Dipika Tuteja

Prevention and reversal of cardiac hypertrophy by soluble epoxide hydrolase inhibitors

Sustained cardiac hypertrophy represents one of the most common causes leading to cardiac failure. There is emerging evidence to implicate the involvement of NF-κB in the development of cardiac hypertrophy. However, several critical questions remain unanswered. We tested the use of soluble epoxide hydrolase (sEH) inhibitors as a means to enhance the biological activities of epoxyeicosatrienoic acids (EETs) to treat cardiac hypertrophy. sEH catalyzes the conversion of EETs to form the corresponding dihydroxyeicosatrienoic acids. Previous data have suggested that EETs may inhibit the activation of NF-κB-mediated gene transcription. We directly demonstrate the beneficial effects of several potent sEH inhibitors (sEHIs) in cardiac hypertrophy. Specifically, we show that sEHIs can prevent the development of cardiac hypertrophy using a murine model of pressure-induced cardiac hypertrophy. In addition, sEHIs reverse the preestablished cardiac hypertrophy caused by chronic pressure overload. We further demonstrate that these compounds potently block the NF-κB activation in cardiac myocytes. Moreover, by using in vivo electrophysiologic recordings, our study shows a beneficial effect of the compounds in the prevention of cardiac arrhythmias that occur in association with cardiac hypertrophy. We conclude that the use of sEHIs to increase the level of the endogenous lipid epoxides such as EETs may represent a viable and completely unexplored avenue to reduce cardiac hypertrophy by blocking NF-κB activation.

Ablation of a Ca2+‐activated K+ channel (SK2 channel) results in action potential prolongation in atrial myocytes and atrial fibrillation

Small conductance Ca2+‐activated K+ channels (SK channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca2+(Ca2+i) with membrane potential. We have recently reported the functional existence of SK2 channels in human and mouse cardiac myocytes. Moreover, we have found that the channel is predominantly expressed in atria compared to the ventricular myocytes. We hypothesize that knockout of SK2 channels may be sufficient to disrupt the intricate balance of the inward and outward currents during repolarization in atrial myocytes. We further predict that knockout of SK2 channels may predispose the atria to tachy‐arrhythmias due to the fact that the late phase of the cardiac action potential is highly susceptible to aberrant excitation. We take advantage of a mouse model with genetic knockout of the SK2 channel gene. In vivo and in vitro electrophysiological studies were performed to probe the functional roles of SK2 channels in the heart. Whole‐cell patch‐clamp techniques show a significant prolongation of the action potential duration prominently in late cardiac repolarization in atrial myocytes from the heterozygous and homozygous null mutant animals. Morover, in vivo electrophysiological recordings show inducible atrial fibrillation in the null mutant mice but not wild‐type animals. No ventricular arrhythmias are detected in the null mutant mice or wild‐type animals. In summary, our data support the important functional roles of SK2 channels in cardiac repolarization in atrial myocytes. Genetic knockout of the SK2 channels results in the delay in cardiac repolarization and atrial arrhythmias.

Cardiac Small Conductance Ca2+-Activated K+ Channel Subunits Form Heteromultimers via the Coiled-Coil Domains in the C Termini of the Channels

Rationale: Ca2+-activated K+ channels are present in a wide variety of cells. We have previously reported the presence of small conductance Ca2+-activated K+ (SK or KCa) channels in human and mouse cardiac myocytes that contribute functionally toward the shape and duration of cardiac action potentials. Three isoforms of SK channel subunits (SK1, SK2, and SK3) are found to be expressed. Moreover, there is differential expression with more abundant SK channels in the atria and pacemaking tissues compared with the ventricles. SK channels are proposed to be assembled as tetramers similar to other K+ channels, but the molecular determinants driving their subunit interaction and assembly are not defined in cardiac tissues. Objective: To investigate the heteromultimeric formation and the domain necessary for the assembly of 3 SK channel subunits (SK1, SK2, and SK3) into complexes in human and mouse hearts. Methods and Results: Here, we provide evidence to support the formation of heteromultimeric complexes among different SK channel subunits in native cardiac tissues. SK1, SK2, and SK3 subunits contain coiled-coil domains (CCDs) in the C termini. In vitro interaction assay supports the direct interaction between CCDs of the channel subunits. Moreover, specific inhibitory peptides derived from CCDs block the Ca2+-activated K+ current in atrial myocytes, which is important for cardiac repolarization. Conclusions: The data provide evidence for the formation of heteromultimeric complexes among different SK channel subunits in atrial myocytes. Because SK channels are predominantly expressed in atrial myocytes, specific ligands of the different isoforms of SK channel subunits may offer a unique therapeutic opportunity to directly modify atrial cells without interfering with ventricular myocytes.

Functional Roles of Cav1.3(α1D) Calcium Channels in Atria Insights Gained From Gene-Targeted Null Mutant Mice

Background— Previous data suggest that L-type Ca 2+ channels containing the Ca v 1.3(α 1D ) subunit are expressed mainly in neurons and neuroendocrine cells, whereas those containing the Ca v 1.2(α 1C ) subunit are found in the brain, vascular smooth muscle, and cardiac tissue. However, our previous report as well as others have shown that Ca v 1.3 Ca 2+ channel–deficient mice ( Ca v 1.3 −/− ) demonstrate sinus bradycardia with a prolonged PR interval. In the present study, we extended our study to examine the role of the Ca v 1.3(α 1D ) Ca 2+ channel in the atria of Ca v 1.3 −/− mice. Methods and Results— We obtained new evidence to demonstrate that there is significant expression of Ca v 1.3 Ca 2+ channels predominantly in the atria compared with ventricular tissues. Whole-cell L-type Ca 2+ currents ( I Ca,L ) recorded from single, isolated atrial myocytes from Ca v 1.3 −/− mice showed a significant depolarizing shift in voltage-dependent activation. In contrast, there were no significant differences in the I Ca,L recorded from ventricular myocytes from wild-type and null mutant mice. We previously documented the hyperpolarizing shift in the voltage-dependent activation of Ca v 1.3 compared with Ca v 1.2 Ca 2+ channel subunits in a heterologous expression system. The lack of Ca v 1.3 Ca 2+ channels in null mutant mice would result in a depolarizing shift in the voltage-dependent activation of I Ca,L in atrial myocytes. In addition, the Ca v 1.3 -null mutant mice showed evidence of atrial arrhythmias, with inducible atrial flutter and fibrillation. We further confirmed the isoform-specific differential expression of Ca v 1.3 versus Ca v 1.2 by in situ hybridization and immunofluorescence confocal microscopy. Conclusions— Using gene-targeted deletion of the Ca v 1.3 Ca 2+ channel, we established the differential distribution of Ca v 1.3 Ca 2+ channels in atrial myocytes compared with ventricles. Our data represent the first report demonstrating important functional roles for Ca v 1.3 Ca 2+ channel in atrial tissues.Background—Previous data suggest that L-type Ca2+ channels containing the Cav1.3(α1D) subunit are expressed mainly in neurons and neuroendocrine cells, whereas those containing the Cav1.2(α1C) subunit are found in the brain, vascular smooth muscle, and cardiac tissue. However, our previous report as well as others have shown that Cav1.3 Ca2+ channel–deficient mice (Cav1.3−/−) demonstrate sinus bradycardia with a prolonged PR interval. In the present study, we extended our study to examine the role of the Cav1.3(α1D) Ca2+ channel in the atria of Cav1.3−/− mice. Methods and Results—We obtained new evidence to demonstrate that there is significant expression of Cav1.3 Ca2+ channels predominantly in the atria compared with ventricular tissues. Whole-cell L-type Ca2+ currents (ICa,L) recorded from single, isolated atrial myocytes from Cav1.3−/− mice showed a significant depolarizing shift in voltage-dependent activation. In contrast, there were no significant differences in the ICa,L recorded from ventricular myocytes from wild-type and null mutant mice. We previously documented the hyperpolarizing shift in the voltage-dependent activation of Cav1.3 compared with Cav1.2 Ca2+ channel subunits in a heterologous expression system. The lack of Cav1.3 Ca2+ channels in null mutant mice would result in a depolarizing shift in the voltage-dependent activation of ICa,L in atrial myocytes. In addition, the Cav1.3-null mutant mice showed evidence of atrial arrhythmias, with inducible atrial flutter and fibrillation. We further confirmed the isoform-specific differential expression of Cav1.3 versus Cav1.2 by in situ hybridization and immunofluorescence confocal microscopy. Conclusions—Using gene-targeted deletion of the Cav1.3 Ca2+ channel, we established the differential distribution of Cav1.3 Ca2+ channels in atrial myocytes compared with ventricles. Our data represent the first report demonstrating important functional roles for Cav1.3 Ca2+ channel in atrial tissues.

Beneficial effects of soluble epoxide hydrolase inhibitors in myocardial infarction model: Insight gained using metabolomic approaches

Myocardial infarction (MI) leading to myocardial cell loss represents one of the common causes leading to cardiac failure. We have previously demonstrated the beneficial effects of several potent soluble epoxide hydrolase (sEH) inhibitors in cardiac hypertrophy. sEH catalizes the conversion of epoxyeicosatrienoic acids (EETs) to form the corresponding dihydroxyeicosatrienoic acids (DHETs). EETs are products of cytochrome P450 epoxygenases that have vasodilatory properties. Additionally, EETs inhibit the activation of nuclear factor (NF)-kappaB-mediated gene transcription. Motivated by the potential to uncover a new class of therapeutic agents for cardiovascular diseases which can be effectively used in clinical setting, we directly tested the biological effects of sEH inhibitors (sEHIs) on the progression of cardiac remodeling using a clinically relevant murine model of MI. We demonstrated that sEHIs were highly effective in the prevention of progressive cardiac remodeling post MI. Using metabolomic profiling of the inflammatory lipid mediators, we documented a significant decrease in EETs/DHETs ratio in MI model predicting a heightened inflammatory state. Treatment with sEHIs resulted in a change in the pattern of lipid mediators from one of inflammation towards resolution. Moreover, the oxylipin profiling showed a striking parallel to the changes in inflammatory cytokines in this model. Our study provides evidence for a possible new therapeutic strategy to improve cardiac function post MI.

Functional roles of Cav1.3(alpha1D) calcium channels in atria: insights gained from gene-targeted null mutant mice.

Background— Previous data suggest that L-type Ca 2+ channels containing the Ca v 1.3(α 1D ) subunit are expressed mainly in neurons and neuroendocrine cells, whereas those containing the Ca v 1.2(α 1C ) subunit are found in the brain, vascular smooth muscle, and cardiac tissue. However, our previous report as well as others have shown that Ca v 1.3 Ca 2+ channel–deficient mice ( Ca v 1.3 −/− ) demonstrate sinus bradycardia with a prolonged PR interval. In the present study, we extended our study to examine the role of the Ca v 1.3(α 1D ) Ca 2+ channel in the atria of Ca v 1.3 −/− mice. Methods and Results— We obtained new evidence to demonstrate that there is significant expression of Ca v 1.3 Ca 2+ channels predominantly in the atria compared with ventricular tissues. Whole-cell L-type Ca 2+ currents ( I Ca,L ) recorded from single, isolated atrial myocytes from Ca v 1.3 −/− mice showed a significant depolarizing shift in voltage-dependent activation. In contrast, there were no significant differences in the I Ca,L recorded from ventricular myocytes from wild-type and null mutant mice. We previously documented the hyperpolarizing shift in the voltage-dependent activation of Ca v 1.3 compared with Ca v 1.2 Ca 2+ channel subunits in a heterologous expression system. The lack of Ca v 1.3 Ca 2+ channels in null mutant mice would result in a depolarizing shift in the voltage-dependent activation of I Ca,L in atrial myocytes. In addition, the Ca v 1.3 -null mutant mice showed evidence of atrial arrhythmias, with inducible atrial flutter and fibrillation. We further confirmed the isoform-specific differential expression of Ca v 1.3 versus Ca v 1.2 by in situ hybridization and immunofluorescence confocal microscopy. Conclusions— Using gene-targeted deletion of the Ca v 1.3 Ca 2+ channel, we established the differential distribution of Ca v 1.3 Ca 2+ channels in atrial myocytes compared with ventricles. Our data represent the first report demonstrating important functional roles for Ca v 1.3 Ca 2+ channel in atrial tissues.Background—Previous data suggest that L-type Ca2+ channels containing the Cav1.3(α1D) subunit are expressed mainly in neurons and neuroendocrine cells, whereas those containing the Cav1.2(α1C) subunit are found in the brain, vascular smooth muscle, and cardiac tissue. However, our previous report as well as others have shown that Cav1.3 Ca2+ channel–deficient mice (Cav1.3−/−) demonstrate sinus bradycardia with a prolonged PR interval. In the present study, we extended our study to examine the role of the Cav1.3(α1D) Ca2+ channel in the atria of Cav1.3−/− mice. Methods and Results—We obtained new evidence to demonstrate that there is significant expression of Cav1.3 Ca2+ channels predominantly in the atria compared with ventricular tissues. Whole-cell L-type Ca2+ currents (ICa,L) recorded from single, isolated atrial myocytes from Cav1.3−/− mice showed a significant depolarizing shift in voltage-dependent activation. In contrast, there were no significant differences in the ICa,L recorded from ventricular myocytes from wild-type and null mutant mice. We previously documented the hyperpolarizing shift in the voltage-dependent activation of Cav1.3 compared with Cav1.2 Ca2+ channel subunits in a heterologous expression system. The lack of Cav1.3 Ca2+ channels in null mutant mice would result in a depolarizing shift in the voltage-dependent activation of ICa,L in atrial myocytes. In addition, the Cav1.3-null mutant mice showed evidence of atrial arrhythmias, with inducible atrial flutter and fibrillation. We further confirmed the isoform-specific differential expression of Cav1.3 versus Cav1.2 by in situ hybridization and immunofluorescence confocal microscopy. Conclusions—Using gene-targeted deletion of the Cav1.3 Ca2+ channel, we established the differential distribution of Cav1.3 Ca2+ channels in atrial myocytes compared with ventricles. Our data represent the first report demonstrating important functional roles for Cav1.3 Ca2+ channel in atrial tissues.

The effects of intracellular Ca2+ on cardiac K+ channel expression and activity: novel insights from genetically altered mice

We tested the hypothesis that chronic changes in intracellular Ca2+ (Ca2+i) can result in changes in ion channel expression; this represents a novel mechanism of crosstalk between changes in Ca2+ cycling proteins and the cardiac action potential (AP) profile. We used a transgenic mouse with cardiac‐specific overexpression of sarcoplasmic reticulum Ca2+ ATPase (SERCA) isoform 1a (SERCA1a OE) with a significant alteration of SERCA protein levels without cardiac hypertrophy or failure. Here, we report significant changes in the expression of a transient outward K+ current (Ito,f), a slowly inactivating K+ current (IK,slow) and the steady state current (ISS) in the transgenic mice with resultant prolongation in cardiac action potential duration (APD) compared with the wild‐type littermates. In addition, there was a significant prolongation of the QT interval on surface electrocardiograms in SERCA1a OE mice. The electrophysiological changes, which correlated with changes in Ca2+i, were further corroborated by measuring the levels of ion channel protein expression. To recapitulate the in vivo experiments, the effects of changes in Ca2+i on ion channel expression were further tested in cultured adult and neonatal mouse cardiac myocytes. We conclude that a primary defect in Ca2+ handling proteins without cardiac hypertrophy or failure may produce profound changes in K+ channel expression and activity as well as cardiac AP.

Functional interaction with filamin A and intracellular Ca2+ enhance the surface membrane expression of a small-conductance Ca2+-activated K+ (SK2) channel

Significance The precise subcellular localization of ion channel proteins is necessary for the proper function of excitable cells. The trafficking of several ion channels is dependent on the interaction of the ion channel proteins with cytoskeletal proteins, underpinned by a number of diseases in which the defect lies with the interacting proteins. Here, we demonstrate the role of filamin A, a cytoskeletal protein, in augmenting the membrane expression of small-conductance, Ca2+-activated K+ channels (KCa2.2 or SK2) in atrial myocytes. We further demonstrate that SK2 channel trafficking is Ca2+-dependent in the presence of another cytoskeletal protein, α-actinin2, thereby establishing the role of filamin A, α-actinin2, and intracellular Ca2+ in trafficking of SK2 channels. The findings may have implications in other excitable cells. For an excitable cell to function properly, a precise number of ion channel proteins need to be trafficked to distinct locations on the cell surface membrane, through a network and anchoring activity of cytoskeletal proteins. Not surprisingly, mutations in anchoring proteins have profound effects on membrane excitability. Ca2+-activated K+ channels (KCa2 or SK) have been shown to play critical roles in shaping the cardiac atrial action potential profile. Here, we demonstrate that filamin A, a cytoskeletal protein, augments the trafficking of SK2 channels in cardiac myocytes. The trafficking of SK2 channel is Ca2+-dependent. Further, the Ca2+ dependence relies on another channel-interacting protein, α-actinin2, revealing a tight, yet intriguing, assembly of cytoskeletal proteins that orchestrate membrane expression of SK2 channels in cardiac myocytes. We assert that changes in SK channel trafficking would significantly alter atrial action potential and consequently atrial excitability. Identification of therapeutic targets to manipulate the subcellular localization of SK channels is likely to be clinically efficacious. The findings here may transcend the area of SK2 channel studies and may have implications not only in cardiac myocytes but in other types of excitable cells.

Cardiac Small Conductance Ca 2+ -Activated K + Channel Subunits Form Heteromultimers via the Coiled-Coil Domains in the C Termini of the Channels

Rationale: Ca2+-activated K+ channels are present in a wide variety of cells. We have previously reported the presence of small conductance Ca2+-activated K+ (SK or KCa) channels in human and mouse cardiac myocytes that contribute functionally toward the shape and duration of cardiac action potentials. Three isoforms of SK channel subunits (SK1, SK2, and SK3) are found to be expressed. Moreover, there is differential expression with more abundant SK channels in the atria and pacemaking tissues compared with the ventricles. SK channels are proposed to be assembled as tetramers similar to other K+ channels, but the molecular determinants driving their subunit interaction and assembly are not defined in cardiac tissues. Objective: To investigate the heteromultimeric formation and the domain necessary for the assembly of 3 SK channel subunits (SK1, SK2, and SK3) into complexes in human and mouse hearts. Methods and Results: Here, we provide evidence to support the formation of heteromultimeric complexes among different SK channel subunits in native cardiac tissues. SK1, SK2, and SK3 subunits contain coiled-coil domains (CCDs) in the C termini. In vitro interaction assay supports the direct interaction between CCDs of the channel subunits. Moreover, specific inhibitory peptides derived from CCDs block the Ca2+-activated K+ current in atrial myocytes, which is important for cardiac repolarization. Conclusions: The data provide evidence for the formation of heteromultimeric complexes among different SK channel subunits in atrial myocytes. Because SK channels are predominantly expressed in atrial myocytes, specific ligands of the different isoforms of SK channel subunits may offer a unique therapeutic opportunity to directly modify atrial cells without interfering with ventricular myocytes.

Pregnancy in Postural Orthostatic Tachycardia Syndrome

Introduction: Postural orthostatic tachycardia syndrome (POTS) is a rare disease characterized by syncope, sinus tachycardia, and orthostasis due to autonomic dysfunction.