficiency of the T-box transcription factor Tbx5 in mouse and man causes congenital heart defects (CHDs) as part of Holt-Oram syndrome (HOS). Here, we show that haploinsufficiency of Tbx5 in mouse results in cellautonomous defects in ventricular relaxation. Tbx5 dosage modulates expression of the sarco(endo)plasmic reticulum Ca 2؉ -ATPase isoform 2a encoded by Atp2a2 and Tbx5 haploinsufficiency in ventricular myocytes results in impaired Ca 2؉ uptake dynamics and Ca 2؉ transient prolongation. We also demonstrate that Tbx5 can activate the Atp2a2 promoter. Furthermore, we find that patients with HOS have significant diastolic filling abnormalities. These results reveal a direct genetic pathway that regulates cardiac diastolic function, implying that patients with structural CHDs may have clinically important underlying anomalies in heart function that merit treatment.
Proceedings of the National Academy of Sciences, 2006
To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic... more To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic (TG) mice were generated with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope). The level of NF-SLN expression (the NF-SLN͞PLN expression ratio) was equivalent to that which induces profound superinhibition when coexpressed with PLN and SERCA2a in HEK-293 cells. In TG hearts, the apparent affinity of SERCA2a for Ca 2؉ was decreased compared with non-TG littermate control hearts. Invasive hemodynamic and echocardiographic analyses revealed impaired cardiac contractility and ventricular hypertrophy in TG mice. Basal PLN phosphorylation was reduced. In isolated papillary muscle subjected to isometric tension, peak amplitudes of Ca 2؉ transients and peak tensions were reduced, whereas decay times of Ca 2؉ transients and relaxation times of tension were increased in TG mice. Isoproterenol largely restored contractility in papillary muscle and stimulated PLN phosphorylation to wild-type levels in intact hearts. No compensatory changes in expression of SERCA2a, PLN, ryanodine receptor, and calsequestrin were observed in TG hearts. Coimmunoprecipitation indicated that overexpressed NF-SLN was bound to both SERCA2a and PLN, forming a ternary complex. These data suggest that NF-SLN overexpression inhibits SERCA2a through stabilization of SERCA2a-PLN interaction in the absence of PLN phosphorylation and through the inhibition of PLN phosphorylation. Inhibition of SERCA2a impairs contractility and calcium cycling, but responsiveness to -adrenergic agonists may prevent progression to heart failure. C a 2ϩ enters cardiomyocytes through L-type Ca 2ϩ channels in the transverse tubules and ryanodine receptor-type Ca 2ϩ release channels in the sarcoplasmic reticulum (SR) to initiate muscle contraction and is removed to induce relaxation by the sarco(endo)plasmic reticulum Ca 2ϩ ATPase (SERCA2a), which transports Ca 2ϩ into the SR lumen, plasma membrane Ca 2ϩ ATPases, and Na ϩ ͞Ca 2ϩ exchangers, which discharge Ca 2ϩ to the extracellular space. SERCA2a is regulated by phospholamban (PLN) and can potentially be regulated by a homologous protein, sarcolipin (SLN) (1, 2).
We hypothesized that cardiac dysfunction was responsible for the high perinatal lethality that we... more We hypothesized that cardiac dysfunction was responsible for the high perinatal lethality that we previously reported in fibrinogen-like protein 2 (Fgl2) knockout (KO) mice. We therefore used ultrasound biomicroscopy to assess left ventricular (LV) cardiac structure and function during development in Fgl2 KO and wild-type (WT) mice. The only deaths observed between embryonic day (E)8.5 (onset of heart beating) and postnatal day (P)28 (weaning) were within 3 days after birth, when 33% of Fgl2 KO pups died. Histopathology and Doppler assessments suggested that death was due to acute congestive cardiac failure without evidence of valvular or other obvious cardiac structural abnormalities. Heart rates in Fgl2 KO embryos were significantly reduced at E8.5 and E17.5, and irregular heart rhythms were significantly more common in Fgl2 KO (21/26) than WT (2/21) embryos at E13.5. Indexes of systolic and/or diastolic cardiac function were also abnormal in KO mice at E13.5 and E17.5, in postnatal mice studied at P1, and in KO mice surviving to P28. M-mode analysis showed no difference in LV diastolic chamber dimension, although posterior wall thickness was thinner at P7 and P28 in Fgl2 KO mice. We conclude that Fgl2 deficiency is not associated with obvious structural cardiac defects but is associated with a high incidence of neonatal death as well as contractile dysfunction and rhythm abnormalities during embryonic and postnatal development in mice.
Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. Her... more Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. Here we show that angiotensin-converting enzyme 2 (ace2) maps to a defined quantitative trait locus (QTL) on the X chromosome in three different rat models of hypertension. In all hypertensive rat strains, ACE2 messenger RNA and protein expression were markedly reduced, suggesting that ace2 is a candidate gene for this QTL. Targeted disruption of ACE2 in mice results in a severe cardiac contractility defect, increased angiotensin II levels, and upregulation of hypoxia-induced genes in the heart. Genetic ablation of ACE on an ACE2 mutant background completely rescues the cardiac phenotype. But disruption of ACER, a Drosophila ACE2 homologue, results in a severe defect of heart morphogenesis. These genetic data for ACE2 show that it is an essential regulator of heart function in vivo.
Proceedings of the National Academy of Sciences, 2006
Thyroid hormone (TH) is critical for cardiac development and heart function. In heart disease, TH... more Thyroid hormone (TH) is critical for cardiac development and heart function. In heart disease, TH metabolism is abnormal, and many biochemical and functional alterations mirror hypothyroidism. Although TH therapy has been advocated for treating heart disease, a clear benefit of TH has yet to be established, possibly because of peripheral actions of TH. To assess the potential efficacy of TH in treating heart disease, type 2 deiodinase (D2), which converts the prohormone thyroxine to active triiodothyronine (T3), was expressed transiently in mouse hearts by using the tetracycline transactivator system. Increased cardiac D2 activity led to elevated cardiac T3 levels and to enhanced myocardial contractility, accompanied by increased Ca 2+ transients and sarcoplasmic reticulum (SR) Ca 2+ uptake. These phenotypic changes were associated with up-regulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) 2a expression as well as decreased Na + /Ca 2+ exchanger, β-myosin heavy chain,...
Proceedings of the National Academy of Sciences of the United States of America, Jan 15, 2015
Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to pro... more Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (PDE3A), protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B(-/-) heart was reversed by blocking cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling. Compared with WT mitochondria, PDE3B(-/-) mitochondria were enriched in antiapoptotic Bcl-2, produced less reactive oxygen species, and more frequently contacted transverse tubules where PDE3B was localized with caveolin-3. Moreover, a PDE3B(-/-) mitochondrial fraction containing connexin-43 and caveolin-3 was ...
Journal of molecular and cellular cardiology, 2014
Cardiac sarcolemmal syntaxin (Syn)-1A interacts with sulfonylurea receptor (SUR) 2A to inhibit AT... more Cardiac sarcolemmal syntaxin (Syn)-1A interacts with sulfonylurea receptor (SUR) 2A to inhibit ATP-sensitive potassium (KATP) channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a ubiquitous endogenous inositol phospholipid, known to bind Kir6.2 subunit to open KATP channels, has recently been shown to directly bind Syn-1A in plasma membrane to form Syn-1A clusters. Here, we sought to determine whether the interaction between Syn-1A and PIP2 interferes with the ability of Syn-1A to bind SUR2A and inhibit KATP channel activity. We found that PIP2 dose-dependently reduced SUR2A binding to GST-Syn-1A by in vitro pulldown assays. FRET studies in intact cells using TIRFM revealed that increasing endogenous PIP2 levels led to increased Syn-1A (-EGFP) cluster formation and a severe reduction in availability of Syn-1A molecules to interact with SUR2A (-mCherry) molecules outside the Syn-1A clusters. Correspondingly, electrophysiological studies employing SUR2A/Kir6.2-expressing HEK cell...
Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca2+ release fro... more Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca2+ release from the sarcoplasmic reticulum (SR) by L-type Ca channels, whose activity is strongly influenced by action potential (AP) profile. The contribution of Ca2+ entry via the Na+/Ca2+ exchanger (NCX) to trigger SR Ca2+ release during ECC in response to an AP remains uncertain. To isolate the contribution of NCX
American journal of physiology. Cell physiology, Jan 15, 2014
We have previously shown that ischemic preconditioning (IPC) protection against necrosis in whole... more We have previously shown that ischemic preconditioning (IPC) protection against necrosis in whole hearts and in both fresh and cultured cardiomyocytes, as well as the improved regulatory volume decrease to hypoosmotic swelling in cardiomyocytes, is abrogated through Cl(-) channel blockade, pointing to a role for enhanced cell volume regulation in IPC. To further define this cardioprotective mechanism, cultured rabbit ventricular cardiomyocytes were preconditioned either by 10-min simulated ischemia (SI) followed by 10-min simulated reperfusion (SR), by 10-min exposure/10-min washout of remote IPC (rIPC) plasma dialysate (from rabbits subjected to repetitive limb ischemia), or by adenoviral transfection with the constitutively active PKC-ε gene. These interventions were done before cardiomyocytes were subjected to either 60- or 75-min SI/60-min SR to assess cell necrosis (by trypan blue staining), 30-min SI to assess ischemic cell swelling, or 30-min hypoosmotic (200 mosM) stress to ...
Journal of the American College of Cardiology, Jan 17, 2011
Our objective was to test the hypothesis that there is a significant diurnal variation for the th... more Our objective was to test the hypothesis that there is a significant diurnal variation for the therapeutic benefit of angiotensin-converting enzyme (ACE) inhibitors on pressure-overload cardiovascular hypertrophy. Physiological and molecular processes exhibit diurnal rhythms that may affect efficacy of disease treatment (chronotherapy). Evidence suggests that the heart primarily remodels during sleep. Although a growing body of clinical and epidemiological evidence suggests that the timing of therapy, such as ACE inhibition, alters diurnal blood pressure patterns in patients with hypertension, the benefits of chronotherapy on myocardial and vascular remodeling have not been studied. We examined the effects of the short-acting ACE inhibitor, captopril, on the structure and function of cardiovascular tissue subjected to pressure overload by transverse aortic constriction (TAC) in mice. Captopril (15 mg/kg intraperitoneally) or placebo was administered at either murine sleep time or wa...
Heart rate is controlled by the opposing activities of sympathetic and parasympathetic inputs to ... more Heart rate is controlled by the opposing activities of sympathetic and parasympathetic inputs to pacemaker myocytes in the sinoatrial node (SAN). Parasympathetic activity on nodal myocytes is mediated by acetylcholine- dependent stimulation of M2 muscarinic receptors and activation of Gi/o signaling. Although regulators of G protein signaling (RGS) proteins are potent inhibitors of Gi/o signaling in many tissues, the RGS
Endoplasmic reticulum (ER) resident protein 44 (ERp44) is a member of the protein disulfide isome... more Endoplasmic reticulum (ER) resident protein 44 (ERp44) is a member of the protein disulfide isomerase family, is induced during ER stress, and may be involved in regulating Ca(2+) homeostasis. However, the role of ERp44 in cardiac development and function is unknown. The aim of this study was to investigate the role of ERp44 in cardiac development and function in mice, zebrafish, and embryonic stem cell (ESC)-derived cardiomyocytes to determine the underlying role of ERp44. We generated and characterized ERp44(-/-) mice, ERp44 morphant zebrafish embryos, and ERp44(-/-) ESC-derived cardiomyocytes. Deletion of ERp44 in mouse and zebrafish caused significant embryonic lethality, abnormal heart development, altered Ca(2+) dynamics, reactive oxygen species generation, activated ER stress gene profiles, and apoptotic cell death. We also determined the cardiac phenotype in pressure overloaded, aortic-banded ERp44(+/-) mice: enhanced ER stress activation and increased mortality, as well as ...
Abstract—Cytokine and extracellular matrix (ECM) homeostasis,are distinct systems that are each d... more Abstract—Cytokine and extracellular matrix (ECM) homeostasis,are distinct systems that are each dysregulated in heart failure. Here we show,that tissue inhibitor of metalloproteinase (TIMP)-3 is a critical regulator of both systems in a mouse,model of left ventricular (LV) dilation and dysfunction. Timp-3,-AB mice. This study provides a basis for anti-TNF and MMP inhibitor combination,therapy in heart disease. (Circ Res. 2005;97:0-0.) Key
Cytokine and extracellular matrix (ECM) homeostasis are distinct systems that are each dysregulat... more Cytokine and extracellular matrix (ECM) homeostasis are distinct systems that are each dysregulated in heart failure. Here we show that tissue inhibitor of metalloproteinase (TIMP)-3 is a critical regulator of both systems in a mouse model of left ventricular (LV) dilation and dysfunction. Timp-3 Ϫ/Ϫ mice develop precipitous LV dilation and dysfunction reminiscent of dilated cardiomyopathy (DCM), culminating in early onset of heart failure by 6 weeks, compared with wild-type aortic-banding (AB). Timp-3 deficiency resulted in increased TNF␣ converting enzyme (TACE) activity within 6 hours after AB leading to enhanced tumor necrosis factor-␣ (TNF␣) processing. In addition, TNF␣ production increased in timp-3 Ϫ/Ϫ -AB myocardium. A significant elevation in gelatinase and collagenase activities was observed 1 week after AB, with localized ECM degradation in timp-3 Ϫ/Ϫ -AB myocardium. Timp-3 Ϫ/Ϫ / tnf␣ Ϫ/Ϫ mice were generated and subjected to AB for comparative analyses with timp-3 Ϫ/Ϫ -AB mice. This revealed the critical role of TNF␣ in the early phase of LV remodeling, de novo expression of Matrix metalloproteinases (MMP)-8 in the absence of TNF␣, and highlighted the importance of interstitial collagenases (MMP-2, MMP-13, and MT1-MMP) for cardiac ECM degradation. Ablation of TNF␣, or limiting MMP activity with a synthetic MMP inhibitor (PD166793), each partially attenuated LV dilation and cardiac dysfunction in timp-3 Ϫ/Ϫ -AB mice. Notably, combining TNF␣ ablation with MMP inhibition completely rescued heart disease in timp-3 Ϫ/Ϫ -AB mice. This study provides a basis for anti-TNF␣ and MMP inhibitor combination therapy in heart disease. (Circ Res. 2005;97:380-390.) Key Words: left ventricular dilation and dysfunction Ⅲ extracellular matrix Ⅲ tissue inhibitor of metalloproteinase-3 Ⅲ matrix metalloproteinase Ⅲ tumor necrosis factor-␣
J-wave ECG patterns are associated with an increased risk of sudden arrhythmic death, and experim... more J-wave ECG patterns are associated with an increased risk of sudden arrhythmic death, and experimental evidence supports a transient outward current (Ito)-mediated mechanism of J-wave formation. This study aimed to determine the frequency of genetic mutations in genes encoding the Ito in patients with J waves on ECG. Comprehensive mutational analysis was performed on Ito-encoding KCNA4, KCND2, and KCND3 genes, as well as the previously described J-wave-associated KCNJ8 gene, in 51 unrelated patients with ECG evidence defining a J-wave syndrome. Only patients with a resuscitated cardiac arrest or type 1 Brugada ECG pattern were included for analysis. A rare genetic mutation of the KCND2 gene, p.D612N, was identified in a single patient. Co-expression of mutant and wild-type KCND2 with KChIP2 in HEK293 cells demonstrated a gain-of-function phenotype, including an increase in peak Ito density of 48% (P<0.05) in the heterozygous state. Using computer modeling, this increase in Ito re...
Anti-(Tityus serrulatus + Tityus bahiensis) and anti-Tityus discrepans venom polyclonal antisera ... more Anti-(Tityus serrulatus + Tityus bahiensis) and anti-Tityus discrepans venom polyclonal antisera were used to investigate whether antigenic differences exist between the venoms of the Brazilian T. serrulatus and the Venezuelan T. discrepans scorpions. Both antisera recognised the toxin-containing electrophoretic fractions of their cognate venoms and also those from Tityus zulianus and Tityus trinitatis venoms on Western blots. The anti-T. discrepans antiserum reacted only weakly with T. serrulatus toxic polypeptides. The effect of T. serrulatus alpha- or beta-toxins on rat skeletal muscle Na+ channels expressed in Xenopus laevis oocytes was abolished by pre-incubating the venom with anti-(T. serrulatus + T. bahiensis) serum but not with anti-T. discrepans serum. Nor did the Brazilian or the Venezuelan sera prevent the reduction in K+ currents by T. serrulatus venom in X. laevis oocytes expressing the rat brain delayed rectifying Shaker K+ channel (Kv1.2). These results indicate that toxins from T. serrulatus and T. discrepans venoms, which primarily target mammalian Na+ channels, are antigenically distinct, although they probably share common epitopes. Our results also suggest that Na+ channel-active toxins are the immunodominant antigens of the T. serrulatus venom.
1. Cardiac hypertrophy and prolongation of the cardiac action potential are hallmark features of ... more 1. Cardiac hypertrophy and prolongation of the cardiac action potential are hallmark features of heart disease. We examined the molecular mechanisms and the functional consequences of this action potential prolongation on calcium handling in right ventricular myocytes obtained from rats 8 weeks following ligation of the left anterior descending coronary artery (post-myocardial infarction (MI) myocytes). 2. Compared with myocytes from sham-operated rats (sham myocytes), post-MI myocytes showed significant reductions in transient outward K+ current (Ito) density (sham 19.7 +/- 1.1 pA pF-1 versus post-MI 11.0 +/- 1.3 pA pF-1; means +/- s.e.m.), inward rectifier K+ current density (sham -13.7 +/- 0.6 pA pF-1 versus post-MI -10.3 +/- 0.9 pA pF-1) and resting membrane potential (sham -84.4 +/- 1.3 mV versus post-MI -74.1 +/- 2.6 mV). Depressed Ito amplitude correlated with significant reductions in Kv4.2 and Kv4.3 mRNA and Kv4.2 protein levels. Kv1.4 mRNA and protein levels were increased and coincided with the appearance of a slow component of recovery from inactivation for Ito. 3. In current-clamp recordings, post-MI myocytes showed a significant increase in [Ca2+]i transient amplitude compared with sham myocytes. Using voltage-clamp depolarizations, no intrinsic differences in Ca2+ handling by the sarcoplasmic reticulum or in L-type Ca2+ channel density (ICa,L) were detected between the groups. 4. Stimulation of post-MI myocytes with an action potential derived from a sham myocyte reduced the [Ca2+] transient amplitude to the sham level and vice versa. 5. The net Ca2+ influx per beat via ICa,L was increased about 2-fold in myocytes stimulated with post-MI action potentials compared with sham action potentials. 6. Our findings demonstrate that reductions in K+ channel expression in post-MI myocytes prolong action potential duration resulting in elevated Ca2+ influx and [Ca2+]i transients.
Proceedings of the National Academy of Sciences, 2008
At the end of every heartbeat, cardiac myocytes must relax to allow filling of the heart. Impaire... more At the end of every heartbeat, cardiac myocytes must relax to allow filling of the heart. Impaired relaxation is a significant factor in heart failure, but all pathways regulating the cardiac relaxation apparatus are not known. Haploinsufficiency of the T-box transcription factor Tbx5 in mouse and man causes congenital heart defects (CHDs) as part of Holt-Oram syndrome (HOS). Here, we show that haploinsufficiency of Tbx5 in mouse results in cellautonomous defects in ventricular relaxation. Tbx5 dosage modulates expression of the sarco(endo)plasmic reticulum Ca 2؉ -ATPase isoform 2a encoded by Atp2a2 and Tbx5 haploinsufficiency in ventricular myocytes results in impaired Ca 2؉ uptake dynamics and Ca 2؉ transient prolongation. We also demonstrate that Tbx5 can activate the Atp2a2 promoter. Furthermore, we find that patients with HOS have significant diastolic filling abnormalities. These results reveal a direct genetic pathway that regulates cardiac diastolic function, implying that patients with structural CHDs may have clinically important underlying anomalies in heart function that merit treatment.
Proceedings of the National Academy of Sciences, 2006
To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic... more To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic (TG) mice were generated with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope). The level of NF-SLN expression (the NF-SLN͞PLN expression ratio) was equivalent to that which induces profound superinhibition when coexpressed with PLN and SERCA2a in HEK-293 cells. In TG hearts, the apparent affinity of SERCA2a for Ca 2؉ was decreased compared with non-TG littermate control hearts. Invasive hemodynamic and echocardiographic analyses revealed impaired cardiac contractility and ventricular hypertrophy in TG mice. Basal PLN phosphorylation was reduced. In isolated papillary muscle subjected to isometric tension, peak amplitudes of Ca 2؉ transients and peak tensions were reduced, whereas decay times of Ca 2؉ transients and relaxation times of tension were increased in TG mice. Isoproterenol largely restored contractility in papillary muscle and stimulated PLN phosphorylation to wild-type levels in intact hearts. No compensatory changes in expression of SERCA2a, PLN, ryanodine receptor, and calsequestrin were observed in TG hearts. Coimmunoprecipitation indicated that overexpressed NF-SLN was bound to both SERCA2a and PLN, forming a ternary complex. These data suggest that NF-SLN overexpression inhibits SERCA2a through stabilization of SERCA2a-PLN interaction in the absence of PLN phosphorylation and through the inhibition of PLN phosphorylation. Inhibition of SERCA2a impairs contractility and calcium cycling, but responsiveness to -adrenergic agonists may prevent progression to heart failure. C a 2ϩ enters cardiomyocytes through L-type Ca 2ϩ channels in the transverse tubules and ryanodine receptor-type Ca 2ϩ release channels in the sarcoplasmic reticulum (SR) to initiate muscle contraction and is removed to induce relaxation by the sarco(endo)plasmic reticulum Ca 2ϩ ATPase (SERCA2a), which transports Ca 2ϩ into the SR lumen, plasma membrane Ca 2ϩ ATPases, and Na ϩ ͞Ca 2ϩ exchangers, which discharge Ca 2ϩ to the extracellular space. SERCA2a is regulated by phospholamban (PLN) and can potentially be regulated by a homologous protein, sarcolipin (SLN) (1, 2).
We hypothesized that cardiac dysfunction was responsible for the high perinatal lethality that we... more We hypothesized that cardiac dysfunction was responsible for the high perinatal lethality that we previously reported in fibrinogen-like protein 2 (Fgl2) knockout (KO) mice. We therefore used ultrasound biomicroscopy to assess left ventricular (LV) cardiac structure and function during development in Fgl2 KO and wild-type (WT) mice. The only deaths observed between embryonic day (E)8.5 (onset of heart beating) and postnatal day (P)28 (weaning) were within 3 days after birth, when 33% of Fgl2 KO pups died. Histopathology and Doppler assessments suggested that death was due to acute congestive cardiac failure without evidence of valvular or other obvious cardiac structural abnormalities. Heart rates in Fgl2 KO embryos were significantly reduced at E8.5 and E17.5, and irregular heart rhythms were significantly more common in Fgl2 KO (21/26) than WT (2/21) embryos at E13.5. Indexes of systolic and/or diastolic cardiac function were also abnormal in KO mice at E13.5 and E17.5, in postnatal mice studied at P1, and in KO mice surviving to P28. M-mode analysis showed no difference in LV diastolic chamber dimension, although posterior wall thickness was thinner at P7 and P28 in Fgl2 KO mice. We conclude that Fgl2 deficiency is not associated with obvious structural cardiac defects but is associated with a high incidence of neonatal death as well as contractile dysfunction and rhythm abnormalities during embryonic and postnatal development in mice.
Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. Her... more Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. Here we show that angiotensin-converting enzyme 2 (ace2) maps to a defined quantitative trait locus (QTL) on the X chromosome in three different rat models of hypertension. In all hypertensive rat strains, ACE2 messenger RNA and protein expression were markedly reduced, suggesting that ace2 is a candidate gene for this QTL. Targeted disruption of ACE2 in mice results in a severe cardiac contractility defect, increased angiotensin II levels, and upregulation of hypoxia-induced genes in the heart. Genetic ablation of ACE on an ACE2 mutant background completely rescues the cardiac phenotype. But disruption of ACER, a Drosophila ACE2 homologue, results in a severe defect of heart morphogenesis. These genetic data for ACE2 show that it is an essential regulator of heart function in vivo.
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